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Sample records for lactis subsp lactis

  1. Tulum Peynirlerinden izole Edilen Lactococcus lactis subsp. lactis YBML9 ve

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    Yasin TUNCER

    2009-04-01

    Full Text Available Bu çalısmanın amacı tulum peynirlerinden izole edilen Lactococcus lactis suslarının fenotipik tanısı ve bu suslar tarafından üretilen bakteriyosinlerin kısmi karakterizasyonlarıdır. Bu amaçla Türkiye'nin sekiz farklı ilinden (Ankara, Antalya, Burdur, Denizli, Erzincan, Isparta, İstanbul ve İzmir yöresel pazarlardan toplanan 60 adet tulum peyniri örneginden 40 adet Lactococcus lactis susu (31 adet L. lactis subsp. lactis ve 9 adet L. lactis subsp. cremoris izole edildi. 40 adet L. lactis susu içerisinden, 2 adet L. lactis subsp. lactis (YBML9 ve YBML21 susu bakteriyosin üretme yeteneginde bulundu. L. lactis subsp. lactis YBML9 ve YBML21 susları tarafından üretilen bakteriyosinler, farklı enzim, pH ve sıcaklık uygulamaları sonucu; sırasıyla nisin ve laktisin 481 olarak tanımlandı.

  2. Comparative Phenotypic and Molecular Genetic Profiling of Wild Lactococcus lactis subsp. lactis Strains of the L. lactis subsp. lactis and L. lactis subsp. cremoris Genotypes, Isolated from Starter-Free Cheeses Made of Raw Milk▿

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    Fernández, Elena; Alegría, Ángel; Delgado, Susana; Martín, M. Cruz; Mayo, Baltasar

    2011-01-01

    Twenty Lactococcus lactis strains with an L. lactis subsp. lactis phenotype isolated from five traditional cheeses made of raw milk with no added starters belonging to the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes (lactis and cremoris genotypes, respectively; 10 strains each) were subjected to a series of phenotypic and genetic typing methods, with the aims of determining their phylogenetic relationships and suitability as starters. Pulsed-field gel electrophoresis (PFGE) analysis of intact genomes digested with SalI and SmaI proved that all strains were different except for three isolates of the cremoris genotype, which showed identical PFGE profiles. Multilocus sequence typing (MLST) analysis using internal sequences of seven loci (namely, atpA, rpoA, pheS, pepN, bcaT, pepX, and 16S rRNA gene) revealed considerable intergenotype nucleotide polymorphism, although deduced amino acid changes were scarce. Analysis of the MLST data for the present strains and others from other dairy and nondairy sources showed that all of them clustered into the cremoris or lactis genotype group, by using both independent and combined gene sequences. These two groups of strains also showed distinctive carbohydrate fermentation and enzyme activity profiles, with the strains in the cremoris group showing broader profiles. However, the profiles of resistance/susceptibility to 16 antibiotics were very similar, showing no atypical resistance, except for tetracycline resistance in three identical cremoris genotype isolates. The numbers and concentrations of volatile compounds produced in milk by the strains belonging to these two groups were clearly different, with the cremoris genotype strains producing higher concentrations of more branched-chain, derived compounds. Together, the present results support the idea that the lactis and cremoris genotypes of phenotypic Lactococcus lactis subsp. lactis actually represent true subspecies. Some strains of the two subspecies

  3. Cloning, Sequencing, and Expression of the Pyruvate Carboxylase Gene in Lactococcus lactis subsp. lactis C2†

    OpenAIRE

    Wang, H.; O'Sullivan, D. J.; Baldwin, K. A.; McKay, L. L.

    2000-01-01

    A functional pyc gene was isolated from Lactococcus lactis subsp. lactis C2 and was found to complement a Pyc defect in L. lactis KB4. The deduced lactococcal Pyc protein was highly homologous to Pyc sequences of other bacteria. The pyc gene was also detected in Lactococcus lactis subsp. cremoris and L. lactis subsp. lactis bv. diacetylactis strains.

  4. Protein Profile and Plasmid Content of Lactococcus lactis subsp. lactis LL52 and Lactococcus lactis subsp. cremoris LC79 Strains under Several Stress Conditions

    OpenAIRE

    LALE, Rahmi; TÜKEL, Çağla; AKÇELİK, Mustafa

    2014-01-01

    Differences in the protein and plasmid content of 2 Lactococcus lactis strains, L. lactis subsp. lactis LL52 and L. lactis subsp. cremoris LC79, under the stresses of high and low temperature, osmotic shock, and low pH were determined. We identified 3 new proteins with molecular masses of 16.0, 29.4, and 45.0 kDa as high temperature stress response specific in strain LL52. High temperature stress did not cause any changes in the protein content of strain LC79. Proteins that were specific for ...

  5. Diversity of the subspecies Bifidobacterium animalis subsp. lactis.

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    Bunesova, Vera; Killer, Jiri; Javurkova, Barbora; Vlkova, Eva; Tejnecky, Vaclav; Musilova, Sarka; Rada, Vojtech

    2017-04-01

    Strains of Bifidobacterium animalis subsp. lactis are well-known health-promoting probiotics used commercially. B. animalis subsp. lactis has been isolated from different sources, and little is known about animal isolates of this taxon. The aim of this study was to examine the genotypic and phenotypic diversity between B. animalis subsp. lactis strains different animal hosts including Cameroon sheep, Barbary sheep, okapi, mouflon, German shepard and to compare to BB12, food isolates and the collection strain DSM 10140. Ten strains of B. animalis subsp. lactis from different sources were characterised by phenotyping, fingerprinting, and multilocus sequence typing (MLST). Regardless of origin, MLST and phylogenetic analyses revealed a close relationship between strains of B. animalis subsp. lactis with commercial and animal origin with the exception of isolates from ovine cheese, mouflon and German Shepard dog. Moreover, isolates from dog and mouflon showed significant differences in fermentation profiles and peptide mass fingerprints (MALDI-TOF). Results indicated phenotypic and genotypic diversity among strains of B. animalis subsp. lactis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Phenotypic variation in Lactococcus lactis subsp. lactis isolates derived from intestinal tracts of marine and freshwater fish.

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    Itoi, S; Yuasa, K; Washio, S; Abe, T; Ikuno, E; Sugita, H

    2009-09-01

    We compared phenotypic characteristics of Lactococcus lactis subsp. lactis derived from different sources including the intestinal tract of marine fish and freshwater fish, and cheese starter culture. In the phylogenetic analysis based on partial 16S rRNA gene nucleotide sequences (1371 bp), freshwater fish-, marine fish- and cheese starter culture-derived strains were identical to that of L. lactis subsp. lactis previously reported. Fermentation profiles determined using the API 50 CH system were similar except for fermentation of several sugars including l-arabinose, mannitol, amygdalin, saccharose, trehalose, inulin and gluconate. The strains did have distinct levels of halotolerance: marine fish-derived strains > cheese starter-derived strain > freshwater fish-derived isolate. Lactococcus lactis subsp. lactis showed extensive diversity in phenotypic adaptation to various environments. The phenotypic properties of these strains suggested that L. lactis subsp. lactis strains from fish intestine have additional functions compared with the cheese starter-derived strain that has previously described. The unique phenotypic traits of the fish intestinal tract-derived L. lactis subsp. lactis might make them useful as a probiotics in aquaculture, and contribute to the development of functional foods and novel food additives, since the strains derived from fish intestines might have additional functions such as antibacterial activity.

  7. Genome Sequence of Lactococcus lactis subsp. lactis NCDO 2118, a GABA-Producing Strain

    DEFF Research Database (Denmark)

    Oliveira, Letícia C; Saraiva, Tessália D L; Soares, Siomar C

    2014-01-01

    Lactococcus lactis subsp. lactis NCDO 2118 is a nondairy lactic acid bacterium, a xylose fermenter, and a gamma-aminobutyric acid (GABA) producer isolated from frozen peas. Here, we report the complete genome sequence of L. lactis NCDO 2118, a strain with probiotic potential activity....

  8. Cadmium tolerant characteristic of a newly isolated Lactococcus lactis subsp. lactis.

    Science.gov (United States)

    Sheng, Yao; Wang, Ying; Yang, Xuan; Zhang, Boyang; He, Xiaoyun; Xu, Wentao; Huang, Kunlun

    2016-12-01

    Environmental contamination caused by heavy metals poses a major threat to the wildlife and human health for their toxicity and intrinsically persistent nature. Some specific food grade bacteria have properties that enable them to eliminate heavy metals from food and water. Lactococcus lactis subsp. lactis, newly isolated from pickles, is a cadmium (Cd) tolerant bacteria. Cd resistant properties of the lactis was evaluated under different Cd stresses. Cd accumulation in different cellular parts was determined by ICP-MS and cell morphology changes were measured by SEM-EDS and TEM-EDS. In addition, functional groups associated with Cd resistance were detected by infrared spectroscopic analysis. The results indicated that Cd mainly accumulated in the cell surface structures including cytoderm and cytomembrane. Functional groups such as OH and NH 2 in the cell surface played essential roles in Cd biosorption. The elements of O, P, S, and N of polysaccharide, membrane protein and phosphatidate in the cell surface structures might be responsible for Cd biosorption for their strong electronegativity. This study indicated that ultrastructural analysis can be a supplemental method to study heavy metal resistance mechanism of microorganism and the newly isolated lactococcus lactis subsp. lactis has great potential to be applied to decontamination of heavy metals. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Comparison of the acidifying activity of Lactococcus lactis subsp. lactis strains isolated from goat's milk and Valdeteja cheese.

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    Alonso-Calleja, C; Carballo, J; Capita, R; Bernardo, A; García-López, M L

    2002-01-01

    This work was carried out to study the acid production by Lactococcus lactis subsp. lactis strains isolated from goat's milk and goat cheese (Valdeteja variety) in order to select a suitable starter culture for industrial goat cheese manufacturing. The titrable acidity of 45 Lactococcus lactis subsp. lactis strains isolated from a home-made batch of Valdeteja cheese with excellent sensory characteristics was measured over a period of 18 h. The strains were divided into two groups depending on the acid production rate: 20 fast acid producer (F) strains and 25 slow acid producer (S) strains. The kinetic parameters (lag phase, maximum acid production rate and value of upper asymptote curve) of the acid production curves for F and S strains were significantly (P titrable acidity of F and S strains were observed after the second hour of incubation. An F strain acetoin producer (Lactococcus lactis subsp. lactis 470Ch2) was selected as autochthonous starter culture for industrial Valdeteja goat cheese manufacturing.

  10. Estudo dos parâmetros da ultrafiltração de permeado de soro de queijo fermentado por Lactococcus lactis subsp. lactis Ultrafiltration conditions of whey permeate fermented by Lactococcus lactis subsp. lactis

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    Viviane BRONSTEIN

    1998-04-01

    Full Text Available Permeado de soro doce, suplementado com extrato de levedura e peptona, foi utilizado como meio de crescimento para Lactococcus lactis subsp. lactis. No final da fase exponencial de crescimento, o meio de cultura fermentado foi submetido a uma ultrafiltração com o objetivo de concentrar o microrganismo. Foram realizados 6 processamentos diferentes, nos quais variou-se as condições iniciais da ultrafiltração, tendo sido avaliados os seguintes parâmetros: porosidade da membrana, pH e número de células viáveis no permeado e no retentado, a fim de ser estudado a influência de cada parâmetro na taxa de permeação da ultrafiltração. As membranas utilizadas foram eficazes como meio de barragem para o microrganismo Lactococcus lactis subsp. lactis, ficando o retentado com uma média celular de 10(8 ufc/ml e o permeado com uma média celular de 10² ufc/ml. Membranas de diferentes porosidades tiveram taxas de fluxo semelhantes. O aumento da concentração celular provocou a diminuição do fluxo. O pH também influenciou a taxa de permeação, havendo um aumento do fluxo quando foi utilizado um pH inicial mais alto.Cheese whey permeate supplemented with yeast extract and peptone was used as a growth medium for the bacteria Lactococcus lactis subsp. lactis. At the end of the exponential growth phase, the fermented growth medium was ultrafiltered to concentrate the microorganism and to evaluate the effect of the membrane porosity, inicial UF pH and cellular concentration in permeation rate during the ultrafiltration process. The membranes used were efficient as a mean of a barrage for the Lactococcus lactis subsp. lactis. On average, the cellular concentrations were 10(8 CFU/mL and 10² CFU/mL for retentate and permeate, respectively. Membranes of different porosities had very similar flux rates. Better flow rates were obtained with inicial UF pH 6,5 and with the minors micrrorganism concentration.

  11. Lactococcus lactis subsp. tructae subsp. nov. isolated from the intestinal mucus of brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss).

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    Pérez, Tania; Balcázar, José Luis; Peix, Alvaro; Valverde, Angel; Velázquez, Encarna; de Blas, Ignacio; Ruiz-Zarzuela, Imanol

    2011-08-01

    The species Lactococcus lactis currently includes three subspecies; L. lactis subsp. lactis and L. lactis subsp. cremoris, isolated from milk sources, and L. lactis subsp. hordniae, isolated from the leafhopper Hordnia circellata. In this study, three strains, designated L105(T), I3 and L101, were isolated from the intestinal mucus of brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss). These strains were closely related to members of the species Lactococcus lactis. Strain L105(T) showed 99.4 % 16S rRNA gene sequence similarity to that of the type strains L. lactis subsp. lactis NCDO 604(T) and L. lactis subsp. hordniae NCDO 2181(T) and showed 99.9 % similarity to the type strain Lactococcus lactis subsp. cremoris NCDO 607(T). Analysis of two housekeeping genes, rpoB and recA, confirmed the close relationship between the novel strains and L. lactis subsp. cremoris with similarities of 99.3 and 99.7 %, respectively. The three strains could, however, be differentiated from their closest relatives on the basis of several phenotypic characteristics, as was the case for L. lactis subsp. lactis and L. lactis subsp. hordniae, which were also closely related on the basis of 16S rRNA, rpoB and recA gene sequence similarities. The strains isolated in this study represent a new subspecies, for which the name Lactococcus lactis subsp. tructae subsp. nov. is proposed. The type strain is L105(T) ( = LMG 24662(T)  = DSM 21502(T)).

  12. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: characterization of the bacteriocin

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    Danielle N. Furtado

    2014-12-01

    Full Text Available Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi was isolated from goat milk, and studied for its antimicrobial activity. The bacteriocin presented a broad spectrum of activity, was sensitive to proteolytic enzymes, resistant to heat and pH extremes, and not affected by the presence of SDS, Tween 20, Tween 80, EDTA or NaCl. Bacteriocin production was dependent on the components of the culture media, especially nitrogen source and salts. When tested by PCR, the bacteriocin gene presented 100% homology to nisin Z gene. These properties indicate that this L. lactis subsp. lactis DF4Mi can be used for enhancement of dairy foods safety and quality.

  13. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Characterization of the bacteriocin

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    Furtado, Danielle N.; Todorov, Svetoslav D.; Landgraf, Mariza; Destro, Maria T.; Franco, Bernadette D.G.M.

    2014-01-01

    Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its antimicrobial activity. The bacteriocin presented a broad spectrum of activity, was sensitive to proteolytic enzymes, resistant to heat and pH extremes, and not affected by the presence of SDS, Tween 20, Tween 80, EDTA or NaCl. Bacteriocin production was dependent on the components of the culture media, especially nitrogen source and salts. When tested by PCR, the bacteriocin gene presented 100% homology to nisin Z gene. These properties indicate that this L. lactis subsp. lactis DF4Mi can be used for enhancement of dairy foods safety and quality. PMID:25763065

  14. Draft Genome Sequence of the Putrescine-Producing Strain Lactococcus lactis subsp. lactis 1AA59

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    del Rio, Beatriz; Linares, Daniel M.; Fernandez, María; Mayo, Baltasar; Martín, M. Cruz

    2015-01-01

    We report here the 2,576,542-bp genome annotated draft assembly sequence of Lactococcus lactis subsp. lactis 1AA59. This strain—isolated from a traditional cheese—produces putrescine, one of the most frequently biogenic amines found in dairy products. PMID:26089428

  15. Introduction of peptidase genes from Lactobacillus delbrueckii subsp. lactis into Lactococcus lactis and controlled expression

    NARCIS (Netherlands)

    Wegmann, U.; Klein, J.R.; Drumm, I.; Kuipers, O.P.; Henrich, B.

    Peptidases PepI, PepL, PepW, and PepG from Lactobacillus delbrueckii subsp, lactis, which have no counterparts in Lactococcus lactis, and peptidase PepQ were examined to determine their potential to confer new peptidolytic properties to lactococci, Controllable expression of the corresponding genes

  16. [A comparison of the properties of bacteriocins formed by Lactococcus lactis subsp. lactis strains of diverse origin].

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    Stoianova, L G; Egorov, N S; Fedorova, G B; Katrukha, G S; Netrusov, A I

    2007-01-01

    Bacteriocins formed by four strains of Lactococcus lactis subsp. lactis have been studied and compared: 729 (a natural strain isolated from milk), 1605 (a mutant of strain 729), F-116 (a recombinant obtained by fusing of protoplasts of the two related strain 729 and 1605), and a nisin-forming strain obtained by adaptive selection at Moscow State University. Antimicrobial activity studies revealed differences between the strains in the effects on individual groups of microorganisms; the activities of the strains were also distinct from that of Nisaplin (a commercial preparation of the bacteriocin nisin). Methods for isolation and purification of bacteriocins have been developed, making it possible to obtain individual components of antibiotic complexes as chromatographically pure preparations. Bacteriocins formed by the strains of Lactococcus lactis subsp. lactis have been identified and differences in their biological and physicochemical properties, established. A novel potent broad-spectrum antibiotic substance distinct from nisin has been isolated from the recombinant strain F-116.

  17. Characterization of a cadmium resistance Lactococcus lactis subsp. lactis strain by antioxidant assays and proteome profiles methods.

    Science.gov (United States)

    Sheng, Yao; Yang, Xuan; Lian, Yuanyuan; Zhang, Boyang; He, Xiaoyun; Xu, Wentao; Huang, Kunlun

    2016-09-01

    Heavy metal contamination poses a major threat to the environment and human health for their potential toxicity and non-biodegradable properties. At present, some probiotics bacteria are reported to have great potential to eliminate heavy metals from food and water. In this study, resistance properties of a newly isolated Lactococcus lactis subsp. lactis for cadmium were studied by antioxidant assays and proteomics analysis. Antioxidant capacity of this strain was significantly activated under cadmium stress indicated by Fenton reaction, DPPH assay, SOD assay and GSH assay. Intracellular antioxidant enzyme systems, such as superoxide dismutase, glutathione reductase and catalase were suggested to play vital roles in the activated antioxidant capacity. The up-regulated cadA was associated with the activated P-type ATPases that plays an important role in cadmium resistance. Proteomics analysis identified 12 over-expressed proteins under 50mg/L cadmium stress and these proteins are abundant in oxidative stress response and energy metabolism regulation, which were considered as consequences as cadmium resistance of the strain. Thus, the probiotics Lactococcus lactis subsp. lactis may resist cadmium stress through antioxidant approach and enhanced energy metabolism. The food grade lactis strain may be applied in metal decontamination in environment and food/feed. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Lactococcus lactis subsp. lactis MA83 Suşunda Aktif Bir Faj Dirençlilik Sisteminin Genetik ve Biyokimyasal Doğası

    OpenAIRE

    Tükel, Çağla; Akçelik, Mustafa

    2003-01-01

    Lactococcus lactis subsp. lactis MA83 susunda fajlann adsorbsiyonu, bu bakteride 32.7 kb büyüklükteki plazmidin varlığında üretilen ekzopolisakkarit materyal tarafından engellendi. Kimyasal analizler sonucunda bu ekzopolisakkarit materyalin ana bileşenlerinin galaktoz, galaktozamin, ramnoz ve fosfat olduğu belirlendi. Ayrıca, L. lactis subsp. lactis MA83 susunda Øla2, Øp78, Ør4 ve Øp81 fajlannın almaç bölgelerinin protein yapıda olduğu saptandı.  

  19. Effect of Potential Probiotic Lactococcus lactis Subsp. lactis on Growth Performance, Intestinal Microbiota, Digestive Enzyme Activities, and Disease Resistance of Litopenaeus vannamei.

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    Adel, Milad; El-Sayed, Abdel-Fattah M; Yeganeh, Sakineh; Dadar, Maryam; Giri, Sib Sankar

    2017-06-01

    The aims of this study were to evaluate the effects of Lactococcus lactis subsp. lactis on the growth, intestinal microbiota, digestive enzyme activity, and disease resistance of Litopenaeus vannamei. Diets containing four different concentrations of L. lactis (0 [basal diet], 10 6 , 10 7 , and 10 8  CFU g -1 ) were fed to white shrimps L. vannamei (average weight 5.89 ± 0.36 g) for 8 weeks. At the end of the feeding trial, shrimps were immersed in Caspian Seawater (10.8 ppt) contaminated with 10 6  CFU ml -1 pathogenic V. anguillarum for 2 h. Results revealed that growth rate, survival, and body protein level were increased with dietary supplementation of L. lactis. The activities of digestive enzymes (cellulose, lipase, amylase, and protease) were significantly higher in the groups fed with diets containing 10 7 or 10 8  CFU g -1 L. lactis than those in the control. The Lactobacillus and Bacillus counts were higher (P lactis-supplemented diets. In addition, higher level of L. lactis supplementation decreased the Vibrio counts. Moreover, L. vannamei fed diet supplemented with 10 8  CFU g -1 of L. lactis exhibited significantly the highest hematocyte count and post-challenge survival rate (79.2 %). Collectively, these results suggest that dietary supplementation of L. lactis subsp. lactis at 10 8  CFU g -1 can promote growth performance, digestive enzyme activity, and disease resistance of L. vannamei.

  20. Quantitative assessment of Lactococcus lactis subsp. cremoris present in artisanal raw cow’s milk cheese

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    Milena Alicja Stachelska

    2018-01-01

    Full Text Available Lactococcus lactis subsp. cremoris belongs to lactic acid bacteria that play a crucial role in cheese production and it is known to be beneficial to human health. The aim of the study was to establish a rapid and accurate quantitative real-time polymerase chain reaction (qPCR method to detect and enumerate L. lactis subsp. cremoris in artisanal raw cow’s milk cheese. Artisanal raw cow’s milk cheese samples were used to check for presence and number of L. lactis subsp. cremoris strains. The method applies a set of target-specific PCR (polymerase chain reaction primers and a fluorogenic probe, and amplifies a part of the LACR_RS01280 gene that encodes the aminoacetone oxidase family flavin adenine dinucleotide (FAD binding enzyme. All 5 L. lactis subsp. cremoris strains examined were found to be qPCR positive. There was no signal recorded for 8 strains which belong to closely related species. The limit of detection amounted to ten copies per reaction and the assay indicated a linear dynamic range of seven logs. This method may be applied in detection and enumeration of L. lactis subsp. cremoris in cheese during its ripening. Moreover, it may be applied to examine the distribution of L. lactis subsp. cremoris during the cheese production and ripening.

  1. Efeito e modo de ação das bacteriocinas produzidas por Lactococcus lactis subsp. lactis ITAL 383, ATCC 11454 e CNRZ 150 contra Listeria innocua LIN 11 Effect and mode of action of the bacterioncin produced by Lactococcus. lactis subsp. lactis ITAL 383, ATCC 11454 e CNRZ 150 against Listeria innocua LIN 11

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    Izildinha MORENO

    1999-01-01

    Full Text Available O efeito e o modo de ação das bacteriocinas produzidas por L. lactis subsp. lactis ITAL 383 e CNRZ 150 são similares à nisina de L. lactis subsp. lactis ATCC 11454. Estas bacteriocinas apresentaram um modo de ação bactericida, causando a lise de células de L. innocua LIN 11, associada ao decréscimo da absorbância e da viabilidade celular. O efeito letal foi maior para células em fase exponencial comparativamente à fase estacionária de crescimento. A adsorção dessas bacteriocinas às células de L. innocua LIN 11 foi muito rápida e influenciada pelo pH do meio de suspensão; adsorção máxima foi verificada a pH 6,0 e logo após o contato inicial. Perda completa de adsorção ocorreu em pH 2,0.The effect and mode of action of the bacteriocin produced by L. lactis subsp. lactis ITAL 383 and CNRZ 150 are similar to the nisin produced by L. lactis subsp. lactis ATCC 11454. It was clearly bactericidal, and caused lysis of a strain of L. innocua LIN 11 detected by the decrease of absorbance values and the cell viability. Their lethal effect was considerably higher during the logarithmic growth when compared to the stationary phase. Adsorption developed rapidly and was influenced by the pH value of the suspension medium. Maximum adsorption was observed at pH 6,0 and immediately after initial contact and loss at pH 2,0.

  2. Inside the adaptation process of Lactobacillus delbrueckii subsp. lactis to bile

    OpenAIRE

    Burns, Patricia; Sánchez García, Borja; Vinderola, Gabriel; Ruas-Madiedo, Patricia; Ruíz García, Lorena; Margolles Barros, Abelardo; Reinheimer, Jorge A.; González de los Reyes-Gavilán, Clara

    2010-01-01

    Progressive adaptation to bile might render some lactobacilli able to withstand physiological bile salt concentrations. In this work, the adaptation to bile was evaluated on previously isolated dairy strains of Lactobacillus delbrueckii subsp. lactis 200 and L. delbrueckii subsp. lactis 200+, a strain derived thereof with stable bile-resistant phenotype. The adaptation to bile was obtained by comparing cytosolic proteomes of both strains grown in the presence or absence of bile. Proteomics we...

  3. Characterization of a Wild, Novel Nisin A-Producing Lactococcus Strain with an L. lactis subsp. cremoris Genotype and an L. lactis subsp. lactis Phenotype, Isolated from Greek Raw Milk

    Science.gov (United States)

    Parapouli, Maria; Delbès-Paus, Céline; Kakouri, Athanasia; Koukkou, Anna-Irini; Montel, Marie-Christine

    2013-01-01

    Several molecular taxonomic studies have revealed that many natural (wild) Lactococcus lactis strains of dairy origin which are phenotypically representative of the L. lactis subspecies lactis cluster genotypically within subspecies cremoris and vice versa. Recently, we isolated two wild nisin-producing (Nis+) L. lactis strains, M78 and M104, of the lactis phenotype from Greek raw milk (J. Samelis, A. Lianou, A. Kakouri, C. Delbès, I. Rogelj, B. B. Matijašic, and M. C. Montel, J. Food Prot. 72:783–790, 2009); strain M78 possess a novel nisin A sequence (GenBank accession number HM219853). In this study, the actual subspecies identity of M78 and M104 isolates was elucidated, using 16S rRNA and acmA (encoding lactococcal N-acetylmuramidase) gene and histidine biosynthesis operon polymorphisms and 16S rRNA and ldh (encoding lactate dehydrogenase) gene phylogenies. Except the acmA gene analysis, molecular tools revealed that isolates M78 and M104 clustered with strains of the cremoris genotype, including the LMG 6897T strain, while they were distant from strains of the lactis genotype, including the LMG 6890T strain. The two wild isolates had identical repetitive sequence-based PCR (rep-PCR), randomly amplified polymorphic DNA (RAPD), plasmid, and whole-cell protein profiles and shared high 16S rRNA (99.9%) and ldh (100%) gene sequence homologies. In contrast, they exhibited identical sugar fermentation and enzymatic patterns which were similar to those of the subspecies lactis LMG 6890T strain. To our knowledge, this is the first complete identification report on a wild L. lactis subsp. cremoris genotype of the lactis phenotype which is capable of nisin A production and, thus, has strong potential for use as a novel dairy starter and/or protective culture. PMID:23542625

  4. Suitability of Lactococcus lactis subsp lactis ATCC 11454 as a protective culture for lightly preserved fish products

    DEFF Research Database (Denmark)

    Wessels, Stephen Wallace; Huss, Hans Henrik

    1996-01-01

    This study is part of strategy to control the human pathogen Listeria monocytogenes in lightly preserved fish products by using food-grade lactic acid bacteria. When the nisin-producing Lactococcus lactis subsp lactis ATCC 11454 was cultured in the same vessel as L-monocytogenes Scott A in brain......-heart infusion broth (BHI) at 30-degrees C, the pathogen declined from 5x10(5) to fewer than 5 cfu ml(-1) within 31 h. The effect was not due to lactic acid inhibition. Growth and nisin production by L- lactis ATCC 11454 were investigated under the conditions of temperature and salt used for light preservation...... and no detectable nisin. On slices of commercial cold-smoked salmon at 10-degrees C, no net propagation pf L-lactis ATCC 11454 could be detected within 21 days. However, when salmon slices were inoculated with L- mycocytogenes at 10(4) cfu g(-1) and a 300-fold excess of washed lactococcus cells, the pathogen...

  5. Differential expression of proteins and genes in the lag phase of Lactococcus lactis subsp lactis grown in synthetic medium and reconstituted skim milk

    DEFF Research Database (Denmark)

    Larsen, N.; Boye, Mette; Jakobsen, Marianne

    2006-01-01

    We investigated protein and gene expression in the lag phase of Lactococcus lactis subsp. lactis CNRZ 157 and compared it to the exponential and stationary phases. By means of two-dimensional polyacrylamide gel electrophoresis, 28 highly expressed lag-phase proteins, implicated in nucleotide meta...

  6. Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation

    Energy Technology Data Exchange (ETDEWEB)

    McIntyre, D.A.; Harlander, S.K. (Univ. of Minnesota, St. Paul (USA))

    1989-03-01

    The objective of this study was to develop a system for electroporating intact cells of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) with a commercially available electroporation unit. Parameters which influenced the efficiency of transformation included growth phase and final concentration of cells, ionic strength of the suspending medium, concentration of plasmid DNA, and the amplitude and duration of the pulse. Washed suspensions of intact cells suspended in deionized distilled water were subjected to one high-voltage electric pulse varying in voltage (300 to 900 V corresponding to field strengths of 5 to 17 kV/cm) and duration (100 {mu}s to 1 s). Transformation efficiencies of 10{sup 3} transformants per {mu}g of DNA were obtained when dense suspensions (final concentration, 5 {times} 10{sup 10} CFU/ml) of stationary-phase cells were subjected to one pulse with a peak voltage of 900 V (field strength, 17 kV/cm) and a pulse duration of 5 ms in the presence of plasmid DNA. Dilution of porated cells in broth medium followed by an expression period of 2 h at 30{degree}C was beneficial in enhancing transformation efficiencies. Plasmids ranging in size from 9.8 to 30.0 kilobase pairs could be transformed by this procedure.

  7. Detection and characterization of bacteriocin-producing Lactococcus lactis strains Detecção e caracterização de Lactococcus lactis produtores de bacteriocinas

    Directory of Open Access Journals (Sweden)

    Izildinha Moreno

    1999-04-01

    Full Text Available One hundred sixty seven strains of Lactococcus lactis were screened for bacteriocin production by well diffusion assay of GM17 agar. Fourteen (8.4% produced antimicrobial activity other than organic acids, bacteriophages or hydrogen peroxide. The frequency of bacteriocin production ranged from 2% in L. lactis subsp. cremoris up to 12% in L. lactis subsp. lactis. Antimicrobial activities were not observed in any strain of L. lactis subsp. lactis var. diacetylactis. Among thirteen bacteriocin-producing strains and two nisin-producing strains (L. lactis subsp. lactis ATCC 11454 and L. lactis subsp. lactis CNRZ 150, eight (53% were characterized as lactose-positive (Lac+ and proteinase-negative (Prt-. The bacteriocin-producing cultures were also characterized on the basis of plasmid content. All strains had 2 to 7 plasmids with molecular weights varying from 0.5 to 28.1 Mdal. Four strains (ITAL 435, ITAL 436, ITAL 437 and ITAL 438 showed identical profiles and the other were quite distinct.Um total de 167 linhagens de L. lactis foi selecionado para os testes de produção de bacteriocinas pelo método de difusão em poços em agar GM17. Desse total, 14 (8.4% produziram substâncias inibidoras que não foram associadas com ácidos orgânicos, peróxido de hidrogênio e bacteriófagos. A frequência de produção de bacteriocinas variou de 2% em L. lactis subsp. cremoris a 12% em L. lactis subsp. lactis. Nenhuma das linhagens de L. lactis subsp. lactis var. diacetylactis produziu substâncias inibidoras. De 13 linhagens produtoras de bacteriocinas e duas de nisina (L. lactis subsp. lactis ATCC 11454 e L. lactis subsp. lactis CNRZ 150, 8 (53% foram caracterizadas como lactose-positivas (Lac+ e proteinase-negativas (Prt-. As linhagens produtoras de bacteriocinas também foram caracterizadas no seu conteúdo de plasmídios. Elas apresentaram de 2 a 7 plasmídios, com pesos moleculares aproximados de 0.5 a 28.1 Mdal. Quatro linhagens (ITAL 435, ITAL 436

  8. Use of a genetically enhanced, pediocin-producing starter culture, Lactococcus lactis subsp. lactis MM217, to control Listeria monocytogenes in cheddar cheese

    NARCIS (Netherlands)

    Buyong, N; Kok, J; Luchansky, JB

    1998-01-01

    Cheddar cheese was prepared with Lactococcus lactis subsp, lactis MM217, a starter culture which contains pMC117 coding for pediocin PA-1, About 75 liters of pasteurized milk (containing ca, 3.6% fat) was inoculated with strain MM217 (ca, 10(6) CFU per ml) and a mixture of three Listeria

  9. Transcriptome profiling of Lactococcus lactis subsp. cremoris CECT 8666 in response to agmatine

    NARCIS (Netherlands)

    Del Rio, Beatriz; Redruello, Begoña; Martin, M Cruz; Fernandez, Maria; de Jong, Anne; Kuipers, Oscar P; Ladero, Victor; Alvarez, Miguel A

    2016-01-01

    The dairy strain Lactococcus lactis subsp. cremoris CECT 8666 (formerly GE2-14) synthesizes the biogenic amine putrescine from agmatine via the agmatine deiminase (AGDI) pathway [1]. The AGDI cluster of L. lactis is composed by five genes aguR, aguB, aguD, aguA and aguC. The last four genes are

  10. Transcriptomic profile of aguR deletion mutant of Lactococcus lactis subsp. cremoris CECT 8666

    NARCIS (Netherlands)

    Del Rio, Beatriz; Linares, Daniel M; Redruello, Begoña; Martin, Maria Cruz; Fernandez, Maria; de Jong, Anne; Kuipers, Oscar P; Ladero, Victor; Alvarez, Miguel A

    2015-01-01

    Lactococcus lactis subsp. cremoris CECT 8666 (formerly GE2-14) is a dairy strain that catabolizes agmatine (a decarboxylated derivative of arginine) into the biogenic amine putrescine by the agmatine deiminase (AGDI) pathway [1]. The AGDI cluster of L. lactis is composed by five genes aguR, aguB,

  11. Enhanced production of nisin by co-culture of Lactococcus lactis sub sp. lactis and Yarrowia lipolytica in molasses based medium.

    Science.gov (United States)

    Ariana, Mehdi; Hamedi, Javad

    2017-08-20

    Nisin is a safe, approved and commercial bacteriocin that is produced by Lactococcus lactis subsp. lactis. Since lactate accumulation in fermentation medium reduces L. lactis growth and nisin production, Yarrowia lipolytica, a lactate consuming yeast and L. lactis subsp. lactis, were simultaneously cultured in a molasses based medium. Y. lipolytica is not able to consume sucrose as carbon source, but rather consumes lactate and hence decrease lactic acid titer by 10% in the medium. Lactic acid consumption, 15% increased pH value and stimulated L. lactis growth. In the mixed culture, nisin production and L. lactis growth were 50% and 49% higher than that of pure culture, respectively. Also the results showed that specific growth rate of L. lactis increased 4 times more than that of the pure culture. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Use of PCR-based methods for rapid differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis.

    Science.gov (United States)

    Torriani, S; Zapparoli, G; Dellaglio, F

    1999-10-01

    Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412(T), which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains.

  13. Use of PCR-Based Methods for Rapid Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis

    Science.gov (United States)

    Torriani, Sandra; Zapparoli, Giacomo; Dellaglio, Franco

    1999-01-01

    Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412T, which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains. PMID:10508059

  14. The effect of nisin from Lactococcus lactis subsp. lactis on refrigerated patin fillet quality

    Science.gov (United States)

    Adilla, S. N.; Utami, R.; Nursiwi, A.; Nurhartadi, E.

    2017-04-01

    The effect of nisin from Lactococcus lactis subsp. lactis with spraying method application on quality of patin fillet during refrigerated storage (4±1°C) was investigated. The quality of patin fillet based on total plate count (TPC), pH, TVB-N, and TBA values during 16 days at 4±1°C. Completely Randomized Design (CDR) was used in one factor (nisin activity) at 0 IU/ml, 500 IU/ml, 1000 IU/ml, and 2000 IU/ml. The observation was done at 0, 4th, 8th, 12th, and 16th days of storage. The result showed that variation of nisin activity significantly affected the quality of fillet according to TPC, pH, and TVB-N values, however no significant difference on the obtained of TBA value. Nisin in 500 IU/ml, 1000 IU/ml, and 2000 IU/ml could extend the shelf-life of fillet until 4th, 8th, and 12th days respectively based on standard in all parameters.

  15. Expression of prophage-encoded endolysins contributes to autolysis of Lactococcus lactis

    NARCIS (Netherlands)

    Visweswaran, Ganesh Ram R.; Kurek, Dorota; Szeliga, Monika; Pastrana, Francisco Romero; Kuipers, Oscar P.; Kok, Jan; Buist, Girbe

    Analysis of autolysis of derivatives of Lactococcus lactis subsp. cremoris MG1363 and subsp. lactis IL1403, both lacking the major autolysin AcmA, showed that L. lactis IL1403 still lysed during growth while L. lactis MG1363 did not. Zymographic analysis revealed that a peptidoglycan hydrolase

  16. From Genome to Phenotype: An Integrative Approach to Evaluate the Biodiversity of Lactococcus lactis

    Science.gov (United States)

    Laroute, Valérie; Tormo, Hélène; Couderc, Christel; Mercier-Bonin, Muriel; Le Bourgeois, Pascal; Cocaign-Bousquet, Muriel; Daveran-Mingot, Marie-Line

    2017-01-01

    Lactococcus lactis is one of the most extensively used lactic acid bacteria for the manufacture of dairy products. Exploring the biodiversity of L. lactis is extremely promising both to acquire new knowledge and for food and health-driven applications. L. lactis is divided into four subspecies: lactis, cremoris, hordniae and tructae, but only subsp. lactis and subsp. cremoris are of industrial interest. Due to its various biotopes, Lactococcus subsp. lactis is considered the most diverse. The diversity of L. lactis subsp. lactis has been assessed at genetic, genomic and phenotypic levels. Multi-Locus Sequence Type (MLST) analysis of strains from different origins revealed that the subsp. lactis can be classified in two groups: “domesticated” strains with low genetic diversity, and “environmental” strains that are the main contributors of the genetic diversity of the subsp. lactis. As expected, the phenotype investigation of L. lactis strains reported here revealed highly diverse carbohydrate metabolism, especially in plant- and gut-derived carbohydrates, diacetyl production and stress survival. The integration of genotypic and phenotypic studies could improve the relevance of screening culture collections for the selection of strains dedicated to specific functions and applications. PMID:28534821

  17. Lactococcus lactis subsp. lactis infection in Bester sturgeon, a cultured hybrid of Huso huso × Acipenser ruthenus, in Taiwan.

    Science.gov (United States)

    Chen, Ming-Hui; Hung, Shao-Wen; Shyu, Ching-Lin; Lin, Cheng-Chung; Liu, Pan-Chen; Chang, Chen-Hsuan; Shia, Wei-Yau; Cheng, Ching-Fu; Lin, Shiun-Long; Tu, Ching-Yu; Lin, Yu-Hsing; Wang, Way-Shyan

    2012-10-01

    Approximately 5300 hybrid sturgeons with an average body weight of 600-800 g were farmed in 3 round tankers measuring 3m in diameter each containing 28,000 L of aerated groundwater. According to the owner's description, the diseased fish had anorexia, pale body color, and reddish spots on the abdomen. The morbidity and lethality rates in this outbreak were about 70% (3706/5300) and 100% (3706/3706), respectively. The clinical examination revealed enteritis, enlarged abdomen, and rapid respiration rate. The gross findings revealed a volume of about 4 mL of ascites. The histopathological examination showed multiple massive, hemorrhagic or coagulative necrotic foci in the liver and spleen. Furthermore, there was diffuse infiltration of glycogen in hepatic cells, and a few polymorphonuclear and mononuclear leucocytes were observed surrounding the spleen. Some bacterial clumps were noted around the necrotic foci. We also observed that there was moderate to severe, acute, multifocal, coagulative necrosis in the renal parenchyma, with some necrotic foci present beneath the margin of the kidney. Additionally, multifocal, coagulative necrosis was found in the pancreas. Results of microbiologic examinations, including biochemical characteristics, PCR amplification of 16S rRNA gene, sequencing and comparison, and phylogenetic analysis, revealed the pathogen of this infection was Lactococcus lactis subsp. lactis, and based on the results of an antimicrobial agent sensitivity test the bacterium was only sensitive to ampicillin and florfenicol. Additionally, results of in vivo experimental infections in hybrid tilapia showed that 1×10(8) and 1×10(9) CFU/mL of our isolate caused death in all fish and LD(50) values ranged from 10(2) to 10(5) CFU/mL. To the best of the authors' knowledge, this is the first reported case of Lactococcus lactis subsp. lactis infection in hybrid sturgeon. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Lactococcus lactis subsp. cremoris strain JFR1 attenuates Salmonella adhesion to human intestinal cells in vitro.

    Science.gov (United States)

    Zhang, Justina Su; Guri, Anilda; Corredig, Milena; Morales-Rayas, Rocio; Hassan, Ashraf; Griffiths, Mansel; LaPointe, Gisèle

    2016-12-01

    Lactococcus lactis subsp. cremoris JFR1 has been studied in reduced fat cheese due to its ability to produce exopolysaccharides (EPS) in situ, contributing to improved textural and organoleptic properties. In this study, the effect of strain JFR1 on virulence gene expression and attachment of Salmonella to HT-29 human colon carcinoma cells was investigated. Overnight cultures of L. lactis subsp. cremoris JFR1 containing EPS, grown in M17 media with 0.5% glucose supplementation, decreased attachment as well as down regulated virulence gene expression in Salmonella enterica subsp. enterica when tested on HT-29 cells. However, EPS isolated from milk fermented with L. lactis subsp. cremoris JFR1 did not affect Salmonella virulence gene expression or attachment to HT-29 cells. These results suggest that EPS does not contribute to the attachment of Salmonella to human intestinal cells. However, the possibility that the isolation process may have affected the structural features of EPS cannot be ruled out. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Milk-derived angiotensin-I-converting enzymeinhibitory peptides generated by Lactobacillus delbrueckii subsp. lactis CRL 581

    Directory of Open Access Journals (Sweden)

    Villegas Josefina M.

    2014-01-01

    Full Text Available Several strains of Lactobacillus helveticus and Lactobacillus delbrueckii subsp. lactis were evaluated for their ability to release angiotensin-I-converting enzyme (ACE inhibitory peptides from α-casein (α-CN and β-casein (β-CN. Casein peptides resulting from L. delbrueckii subsp. lactis CRL 581-mediated hydrolysis exhibited the highest ACE-inhibitory (ACEI activities, with values of 53 and 40% for α-CN and β-CN, respectively. The casein hydrolysates were fractionated by reversedphase high pressure liquid chromatography and some of the active peptides were identified by mass spectrometry. The fraction with the highest ACEI activity arose from β-CN and contained a mixture of the β-CN f194-206 (QEPVLGPVRGPFP and f198-206 (LGPVRGPFP peptides. Furthermore, the ACEI tripeptide IPP was identified in all β-CN hydrolysates; L. delbrueckii subsp. lactis CRL 581 produced the highest amount of this peptide. The bioactive peptides released by CRL 581 strain may be used in the formulation of functional foods and nutraceuticals, representing a healthier and natural alternative for regulating blood pressure.

  20. Analyses of the probiotic property and stress resistance-related genes of Lactococcus lactis subsp. lactis NCDO 2118 through comparative genomics and in vitro assays.

    Science.gov (United States)

    Oliveira, Letícia C; Saraiva, Tessália D L; Silva, Wanderson M; Pereira, Ulisses P; Campos, Bruno C; Benevides, Leandro J; Rocha, Flávia S; Figueiredo, Henrique C P; Azevedo, Vasco; Soares, Siomar C

    2017-01-01

    Lactococcus lactis subsp. lactis NCDO 2118 was recently reported to alleviate colitis symptoms via its anti-inflammatory and immunomodulatory activities, which are exerted by exported proteins that are not produced by L. lactis subsp. lactis IL1403. Here, we used in vitro and in silico approaches to characterize the genomic structure, the safety aspects, and the immunomodulatory activity of this strain. Through comparative genomics, we identified genomic islands, phage regions, bile salt and acid stress resistance genes, bacteriocins, adhesion-related and antibiotic resistance genes, and genes encoding proteins that are putatively secreted, expressed in vitro and absent from IL1403. The high degree of similarity between all Lactococcus suggests that the Symbiotic Islands commonly shared by both NCDO 2118 and KF147 may be responsible for their close relationship and their adaptation to plants. The predicted bacteriocins may play an important role against the invasion of competing strains. The genes related to the acid and bile salt stresses may play important roles in gastrointestinal tract survival, whereas the adhesion proteins are important for persistence in the gut, culminating in the competitive exclusion of other bacteria. Finally, the five secreted and expressed proteins may be important targets for studies of new anti-inflammatory and immunomodulatory proteins. Altogether, the analyses performed here highlight the potential use of this strain as a target for the future development of probiotic foods.

  1. Cell growth and resistance of Lactococcus lactis subsp. lactis TOMSC161 following freezing, drying and freeze-dried storage are differentially affected by fermentation conditions.

    Science.gov (United States)

    Velly, H; Fonseca, F; Passot, S; Delacroix-Buchet, A; Bouix, M

    2014-09-01

    To investigate the effects of fermentation parameters on the cell growth and on the resistance to each step of the freeze-drying process of Lactococcus lactis subsp. lactis TOMSC161, a natural cheese isolate, using a response surface methodology. Cells were cultivated at different temperatures (22, 30 and 38°C) and pH (5·6, 6·2 and 6·8) and were harvested at different growth phases (0, 3 and 6 h of stationary phase). Cultivability and acidification activity losses of Lc. lactis were quantified after freezing, drying, 1 and 3 months of storage at 4 and 25°C. Lactococcus lactis was not damaged by freezing but was sensitive to drying and to ambient temperature storage. Moreover, the fermentation temperature and the harvesting time influenced the drying resistance of Lc. lactis. Lactococcus lactis cells grown in a whey-based medium at 32°C, pH 6·2 and harvested at late stationary phase exhibited both an optimal growth and the highest resistance to freeze-drying and storage. A better insight on the individual and interaction effects of fermentation parameters made it possible the freeze-drying and storage preservation of a sensitive strain of technological interest. Evidence on the particularly damaging effect of the drying step and the high-temperature storage is presented. © 2014 The Society for Applied Microbiology.

  2. Predictive modeling of Bifidobacterium animalis subsp. lactis Bb-12 growth in cow’s, goat’s and soy milk

    Directory of Open Access Journals (Sweden)

    Vedran Slačanac

    2013-11-01

    Full Text Available The aim of this study was to use a predictive model to analyse the growth of a probiotic strain Bifidobacterium animalis subsp. lactis Bb-12 in cow’s, goat’s and soy milk. The Gompertz model was used, and the suitability of the model was estimated by the Schnute algorithm. Except for the analysis of Bifidobacterium animalis subsp. lactis Bb-12 growth, the Gompertz model was also used for the analysis of pH changes during the fermentation process. Experimental results, as well as the values of kinetic parameters obtained in this study, showed that the highest growth rate of Bifidobacterium animalis subsp. lactis Bb-12 was obtained in goat’s milk, and the lowest in soy milk. Contrary to the growth of Bifidobacterium animalis subsp. lactis Bb-12, pH decreased faster in soy milk than in cow’s milk. The highest rate of pH decrease was also observed in goat’s milk, which is in correspondence with results of various previous studies. The Gompertz model proved to be highly suitable for analysing the course and the fermentation kinetics in these three kinds of milk, and might be used to analyse the growth kinetics of other probiotic and starter cultures in milk.

  3. Cloning, Expression, and Functional Characterization of Secondary Amino Acid Transporters of Lactococcus lactis

    NARCIS (Netherlands)

    Trip, Hein; Mulder, Niels L.; Lolkema, Juke S.

    Fourteen genes encoding putative secondary amino acid transporters were identified in the genomes of Lactococcus lactis subsp. cremoris strains MG1363 and SK11 and L. lactis subsp. lactis strains IL1403 and KF147, 12 of which were common to all four strains. Amino acid uptake in L. lactis cells

  4. Inside the adaptation process of Lactobacillus delbrueckii subsp. lactis to bile.

    Science.gov (United States)

    Burns, Patricia; Sánchez, Borja; Vinderola, Gabriel; Ruas-Madiedo, Patricia; Ruiz, Lorena; Margolles, Abelardo; Reinheimer, Jorge; de los Reyes-Gavilán, Clara G

    2010-08-15

    Progressive adaptation to bile might render some lactobacilli able to withstand physiological bile salt concentrations. In this work, the adaptation to bile was evaluated on previously isolated dairy strains of Lactobacillus delbrueckii subsp. lactis 200 and L. delbrueckii subsp. lactis 200+, a strain derived thereof with stable bile-resistant phenotype. The adaptation to bile was obtained by comparing cytosolic proteomes of both strains grown in the presence or absence of bile. Proteomics were complemented with physiological studies on both strains focusing on glycolytic end-products, the ability to adhere to the human intestinal epithelial cell line HT29-MTX and survival to simulated gastrointestinal conditions. Protein pattern comparison of strains grown with and without bile allowed us to identify 9 different proteins whose production was regulated by bile in both strains, and 17 proteins that showed differences in their levels between the parental and the bile-resistant derivative. These included general stress response chaperones, proteins involved in transcription and translation, in peptidoglycan/exopolysaccharide biosynthesis, in the lipid and nucleotide metabolism and several glycolytic and pyruvate catabolism enzymes. Differences in the level of metabolic end-products of the sugar catabolism were found between the strains 200 and 200+. A decrease in the adhesion of both strains to the intestinal cell line was detected in the presence of bile. In simulated gastric and intestinal juices, a protective effect was exerted by milk improving the survival of both microorganisms. These results indicate that bile tolerance in L. delbrueckii subsp. lactis involves several mechanisms responding to the deleterious impact of bile salts on bacterial physiology. Copyright 2010 Elsevier B.V. All rights reserved.

  5. Transcriptome profiling of Lactococcus lactis subsp. cremoris CECT 8666 in response to agmatine

    Directory of Open Access Journals (Sweden)

    Beatriz del Rio

    2016-03-01

    Full Text Available The dairy strain Lactococcus lactis subsp. cremoris CECT 8666 (formerly GE2-14 synthesizes the biogenic amine putrescine from agmatine via the agmatine deiminase (AGDI pathway [1]. The AGDI cluster of L. lactis is composed by five genes aguR, aguB, aguD, aguA and aguC. The last four genes are co-transcribed as a single policistronic mRNA forming the catabolic operon aguBDAC, which encodes the proteins necessary for agmatine uptake and its conversion into putrescine [1,2]. The first gene of the cluster, aguR, encodes a transmembrane protein that functions as a one-component signal transduction system that senses the agmatine concentration of the medium and accordingly regulates the transcription of aguBDAC [2]. The catabolic operon aguBDAC is transcriptionally activated by agmatine [2] and transcriptionally regulated by carbon catabolite repression (CCR via glucose, but not by other sugars such as lactose or galactose [1,3]. On the contrary, the transcription of the aguR regulatory gene is not subject to CCR regulation [1,3] nor is regulated by agmatine [2]. In this study we report the transcriptional profiling of L. lactis subsp. cremoris CECT 8666 grown in M17 medium with galactose (GalM17 as carbon source and supplemented with agmatine, compared to that of the strain grown in the same culture medium without agmatine. The transcriptional profiling data of agmatine-regulated genes were deposited in the Gene Expression Omnibus (GEO database under Accession no. GSE74808. Keywords: Lactococcus lactis, Biogenic amines, Putrescine, Agmatine deiminase, Agmatine

  6. High-Resolution Amplified Fragment Length Polymorphism Typing of Lactococcus lactis Strains Enables Identification of Genetic Markers for Subspecies-Related Phenotypes▿

    Science.gov (United States)

    Kütahya, Oylum Erkus; Starrenburg, Marjo J. C.; Rademaker, Jan L. W.; Klaassen, Corné H. W.; van Hylckama Vlieg, Johan E. T.; Smid, Eddy J.; Kleerebezem, Michiel

    2011-01-01

    A high-resolution amplified fragment length polymorphism (AFLP) methodology was developed to achieve the delineation of closely related Lactococcus lactis strains. The differentiation depth of 24 enzyme-primer-nucleotide combinations was experimentally evaluated to maximize the number of polymorphisms. The resolution depth was confirmed by performing diversity analysis on 82 L. lactis strains, including both closely and distantly related strains with dairy and nondairy origins. Strains clustered into two main genomic lineages of L. lactis subsp. lactis and L. lactis subsp. cremoris type-strain-like genotypes and a third novel genomic lineage rooted from the L. lactis subsp. lactis genomic lineage. Cluster differentiation was highly correlated with small-subunit rRNA homology and multilocus sequence analysis (MLSA) studies. Additionally, the selected enzyme-primer combination generated L. lactis subsp. cremoris phenotype-specific fragments irrespective of the genotype. These phenotype-specific markers allowed the differentiation of L. lactis subsp. lactis phenotype from L. lactis subsp. cremoris phenotype strains within the same L. lactis subsp. cremoris type-strain-like genomic lineage, illustrating the potential of AFLP for the generation of phenotype-linked genetic markers. PMID:21666014

  7. Use of PCR-Based Methods for Rapid Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis

    OpenAIRE

    Torriani, Sandra; Zapparoli, Giacomo; Dellaglio, Franco

    1999-01-01

    Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy pr...

  8. Lactose-mediated carbon catabolite repression of putrescine production in dairy Lactococcus lactis is strain dependent.

    Science.gov (United States)

    del Rio, Beatriz; Ladero, Victor; Redruello, Begoña; Linares, Daniel M; Fernández, Maria; Martín, Maria Cruz; Alvarez, Miguel A

    2015-06-01

    Lactococcus lactis is the lactic acid bacterial (LAB) species most widely used as a primary starter in the dairy industry. However, several strains of L. lactis produce the biogenic amine putrescine via the agmatine deiminase (AGDI) pathway. We previously reported the putrescine biosynthesis pathway in L. lactis subsp. cremoris GE2-14 to be regulated by carbon catabolic repression (CCR) via glucose but not lactose (Linares et al., 2013). The present study shows that both these sugars repress putrescine biosynthesis in L. lactis subsp. lactis T3/33, a strain isolated from a Spanish artisanal cheese. Furthermore, we demonstrated that both glucose and lactose repressed the transcriptional activity of the aguBDAC catabolic genes of the AGDI route. Finally, a screening performed in putrescine-producing dairy L. lactis strains determined that putrescine biosynthesis was repressed by lactose in all the L. lactis subsp. lactis strains tested, but in only one L. lactis subsp. cremoris strain. Given the obvious importance of the lactose-repression in cheese putrescine accumulation, it is advisable to consider the diversity of L. lactis in this sense and characterize consequently the starter cultures to select the safest strains. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Different effects of two newly-isolated probiotic Lactobacillus plantarum 15HN and Lactococcus lactis subsp. Lactis 44Lac strains from traditional dairy products on cancer cell lines.

    Science.gov (United States)

    Haghshenas, Babak; Abdullah, Norhafizah; Nami, Yousef; Radiah, Dayang; Rosli, Rozita; Khosroushahi, Ahmad Yari

    2014-12-01

    Lactobacillus and Lactococcus strains isolated from food products can be introduced as probiotics because of their health-promoting characteristics and non-pathogenic nature. This study aims to perform the isolation, molecular identification, and probiotic characterization of Lactobacillus and Lactococcus strains from traditional Iranian dairy products. Primary probiotic assessments indicated high tolerance to low pH and high bile salt conditions, high anti-pathogenic activities, and susceptibility to high consumption antibiotics, thus proving that both strains possess probiotic potential. Cytotoxicity assessments were used to analyze the effects of the secreted metabolite on different cancer cell lines, including HT29, AGS, MCF-7, and HeLa, as well as a normal human cell line (HUVEC). Results showed acceptable cytotoxic properties for secreted metabolites (40 μg/ml dry weight) of Lactococcus lactis subsp. Lactis 44Lac. Such performance was similar to that of Taxol against all of the treated cancer cell lines; however, the strain exhibited no toxicity on the normal cell line. Cytotoxic assessments through flow cytometry and fluorescent microscopy demonstrated that apoptosis is the main cytotoxic mechanism for secreted metabolites of L. lactis subsp. Lactis 44Lac. By contrast, the effects of protease-treated metabolites on the AGS cell line verified the protein nature of anti-cancer metabolites. However, precise characterizations and in vitro/in vivo investigations on purified proteins should be conducted before these metabolites are introduced as potential anti-cancer therapeutics. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Complete sequences of four plasmids of Lactococcus lactis subsp cremoris SK11 reveal extensive adaptation to the dairy environment

    NARCIS (Netherlands)

    Siezen, R.J.; Renckens, B.; Swam, van I.; Peters, S.; Kranenburg, van R.; Kleerebezem, M.; Vos, de W.M.

    2005-01-01

    Lactococcus lactis strains are known to carry plasmids encoding industrially important traits. L. lactis subsp. cremoris SK11 is widely used by the dairy industry in cheese making. Its complete plasmid complement was sequenced and found to contain the plasmids pSK11A (10,372 bp), pSK11B (13,332 bp),

  11. Genome Sequence of the Cheese-Starter Strain Lactobacillus delbrueckii subsp. lactis CRL 581.

    Science.gov (United States)

    Hebert, Elvira María; Raya, Raúl R; Brown, Lucía; Font de Valdez, Graciela; Savoy de Giori, Graciela; Taranto, María Pía

    2013-08-08

    We report the genome sequence of Lactobacillus delbrueckii subsp. lactis CRL 581 (1,911,137 bp, GC 49.7%), a proteolytic strain isolated from a homemade Argentinian hard cheese which has a key role in bacterial nutrition and releases bioactive health-beneficial peptides from milk proteins.

  12. Transcriptomic profile of aguR deletion mutant of Lactococcus lactis subsp. cremoris CECT 8666

    Science.gov (United States)

    del Rio, Beatriz; Linares, Daniel M.; Redruello, Begoña; Martin, Maria Cruz; Fernandez, Maria; de Jong, Anne; Kuipers, Oscar P.; Ladero, Victor; Alvarez, Miguel A.

    2015-01-01

    Lactococcus lactis subsp. cremoris CECT 8666 (formerly GE2-14) is a dairy strain that catabolizes agmatine (a decarboxylated derivative of arginine) into the biogenic amine putrescine by the agmatine deiminase (AGDI) pathway [1]. The AGDI cluster of L. lactis is composed by five genes aguR, aguB, aguD, aguA and aguC. The last four genes are responsible for the deamination of agmatine to putrescine and are co-transcribed as a single policistronic mRNA forming the catabolic operon aguBDAC[1]. aguR encodes a transmembrane protein that functions as a one-component signal transduction system that senses the agmatine concentration of the medium and accordingly regulates the transcription of aguBDAC[2], which is also transcriptionally regulated by carbon catabolic repression (CCR) via glucose, but not by other sugars such as lactose and galactose [1], [3]. Here we report the transcriptional profiling of the aguR gene deletion mutant (L. lactis subsp. cremoris CECT 8666 ∆aguR) [2] compared to the wild type strain, both grown in M17 medium with galactose as carbon source and supplemented with agmatine. The transcriptional profiling data of AguR-regulated genes were deposited in the Gene Expression Omnibus (GEO) database under accession no. GSE59514. PMID:26697381

  13. Transcriptomic profile of aguR deletion mutant of Lactococcus lactis subsp. cremoris CECT 8666

    Directory of Open Access Journals (Sweden)

    Beatriz del Rio

    2015-12-01

    Full Text Available Lactococcus lactis subsp. cremoris CECT 8666 (formerly GE2-14 is a dairy strain that catabolizes agmatine (a decarboxylated derivative of arginine into the biogenic amine putrescine by the agmatine deiminase (AGDI pathway [1]. The AGDI cluster of L. lactis is composed by five genes aguR, aguB, aguD, aguA and aguC. The last four genes are responsible for the deamination of agmatine to putrescine and are co-transcribed as a single policistronic mRNA forming the catabolic operon aguBDAC [1]. aguR encodes a transmembrane protein that functions as a one-component signal transduction system that senses the agmatine concentration of the medium and accordingly regulates the transcription of aguBDAC [2], which is also transcriptionally regulated by carbon catabolic repression (CCR via glucose, but not by other sugars such as lactose and galactose [1,3]. Here we report the transcriptional profiling of the aguR gene deletion mutant (L. lactis subsp. cremoris CECT 8666 ∆aguR [2] compared to the wild type strain, both grown in M17 medium with galactose as carbon source and supplemented with agmatine. The transcriptional profiling data of AguR-regulated genes were deposited in the Gene Expression Omnibus (GEO database under accession no. GSE59514.

  14. Transcriptome analysis of Lactococcus lactis subsp. lactis during milk acidification as affected by dissolved oxygen and the redox potential.

    Science.gov (United States)

    Larsen, Nadja; Moslehi-Jenabian, Saloomeh; Werner, Birgit Brøsted; Jensen, Maiken Lund; Garrigues, Christel; Vogensen, Finn Kvist; Jespersen, Lene

    2016-06-02

    Performance of Lactococcus lactis as a starter culture in dairy fermentations depends on the levels of dissolved oxygen and the redox state of milk. In this study the microarray analysis was used to investigate the global gene expression of L. lactis subsp. lactis DSM20481(T) during milk acidification as affected by oxygen depletion and the decrease of redox potential. Fermentations were carried out at different initial levels of dissolved oxygen (dO2) obtained by milk sparging with oxygen (high dO2, 63%) or nitrogen (low dO2, 6%). Bacterial exposure to high initial oxygen resulted in overexpression of genes involved in detoxification of reactive oxygen species (ROS), oxidation-reduction processes, biosynthesis of trehalose and down-regulation of genes involved in purine nucleotide biosynthesis, indicating that several factors, among them trehalose and GTP, were implicated in bacterial adaptation to oxidative stress. Generally, transcriptional changes were more pronounced during fermentation of oxygen sparged milk. Genes up-regulated in response to oxygen depletion were implicated in biosynthesis and transport of pyrimidine nucleotides, branched chain amino acids and in arginine catabolic pathways; whereas genes involved in salvage of nucleotides and cysteine pathways were repressed. Expression pattern of genes involved in pyruvate metabolism indicated shifts towards mixed acid fermentation after oxygen depletion with production of specific end-products, depending on milk treatment. Differential expression of genes, involved in amino acid and pyruvate pathways, suggested that initial oxygen might influence the release of flavor compounds and, thereby, flavor development in dairy fermentations. The knowledge of molecular responses involved in adaptation of L. lactis to the shifts of redox state and pH during milk fermentations is important for the dairy industry to ensure better control of cheese production. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Gene Replacement and Fluorescent Labeling to Study the Functional Role of Exopolysaccharides in Bifidobacterium animalis subsp. lactis

    Directory of Open Access Journals (Sweden)

    Nuria Castro-Bravo

    2017-07-01

    Full Text Available An extracellular layer of exopolysaccharides (EPS covers the surface of some Bifidobacterium animalis subsp. lactis strains, which could be of relevance for its probiotic performance. In order to understand the functional characteristics of B. animalis subsp. lactis, two isogenic strains that differ in their EPS-producing phenotype, due to a single mutation in the gene Balat_1410, were studied. By means of a double crossover recombination strategy, successfully used for the first time in bifidobacteria, Balat_1410 in the type strain B. animalis subsp. lactis DSM10140 was replaced by a mutated gene containing a non-synonymous mutation previously associated with the appearance of a mucoid-ropy phenotype. Nuclear magnetic resonance and SEC-MALS analyses showed that the novel strain harboring the mutation acquired a ropy phenotype, due to the production of a high molecular weight (HMW-EPS that is not produced in the wild-type strain. Fluorescence labeling of both strains with two fluorescent proteins, m-Cherry and Green Fluorescent Protein, was achieved by expressing the corresponding genes under the control of a native selected promoter (the elongation factor Tu promoter. Remarkably, qualitative and quantitative fluorescence analyses demonstrated that the ropy strain displays a lower capability to adhere to human intestinal epithelial cells. In addition, the presence of the HMW-EPS reduced the capability of the producing strain to form biofilms upon three different abiotic surfaces. This work also highlights the fact that different EPS confer variable functional characteristics to the bifidobacterial surface, which may be relevant for the performance of B. animalis subsp. lactis as a probiotic. The construction of molecular tools allowing the functional characterization of surface structures in next generation probiotics is still a challenging issue that deserves further attention, given the relevant role that such molecules must play in the

  16. POTENTIAL OF Lactococcus lactis subsp. lactis MTCC 3041 AS A BIOPRESERVATIVE

    Directory of Open Access Journals (Sweden)

    Neha Sharma

    2013-10-01

    Full Text Available Lactic acid bacteria especially in developing countries can be exploited against frequently occurring spoilage organisms of fresh fruits and vegetables in addition to pathogens. Keeping in views this antagonism imparted by bacteria Lactococci, the present study was taken and effectiveness of bacteriocin of Lactococci was also studied in preservatives and enzymes. Lactic acid bacteria Lactococcus lactis subs. Lactis MTCC 3041 was used as bacteriocin producer strain. Isolation of most frequently occurring spoilage organisms from spoiled Mango and Kinnow was done by microbiological procedures and were identified by microscopic studies as Isolate 1 and Isolate 2. It has limited use in processed salted food as no zone of inhibition was observed at and above 5% NaCl (w/v.0.3% (w/v is the minimum concentration of KMS that provides stress to the microorganism for the production of bacteriocin. It is not suitable for food having sodium benzoate as preservative as with increase in concentration growth of Lactococcus lactis decreases. Presence of bacteriocin hinders the growth of the isolate 1 as fresh weight of the mycelium in test sample is 7.09% less than the control. Being non-pathogenic this organism can be safely used against spoilage organisms in addition to food borne pathogens.

  17. Expression of prophage-encoded endolysins contributes to autolysis of Lactococcus lactis.

    Science.gov (United States)

    Visweswaran, Ganesh Ram R; Kurek, Dorota; Szeliga, Monika; Pastrana, Francisco Romero; Kuipers, Oscar P; Kok, Jan; Buist, Girbe

    2017-02-01

    Analysis of autolysis of derivatives of Lactococcus lactis subsp. cremoris MG1363 and subsp. lactis IL1403, both lacking the major autolysin AcmA, showed that L. lactis IL1403 still lysed during growth while L. lactis MG1363 did not. Zymographic analysis revealed that a peptidoglycan hydrolase activity of around 30 kDa is present in cell extracts of L. lactis IL1403 that could not be detected in strain MG1363. A comparison of all genes encoding putative peptidoglycan hydrolases of IL1403 and MG1363 led to the assumption that one or more of the 99 % homologous 27.9-kDa endolysins encoded by the prophages bIL285, bIL286 and bIL309 could account for the autolysis phenotype of IL1403. Induced expression of the endolysins from bIL285, bIL286 or bIL309 in L. lactis MG1363 resulted in detectable lysis or lytic activity. Prophage deletion and insertion derivatives of L. lactis IL1403 had a reduced cell lysis phenotype. RT-qPCR and zymogram analysis showed that each of these strains still expressed one or more of the three phage lysins. A homologous gene and an endolysin activity were also identified in the natural starter culture L. lactis subsp. cremoris strains E8, Wg2 and HP, and the lytic activity could be detected under growth conditions that were identical as those used for IL1403. The results presented here show that these endolysins of L. lactis are expressed during normal growth and contribute to autolysis without production of (lytic) phages. Screening for natural strains expressing homologous endolysins could help in the selection of strains with enhanced autolysis and, thus, cheese ripening properties.

  18. Alternative lactose catabolic pathway in Lactococcus lactis IL1403

    NARCIS (Netherlands)

    Aleksandrzak-Piekarczyk, T; Kok, J; Renault, P; Bardowski, J

    2005-01-01

    In this study, we present a glimpse of the diversity of Lactococcus lactis subsp. lactis IL1403 beta-galactosidase phenotype-negative mutants isolated by negative selection on solid media containing cellobiose or lactose and X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside), and we

  19. Insights into physiological traits of Bifidobacterium animalis subsp. lactis BB-12 through membrane proteome analysis

    DEFF Research Database (Denmark)

    Gilad, Ofir; Hjernø, Karin; Østerlund, Eva Christina

    2012-01-01

    Bifidobacterium animalis subsp. lactis BB-12 is a widely used probiotic strain associated with a variety of health-promoting traits. There is, however, only limited knowledge available regarding the membrane proteome and the proteins involved in oligosaccharide transport in BB-12. We applied two...

  20. Comparative and functional genomics of the Lactococcus lactis taxon; insights into evolution and niche adaptation.

    Science.gov (United States)

    Kelleher, Philip; Bottacini, Francesca; Mahony, Jennifer; Kilcawley, Kieran N; van Sinderen, Douwe

    2017-03-29

    Lactococcus lactis is among the most widely studied lactic acid bacterial species due to its long history of safe use and economic importance to the dairy industry, where it is exploited as a starter culture in cheese production. In the current study, we report on the complete sequencing of 16 L. lactis subsp. lactis and L. lactis subsp. cremoris genomes. The chromosomal features of these 16 L. lactis strains in conjunction with 14 completely sequenced, publicly available lactococcal chromosomes were assessed with particular emphasis on discerning the L. lactis subspecies division, evolution and niche adaptation. The deduced pan-genome of L. lactis was found to be closed, indicating that the representative data sets employed for this analysis are sufficient to fully describe the genetic diversity of the taxon. Niche adaptation appears to play a significant role in governing the genetic content of each L. lactis subspecies, while (differential) genome decay and redundancy in the dairy niche is also highlighted.

  1. Cell-Wall-Bound Proteinase of Lactobacillus delbrueckii subsp. lactis ACA-DC 178: Characterization and Specificity for β-Casein

    Science.gov (United States)

    Tsakalidou, E.; Anastasiou, R.; Vandenberghe, I.; van Beeumen, J.; Kalantzopoulos, G.

    1999-01-01

    Lactobacillus delbrueckii subsp. lactis ACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca2+-free buffer. The crude proteinase extract shows its highest activity at pH 6.0 and 40°C. It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the enzyme is similar to the lactococcal PI-type proteinases, since it hydrolyzes β-casein mainly and α- and κ-caseins to a much lesser extent. The cell-wall-bound proteinase from L. delbrueckii subsp. lactis ACA-DC 178 liberates four main peptides from β-casein, which have been identified. PMID:10223997

  2. Microencapsulation of Bifidobacterium animalis subsp. lactis and Lactobacillus acidophilus in cocoa butter using spray chilling technology

    Science.gov (United States)

    Pedroso, D.L.; Dogenski, M.; Thomazini, M.; Heinemann, R.J.B.; Favaro-Trindade, C.S.

    2013-01-01

    In the present study, the cells of Bifidobacterium animalis subsp. lactis (BI-01) and Lactobacillus acidophilus (LAC-04) were encapsulated in cocoa butter using spray-chilling technology. Survival assays were conducted to evaluate the resistance of the probiotics to the spray-chilling process, their resistance to the simulated gastric and intestinal fluids (SGF and SIF), and their stability during 90 days of storage. The viability of the cells was not affected by microencapsulation. The free and encapsulated cells of B. animalis subsp. lactis were resistant to both SGF and SIF. The micro-encapsulated cells of L. acidophilus were more resistant to SGF and SIF than the free cells; the viability of the encapsulated cells was enhanced by 67%, while the free cells reached the detection limit of the method (103 CFU/g). The encapsulated probiotics were unstable when they were stored at 20 °C. The population of encapsulated L. acidophilus decreased drastically when they were stored at 7 °C; only 20% of cells were viable after 90 days of storage. The percentage of viable cells of the encapsulated B. animalis subsp.lactis, however, was 72% after the same period of storage. Promising results were obtained when the microparticles were stored at −18 °C; the freeze granted 90 days of shelf life to the encapsulated cells. These results suggest that the spray-chilling process using cocoa butter as carrier protects L. acidophilus from gastrointestinal fluids. However, the viability of the cells during storage must be improved. PMID:24516445

  3. Microencapsulation of Bifidobacterium animalis subsp. lactis and Lactobacillus acidophilus in cocoa butter using spray chilling technology

    Directory of Open Access Journals (Sweden)

    D.L. Pedroso

    2013-09-01

    Full Text Available In the present study, the cells of Bifidobacterium animalis subsp. lactis (BI-01 and Lactobacillus acidophilus (LAC-04 were encapsulated in cocoa butter using spray-chilling technology. Survival assays were conducted to evaluate the resistance of the probiotics to the spray-chilling process, their resistance to the simulated gastric and intestinal fluids (SGF and SIF, and their stability during 90 days of storage. The viability of the cells was not affected by microencapsulation. The free and encapsulated cells of B. animalis subsp. lactis were resistant to both SGF and SIF. The micro-encapsulated cells of L. acidophilus were more resistant to SGF and SIF than the free cells; the viability of the encapsulated cells was enhanced by 67%, while the free cells reached the detection limit of the method (10³ CFU/g. The encapsulated probiotics were unstable when they were stored at 20 °C. The population of encapsulated L. acidophilus decreased drastically when they were stored at 7 °C; only 20% of cells were viable after 90 days of storage. The percentage of viable cells of the encapsulated B. animalis subsp.lactis, however, was 72% after the same period of storage. Promising results were obtained when the microparticles were stored at -18 °C; the freeze granted 90 days of shelf life to the encapsulated cells. These results suggest that the spray-chilling process using cocoa butter as carrier protects L. acidophilus from gastrointestinal fluids. However, the viability of the cells during storage must be improved.

  4. Functional cream cheese supplemented with Bifidobacterium animalis subsp. lactis DSM 10140 and Lactobacillus reuteri DSM 20016 and prebiotics.

    Science.gov (United States)

    Speranza, Barbara; Campaniello, Daniela; Monacis, Noemi; Bevilacqua, Antonio; Sinigaglia, Milena; Corbo, Maria Rosaria

    2018-06-01

    The aim of this study was to develop a functional fresh cream cheese with Bifidobacterium animalis subsp. lactis DSM 10140 or Lactobacillus reuteri DSM 20016 and prebiotics (inulin, FOS and lactulose). The research was divided into two steps: in vitro evaluation of the effects of prebiotic compounds; validation at laboratory level with production of functional cream mini-cheeses. Prebiotics showed a protective effect: B. animalis subsp. lactis DSM 10140 cultivability on Petri dishes was positively influenced by lactulose, whereas fructooligosaccharides (FOS) were the prebiotic compounds able to prolong Lb. reuteri DSM 20016 cultivability. At 30 °C, a prolongation of the death time (more than 300 days) was observed, while the controls showed death time values about 100 days. At 45 °C, death time values increased from 32.2 (control) to 33, 35, and 38 days in the samples added with FOS, inulin and lactulose, respectively. Lactulose and FOS were chosen to be added to cream mini-cheeses inoculated with B. animalis subsp. lactis DSM 10140 and Lb. reuteri DSM 20016, respectively; the proposed functional cream cheese resulted in a product with favourable conditions for the viability of both probiotics which maintained cultivable cells above the recommended level during 28 days of storage at 4 °C with good sensory characteristics. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Lactobacillus delbrueckii subsp lactis CIDCA 133 modulates response of human epithelial and dendritic cells infected with Bacillus cereus.

    Science.gov (United States)

    Rolny, I S; Tiscornia, I; Racedo, S M; Pérez, P F; Bollati-Fogolín, M

    2016-11-30

    It is known that probiotic microorganisms are able to modulate pathogen virulence. This ability is strain dependent and involves multiple interactions between microorganisms and relevant host's cell populations. In the present work we focus on the effect of a potentially probiotic lactobacillus strain (Lactobacillus delbrueckii subsp. lactis CIDCA 133) in an in vitro model of Bacillus cereus infection. Our results showed that infection of intestinal epithelial HT-29 cells by B. cereus induces nuclear factor kappa B (NF-κB) pathway. Noteworthy, the presence of strain L. delbrueckii subsp.lactis CIDCA 133 increases stimulation. However, B. cereus-induced interleukin (IL)-8 production by epithelial cells is partially abrogated by L. delbrueckii subsp. lactis CIDCA 133. These findings suggest that signalling pathways other than that of NF-κB are involved. In a co-culture system (HT-29 and monocyte-derived dendritic cells), B. cereus was able to translocate from the epithelial (upper) to the dendritic cell compartment (lower). This translocation was partially abrogated by the presence of lactobacilli in the upper compartment. In addition, infection of epithelial cells in the co-culture model, led to an increase in the expression of CD86 by dendritic cells. This effect could not be modified in the presence of lactobacilli. Interestingly, infection of enterocytes with B. cereus triggers production of proinflammatory cytokines by dendritic cells (IL-8, IL-6 and tumour necrosis factor alpha (TNF-α)). The production of TNF-α (a protective cytokine in B. cereus infections) by dendritic cells was increased in the presence of lactobacilli. The present work demonstrates for the first time the effect of L. delbrueckii subsp. lactis CIDCA 133, a potentially probiotic strain, in an in vitro model of B. cereus infection. The presence of the probiotic strain modulates cell response both in infected epithelial and dendritic cells thus suggesting a possible beneficial effect of

  6. Safety of Bifidobacterium animalis Subsp. Lactis (B. lactis) Strain BB-12-Supplemented Yogurt in Healthy Children.

    Science.gov (United States)

    Tan, Tina P; Ba, Zhaoyong; Sanders, Mary E; D'Amico, Frank J; Roberts, Robert F; Smith, Keisha H; Merenstein, Daniel J

    2017-02-01

    Probiotics are live microorganisms that may provide health benefits to the individual when consumed in sufficient quantities. For studies conducted on health or disease endpoints on probiotics in the United States, the Food and Administration has required those studies to be conducted as investigational new drugs. This phase I, double-blinded, randomized, controlled safety study represents the first requirement of this pathway. The purpose of the study was to determine the safety of Bifidobacterium animalis subsp. lactis (B lactis) strain BB-12 (BB-12)-supplemented yogurt when consumed by a generally healthy group of children. The secondary aim was to assess the effect of BB-12-supplemented yogurt on the gut microbiota of the children. Sixty children ages 1 to 5 years were randomly assigned to consume 4 ounces of either BB-12-supplemented yogurt or nonsupplemented control yogurt daily for 10 days. The primary outcome was to assess safety and tolerability, as determined by the number of reported adverse events. A total of 186 nonserious adverse events were reported, with no significant differences between the control and BB-12 groups. No significant changes due to probiotic treatment were observed in the gut microbiota of the study cohort. BB-12-supplemented yogurt is safe and well-tolerated when consumed by healthy children. The present study will form the basis for future randomized clinical trials investigating the potential effects of BB-12-supplemented yogurt in different disease states.

  7. The pyrimidine operon pyrRPB-carA from Lactococcus lactis

    DEFF Research Database (Denmark)

    Martinussen, Jan; Schallert, J.; Andersen, Birgit

    2001-01-01

    The four genes pyrR, pyrP, pyrB, and carA were found to constitute an operon in Lactococcus lactis subsp, lactis MG1363. The functions of the different genes were established by mutational analysis. The first gene in the operon is the pyrimidine regulatory gene, pyrR, which is responsible...

  8. EXPRESSION OF A CHITINASE GENE FROM SERRATIA-MARCESCENS IN LACTOCOCCUS-LACTIS AND LACTOBACILLUS-PLANTARUM

    NARCIS (Netherlands)

    BRURBERG, MB; HAANDRIKMAN, AJ; LEENHOUTS, KJ; VENEMA, G; NES, IF

    1994-01-01

    A chitinase gene from the Gram-negative bacterium Serratia marcescens BJL200 was cloned in Lactococcus lactis subsp. lactis MG1363 and in the silage inoculum strain Lactobacillus plantarum E19b. The chitinase gene was expressed as an active enzyme at a low level in Lactococcus lactis, when cloned in

  9. Anti-inflammatory properties of fermented soy milk with Lactococcus lactis subsp. lactis S-SU2 in murine macrophage RAW264.7 cells and DSS-induced IBD model mice.

    Science.gov (United States)

    Kawahara, Miho; Nemoto, Maki; Nakata, Toru; Kondo, Saya; Takahashi, Hajime; Kimura, Bon; Kuda, Takashi

    2015-06-01

    Six lactic acid bacteria strains (four Lactobacillus plantarum strains and one each of Lactococcus lactis subsp. lactis and Pediococcus pentosaceus) have been isolated and shown to possess anti-oxidant activity. In this study, we determined their acid, bile, salt resistance, and adhesion activity on human enterocyte-like HT-29-Luc and Caco-2 cells. An isolate Lc. lactis S-SU2 showed highest bile resistance and adhesion activity compared to type strains. S-SU2 could ferment both 10% skimmed milk and soy milk while the type strain could not ferment soy milk. Soy milk fermented with S-SU2 showed an increased nitric oxide (NO) secretion in the mouse macrophage RAW264.7 cells without bacterial lipopolysaccharide (LPS). Furthermore, the inhibitory effects of the fermented soy milk on Escherichia coli O111 LPS-induced NO secretion were higher than those of fresh soy milk. Inflammatory bowel disease (IBD) was induced in mice fed either 5% (w/v) dextran sodium sulfate (DSS) in drinking water or 50% soy milk in drinking water. Shortening of colon length, breaking of epithelial cells, lowering liver and thymus weights, and enlargement of spleen are some of the characteristics observed in the IBD, which were prevented by the use of soy milk fermented with Lc. lactis S-SU2. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Evaluation of Lactococcus lactis Isolates from Nondairy Sources with Potential Dairy Applications Reveals Extensive Phenotype-Genotype Disparity and Implications for a Revised Species.

    Science.gov (United States)

    Cavanagh, Daniel; Casey, Aidan; Altermann, Eric; Cotter, Paul D; Fitzgerald, Gerald F; McAuliffe, Olivia

    2015-06-15

    Lactococcus lactis is predominantly associated with dairy fermentations, but evidence suggests that the domesticated organism originated from a plant niche. L. lactis possesses an unusual taxonomic structure whereby strain phenotypes and genotypes often do not correlate, which in turn has led to confusion in L. lactis classification. A bank of L. lactis strains was isolated from various nondairy niches (grass, vegetables, and bovine rumen) and was further characterized on the basis of key technological traits, including growth in milk and key enzyme activities. Phenotypic analysis revealed all strains from nondairy sources to possess an L. lactis subsp. lactis phenotype (lactis phenotype); however, seven of these strains possessed an L. lactis subsp. cremoris genotype (cremoris genotype), determined by two separate PCR assays. Multilocus sequence typing (MLST) showed that strains with lactis and cremoris genotypes clustered together regardless of habitat, but it highlighted the increased diversity that exists among "wild" strains. Calculation of average nucleotide identity (ANI) and tetranucleotide frequency correlation coefficients (TETRA), using the JSpecies software tool, revealed that L. lactis subsp. cremoris and L. lactis subsp. lactis differ in ANI values by ∼14%, below the threshold set for species circumscription. Further analysis of strain TIFN3 and strains from nonindustrial backgrounds revealed TETRA values of lactis taxonomy. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  11. Assessment of stress tolerance acquisition in the heat-tolerant derivative strains of Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus rhamnosus GG.

    Science.gov (United States)

    Aakko, J; Sánchez, B; Gueimonde, M; Salminen, S

    2014-07-01

    The purpose of this study was to investigate the heat-shock response at molecular level in Lactobacillus rhamnosus GG, Bifidobacterium animalis subsp. lactis BB-12 and their heat-tolerant derivatives and to characterize the changes that make the derivatives more robust in terms of heat stress. The study strains were exposed for 2 h to a heat-shock treatment, Bif. animalis subsp. lactis BB-12 and its derivative at 50°C and the Lact. rhamnosus GG and its derivative at 60°C. Protein synthesis before and after heat shock was examined using proteomics and RT-qPCR. The analysis revealed that the regulation of seven proteins in both strain pairs was modified as a response to heat or between the original and the derivative strain. The comparison of wild-type strains and the heat-tolerant derivatives suggests that the acquisition of heat tolerance in the Bif. animalis subsp. lactis BB-12 derivative is due to a slightly increased constitutive level of chaperones, while in Lact. rhamnosus GG derivative, the main reason seems to be a higher ability to induce the production of chaperones. This study revealed possible markers of heat tolerance in B. lactis and Lact. rhamnosus strains. This study increases our knowledge on how Lactobacillus and Bifidobacterium strains may acquire heat tolerance. These findings may be useful for improving the heat tolerance of existing probiotic strains as well as screening new heat-tolerant strains. © 2014 The Society for Applied Microbiology.

  12. Lactococcus lactis Subsp. Lactis Suşlarında Yüksek Sıklıkta Konjugal Transfer Sistemlerinin Analizi

    Directory of Open Access Journals (Sweden)

    Çağla Tükel

    2015-02-01

    Full Text Available Bu çalışmada L. lactis subsp. lactis suşlarında laktoz fermentasyonu özelliğini kodlayan altı farklı plazmidin yüksek sıklıkta konjugal aktarım yeteneği araştırıldı. Bu plazmidlerin konjugal transfer sıklıkları; iki seks faktörünün interaksiyonuna bağlı olarak (Clu ve Agg, Clu-/Agg-, Agg+ x Clu-/Agg+, Agg- ya da Clu+/Agg- x Clu-/Agg- konjugasyon eşleri için 1.5x10-5–1.0x10-7 ve Clu+/Agg- x Clu-/Agg+ konjugasyon eşleri için 7.1x10-2-2.7x10-3 oranlarında değişim gösterdi. Laktoz plazmidlerinin stabiliteleri ise; doğal suşlarda %82-96, MG1390 alıcı suşu için tanımlanan konjugantlarda %77-98 ve MCL8060 alıcı suşu için tanımlanan konjugantlarda ise %44-67 arasında saptandı.

  13. Influence of the addition of Lactobacillus acidophilus La-05, Bifidobacterium animalis subsp. lactis Bb-12 and inulin on the technological, physicochemical, microbiological and sensory features of creamy goat cheese.

    Science.gov (United States)

    Barbosa, Ilsa C; Oliveira, Maria E G; Madruga, Marta S; Gullón, Beatriz; Pacheco, Maria T B; Gomes, Ana M P; Batista, Ana S M; Pintado, Maria M E; Souza, Evandro L; Queiroga, Rita C R E

    2016-10-12

    The effects of the addition of Lactobacillus acidophilus LA-05, Bifidobacterium animalis subsp. lactis BB-12 and inulin on the quality characteristics of creamy goat cheese during refrigerated storage were evaluated. The manufactured cheeses included the addition of starter culture (Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris - R-704) (CC); starter culture, L. acidophilus LA-05 and inulin (CLA); starter culture, B. lactis BB-12 and inulin (CBB); or starter culture, L. acidophilus LA-05, B. lactis BB-12 and inulin (CLB). In the synbiotic cheeses (CLA, CBB and CLB), the counts of L. acidophilus LA-05 and B. lactis BB-12 were greater than 6log CFU g -1 , the amount of inulin was greater than 6 g per 100 g, and the firmness was reduced. The cheeses evaluated had high brightness values (L*), with a predominance of yellow (b*). CC had higher contents of proteins, lipids and minerals compared to the other cheeses. There was a decrease in the amount of short-chain fatty acids (SCFAs) and an increase of medium-chain (MCFAs) and long-chain fatty acids (LCFAs) in the synbiotic cheeses compared to CC. The amount of conjugated linoleic acid increased in CLA, CBB and CLB. The highest depth of proteolysis and the greatest changes in the release of free amino acids were found in CLB. The addition of inulin and probiotics, alone or in co-culture, did not affect the cheese acceptance. Inulin and probiotics can be used together for the production of creamy goat cheese without negatively affecting the general quality characteristics of the product, and to add value because of its synbiotic potential.

  14. Strains of Lactococcus lactis with a partial pyrimidine requirement show sensitivity toward aspartic acid

    DEFF Research Database (Denmark)

    Wadskov-Hansen, Steen Lyders Lerche; Martinussen, Jan

    2009-01-01

    The growth rate of the widely used laboratory strain Lactococcus lactis subsp. cremoris LM0230 was reduced if aspartic acid were present in the growth medium. The strain LM0230 is a plasmid- and phage-cured derivative of L. lactis subsp. cremoris C2, the ancestor of the original dairy isolate L...... with the wild-type strain, and this varied with the concentration of aspartic acid. The observed effect of aspartate could be explained by the accumulation of the toxic pyrimidine de novo pathway intermediate, carbamoyl aspartate. Assays of the pyrimidine biosynthetic enzymes of L. lactis LM0230 showed...... that the partial pyrimidine requirement can be explained by a low specific activity of the pyrimidine biosynthetic enzymes. In conclusion, L. lactis LM0230 during the process of plasmid- and prophage-curing has acquired a partial pyrimidine requirement resulting in sensitivity toward aspartic acid....

  15. Safety of Bifidobacterium animalis subsp. lactis (B. lactis) strain BB-12®-supplemented yogurt in healthy children

    Science.gov (United States)

    Tan, Tina P.; Ba, Zhaoyong; Sanders, Mary Ellen; D’Amico, Frank J.; Roberts, Robert F.; Smith, Keisha Herbin; Merenstein, Daniel J.

    2016-01-01

    Objectives Probiotics are live microorganisms that may provide health benefits to the individual when consumed in sufficient quantities. For studies conducted on health or disease endpoints on probiotics in the United States, the Food and Administration (FDA) has required those studies to be conducted as investigational new drugs. This phase I, double-blinded, randomized, controlled safety study represents the first requirement of this pathway. The purpose of the study was to determine the safety of Bifidobacterium animalis subsp. lactis (B. lactis) strain BB-12® (BB-12®)-supplemented yogurt when consumed by a generally healthy group of children. The secondary aim was to assess the effect of BB-12®-supplemented yogurt on the gut microbiota of the children. Methods Sixty children aged 1–5 years were randomly assigned to consume four ounces of either BB-12®-supplemented yogurt or non-supplemented control yogurt daily for 10 days. The primary outcome was to assess safety and tolerability, as determined by the number of reported adverse events. Results A total of 186 non-serious adverse events were reported, with no significant differences between the control and BB-12® groups. No significant changes due to probiotic treatment were observed in the gut microbiota of the study cohort. Conclusions BB-12®-supplemented yogurt is safe and well-tolerated when consumed by healthy children. This study will form the basis for future randomized clinical trials investigating the potential effects of BB-12®-supplemented yogurt in different disease states. PMID:28114246

  16. Genome Sequence of Lactobacillus delbrueckii subsp. lactis CNRZ327, a Dairy Bacterium with Anti-Inflammatory Properties.

    Science.gov (United States)

    El Kafsi, Hela; Binesse, Johan; Loux, Valentin; Buratti, Julien; Boudebbouze, Samira; Dervyn, Rozenn; Hammani, Amal; Maguin, Emmanuelle; van de Guchte, Maarten

    2014-07-17

    Lactobacillus delbrueckii subsp. lactis CNRZ327 is a dairy bacterium with anti-inflammatory properties both in vitro and in vivo. Here, we report the genome sequence of this bacterium, which appears to contain no less than 215 insertion sequence (IS) elements, an exceptionally high number regarding the small genome size of the strain. Copyright © 2014 El Kafsi et al.

  17. Unleashing Natural Competence in Lactococcus lactis by Induction of the Competence Regulator ComX

    Science.gov (United States)

    Mulder, Joyce; Wels, Michiel; Kuipers, Oscar P.; Bron, Peter A.

    2017-01-01

    ABSTRACT In biotechnological workhorses like Streptococcus thermophilus and Bacillus subtilis, natural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starter Lactococcus lactis, natural competence has not been established to date. However, in silico analysis of the complete genome sequences of 43 L. lactis strains revealed complete late competence gene sets in 2 L. lactis subsp. cremoris strains (KW2 and KW10) and at least 10 L. lactis subsp. lactis strains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulator comX in L. lactis subsp. lactis KF147 resulted in the induction of expression of the canonical competence regulon and elicited a state of natural competence in this strain. In contrast, comX expression in L. lactis NZ9000, which was predicted to encode an incomplete competence gene set, failed to induce natural competence. Moreover, mutagenesis of the comEA-EC operon in strain KF147 abolished the comX-driven natural competence, underlining the involvement of the competence machinery. Finally, introduction of nisin-inducible comX expression into nisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence in these strains also, expanding this phenotype to other L. lactis strains of both subspecies. IMPORTANCE Specific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the species Lactococcus lactis are very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end product. The activation of natural competence in this industrially

  18. Fate of Lactococcus lactis starter cultures during late ripening in cheese models.

    Science.gov (United States)

    Ruggirello, Marianna; Cocolin, Luca; Dolci, Paola

    2016-10-01

    The presence of Lactococcus lactis, commonly employed as starter culture, was, recently, highlighted and investigated during late cheese ripening. Thus, the main goal of the present study was to assess the persistence and viability of this microorganism throughout manufacturing and ripening of model cheeses. Eight commercial starters, constituted of L. lactis subsp. lactis and L. lactis subsp. cremoris, were inoculated in pasteurized milk in order to manufacture miniature cheeses, ripened for six months. Samples were analysed at different steps (milk after inoculum, curd after cutting, curd after pressing and draining, cheese immediately after salting and cheese at 7, 15, 30, 60, 90, 120, 150 and 180 days of ripening) and submitted to both culture-dependent (traditional plating on M17) and -independent analysis (reverse transcription-quantitative PCR). On the basis of direct RNA analysis, L. lactis populations were detected in all miniature cheeses up to the sixth month of ripening, confirming the presence of viable cells during the whole ripening process, including late stages. Noteworthy, L. lactis was detected by RT-qPCR in cheese samples also when traditional plating failed to indicate its presence. This discrepancy could be explain with the fact that lactococci, during ripening process, enter in a stressed physiological state (viable not culturable, VNC), which might cause their inability to grow on synthetic medium despite their viability in cheese matrix. Preliminary results obtained by "resuscitation" assays corroborated this hypothesis and 2.5% glucose enrichment was effective to recover L. lactis cells in VNC state. The capability of L. lactis to persist in late ripening, and the presence of VNC cells which are known to shift their catabolism to peptides and amino acids consumption, suggests a possible technological role of this microorganism in cheese ripening with a possible impact on flavour formation. Copyright © 2016 Elsevier Ltd. All rights

  19. Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) stimulates murine macrophages infected with Citrobacter rodentium.

    Science.gov (United States)

    Hugo, Ayelén A; Rolny, Ivanna S; Romanin, David; Pérez, Pablo F

    2017-03-01

    Citrobacter rodentium is a specific murine enteropathogen which causes diarrheal disease characterized by colonic hyperplasia and intestinal inflammation. Recruitment of neutrophils and macrophages constitute a key step to control the infection. Since modulation of the activity of professional phagocytic cells could contribute to improve host´s defences against C. rodentium, we investigated the effect of Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) on the interaction between murine macrophages (RAW 264.7) and C. rodentium. Phagocytosis, surface molecules and inducible nitric oxide synthase (iNOs) expression were determined by flow cytometry. Reactive oxygen species (ROS) were assessed by fluorescence microscopy. The presence of lactobacilli increased phagocytosis of C. rodentium whereas C. rodentium had no effect on lactobacilli internalization. Survival of internalized C. rodentium diminished when strain CIDCA 133 was present. CD-86, MHCII, iNOs expression and nitrite production were increased when C. rodentium and lactobacilli were present even though strain CIDCA 133 alone had no effect. Strain CIDCA 133 led to a strong induction of ROS activity which was not modified by C. rodentium. Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) is able to increase the activation of murine macrophages infected with C. rodentium. The sole presence of lactobacilli is enough to modify some stimulation markers (e.g. ROS induction) whereas other markers require the presence of both bacteria; thus, indicating a synergistic effect.

  20. Characterization of the mature cell surface proteinase of Lactobacillus delbrueckii subsp. lactis CRL 581.

    Science.gov (United States)

    Villegas, Josefina M; Brown, Lucía; Savoy de Giori, Graciela; Hebert, Elvira M

    2015-05-01

    The cell envelope-associated proteinase (CEP) of Lactobacillus delbrueckii subsp. lactis CRL 581 (PrtL) has an essential role in bacterial growth, contributes to the flavor and texture development of fermented products, and can release bioactive health-beneficial peptides during milk fermentation. The genome of L. delbrueckii subsp. lactis CRL 581 possesses only one gene that encodes PrtL, which consists of 1924 amino acids and is a multidomain protein anchored to the cell via its W domain. PrtL was extracted from the cell under high ionic strength conditions using NaCl, suggesting an electrostatic interaction between the proteinase and the cell envelope. The released PrtL was purified and biochemically characterized; its activity was maximal at temperatures between 37 and 40 °C and at pH between 7 and 8. Under optimal conditions, PrtL exhibited higher affinity for succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide than for succinyl-alanyl-glutamyl-prolyl-phenylalanine-p-nitroanilide, while methoxy-succinyl-arginyl-prolyl-tyrosyl-p-nitroanilide was not degraded. A similar α- and β-casein degradation pattern was observed with the purified and the cell envelope-bound proteinase. Finally, on the basis of its specificity towards caseins and the unique combination of amino acids at residues thought to be involved in substrate specificity, PrtL can be classified as a representative of a new group of CEP.

  1. Lactococcus lactis ssp. lactis as Potential Functional Starter Culture

    Directory of Open Access Journals (Sweden)

    Jelena Cvrtila

    2014-01-01

    Full Text Available The aim of this study is to identify and characterise potential autochthonous functional starter cultures in homemade horsemeat sausage. The dominant microflora in the samples of horsemeat sausage were lactic acid bacteria (LAB, followed by micrococci. Among the LAB, Lactococcus lactis ssp. lactis and Lactobacillus plantarum were the dominant species, and since the first is not common in fermented sausages, we characterised it as a potential functional starter culture. Lactococcus lactis ssp. lactis produced a significant amount of lactic acid, displayed good growth capability at 12, 18 and 22 °C, growth in the presence of 5 % NaCl, good viability after lyophilisation and in simulated gastric and small intestinal juice, antimicrobial activity against test pathogens, and good adhesive properties in vitro.

  2. Continuous D-lactic acid production by a novel thermotolerant Lactobacillus delbrueckii subsp. lactis QU 41.

    Science.gov (United States)

    Tashiro, Yukihiro; Kaneko, Wataru; Sun, Yanqi; Shibata, Keisuke; Inokuma, Kentaro; Zendo, Takeshi; Sonomoto, Kenji

    2011-03-01

    We isolated and characterized a D-lactic acid-producing lactic acid bacterium (D-LAB), identified as Lactobacillus delbrueckii subsp. lactis QU 41. When compared to Lactobacillus coryniformis subsp. torquens JCM 1166 (T) and L. delbrueckii subsp. lactis JCM 1248 (T), which are also known as D-LAB, the QU 41 strain exhibited a high thermotolerance and produced D-lactic acid at temperatures of 50 °C and higher. In order to optimize the culture conditions of the QU 41 strain, we examined the effects of pH control, temperature, neutralizing reagent, and initial glucose concentration on D-lactic acid production in batch cultures. It was found that the optimal production of 20.1 g/l D-lactic acid was acquired with high optical purity (>99.9% of D-lactic acid) in a pH 6.0-controlled batch culture, by adding ammonium hydroxide as a neutralizing reagent, at 43 °C in MRS medium containing 20 g/l glucose. As a result of product inhibition and low cell density, continuous cultures were investigated using a microfiltration membrane module to recycle flow-through cells in order to improve D-lactic acid productivity. At a dilution rate of 0.87 h(-1), the high cell density continuous culture exhibited the highest D-lactic acid productivity of 18.0 g/l/h with a high yield (ca. 1.0 g/g consumed glucose) and a low residual glucose (<0.1 g/l) in comparison with systems published to date.

  3. DEGRADATION AND DEBITTERING OF A TRYPTIC DIGEST FROM BETA-CASEIN BY AMINOPEPTIDASE-N FROM LACTOCOCCUS-LACTIS SUBSP CREMORIS WG2

    NARCIS (Netherlands)

    TAN, PST; VANKESSEL, TAJM; VANDEVEERDONK, FLM; ZUURENDONK, PF; BRUINS, AP; KONINGS, WN

    The mode of action of purified aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2 on a complex peptide mixture of a tryptic digest from bovine beta-casein was analyzed. The oligopeptides produced in the tryptic digest before and after aminopeptidase N treatment were identified by analysis

  4. Proteome analysis of the purine stimulon from Lactococcus lactis

    DEFF Research Database (Denmark)

    Beyer, N.H.; Roepstorff, P.; Hammer, Karin

    2003-01-01

    kinase) and translation elongation factors (GTPases: EF-TU, EF-G). Two Dcu proteins could not be identified. Out of 28 proteins subjected to mass spectrometry, 19 could be readily identified despite the fact that only the genome sequence of a strain of L. lactis subsp. lactis was available. The two...... subspecies share about 85% sequence identity, comparable to the genetic distance between Escherichia coli and Salmonella typhimurium. A success rate of 68% indicates that it may be feasible to perform proteomics based upon genomic sequences of relatives outside the genus....

  5. Safety of Bifidobacterium animalis subsp. lactis (B. lactis) strain BB-12-supplemented yogurt in healthy adults on antibiotics: a phase I safety study.

    Science.gov (United States)

    Merenstein, Daniel J; Tan, Tina P; Molokin, Aleksey; Smith, Keisha Herbin; Roberts, Robert F; Shara, Nawar M; Mete, Mihriye; Sanders, Mary Ellen; Solano-Aguilar, Gloria

    2015-01-01

    Probiotics are live microorganisms that, when administered in sufficient doses, provide health benefits on the host. The United States Food and Drug Administration (FDA) requires phase I safety studies for probiotics when the intended use of the product is as a drug. The purpose of the study was to determine the safety of Bifidobacterium animalis subsp lactis (B. lactis) strain BB-12 (BB-12)-supplemented yogurt when consumed by a generally healthy group of adults who were prescribed a 10-day course of antibiotics for a respiratory infection. Secondary aims were to assess the ability of BB-12 to affect the expression of whole blood immune markers associated with cell activation and inflammatory response. A phase I, double-blinded, randomized controlled study was conducted in compliance with FDA guidelines for an Investigational New Drug (IND). Forty participants were randomly assigned to consume 4 ounces of either BB-12 -supplemented yogurt or non-supplemented control yogurt daily for 10 d. The primary outcome was to assess safety and tolerability, assessed by the number of reported adverse events. A total of 165 non-serious adverse events were reported, with no differences between the control and BB-12 groups. When compared to the control group, B lactis fecal levels were modestly higher in the BB-12-supplemented group. In a small subset of patients, changes in whole blood expression of genes associated with regulation and activation of immune cells were detected in the BB-12-supplemented group. BB-12-supplemented yogurt is safe and well tolerated when consumed by healthy adults concurrently taking antibiotics. This study will form the basis for future randomized clinical trials investigating the potential immunomodulatory effects of BB-12-supplemented yogurt in a variety of disease states.

  6. Genome Sequence Analysis of the Biogenic Amine-Producing Strain Lactococcus lactis subsp. cremoris CECT 8666 (Formerly GE2-14)

    Science.gov (United States)

    del Rio, Beatriz; Linares, Daniel M.; Fernandez, Maria; Mayo, Baltasar; Martin, M. Cruz; Alvarez, Miguel A.

    2014-01-01

    We here report a 2,801,031-bp annotated draft assembly for the Lactococcus lactis subsp. cremoris GE2-14 genome. This dairy strain produces the biogenic amine putrescine. This sequence may help identify the mechanisms regulating putrescine biosynthesis and throw light on ways to reduce its presence in fermented foods. PMID:25342694

  7. Proton Motive Force-Driven and ATP-Dependent Drug Extrusion Systems in Multidrug-Resistant Lactococcus lactis

    NARCIS (Netherlands)

    BOLHUIS, H; MOLENAAR, D; POELARENDS, G; VANVEEN, HW; POOLMAN, B; DRIESSEN, AJM; KONINGS, WN

    1994-01-01

    Three mutants of Lactococcus lactis subsp. lactis MG1363, termed Eth(R), Dau(R), and Rho(R), were selected for resistance to high concentrations of ethidium bromide, daunomycin, and rhodamine 6G, respectively. These mutants were found to be cross resistant to a number of structurally and

  8. Interaction between the genomes of Lactococcus lactis and phages of the P335 species

    Science.gov (United States)

    Kelly, William J.; Altermann, Eric; Lambie, Suzanne C.; Leahy, Sinead C.

    2013-01-01

    Phages of the P335 species infect Lactococcus lactis and have been particularly studied because of their association with strains of L. lactis subsp. cremoris used as dairy starter cultures. Unlike other lactococcal phages, those of the P335 species may have a temperate or lytic lifestyle, and are believed to originate from the starter cultures themselves. We have sequenced the genome of L. lactis subsp. cremoris KW2 isolated from fermented corn and found that it contains an integrated P335 species prophage. This 41 kb prophage (Φ KW2) has a mosaic structure with functional modules that are highly similar to several other phages of the P335 species associated with dairy starter cultures. Comparison of the genomes of 26 phages of the P335 species, with either a lytic or temperate lifestyle, shows that they can be divided into three groups and that the morphogenesis gene region is the most conserved. Analysis of these phage genomes in conjunction with the genomes of several L. lactis strains shows that prophage insertion is site specific and occurs at seven different chromosomal locations. Exactly how induced or lytic phages of the P335 species interact with carbohydrate cell surface receptors in the host cell envelope remains to be determined. Genes for the biosynthesis of a variable cell surface polysaccharide and for lipoteichoic acids (LTAs) are found in L. lactis and are the main candidates for phage receptors, as the genes for other cell surface carbohydrates have been lost from dairy starter strains. Overall, phages of the P335 species appear to have had only a minor role in the adaptation of L. lactis subsp. cremoris strains to the dairy environment, and instead they appear to be an integral part of the L. lactis chromosome. There remains a great deal to be discovered about their role, and their contribution to the evolution of the bacterial genome. PMID:24009606

  9. Sequencing and Transcriptional Analysis of the Biosynthesis Gene Cluster of Putrescine-Producing Lactococcus lactis ▿ †

    Science.gov (United States)

    Ladero, Victor; Rattray, Fergal P.; Mayo, Baltasar; Martín, María Cruz; Fernández, María; Alvarez, Miguel A.

    2011-01-01

    Lactococcus lactis is a prokaryotic microorganism with great importance as a culture starter and has become the model species among the lactic acid bacteria. The long and safe history of use of L. lactis in dairy fermentations has resulted in the classification of this species as GRAS (General Regarded As Safe) or QPS (Qualified Presumption of Safety). However, our group has identified several strains of L. lactis subsp. lactis and L. lactis subsp. cremoris that are able to produce putrescine from agmatine via the agmatine deiminase (AGDI) pathway. Putrescine is a biogenic amine that confers undesirable flavor characteristics and may even have toxic effects. The AGDI cluster of L. lactis is composed of a putative regulatory gene, aguR, followed by the genes (aguB, aguD, aguA, and aguC) encoding the catabolic enzymes. These genes are transcribed as an operon that is induced in the presence of agmatine. In some strains, an insertion (IS) element interrupts the transcription of the cluster, which results in a non-putrescine-producing phenotype. Based on this knowledge, a PCR-based test was developed in order to differentiate nonproducing L. lactis strains from those with a functional AGDI cluster. The analysis of the AGDI cluster and their flanking regions revealed that the capacity to produce putrescine via the AGDI pathway could be a specific characteristic that was lost during the adaptation to the milk environment by a process of reductive genome evolution. PMID:21803900

  10. Plasmids of Raw Milk Cheese Isolate Lactococcus lactis subsp. lactis Biovar diacetylactis DPC3901 Suggest a Plant-Based Origin for the Strain ▿ †

    Science.gov (United States)

    Fallico, Vincenzo; McAuliffe, Olivia; Fitzgerald, Gerald F.; Ross, R. Paul

    2011-01-01

    The four-plasmid complement of the raw milk cheese isolate Lactococcus lactis subsp. lactis biovar diacetylactis DPC3901 was sequenced, and some genetic features were functionally analyzed. The complete sequences of pVF18 (18,977 bp), pVF21 (21,739 bp), pVF22 (22,166 bp), and pVF50 (53,876 bp) were obtained. Each plasmid contained genes not previously described for Lactococcus, in addition to genes associated with plant-derived lactococcal strains. Most of the novel genes were found on pVF18 and encoded functions typical of bacteria associated with plants, such as activities of plant cell wall modification (orf11 and orf25). In addition, a predicted high-affinity regulated system for the uptake of cobalt was identified (orf19 to orf21 [orf19-21]), which has a single database homolog on a plant-derived Leuconostoc plasmid and whose functionality was demonstrated following curing of pVF18. pVF21 and pVF22 encode additional metal transporters, which, along with orf19-21 of pVF18, could enhance host ability to uptake growth-limiting amounts of biologically essential ions within the soil. In addition, vast regions from pVF50 and pVF21 share significant homology with the plant-derived lactococcal plasmid pGdh442, which is indicative of extensive horizontal gene transfer and recombination between these plasmids and suggests a common plant niche for their hosts. Phenotypes associated with these regions include glutamate dehydrogenase activity and Na+ and K+ transport. The presence of numerous plant-associated markers in L. lactis DPC3901 suggests a plant origin for the raw milk cheese isolate and provides for the first time the genetic basis to support the concept of the plant-milk transition for Lactococcus strains. PMID:21803914

  11. Structural basis for arabinoxylo‐oligosaccharide capture by the probiotic Bifidobacterium animalis subsp. lactis Bl‐04

    DEFF Research Database (Denmark)

    Hansen, Morten Ejby; Fredslund, Folmer; Vujicic‐Zagar, Andreja

    2013-01-01

    Glycan utilization plays a key role in modulating the composition of the gut microbiota, but molecular insight into oligosaccharide uptake by this microbial community is lacking. Arabinoxylo‐oligosaccharides (AXOS) are abundant in the diet, and are selectively fermented by probiotic bifidobacteria...... in the colon. Here we show how selectivity for AXOS uptake is established by the probiotic strain Bifidobacterium animalis subsp. lactis Bl‐04. The binding protein BlAXBP, which is associated with an ATP‐binding cassette (ABC) transporter that mediates the uptake of AXOS, displays an exceptionally broad...

  12. Lactococcus lactis is diploid

    DEFF Research Database (Denmark)

    Michelsen, Ole; Jensen, Peter Ruhdal

    As part of a collaboration with Danish Dairy Research Foundation we are interested in the DNA replication of Lactococcus lactis. For that we implemented flowcytometric analysis for these studies. The L. lactis does not respond to inhibition by rifampicin by finishing ongoing replication forks. We....... This unexpected result has been confirmed by radioactive labelling of slow growing cultures of Lactococcus lactis, which also showed the presence of two chromosomes. We therefore conclude that Lactococcus lactis is the first diploid bacterium found....... therefore turned to slow growing cultures in order to obtain information about the DNA replication in the cell cycle. From these studies we have obtained evidence that suggest that slow growing L. lactis are born with two chromosomes in contrast to other studied bacteria, which are born with one chromosome...

  13. Analysis of heat shock gene expression in Lactococcus lactis MG1363

    DEFF Research Database (Denmark)

    Arnau, José; Sørensen, Kim; Appel, Karen Fuglede

    1996-01-01

    The induction of the heat shock response in Lactococcus lactis subsp. cremoris strain MG1363 was analysed at the RNA level using a novel RNA isolation procedure to prevent degradation. Cloning of the dnaJ and groEL homologous was carried out. Nothern blot analysis showed a similar induction pattern...... in the heat shock response in L. lactis MG1363 is presented. A gene located downstream of the dnaK operon in strain MG1363, named orf4, was shown not to be regulated by heat shock....

  14. High efficiency electrotransformation of Lactococcus lactis spp. lactis cells pretreated with lithium acetate and dithiothreitol

    Directory of Open Access Journals (Sweden)

    Filioussis George

    2007-03-01

    Full Text Available Abstract Background A goal for the food industry has always been to improve strains of Lactococcus lactis and stabilize beneficial traits. Genetic engineering is used extensively for manipulating this lactic acid bacterium, while electropolation is the most widely used technique for introducing foreign DNA into cells. The efficiency of electrotransformation depends on the level of electropermealization and pretreatment with chemicals which alter cell wall permeability, resulting in improved transformation efficiencies is rather common practice in bacteria as in yeasts and fungi. In the present study, treatment with lithium acetate (LiAc and dithiothreitol (DTT in various combinations was applied to L. lactis spp. lactis cells of the early-log phase prior to electroporation with plasmid pTRKH3 (a 7.8 kb shuttle vector, suitable for cloning into L. lactis. Two strains of L. lactis spp. lactis were used, L. lactis spp. lactis LM0230 and ATCC 11454. To the best of our knowledge these agents have never been used before with L. lactis or other bacteria. Results Electrotransformation efficiencies of up to 105 transformants per μg DNA have been reported in the literature for L. lactis spp.lactis LM0230. We report here that treatment with LiAc and DDT before electroporation increased transformation efficiency to 225 ± 52.5 × 107 transformants per μg DNA, while with untreated cells or treated with LiAc alone transformation efficiency approximated 1.2 ± 0.5 × 105 transformants per μg DNA. Results of the same trend were obtained with L. lactis ATCC 11454, although transformation efficiency of this strain was significantly lower. No difference was found in the survival rate of pretreated cells after electroporation. Transformation efficiency was found to vary directly with cell density and that of 1010 cells/ml resulted in the highest efficiencies. Following electrotransformation of pretreated cells with LiAc and DDT, pTRKH3 stability was examined

  15. Technological characterization and survival of the exopolysaccharide-producing strain Lactobacillus delbrueckii subsp. lactis 193 and its bile-resistant derivative 193+ in simulated gastric and intestinal juices.

    Science.gov (United States)

    Burns, Patricia; Vinderola, Gabriel; Reinheimer, Jorge; Cuesta, Isabel; de Los Reyes-Gavilán, Clara G; Ruas-Madiedo, Patricia

    2011-08-01

    The capacity of lactic acid bacteria to produce exopolysaccharides (EPS) conferring microorganisms a ropy phenotype could be an interesting feature from a technological point of view. Progressive adaptation to bile salts might render some lactobacilli able to overcome physiological gut barriers but could also modify functional properties of the strain, including the production of EPS. In this work some technological properties and the survival ability in simulated gastrointestinal conditions of Lactobacillus delbrueckii subsp. lactis 193, and Lb. delbrueckii subsp. lactis 193+, a strain with stable bile-resistant phenotype derived thereof, were characterized in milk in order to know whether the acquisition of resistance to bile could modify some characteristics of the microorganism. Both strains were able to grow and acidify milk similarly; however the production of ethanol increased at the expense of the aroma compound acetaldehyde in milk fermented by the strain 193+, with respect to milk fermented by the strain 193. Both microorganisms produced a heteropolysaccharide composed of glucose and galactose, and were able to increase the viscosity of fermented milks. In spite of the higher production yield of EPS by the bile-resistant strain 193+, it displayed a lower ability to increase viscosity than Lb. delbrueckii subsp. lactis 193. Milk increased survival in simulated gastric juice; the presence of bile improved adhesion to the intestinal cell line HT29-MTX in both strains. However, the acquisition of a stable resistance phenotype did not improve survival in simulated gastric and intestinal conditions or the adhesion to the intestinal cell line HT29-MTX. Thus, Lb. delbrueckii subsp. lactis 193 presents suitable technological properties for the manufacture of fermented dairy products; the acquisition of a stable bile-resistant phenotype modified some properties of the microorganism. This suggests that the possible use of bile-resistant derivative strains should be

  16. Development of a pentaplex PCR assay for the simultaneous detection of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. helveticus, L. fermentum in whey starter for Grana Padano cheese.

    Science.gov (United States)

    Cremonesi, Paola; Vanoni, Laura; Morandi, Stefano; Silvetti, Tiziana; Castiglioni, Bianca; Brasca, Milena

    2011-03-30

    A pentaplex PCR assay for the rapid, selective and simultaneous detection of Lactobacillus helveticus, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and L. fermentum, was developed. The target sequences were a group of genes coding for beta-galactosidase production (S. thermophilus and L. delbrueckii subsp. bulgaricus), for cell-enveloped associated proteinase synthesis (L. helveticus), for dipeptide transport system production (L. delbrueckii subsp. lactis) and for arginine-ornithine antiporter protein production (L. fermentum). The analytical specificity of the assay was evaluated with 5 reference strains and 140 lactic acid bacterial strains derived from raw milk cheeses and belonging to the Lactobacillus, Streptococcus, Lactococcus and Enterococcus genera. The identification limit for each target strain was 10(3)CFU/ml. This new molecular assay was used to investigate the LAB population by direct extraction of DNA from the 12 whey cultures for Grana Padano. The pentaplex PCR assay revealed a good correspondence with microbiological analyses and allowed to identify even minor LAB community members which, can be out-competed in vitro by numerically more abundant microbial species. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Genes but not genomes reveal bacterial domestication of Lactococcus lactis.

    Directory of Open Access Journals (Sweden)

    Delphine Passerini

    Full Text Available BACKGROUND: The population structure and diversity of Lactococcus lactis subsp. lactis, a major industrial bacterium involved in milk fermentation, was determined at both gene and genome level. Seventy-six lactococcal isolates of various origins were studied by different genotyping methods and thirty-six strains displaying unique macrorestriction fingerprints were analyzed by a new multilocus sequence typing (MLST scheme. This gene-based analysis was compared to genomic characteristics determined by pulsed-field gel electrophoresis (PFGE. METHODOLOGY/PRINCIPAL FINDINGS: The MLST analysis revealed that L. lactis subsp. lactis is essentially clonal with infrequent intra- and intergenic recombination; also, despite its taxonomical classification as a subspecies, it displays a genetic diversity as substantial as that within several other bacterial species. Genome-based analysis revealed a genome size variability of 20%, a value typical of bacteria inhabiting different ecological niches, and that suggests a large pan-genome for this subspecies. However, the genomic characteristics (macrorestriction pattern, genome or chromosome size, plasmid content did not correlate to the MLST-based phylogeny, with strains from the same sequence type (ST differing by up to 230 kb in genome size. CONCLUSION/SIGNIFICANCE: The gene-based phylogeny was not fully consistent with the traditional classification into dairy and non-dairy strains but supported a new classification based on ecological separation between "environmental" strains, the main contributors to the genetic diversity within the subspecies, and "domesticated" strains, subject to recent genetic bottlenecks. Comparison between gene- and genome-based analyses revealed little relationship between core and dispensable genome phylogenies, indicating that clonal diversification and phenotypic variability of the "domesticated" strains essentially arose through substantial genomic flux within the dispensable

  18. Riboflavin Production in Lactococcus lactis: Potential for In Situ Production of Vitamin-Enriched Foods

    Science.gov (United States)

    Burgess, Catherine; O'Connell-Motherway, Mary; Sybesma, Wilbert; Hugenholtz, Jeroen; van Sinderen, Douwe

    2004-01-01

    This study describes the genetic analysis of the riboflavin (vitamin B2) biosynthetic (rib) operon in the lactic acid bacterium Lactococcus lactis subsp. cremoris strain NZ9000. Functional analysis of the genes of the L. lactis rib operon was performed by using complementation studies, as well as by deletion analysis. In addition, gene-specific genetic engineering was used to examine which genes of the rib operon need to be overexpressed in order to effect riboflavin overproduction. Transcriptional regulation of the L. lactis riboflavin biosynthetic process was investigated by using Northern hybridization and primer extension, as well as the analysis of roseoflavin-induced riboflavin-overproducing L. lactis isolates. The latter analysis revealed the presence of both nucleotide replacements and deletions in the regulatory region of the rib operon. The results presented here are an important step toward the development of fermented foods containing increased levels of riboflavin, produced in situ, thus negating the need for vitamin fortification. PMID:15466513

  19. The Plasmid Complement of Lactococcus lactis UC509.9 Encodes Multiple Bacteriophage Resistance Systems

    Science.gov (United States)

    Ainsworth, Stuart; Mahony, Jennifer

    2014-01-01

    Lactococcus lactis subsp. cremoris strains are used globally for the production of fermented dairy products, particularly hard cheeses. Believed to be of plant origin, L. lactis strains that are used as starter cultures have undergone extensive adaptation to the dairy environment, partially through the acquisition of extrachromosomal DNA in the form of plasmids that specify technologically important phenotypic traits. Here, we present a detailed analysis of the eight plasmids of L. lactis UC509.9, an Irish dairy starter strain. Key industrial phenotypes were mapped, and genes that are typically associated with lactococcal plasmids were identified. Four distinct, plasmid-borne bacteriophage resistance systems were identified, including two abortive infection systems, AbiB and AbiD1, thereby supporting the observed phage resistance of L. lactis UC509.9. AbiB escape mutants were generated for phage sk1, which were found to carry mutations in orf6, which encodes the major capsid protein of this phage. PMID:24814781

  20. Formation and conversion of oxygen metabolites by Lactococcus lactis subsp lactis ATCC 19435 under different growth conditions

    NARCIS (Netherlands)

    Niel, van E.W.J.; Hofvendahl, K.; Hahn Hagerdal, B.

    2002-01-01

    A semidefined medium based on Casamino Acids allowed Lactococcus lactis ATCC 19435 to grow in the presence of oxygen at a slow rate (0.015 h-1). Accumulation of H2O2 in the culture prevented a higher growth rate. Addition of asparagine to the medium increased the growth rate, whereby H2O2

  1. Identification and characterization of tetracycline resistance in Lactococcus lactis isolated from Polish raw milk and fermented artisanal products.

    Science.gov (United States)

    Zycka-Krzesinska, Joanna; Boguslawska, Joanna; Aleksandrzak-Piekarczyk, Tamara; Jopek, Jakub; Bardowski, Jacek K

    2015-10-15

    To assess the occurrence of antibiotic-resistant Lactic Acid Bacteria (LAB) in Polish raw milk and fermented artisanal products, a collection comprising 500 isolates from these products was screened. Among these isolates, six strains (IBB28, IBB160, IBB161, IBB224, IBB477 and IBB487) resistant to tetracycline were identified. The strains showing atypical tetracycline resistance were classified as Lactococcus lactis: three of them were identified as L. lactis subsp. cremoris (IBB224, IBB477 and IBB487) and the other three (IBB28, IBB160, IBB161) were identified as L. lactis subsp. lactis. The mechanism involving Ribosomal Protection Proteins (RPP) was identified as responsible for tetracycline resistance. Three of the tested strains (IBB28, IBB160 and IBB224) had genes encoding the TetS protein, whereas the remaining three (IBB161, IBB477 and IBB487) expressed TetM. The results also demonstrated that the genes encoding these proteins were located on genetic mobile elements. The tet(S) gene was found to be located on plasmids, whereas tet(M) was found within the Tn916 transposon. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Structure and properties of the metastable bacteriocin Lcn972 from Lactococcus lactis

    Science.gov (United States)

    Turner, David L.; Lamosa, Pedro; Rodríguez, Ana; Martínez, Beatriz

    2013-01-01

    Lactococcus lactis subsp. lactis IPLA 972 produces a polypeptide bacteriocin of 7.5 kDa which has a bactericidal effect on sensitive lactococci, inhibiting septum formation in dividing cells. The active form is a monomer that is metastable under normal conditions but is stabilised by glycerol. The NMR structure of Lcn972 shows a β-sandwich comprising two three-stranded antiparallel β-sheets. Detaching the final strand could allow the sandwich to open, and the irreversible unfolding leads to a loss of antibacterial activity. Covalent linkage of the final strand should increase the stability of Lcn972 and facilitate the study of its interaction with lipid II.

  3. Expanding the molecular toolbox for Lactococcus lactis: construction of an inducible thioredoxin gene fusion expression system

    LENUS (Irish Health Repository)

    Douillard, Francois P

    2011-08-09

    Abstract Background The development of the Nisin Inducible Controlled Expression (NICE) system in the food-grade bacterium Lactococcus lactis subsp. cremoris represents a cornerstone in the use of Gram-positive bacterial expression systems for biotechnological purposes. However, proteins that are subjected to such over-expression in L. lactis may suffer from improper folding, inclusion body formation and\\/or protein degradation, thereby significantly reducing the yield of soluble target protein. Although such drawbacks are not specific to L. lactis, no molecular tools have been developed to prevent or circumvent these recurrent problems of protein expression in L. lactis. Results Mimicking thioredoxin gene fusion systems available for E. coli, two nisin-inducible expression vectors were constructed to over-produce various proteins in L. lactis as thioredoxin fusion proteins. In this study, we demonstrate that our novel L. lactis fusion partner expression vectors allow high-level expression of soluble heterologous proteins Tuc2009 ORF40, Bbr_0140 and Tuc2009 BppU\\/BppL that were previously insoluble or not expressed using existing L. lactis expression vectors. Over-expressed proteins were subsequently purified by Ni-TED affinity chromatography. Intact heterologous proteins were detected by immunoblotting analyses. We also show that the thioredoxin moiety of the purified fusion protein was specifically and efficiently cleaved off by enterokinase treatment. Conclusions This study is the first description of a thioredoxin gene fusion expression system, purposely developed to circumvent problems associated with protein over-expression in L. lactis. It was shown to prevent protein insolubility and degradation, allowing sufficient production of soluble proteins for further structural and functional characterization.

  4. Production of Angiotensin-I-Converting-Enzyme-Inhibitory Peptides in Fermented Milks Started by Lactobacillus delbrueckii subsp. bulgaricus SS1 and Lactococcus lactis subsp. cremoris FT4

    Science.gov (United States)

    Gobbetti, M.; Ferranti, P.; Smacchi, E.; Goffredi, F.; Addeo, F.

    2000-01-01

    Two fermented milks containing angiotensin-I-converting-enzyme (ACE)-inhibitory peptides were produced by using selected Lactobacillus delbrueckii subsp. bulgaricus SS1 and L. lactis subsp. cremoris FT4. The pH 4.6-soluble nitrogen fraction of the two fermented milks was fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest ACE-inhibitory indexes were further purified, and the related peptides were sequenced by tandem fast atom bombardment-mass spectrometry. The most inhibitory fractions of the milk fermented by L. delbrueckii subsp. bulgaricus SS1 contained the sequences of β-casein (β-CN) fragment 6-14 (f6-14), f7-14, f73-82, f74-82, and f75-82. Those from the milk fermented by L. lactis subsp. cremoris FT4 contained the sequences of β-CN f7-14, f47-52, and f169-175 and κ-CN f155-160 and f152-160. Most of these sequences had features in common with other ACE-inhibitory peptides reported in the literature. In particular, the β-CN f47-52 sequence had high homology with that of angiotensin-II. Some of these peptides were chemically synthesized. The 50% inhibitory concentrations (IC50s) of the crude purified fractions containing the peptide mixture were very low (8.0 to 11.2 mg/liter). When the synthesized peptides were used individually, the ACE-inhibitory activity was confirmed but the IC50s increased considerably. A strengthened inhibitory effect of the peptide mixtures with respect to the activity of individual peptides was presumed. Once generated, the inhibitory peptides were resistant to further proteolysis either during dairy processing or by trypsin and chymotrypsin. PMID:10966406

  5. Consumption of Yogurt Containing Probiotic Bifidobacterium Lactis Reduces Streptococcus mutans in Orthodontic Patients

    Directory of Open Access Journals (Sweden)

    Armelia Sari Widyarman

    2018-01-01

    Full Text Available Background: Probiotic bacteria is commonly used as a food supplement intended to benefit the host by improving intestinal bacterial balance. Probiotics have also been investigated from the perspective of oral health. Objectives: The purpose of this study was to investigate the effect of daily intake of yogurt containing probiotic Bifidobacterium animalis subsp. lactis BB-12 (B. lactis on salivary Streptococcus mutans (S. mutans counts in patients undergoing fixed orthodontic treatment. Methods: Saliva samples were collected from each subject (n = 7; mean age, 21 years using spitting method in centrifuge tubes at baseline and two weeks after daily probiotic yogurt consumption. B. lactis BB-12 and S. mutans ATCC 25175 were cultured in BHI-broth (37ºC, anaerobic conditions. After 48-h incubation, the number of colonies on each dilution plate was used to extrapolate a standard curve. The total number of target DNA molecules were identified using real-time PCR followed by SYBR Green reagents and 16S rRNA gene specific primers S. mutans and B. lactis BB-12. Data were analyzed statistically using paired-sample t-tests. Results: Statistical evaluation indicated that there was a significant reduction in the presence of S. mutans before probiotic yogurt consumption, (4.73 ± 1.43 log10 CFU/mL and after two weeks of daily consumption of probiotic yogurt, (4.03 ± 0.77 log10 CFU/mL, p=0.001. Moreover, no B. lactis was found in the saliva of any of the subjects before probiotic consumption, but after two weeks of consumption, B. lactis was found in the saliva of four subjects. Conclusions: Consuming probiotic yogurt containing B. lactis reduced the quantity of S. mutans in the saliva of subjects during fixed orthodontic treatment. Thus, the probiotic bacteria could be beneficial in improving oral health.

  6. Cloning and Characterization of upp, a Gene Encoding Uracil Phosphoribosyltransferase from Lactococcus lactis

    DEFF Research Database (Denmark)

    Martinussen, Jan; Hammer, Karin

    1994-01-01

    Uracil phosphoribosyltransferase catalyzes the key reaction in the salvage of uracil in many microorganisms. The gene encoding uracil phosphoribosyltransferase (upp) was cloned from Lactococcus lactis subsp. cremoris MG1363 by complementation of an Escherichia coli mutant. The gene was sequenced...

  7. Expanding the molecular toolbox for Lactococcus lactis: construction of an inducible thioredoxin gene fusion expression system

    OpenAIRE

    Cambillau Christian; O'Connell-Motherway Mary; Douillard François P; van Sinderen Douwe

    2011-01-01

    Abstract Background The development of the Nisin Inducible Controlled Expression (NICE) system in the food-grade bacterium Lactococcus lactis subsp. cremoris represents a cornerstone in the use of Gram-positive bacterial expression systems for biotechnological purposes. However, proteins that are subjected to such over-expression in L. lactis may suffer from improper folding, inclusion body formation and/or protein degradation, thereby significantly reducing the yield of soluble target protei...

  8. Engineering of sugar metabolism in Lactococcus lactis

    NARCIS (Netherlands)

    Pool, Weia Arianne

    2008-01-01

    Short English Summary Lactococcus lactis is a lactic acid bacterium used in the dairy industry. This thesis decribes the genetic engineering performed on the sugar metabolism of L. lactis. Besides our fundamental interest for sugar metabolism and its regulation in L. lactis, this project had the

  9. Purification and partial characterization of bacteriocin produced by Lactococcus lactis ssp. lactis LL171.

    Science.gov (United States)

    Kumari, Archana; Akkoç, Nefise; Akçelik, Mustafa

    2012-04-01

    Lactic acid bacteria (LAB) are possessing ability to synthesize antimicrobial compounds (like bacteriocin) during their growth. In this regard, novel bacteriocin compound secreting capability of LAB isolated from Tulum Cheese in Turkey was demonstrated. The synthesized bacteriocin was purified by ammonium sulphate precipitation, dialysis and gel filtration. The molecular weight (≈3.4 kDa) of obtained bacteriocin was confirmed by SDS-PAGE, which revealed single peptide band. Molecular identification of LAB strain isolated from Tulum Cheese was conducted using 16S rDNA gene sequencing as Lactococcus lactis ssp. lactis LL171. The amino acid sequences (KKIDTRTGKTMEKTEKKIELSLKNMKTAT) of the bacteriocin from Lactococcus lactis ssp. lactis LL171 was found unique and novel than reported bacteriocins. Further, the bacteriocin was possessed the thermostable property and active at wide range of pH values from 1 to 11. Thus, bacteriocin reported in this study has the potential applications property as food preservative agent.

  10. Coculture-inducible bacteriocin biosynthesis of different probiotic strains by dairy starter culture Lactococcus lactis

    Directory of Open Access Journals (Sweden)

    Blaženka Kos

    2011-12-01

    Full Text Available Bacteriocins produced by probiotic strains effectively contribute to colonization ability of probiotic strains and facilitate their establishment in the competitive gut environment and also protect the gut from gastrointestinal pathogens. Moreover, bacteriocins have received considerable attention due to their potential application as biopreservatives, especially in dairy industry. Hence, the objective of this research was to investigate antimicrobial activity of probiotic strains Lactobacillus helveticus M92, Lactobacillus plantarum L4 and Enterococcus faecium L3, with special focus on their bacteriocinogenic activity directed towards representatives of the same or related bacterial species, and towards distant microorganisms including potential food contaminants or causative agents of gut infections. In order to induce bacteriocin production, probiotic cells were cocultivated with Lactococcus lactis subsp. lactis LMG 9450, one of the most important starter cultures in cheese production. The presence of bacteriocin coding genes was investigated by PCR amplification with sequence-specific primers for helveticin and was confirmed for probiotic strain L. helveticus M92. All examined probiotic strains have shown bacteriocinogenic activity against Staphylococcus aureus 3048, Staphylococcus aureus K-144, Escherichia coli 3014, Salmonella enterica serovar Typhimurium FP1, Bacillus subtilis ATCC 6633, Bacillus cereus TM2, which is an important functional treat of probiotic strains significant in competitive exclusion mechanism which provides selective advantage of probiotic strains against undesirable microorganisms in gastrointestinal tract of the host. According to obtained results, living cells of starter culture Lc. lactis subsp. lactis LMG 9450 induced bacteriocin production by examined probiotic strains but starter culture itself was not sensitive to bacteriocin activity.

  11. Liposome-enhanced transformation of Streptococcus lactis and plasmid transfer by intergeneric protoplast fusion of Streptococcus lactis and Bacillus subtilis

    NARCIS (Netherlands)

    Vossen, Jos M.B.M. van der; Kok, Jan; Lelie, Daniel van der; Venema, Gerhardus

    An efficient protoplast transformation system and a procedure of plasmid transfer by means of protoplast fusion is described for Streptococcus lactis. Protoplasts of S. lactis IL1403 and S. lactis MG1363 were transformed by pGK12 [2.9 MDa erythromycin resistance (Emr)] with an efficiency of 3 × 10^5

  12. Production of the small heat shock protein Lo18 from Oenococcus oeni in Lactococcus lactis improves its stress tolerance.

    Science.gov (United States)

    Weidmann, Stéphanie; Maitre, Magali; Laurent, Julie; Coucheney, Françoise; Rieu, Aurélie; Guzzo, Jean

    2017-04-17

    Lactococcus lactis is a lactic acid bacterium widely used in cheese and fermented milk production. During fermentation, L. lactis is subjected to acid stress that impairs its growth. The small heat shock protein (sHsp) Lo18 from the acidophilic species Oenococcus oeni was expressed in L. lactis. This sHsp is known to play an important role in protein protection and membrane stabilization in O. oeni. The role of this sHsp could be studied in L. lactis, since no gene encoding for sHsp has been detected in this species. L. lactis subsp. cremoris strain MG1363 was transformed with the pDLhsp18 plasmid, which is derived from pDL278 and contains the hsp18 gene (encoding Lo18) and its own promoter sequence. The production of Lo18 during stress conditions was checked by immunoblotting and the cellular distribution of Lo18 in L. lactis cells after heat shock was determined. Our results clearly indicated a role for Lo18 in cytoplasmic protein protection and membrane stabilization during stress. The production of sHsp in L. lactis improved tolerance to heat and acid conditions in this species. Finally, the improvement of the L. lactis survival in milk medium thanks to Lo18 was highlighted, suggesting an interesting role of this sHsp. These findings suggest that the expression of a sHsp by a L. lactis strain results in greater resistance to stress, and, can consequently enhance the performances of industrial strains. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Inhibitory Effect of Lactococcus lactis HY 449 on Cariogenic Biofilm.

    Science.gov (United States)

    Kim, Young-Jae; Lee, Sung-Hoon

    2016-11-28

    Dental caries is caused by cariogenic biofilm, an oral biofilm including Streptococcus mutans . Recently, the prevention of dental caries using various probiotics has been attempted. Lactococcus lactis HY 449 is a probiotic bacterium. The aim of this study was to investigate the effect of L. lactis HY 449 on cariogenic biofilm and to analyze its inhibitory mechanisms. Cariogenic biofilm was formed in the presence or absence of L. lactis HY 449 and L. lactis ATCC 19435, and analyzed with a confocal laser microscope. The formation of cariogenic biofilm was reduced in cultures spiked with both L. lactis strains, and L. lactis HY 449 exhibited more inhibitory effects than L. lactis ATCC 19435. In order to analyze and to compare the inhibitory mechanisms, the antibacterial activity of the spent culture medium from both L. lactis strains against S. mutans was investigated, and the expression of glucosyltransferases ( gtfs ) of S. mutans was then analyzed by real-time RT-PCR. In addition, the sucrose fermentation ability of both L. lactis strains was examined. Both L. lactis strains showed antibacterial activity and inhibited the expression of gtfs , and the difference between both strains did not show. In the case of sucrose-fermenting ability, L. lactis HY 449 fermented sucrose but L. lactis ATCC 19435 did not. L. lactis HY 449 inhibited the uptake of sucrose and the gtfs expression of S. mutans , whereby the development of cariogenic biofilm may be inhibited. In conclusion, L. lactis HY 449 may be a useful probiotic for the prevention of dental caries.

  14. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Application in the control of Listeria monocytogenes in fresh Minas-type goat cheese

    Directory of Open Access Journals (Sweden)

    Danielle N. Furtado

    2015-03-01

    Full Text Available Listeria monocytogenes is a pathogen frequently found in dairy products. Its control in fresh cheeses is difficult, due to the psychrotrophic properties and salt tolerance. Bacteriocinogenic lactic acid bacteria (LAB with proven in vitro antilisterial activity can be an innovative technological approach but their application needs to be evaluated by means of in situ tests. In this study, a novel bacteriocinogenic Lactococcus lactis strain (Lc. lactis DF4Mi, isolated from raw goat milk, was tested for control of growth of L. monocytogenes in artificially contaminated fresh Minas type goat cheese during storage under refrigeration. A bacteriostatic effect was achieved, and counts after 10 days were 3 log lower than in control cheeses with no added LAB. However, this effect did not differ significantly from that obtained with a non-bacteriocinogenic Lc. lactis strain. Addition of nisin (12.5 mg/kg caused a rapid decrease in the number of viable L. monocytogenes in the cheeses, suggesting that further studies with the purified bacteriocin DF4Mi may open new possibilities for this strain as biopreservative in dairy products.

  15. Stability of free and immobilized Lactobacillus acidophilus and Bifidobacterium lactis in acidified milk and of immobilized B. lactis in yoghurt Estabilidade de Lactobacillus acidophilus e Bifidobacterium lactis nas formas livre e imobilizada em leite acidificado e de B. lactis imobilizado em iogurte

    Directory of Open Access Journals (Sweden)

    Carlos Raimundo Ferreira Grosso

    2004-06-01

    Full Text Available This study evaluated the stability of Bifidobacterium lactis (Bb-12 and of Lactobacillus acidophilus (La-05 both free and immobilized in calcium alginate, in milk and in acidified milk (pH 5.0, 4.4 and 3.8. The stability of immobilized B. lactis in yoghurt (fermented to pH 4.2, during 28 days of refrigerated storage was also evaluated. The efficiency of two culture media (modified MRS agar and Reinforced Clostridial Agar plus Prussian Blue for counting of B. lactis in yoghurt was determined. Lee's agar was used to count Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus when B. lactis were counted in the MRS medium. B. lactis and L. acidophilus in both free and immobilized forms presented satisfactory rates of survival in milk and acidified milk because the average reduction of the population was only one log cycle after 21 days of storage. The number of viable cells of immobilized B. lactis in yoghurt presented a gradual decline throughout the storage period, passing from 10(8 cfu/ml to no count after 28 days of storage. When the cultures were not in equilibrium just the selective medium was efficient in counting B. lactis in yoghurt. The results showed that both microorganisms can be added to milk and acidified milk, because their population was only slightly affected during storage. The presence of traditional culture of yoghurt seems to be harmful for survival of immobilized B. lactis and the immobilization in calcium alginate failed as an effective barrier to protect the cells in all analysed treatments.Este trabalho avaliou a estabilidade de Bifidobacterium lactis (Bb-12 e de Lactobacillus acidophilus (La-05 nas formas livre e imobilizada em alginato de cálcio, em leite e leite acidificado (pHs 5.0, 4.4 e 3.8, e a estabilidade de B. lactis imobilizado em iogurte (fermentado até pH 4.2, durante 28 dias de estocagem refrigerada. Também foi estudada a eficiência de dois meios de cultura (ágar MRS modificado e

  16. Transcriptome analysis and related databases of Lactococcus lactis

    NARCIS (Netherlands)

    Kuipers, Oscar P.; Jong, Anne de; Baerends, Richard J.S.; Hijum, Sacha A.F.T. van; Zomer, Aldert L.; Karsens, Harma A.; Hengst, Chris D. den; Kramer, Naomi E.; Buist, Girbe; Kok, Jan

    Several complete genome sequences of Lactococcus lactis and their annotations will become available in the near future, next to the already published genome sequence of L. lactis ssp. lactis IL1403. This will allow intraspecies comparative genomics studies as well as functional genomics studies

  17. Licheniocin 50.2 and Bacteriocins from Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 Inhibit Biofilms of Coagulase Negative Staphylococci and Listeria monocytogenes Clinical Isolates.

    Science.gov (United States)

    Cirkovic, Ivana; Bozic, Dragana D; Draganic, Veselin; Lozo, Jelena; Beric, Tanja; Kojic, Milan; Arsic, Biljana; Garalejic, Eliana; Djukic, Slobodanka; Stankovic, Slavisa

    2016-01-01

    Coagulase negative staphylococci (CoNS) and Listeria monocytogenes have important roles in pathogenesis of various genital tract infections and fatal foetomaternal infections, respectively. The aim of our study was to investigate the inhibitory effects of two novel bacteriocins on biofilms of CoNS and L. monocytogenes genital isolates. The effects of licheniocin 50.2 from Bacillus licheniformis VPS50.2 and crude extract of bacteriocins produced by Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 (BGBU1-4 crude extract) were evaluated on biofilm formation and formed biofilms of eight CoNS (four S. epidermidis, two S. hominis, one S. lugdunensis and one S. haemolyticus) and 12 L. monocytogenes genital isolates. Licheniocin 50.2 and BGBU1-4 crude extract inhibited the growth of both CoNS and L. monocytogenes isolates, with MIC values in the range between 200-400 AU/ml for licheniocin 50.2 and 400-3200 AU/ml for BGBU1-4 crude extract. Subinhibitory concentrations (1/2 × and 1/4 × MIC) of licheniocin 50.2 inhibited biofilm formation by all CoNS isolates (p < 0.05, respectively), while BGBU1-4 crude extract inhibited biofilm formation by all L. monocytogenes isolates (p < 0.01 and p < 0.05, respectively). Both bacteriocins in concentrations of 100 AU/mL and 200 AU/mL reduced the amount of 24 h old CoNS and L. monocytogenes biofilms (p < 0.05, p < 0.01, p < 0.001). This study suggests that novel bacteriocins have potential to be used for genital application, to prevent biofilm formation and/or to eradicate formed biofilms, and consequently reduce genital and neonatal infections by CoNS and L. monocytogenes.

  18. Detection and viability of Lactococcus lactis throughout cheese ripening.

    Directory of Open Access Journals (Sweden)

    Marianna Ruggirello

    Full Text Available Recent evidences highlighted the presence of Lactococcus lactis during late cheese ripening. For this reason, the role of this microorganism, well known as dairy starter, should be reconsidered throughout cheese manufacturing and ripening. Thus, the main objective of this study was to develop a RT-qPCR protocol for the detection, quantification and determination of the viability of L. lactis in ripened cheese samples by direct analysis of microbial nucleic acids. Standard curves were constructed for the specific quantification of L. lactis in cheese matrices and good results in terms of selectivity, correlation coefficient and efficiency were obtained. Thirty-three ripened cheeses were analyzed and, on the basis of RNA analysis, twelve samples showed 106 to 108 CFU of L. lactis per gram of product, thirteen from 103 to 105 CFU/g, and in eight cheeses, L. lactis was not detected. Traditional plating on M17 medium led to loads ranging from 105 to 109 CFU/g, including the cheese samples where no L. lactis was found by RT-qPCR. From these cheeses, none of the colonies isolated on M17 medium was identified as L. lactis species. These data could be interpreted as a lack of selectivity of M17 medium where colony growth is not always related to lactococcal species. At the same time, the absence or low abundance of L. lactis isolates on M17 medium from cheese where L. lactis was detected by RT-qPCR support the hypothesis that L. lactis starter populations are mainly present in viable but not culturable state during ripening and, for this reason, culture-dependent methods have to be supplemented with direct analysis of cheese.

  19. Detection and Viability of Lactococcus lactis throughout Cheese Ripening

    Science.gov (United States)

    Cocolin, Luca

    2014-01-01

    Recent evidences highlighted the presence of Lactococcus lactis during late cheese ripening. For this reason, the role of this microorganism, well known as dairy starter, should be reconsidered throughout cheese manufacturing and ripening. Thus, the main objective of this study was to develop a RT-qPCR protocol for the detection, quantification and determination of the viability of L. lactis in ripened cheese samples by direct analysis of microbial nucleic acids. Standard curves were constructed for the specific quantification of L. lactis in cheese matrices and good results in terms of selectivity, correlation coefficient and efficiency were obtained. Thirty-three ripened cheeses were analyzed and, on the basis of RNA analysis, twelve samples showed 106 to 108 CFU of L. lactis per gram of product, thirteen from 103 to 105 CFU/g, and in eight cheeses, L. lactis was not detected. Traditional plating on M17 medium led to loads ranging from 105 to 109 CFU/g, including the cheese samples where no L. lactis was found by RT-qPCR. From these cheeses, none of the colonies isolated on M17 medium was identified as L. lactis species. These data could be interpreted as a lack of selectivity of M17 medium where colony growth is not always related to lactococcal species. At the same time, the absence or low abundance of L. lactis isolates on M17 medium from cheese where L. lactis was detected by RT-qPCR support the hypothesis that L. lactis starter populations are mainly present in viable but not culturable state during ripening and, for this reason, culture-dependent methods have to be supplemented with direct analysis of cheese. PMID:25503474

  20. Genotypic and Phenotypic Analysis of Dairy Lactococcus lactis Biodiversity in Milk: Volatile Organic Compounds as Discriminating Markers

    Science.gov (United States)

    Dhaisne, Amandine; Guellerin, Maeva; Laroute, Valérie; Laguerre, Sandrine; Le Bourgeois, Pascal; Loubiere, Pascal

    2013-01-01

    The diversity of nine dairy strains of Lactococcus lactis subsp. lactis in fermented milk was investigated by both genotypic and phenotypic analyses. Pulsed-field gel electrophoresis and multilocus sequence typing were used to establish an integrated genotypic classification. This classification was coherent with discrimination of the L. lactis subsp. lactis bv. diacetylactis lineage and reflected clonal complex phylogeny and the uniqueness of the genomes of these strains. To assess phenotypic diversity, 82 variables were selected as important dairy features; they included physiological descriptors and the production of metabolites and volatile organic compounds (VOCs). Principal-component analysis (PCA) demonstrated the phenotypic uniqueness of each of these genetically closely related strains, allowing strain discrimination. A method of variable selection was developed to reduce the time-consuming experimentation. We therefore identified 20 variables, all associated with VOCs, as phenotypic markers allowing discrimination between strain groups. These markers are representative of the three metabolic pathways involved in flavor: lipolysis, proteolysis, and glycolysis. Despite great phenotypic diversity, the strains could be divided into four robust phenotypic clusters based on their metabolic orientations. Inclusion of genotypic diversity in addition to phenotypic characters in the classification led to five clusters rather than four being defined. However, genotypic characters make a smaller contribution than phenotypic variables (no genetic distances selected among the most contributory variables). This work proposes an original method for the phenotypic differentiation of closely related strains in milk and may be the first step toward a predictive classification for the manufacture of starters. PMID:23709512

  1. Engineering of EPA/DHA omega-3 fatty acid production by Lactococcus lactis subsp. cremoris MG1363.

    Science.gov (United States)

    Amiri-Jami, Mitra; Lapointe, Gisele; Griffiths, Mansel W

    2014-04-01

    Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to be of major importance in human health. Therefore, these essential polyunsaturated fatty acids have received considerable attention in both human and farm animal nutrition. Currently, fish and fish oils are the main dietary sources of EPA/DHA. To generate sustainable novel sources for EPA and DHA, the 35-kb EPA/DHA synthesis gene cluster was isolated from a marine bacterium, Shewanella baltica MAC1. To streamline the introduction of the genes into food-grade microorganisms such as lactic acid bacteria, unnecessary genes located upstream and downstream of the EPA/DHA gene cluster were deleted. Recombinant Escherichia coli harboring the 20-kb gene cluster produced 3.5- to 6.1-fold more EPA than those carrying the 35-kb DNA fragment coding for EPA/DHA synthesis. The 20-kb EPA/DHA gene cluster was cloned into a modified broad-host-range low copy number vector, pIL252m (4.7 kb, Ery) and expressed in Lactococcus lactis subsp. cremoris MG1363. Recombinant L. lactis produced DHA (1.35 ± 0.5 mg g(-1) cell dry weight) and EPA (0.12 ± 0.04 mg g(-1) cell dry weight). This is believed to be the first successful cloning and expression of EPA/DHA synthesis gene cluster in lactic acid bacteria. Our findings advance the future use of EPA/DHA-producing lactic acid bacteria in such applications as dairy starters, silage adjuncts, and animal feed supplements.

  2. Lactococcus lactis - a diploid bacterium

    DEFF Research Database (Denmark)

    Michelsen, Ole; Hansen, Flemming G.; Jensen, Peter Ruhdal

    the next division. Thus, the regions of the chromosome that are the last to be replicated are haploid even in fast-growing bacteria. In contrast to this general rule for bacteria, we found that Lactococcus lactis, a bacterium which has been exploited for thousands of years for the production of fermented...... milk products, is born with two complete non-replicating chromosomes. L. lactis therefore remain diploid throughout its entire life cycle....

  3. Metabolic Profiling of Lactococcus lactis Under Different Culture Conditions

    Directory of Open Access Journals (Sweden)

    Normah Mohd Noor

    2012-07-01

    Full Text Available Gas chromatography mass spectrometry (GC-MS and headspace gas chromatography mass spectrometry (HS/GC-MS were used to study metabolites produced by Lactococcus lactis subsp. cremoris MG1363 grown at a temperature of 30 °C with and without agitation at 150 rpm, and at 37 °C without agitation. It was observed that L. lactis produced more organic acids under agitation. Primary alcohols, aldehydes, ketones and polyols were identified as the corresponding trimethylsilyl (TMS derivatives, whereas amino acids and organic acids, including fatty acids, were detected through methyl chloroformate derivatization. HS analysis indicated that branched-chain methyl aldehydes, including 2-methylbutanal, 3-methylbutanal, and 2-methylpropanal are degdradation products of isoleucine, leucine or valine. Multivariate analysis (MVA using partial least squares discriminant analysis (PLS-DA revealed the major differences between treatments were due to changes of amino acids and fermentation products.

  4. Dynamics of pyruvate metabolism in Lactococcus lactis

    DEFF Research Database (Denmark)

    Melchiorsen, Claus Rix; Jensen, Niels B.S.; Christensen, Bjarke

    2001-01-01

    The pyruvate metabolism in the lactic acid bacterium Lactococcus lactis was studied in anaerobic cultures under transient conditions. During growth of L. lactis in continuous culture at high dilution rate, homolactic product formation was observed, i.e., lactate was produced as the major end...... product. At a lower dilution rate, the pyruvate metabolism shifted towards mixed acid-product formation where formate, acetate, and ethanol were produced in addition to lactate. The regulation of the shift in pyruvate metabolism was investigated by monitoring the dynamic behavior of L. lactis...

  5. Heterologous Expression and Characterization of an N-Acetyl-beta-D-hexosaminidase from Lactococcus lactis ssp. lactis IL1403

    Czech Academy of Sciences Publication Activity Database

    Nguyen, A. H.; Nguyen, T.-H.; Křen, Vladimír; Eijsink, V. G. H.; Haltrich, D.; Peterbauer, C.

    2012-01-01

    Roč. 60, č. 12 (2012), s. 3275-3281 ISSN 0021-8561 R&D Projects: GA ČR(CZ) GAP207/11/0629 Keywords : N-acetyl-beta-D-hexosaminidase * Lactococcus lactis ssp lactis IL1403 * pNP-GlcNAc Subject RIV: CE - Biochemistry Impact factor: 2.906, year: 2012

  6. Lytic Infection of Lactococcus lactis by Bacteriophages Tuc2009 and c2 Triggers Alternative Transcriptional Host Responses

    NARCIS (Netherlands)

    Ainsworth, S.; Zomer, A.L.; Mahony, J.; Sinderen, D. van

    2013-01-01

    Here we present an entire temporal transcriptional profile of Lactococcus lactis subsp. cremoris UC509.9 undergoing lytic infection with two distinct bacteriophages, Tuc2009 and c2. Furthermore, corresponding high-resolution whole-phage genome tiling arrays of both bacteriophages were performed

  7. Spray-drying process preserves the protective capacity of a breast milk-derived Bifidobacterium lactis strain on acute and chronic colitis in mice

    Science.gov (United States)

    Burns, Patricia; Alard, Jeanne; Hrdỳ, Jiri; Boutillier, Denise; Páez, Roxana; Reinheimer, Jorge; Pot, Bruno; Vinderola, Gabriel; Grangette, Corinne

    2017-01-01

    Gut microbiota dysbiosis plays a central role in the development and perpetuation of chronic inflammation in inflammatory bowel disease (IBD) and therefore is key target for interventions with high quality and functional probiotics. The local production of stable probiotic formulations at limited cost is considered an advantage as it reduces transportation cost and time, thereby increasing the effective period at the consumer side. In the present study, we compared the anti-inflammatory capacities of the Bifidobacterium animalis subsp. lactis (B. lactis) INL1, a probiotic strain isolated in Argentina from human breast milk, with the commercial strain B. animalis subsp. lactis BB12. The impact of spray-drying, a low-cost alternative of bacterial dehydration, on the functionality of both bifidobacteria was also investigated. We showed for both bacteria that the spray-drying process did not impact on bacterial survival nor on their protective capacities against acute and chronic colitis in mice, opening future perspectives for the use of strain INL1 in populations with IBD. PMID:28233848

  8. Adaptation of Lactococcus lactis to its environment : a genomics approach

    NARCIS (Netherlands)

    Zomer, Albertus Lambert

    2007-01-01

    This thesis describes a number of strategies of Lactococcus lactis to adapt to its ever-changing environment. Although the complete genome sequence of L. lactis subspecies lactis IL1403, became available when this research was started, the genome sequence of the lactic acid bacterial paradigm, L.

  9. Preferential localization of Lactococcus lactis cells entrapped in a caseinate/alginate phase separated system.

    Science.gov (United States)

    Léonard, Lucie; Gharsallaoui, Adem; Ouaali, Fahima; Degraeve, Pascal; Waché, Yves; Saurel, Rémi; Oulahal, Nadia

    2013-09-01

    This study aimed to entrap bioprotective lactic acid bacteria in a sodium caseinate/sodium alginate aqueous two-phase system. Phase diagram at pH=7 showed that sodium alginate and sodium caseinate were not miscible when their concentrations exceeded 1% (w/w) and 6% (w/w), respectively. The stability of the caseinate/alginate two-phase system was also checked at pH values of 6.0 and 5.5. Lactococcus lactis subsp. lactis LAB3 cells were added in a 4% (w/w) caseinate/1.5% (w/w) alginate two-phase system at pH=7. Fluorescence microscopy allowed to observe that the caseinate-rich phase formed droplets dispersed in a continuous alginate-rich phase. The distribution of bacteria in such a system was observed by epifluorescence microscopy: Lc. lactis LAB3 cells stained with Live/Dead(®) Baclight kit™ were located exclusively in the protein phase. Since zeta-potential measurements indicated that alginate, caseinate and bacterial cells all had an overall negative charge at pH 7, the preferential adhesion of LAB cells was assumed to be driven by hydrophobic effect or by depletion phenomena in such biopolymeric systems. Moreover, LAB cells viability was significantly higher in the ternary mixture obtained in the presence of both caseinate and alginate than in single alginate solution. Caseinate/alginate phase separated systems appeared thus well suited for Lc. lactis LAB3 cells entrapment. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Glucose metabolism in Lactococcus lactis MG1363 under different aeration conditions: Requirement of acetate to sustain growth under microaerobic conditions

    DEFF Research Database (Denmark)

    Nordkvist, Mikkel; Jensen, N.B.S.; Villadsen, John

    2003-01-01

    Lactococcus lactis subsp. lactis MG1363 was grown in batch cultures on a defined medium with glucose as the energy source under different aeration conditions, namely, anaerobic conditions, aerobic conditions, and microaerobic conditions with a dissolved oxygen tension of 5% (when saturation...... resulted in acetate, CO2, and acetoin replacing formate and ethanol as end products. Under microaerobic conditions, growth came to a gradual halt, although more than 60% of the glucose was still left. A decline in growth was not observed during microaerobic cultivation when acetate was added to the medium...

  11. Short communication: Genotypic and phenotypic identification of environmental streptococci and association of Lactococcus lactis ssp. lactis with intramammary infections among different dairy farms.

    Science.gov (United States)

    Werner, B; Moroni, P; Gioia, G; Lavín-Alconero, L; Yousaf, A; Charter, M E; Carter, B Moslock; Bennett, J; Nydam, D V; Welcome, F; Schukken, Y H

    2014-11-01

    Lactococcus species are counted among a large and closely related group of environmental streptococci and streptococci-like bacteria that include bovine mastitis pathogenic Streptococcus, Enterococcus, and Aerococcus species. Phenotypic and biochemical identification methods can be inaccurate and unreliable for species within this group, particularly for Lactococcus spp. As a result, the incidence of Lactococcus spp. on the farm may have been historically underreported and consequently little is known about the clinical importance of this genus as a mastitis pathogen. We used molecular genetic identification methods to accurately differentiate 60 environmental streptococci and streptococci-like bacteria isolated from cows with high somatic cell count and chronic intramammary infection (IMI; >2 somatic cell scores above 4) among 5 geographically distinct farms in New York and Minnesota that exhibited an observed increase in IMI. These isolates were phenotypically identified as Streptococcus uberis and Streptococcus spp. Genetic methods identified 42 isolates (70%) as Lactococcus lactis ssp. lactis, including all 10 isolates originally phenotypically identified as Streptococcus uberis. Antibiotic inhibition testing of all Lc. lactis ssp. lactis showed that 7 isolates were resistant to tetracycline. In the present study, a predominance of Lc. lactis ssp. lactis was identified in association with chronic, clinical bovine IMI among all 5 farms and characterized antimicrobial resistance for treatment therapies. Routine use by mastitis testing labs of molecular identification methods for environmental streptococci and streptococci-like bacteria can further define the role and prevalence of Lc. lactis ssp. lactis in association with bovine IMI and may lead to more targeted therapies. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  12. Combined Transcriptome and Proteome Analysis of Bifidobacterium animalis subsp. lactis BB-12 Grown on Xylo-Oligosaccharides and a Model of Their Utilization

    DEFF Research Database (Denmark)

    Gilad, Ofir; Jacobsen, Susanne; Stuer-Lauridsen, B.

    2010-01-01

    -documented and widely used probiotic strain B. animalis subsp. lactis BB-12, a combined proteomic and transcriptomic approach was applied, involving DNA microarrays, real-time quantitative PCR (qPCR), and two-dimensional difference gel electrophoresis (2D-DIGE) analyses of samples obtained from cultures grown on either...... of these (beta-D-xylosidase, sugar-binding protein, and xylose isomerase) showed higher abundance on XOS. Based on the obtained results, a model for the catabolism of XOS in BB-12 is suggested, according to which the strain utilizes an ABC (ATP-binding cassette) transport system (probably for oligosaccharides...

  13. Early adaptation to oxygen is key to the industrially important traits of Lactococcus lactis ssp. cremoris during milk fermentation.

    Science.gov (United States)

    Cretenet, Marina; Le Gall, Gwenaëlle; Wegmann, Udo; Even, Sergine; Shearman, Claire; Stentz, Régis; Jeanson, Sophie

    2014-12-03

    Lactococcus lactis is the most used species in the dairy industry. Its ability to adapt to technological stresses, such as oxidative stress encountered during stirring in the first stages of the cheese-making process, is a key factor to measure its technological performance. This study aimed to understand the response to oxidative stress of Lactococcus lactis subsp. cremoris MG1363 at the transcriptional and metabolic levels in relation to acidification kinetics and growth conditions, especially at an early stage of growth. For those purposes, conditions of hyper-oxygenation were initially fixed for the fermentation. Kinetics of growth and acidification were not affected by the presence of oxygen, indicating a high resistance to oxygen of the L. lactis MG1363 strain. Its resistance was explained by an efficient consumption of oxygen within the first 4 hours of culture, leading to a drop of the redox potential. The efficient consumption of oxygen by the L. lactis MG1363 strain was supported by a coherent and early adaptation to oxygen after 1 hour of culture at both gene expression and metabolic levels. In oxygen metabolism, the over-expression of all the genes of the nrd (ribonucleotide reductases) operon or fhu (ferrichrome ABC transports) genes was particularly significant. In carbon metabolism, the presence of oxygen led to an early shift at the gene level in the pyruvate pathway towards the acetate/2,3-butanediol pathway confirmed by the kinetics of metabolite production. Finally, the MG1363 strain was no longer able to consume oxygen in the stationary growth phase, leading to a drastic loss of culturability as a consequence of cumulative stresses and the absence of gene adaptation at this stage. Combining metabolic and transcriptomic profiling, together with oxygen consumption kinetics, yielded new insights into the whole genome adaptation of L. lactis to initial oxidative stress. An early and transitional adaptation to oxidative stress was revealed for L

  14. The Evolution of gene regulation research in Lactococcus lactis.

    Science.gov (United States)

    Kok, Jan; van Gijtenbeek, Lieke A; de Jong, Anne; van der Meulen, Sjoerd B; Solopova, Ana; Kuipers, Oscar P

    2017-08-01

    Lactococcus lactis is a major microbe. This lactic acid bacterium (LAB) is used worldwide in the production of safe, healthy, tasteful and nutritious milk fermentation products. Its huge industrial importance has led to an explosion of research on the organism, particularly since the early 1970s. The upsurge in the research on L. lactis coincided not accidentally with the advent of recombinant DNA technology in these years. The development of methods to take out and re-introduce DNA in L. lactis, to clone genes and to mutate the chromosome in a targeted way, to control (over)expression of proteins and, ultimately, the availability of the nucleotide sequence of its genome and the use of that information in transcriptomics and proteomics research have enabled to peek deep into the functioning of the organism. Among many other things, this has provided an unprecedented view of the major gene regulatory pathways involved in nitrogen and carbon metabolism and their overlap, and has led to the blossoming of the field of L. lactis systems biology. All of these advances have made L. lactis the paradigm of the LAB. This review will deal with the exciting path along which the research on the genetics of and gene regulation in L. lactis has trodden. © FEMS 2017.

  15. Effect of autochthonous bacteriocin-producing Lactococcus lactis on bacterial population dynamics and growth of halotolerant bacteria in Brazilian charqui.

    Science.gov (United States)

    Biscola, Vanessa; Abriouel, Hikmate; Todorov, Svetoslav Dimitrov; Capuano, Verena Sant'Anna Cabral; Gálvez, Antonio; Franco, Bernadette Dora Gombossy de Melo

    2014-12-01

    Charqui is a fermented, salted and sun-dried meat product, widely consumed in Brazil and exported to several countries. Growth of microorganisms in this product is unlikely due to reduced Aw, but halophilic and halotolerant bacteria may grow and cause spoilage. Charqui is a good source of lactic acid bacteria able to produce antimicrobial bacteriocins. In this study, an autochthonous bacteriocinogenic strain (Lactococcus lactis subsp. lactis 69), isolated from charqui, was added to the meat used for charqui manufacture and evaluated for its capability to prevent the growth of spoilage bacteria during storage up to 45 days. The influence of L. lactis 69 on the bacterial diversity during the manufacturing of the product was also studied, using denaturing gradient gel electrophoresis (DGGE). L. lactis 69 did not affect the counts and diversity of lactic acid bacteria during manufacturing and storage, but influenced negatively the populations of halotolerant microorganisms, reducing the spoilage potential. The majority of tested virulence genes was absent, evidencing the safety and potential technological application of this strain as an additional hurdle to inhibit undesirable microbial growth in this and similar fermented meat products. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Combination of Metabolomic and Proteomic Analysis Revealed Different Features among Lactobacillus delbrueckii Subspecies bulgaricus and lactis Strains While In Vivo Testing in the Model Organism Caenorhabditis elegans Highlighted Probiotic Properties

    Directory of Open Access Journals (Sweden)

    Elena Zanni

    2017-06-01

    Full Text Available Lactobacillus delbrueckii represents a technologically relevant member of lactic acid bacteria, since the two subspecies bulgaricus and lactis are widely associated with fermented dairy products. In the present work, we report the characterization of two commercial strains belonging to L. delbrueckii subspecies bulgaricus, lactis and a novel strain previously isolated from a traditional fermented fresh cheese. A phenomic approach was performed by combining metabolomic and proteomic analysis of the three strains, which were subsequently supplemented as food source to the model organism Caenorhabditis elegans, with the final aim to evaluate their possible probiotic effects. Restriction analysis of 16S ribosomal DNA revealed that the novel foodborne strain belonged to L. delbrueckii subspecies lactis. Proteomic and metabolomic approaches showed differences in folate, aminoacid and sugar metabolic pathways among the three strains. Moreover, evaluation of C. elegans lifespan, larval development, brood size, and bacterial colonization capacity demonstrated that L. delbrueckii subsp. bulgaricus diet exerted beneficial effects on nematodes. On the other hand, both L. delbrueckii subsp. lactis strains affected lifespan and larval development. We have characterized three strains belonging to L. delbrueckii subspecies bulgaricus and lactis highlighting their divergent origin. In particular, the two closely related isolates L. delbrueckii subspecies lactis display different galactose metabolic capabilities. Moreover, the L. delbrueckii subspecies bulgaricus strain demonstrated potential probiotic features. Combination of omic platforms coupled with in vivo screening in the simple model organism C. elegans is a powerful tool to characterize industrially relevant bacterial isolates.

  17. Combination of Metabolomic and Proteomic Analysis Revealed Different Features among Lactobacillus delbrueckii Subspecies bulgaricus and lactis Strains While In Vivo Testing in the Model Organism Caenorhabditis elegans Highlighted Probiotic Properties.

    Science.gov (United States)

    Zanni, Elena; Schifano, Emily; Motta, Sara; Sciubba, Fabio; Palleschi, Claudio; Mauri, Pierluigi; Perozzi, Giuditta; Uccelletti, Daniela; Devirgiliis, Chiara; Miccheli, Alfredo

    2017-01-01

    Lactobacillus delbrueckii represents a technologically relevant member of lactic acid bacteria, since the two subspecies bulgaricus and lactis are widely associated with fermented dairy products. In the present work, we report the characterization of two commercial strains belonging to L. delbrueckii subspecies bulgaricus , lactis and a novel strain previously isolated from a traditional fermented fresh cheese. A phenomic approach was performed by combining metabolomic and proteomic analysis of the three strains, which were subsequently supplemented as food source to the model organism Caenorhabditis elegans , with the final aim to evaluate their possible probiotic effects. Restriction analysis of 16S ribosomal DNA revealed that the novel foodborne strain belonged to L. delbrueckii subspecies lactis . Proteomic and metabolomic approaches showed differences in folate, aminoacid and sugar metabolic pathways among the three strains. Moreover, evaluation of C. elegans lifespan, larval development, brood size, and bacterial colonization capacity demonstrated that L. delbrueckii subsp. bulgaricus diet exerted beneficial effects on nematodes. On the other hand, both L. delbrueckii subsp. lactis strains affected lifespan and larval development. We have characterized three strains belonging to L. delbrueckii subspecies bulgaricus and lactis highlighting their divergent origin. In particular, the two closely related isolates L. delbrueckii subspecies lactis display different galactose metabolic capabilities. Moreover, the L. delbrueckii subspecies bulgaricus strain demonstrated potential probiotic features. Combination of omic platforms coupled with in vivo screening in the simple model organism C. elegans is a powerful tool to characterize industrially relevant bacterial isolates.

  18. INHIBITION OF STAPHYLOCOCCUS AUREUS BY LACTIC ACID BACTERIA AND / OR BIFIDOBACTERIUM LACTIS DURING MILK FERMENTATION AND STORAGE

    Directory of Open Access Journals (Sweden)

    Khalaf S. Al-Delaimy

    2013-02-01

    Full Text Available Survival and inhibition of Staphylococcus aureus by the lactic acid bacteria (LAB starter culture (Sterptococcus thermophillus and Lactobacillus delbrukii subsp. bulgaricus and/ or probiotic bacteria Bifidobacterium lactis during milk fermentation to yoghurt and storage up to 12 days was studied. Adding S. aureus (initial count log 6.64/ ml with LAB (initial count log 6.8/ ml in milk during yoghurt processing and storage resulted in no significant change in the counts of both S. aureus and LAB during fermentation period of 4 hrs at 45° C. A steady decrease in S. aureus count during storage at 25° C and 4° C was observed reaching a complete (100 % inhibition after 9 and 12 days, respectively, with no significant increase in LAB count. Adding S. aureus (initial count log 6.62/ ml with B. lactis (initial count log 6.83/ ml in milk for 4 hr at45° C, no significant changes in the counts of both bacteria were found. After storage at 25° C and at 4° C a sharp decline in the S. aureus count with a 100 % inhibition after 6 and 9 days with approximately two log and one log increase in B. lactis counts consecutively. In general similar result was observed when adding S. aureus together with LAB and B. lactis in milk during fermentation and storage. pH values decreased during milk fermentation and storage from initially 6.55-6.64 to around 4 in most milk samples. The results of this study show that S. aureus was completely inhibited by LAB and/or B. lactis after milk fermentation to yoghurt and storage at room temperature and refrigeration for 6-9 days. It is therefore recommended to add the probiotic B. lactis with LAB to milk for yoghurt processing.

  19. The riboflavin transporter RibU in Lactococcus lactis : Molecular characterization of gene expression and the transport mechanism

    NARCIS (Netherlands)

    Burgess, CM; Slotboom, DJ; Geertsma, ER; Duurkens, Hinderika; Poolman, B; van Sinderen, D

    This study describes the characterization of the riboflavin transport protein RibU in the lactic acid bacterium Lactococcus lactis subsp. cremoris NZ9000. RibU is predicted to contain five membrane-spanning segments and is a member of a novel transport protein family, not described in the Transport

  20. Transforming Lactococcus lactis into a microbial cell factory

    DEFF Research Database (Denmark)

    Petersen, Kia Vest

    the potential of Lactococcus lactis as a platform organism for production of biofuels and-chemicals with a focus on characterization and optimization of the xylose metabolism. The plant isolate L. lactis KF147 was selected as the potential platform organism due to its natural ability to utilize both the pentose....... To simplify further analysis arcA encoding the arginine deiminase was deleted, thus eliminating the arginine catabolism. We found that in L. lactis KF147 xylose is metabolized through two pathways namely the phosphoketolase pathway and the non-oxidative part of the pentose phosphate pathway. The only products......, and ethanol. Three adaptive mutations were identified in AD29. Two is by all accounts involved in regulatory mechanisms either to stress (yhfB) or more globally (ytgF), and the last facilitate improved uptake of xylose (ptnC). Based on the above findings we conclude that L. lactis KF147 possesses many...

  1. Probiotic Lactococcus lactis: A Review

    Directory of Open Access Journals (Sweden)

    Priti Khemariya

    2017-07-01

    Full Text Available Lactococcus lactis plays a critical role in food, dairy and health sectors. In food and dairy industries, it is found in production processes of various fermented products such as sausages, pickled vegetables, beverages such as beer and wine, breads, soymilk kefir, sour milk, butter, cream, fresh cheese and different types of cheeses, like Cheddar, Colby, Cottage cheese, Camembert, cream cheese, Roquefort and Brie. Additionally, there is an increasing interest towards the possible health benefits of the probiotic activity of this organism which generally is species and strain specific and depends upon the survival in gastrointestinal tract with sufficient number. Certain strains have the ability to produce antimicrobial peptide called nisin which exhibits preservative potential. Therefore, application of bacteriocinogenic Lactococcus lactis in food and dairy sectors to preserve foods as a natural way and contributing health promoting attributes due to probiotic activity would definitely fulfil today’s consumer demands. This paper aimed to review the adaptation, antibiotic resistance, therapeutic and preservation potential of bacteriocinogenic and probiotic Lactococcus lactis.

  2. Lactococcus lactis Diversity in Undefined Mixed Dairy Starter Cultures as Revealed by Comparative Genome Analyses and Targeted Amplicon Sequencing of epsD.

    Science.gov (United States)

    Frantzen, Cyril A; Kleppen, Hans Petter; Holo, Helge

    2018-02-01

    Undefined mesophilic mixed (DL) starter cultures are used in the production of continental cheeses and contain unknown strain mixtures of Lactococcus lactis and leuconostocs. The choice of starter culture affects the taste, aroma, and quality of the final product. To gain insight into the diversity of Lactococcus lactis strains in starter cultures, we whole-genome sequenced 95 isolates from three different starter cultures. Pan-genomic analyses, which included 30 publically available complete genomes, grouped the strains into 21 L. lactis subsp . lactis and 28 L. lactis subsp. cremoris lineages. Only one of the 95 isolates grouped with previously sequenced strains, and the three starter cultures showed no overlap in lineage distributions. The culture diversity was assessed by targeted amplicon sequencing using purR , a core gene, and epsD , present in 93 of the 95 starter culture isolates but absent in most of the reference strains. This enabled an unprecedented discrimination of starter culture Lactococcus lactis and revealed substantial differences between the three starter cultures and compositional shifts during the cultivation of cultures in milk. IMPORTANCE In contemporary cheese production, standardized frozen seed stock starter cultures are used to ensure production stability, reproducibility, and quality control of the product. The dairy industry experiences significant disruptions of cheese production due to phage attacks, and one commonly used countermeasure to phage attack is to employ a starter rotation strategy, in which two or more starters with minimal overlap in phage sensitivity are used alternately. A culture-independent analysis of the lactococcal diversity in complex undefined starter cultures revealed large differences between the three starter cultures and temporal shifts in lactococcal composition during the production of bulk starters. A better understanding of the lactococcal diversity in starter cultures will enable the development of

  3. Exopolysaccharide-producing Bifidobacterium animalis subsp. lactis strains and their polymers elicit different responses on immune cells from blood and gut associated lymphoid tissue.

    Science.gov (United States)

    Hidalgo-Cantabrana, Claudio; Nikolic, Milica; López, Patricia; Suárez, Ana; Miljkovic, Marija; Kojic, Milan; Margolles, Abelardo; Golic, Natasa; Ruas-Madiedo, Patricia

    2014-04-01

    The effect of exopolysaccharide (EPS) producing bifidobacteria, and the EPS derived thereof, on the modulation of immune response was evaluated. Cells isolated from gut associated lymphoid tissue (GALT) and from peripheral blood mononuclear cells (PBMC) of naïve rats were used. The proliferation and cytokine production of these immune cells in the presence of the three isogenic Bifidobacterium animalis subsp. lactis strains (A1, A1dOx and A1dOxR), as well as their purified polymers, were in vitro analysed. The cytokine pattern produced by immune cells isolated from GALT showed that most levels remained stable in the presence of the three strains or their corresponding polymers. However, in PBMC the UV-inactivated bacteria induced higher levels of the ratios IFNγ/IL-17, TNFα/IL-10 and TNFα/TGFβ, and no variation in the ratio IFNγ/IL-4. Thus, B. animalis subsp. lactis strains were able to activate blood monocytes as well as T lymphocytes towards a mild inflammatory Th1 response. Furthermore, only the EPS-A1dOxR was able to stimulate a response in a similar way than its EPS-producing bacterium. Our work supports the notion that some bifidobacterial EPS could play a role in mediating the dialog of these microorganisms with the immune system. In addition, this study emphasizes the effect that the origin of the immune cells has in results obtained; this could explain the great amount of contradiction found in literature about the immunomodulation capability of EPS from probiotic bacteria. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Chorioamnionitis due to Lactococcus lactis cremoris: A case report

    Directory of Open Access Journals (Sweden)

    F. Azouzi

    2015-07-01

    Full Text Available Lactococcus lactis cremoris is rarely involved in human pathology. A thirty two-year old pregnant woman with premature rupture of membrane history presented with chorioamnionitis due to L. lactis cremoris. She underwent an emergency caesarian section and was treated with antibiotics including the association of amoxicillin and clavulanic acid. She was completely recovered. This is the first case to our knowledge of chorioamnionitis due to this organism. Keywords: Chorioamnionitis, Premature rupture of membranes, Lactococcus lactis cremoris

  5. Analysis of the human intestinal epithelial cell transcriptional response to Lactobacillus acidophilus, Lactobacillus salivarius, Bifidobacterium lactis and Escherichia coli

    DEFF Research Database (Denmark)

    Putaala, H; Barrangou, R; Leyer, G J

    2010-01-01

    a comparative analysis of the global in vitro transcriptional response of human intestinal epithelial cells to Lactobacillus acidophilus NCFM™, Lactobacillus salivarius Ls-33, Bifidobacterium animalis subsp. lactis 420, and enterohaemorrhagic Escherichia coli O157:H7 (EHEC). Interestingly, L. salivarius Ls-33...

  6. Phage-resistance linked to cell heterogeneity in the commercial strain Lactobacillus delbrueckii subsp. lactis Ab1.

    Science.gov (United States)

    Suárez, Viviana B; Maciel, Natalia; Guglielmotti, Daniela; Zago, Miriam; Giraffa, Giorgio; Reinheimer, Jorge

    2008-12-10

    The aim of this work was to study the relationship between the cell morphological heterogeneity and the phage-resistance in the commercial strain Lactobacillus delbrueckii subsp. lactis Ab1. Two morphological variants (named C and T) were isolated from this strain. Phage-resistant derivatives were isolated from them and the percentage of occurrence of confirmed phage-resistant cells was 0.001% of the total cellular population. Within these phage-resistant cell derivatives there were T (3 out of 4 total isolates) and C (1 out of 4 total isolates) variants. The study of some technological properties (e.g. proteolytic and acidifying activities) demonstrated that most of phage-resistant derivatives were not as good as the parental strain. However, for one derivative (a T variant), the technological properties were better than those of the parental strain. On the other hand, it was possible to determinate that the system of phage-resistance in the T variants was interference in adsorption step, with adsorption rates M.

  7. Nisin Z produced by Lactococcus lactis from bullfrog hatchery is active against Citrobacter freundii, a red-leg syndrome related pathogen.

    Science.gov (United States)

    Quintana, Gabriel; Niederle, Maria V; Minahk, Carlos J; Picariello, Gianluca; Nader-Macías, María E F; Pasteris, Sergio E

    2017-09-27

    Lactococcus lactis subsp. lactis CRL 1584 isolated from a bullfrog hatchery produces a bacteriocin that inhibits both indigenous Citrobacter freundii (a Red-Leg Syndrome related pathogen) and Lactobacillus plantarum, and Listeria monocytogenes as well. Considering that probiotics requires high cell densities and/or bacteriocin concentrations, the effect of the temperature on L. lactis growth and bacteriocin production was evaluated to find the optimal conditions. Thus, the growth rate was maximal at 36 °C, whereas the highest biomass and bacteriocin activity was achieved between 20 and 30 °C and 20-25 °C, respectively. The bacteriocin synthesis was closely growth associated reaching the maximal values at the end of the exponential phase. Since bacteriocins co-production has been evidenced in bacterial genera, a purification of the bacteriocin/s from L. lactis culture supernatants was carried out. The active fraction was purified by cationic-exchange chromatography and then, a RP-HPLC was carried out. The purified sample was a peptide with a 3353.05 Da, a molecular mass that matches nisin Z, which turned out to be the only bacteriocin produced by L. lactis CRL 1584. Nisin Z showed bactericidal effect on C. freundii and L. monocytogenes, which increased in the presence L-lactic acid + H 2 O 2 . This is the first report on nisin Z production by L. lactis from a bullfrog hatchery that resulted active on a Gram-negative pathogen. This peptide has potential probiotic for raniculture and as food biopreservative for bullfrog meat.

  8. Expression of six peptidases from Lactobacillus helveticus in Lactococcus lactis.

    Science.gov (United States)

    Luoma, S; Peltoniemi, K; Joutsjoki, V; Rantanen, T; Tamminen, M; Heikkinen, I; Palva, A

    2001-03-01

    For development of novel starter strains with improved proteolytic properties, the ability of Lactococcus lactis to produce Lactobacillus helveticus aminopeptidase N (PepN), aminopeptidase C (PepC), X-prolyl dipeptidyl aminopeptidase (PepX), proline iminopeptidase (PepI), prolinase (PepR), and dipeptidase (PepD) was studied by introducing the genes encoding these enzymes into L. lactis MG1363 and its derivatives. According to Northern analyses and enzyme activity measurements, the L. helveticus aminopeptidase genes pepN, pepC, and pepX are expressed under the control of their own promoters in L. lactis. The highest expression level, using a low-copy-number vector, was obtained with the L. helveticus pepN gene, which resulted in a 25-fold increase in PepN activity compared to that of wild-type L. lactis. The L. helveticus pepI gene, residing as a third gene in an operon in its host, was expressed in L. lactis under the control of the L. helveticus pepX promoter. The genetic background of the L. lactis derivatives tested did not affect the expression level of any of the L. helveticus peptidases studied. However, the growth medium used affected both the recombinant peptidase profiles in transformant strains and the resident peptidase activities. The levels of expression of the L. helveticus pepD and pepR clones under the control of their own promoters were below the detection limit in L. lactis. However, substantial amounts of recombinant pepD and PepR activities were obtained in L. lactis when pepD and pepR were expressed under the control of the inducible lactococcal nisA promoter at an optimized nisin concentration.

  9. Autolysis of Lactococcus lactis is influenced by proteolysis

    NARCIS (Netherlands)

    Buist, G; Venema, G; Kok, J.

    1998-01-01

    The autolysin AcmA of Lactococcus lactis was shown to be degraded by the extracellular Lactococcal proteinase PrtP. Autolysis, as evidenced by reduction in optical density of a stationary-phase culture and concomitant release of intracellular proteins, was greatly reduced when L. lactis MG1363 cells

  10. A comparative study between inhibitory effect of L. lactis and nisin on important pathogenic bacteria in Iranian UF Feta cheese

    Directory of Open Access Journals (Sweden)

    Saeed Mirdamadi

    2015-02-01

    Full Text Available   Introduction : In the present study, the inhibitory effect of nisin-producing Lactococcus lactis during co-culture and pure standard nisin were assessed against selected foodborne pathogenes in growth medium and Iranian UF Feta cheese. In comparison L lactis, not only proves flavor but also plays a better role in microbial quality of Iranian UF Feta cheese as a model of fermented dairy products.   Materials and method s: L. lactis subsp. lactis as nisin producer strain, Listeria monocytogenes, Escherichia coli and Staphylococcus aureus as pathogenic strains were inoculated in Ultra-Filtered Feta cheese. Growth curve of bacterial strains were studied by colony count method in growth medium and UF Feta cheese separately and during co-culture with L. lactis. Nisin production was determined by agar diffusion assay method against susceptible test strain and confirmed by RP-HPLC analysis method.   Results : Counts of L. monocytogenes decreased in cheese sample containing L. lactis and standard nisin, to 103 CFU/g after 7 days and it reached to undetectable level within 2 weeks. S. aureus counts remained at its initial number, 105 CFU/g, after 7 days then decreased to 104 CFU/g on day 14 and it was not detectable on day 28. E. coli numbers increased in both treatments after 7 days and then decreased to 104 CFU/g after 28 days. Despite the increasing number of E. coli in growth medium containing nisin, due to the synergistic effect of nisin and other metabolites produced by Lactococcus lactis and starter cultures, the number of E. coli decreased with slow rate . Discussion and conclusion : The results showed, L. monocytogenes was inhibited by L. lactis before entering the logarithmic phase during co-culture. S. aureus was also inhibited during co-culture, but it showed less sensitivity in comparison with L. monocytogenes. However, the number of E. coli remained steady in co-culture with L. lactis. Also, we found that, in all cheese samples, E

  11. Antimicrobial susceptibilities and random amplified polymorphic DNA-PCR fingerprint characterization of Lactococcus lactis ssp. lactis and Lactococcus garvieae isolated from bovine intramammary infections.

    Science.gov (United States)

    Plumed-Ferrer, C; Barberio, A; Franklin-Guild, R; Werner, B; McDonough, P; Bennett, J; Gioia, G; Rota, N; Welcome, F; Nydam, D V; Moroni, P

    2015-09-01

    In total, 181 streptococci-like bacteria isolated from intramammary infections (IMI) were submitted by a veterinary clinic to Quality Milk Production Services (QMPS, Cornell University, Ithaca, NY). The isolates were characterized by sequence analysis, and 46 Lactococcus lactis ssp. lactis and 47 Lactococcus garvieae were tested for susceptibility to 17 antibiotics. No resistant strains were found for β-lactam antibiotics widely used in clinical practice (penicillin, ampicillin, and amoxicillin), and all minimum inhibitory concentrations (MIC) were far from the resistance breakpoints. Eight strains had MIC intermediate to cefazolin. The random amplification of polymorphic DNA (RAPD)-PCR fingerprint patterns showed a slightly higher heterogeneity for Lc. lactis ssp. lactis isolates than for Lc. garvieae isolates. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  12. Impact of Bifidobacterium animalis subsp. lactis BB-12 and, Lactobacillus acidophilus LA-5-containing yoghurt, on fecal bacterial counts of healthy adults.

    Science.gov (United States)

    Savard, Patricia; Lamarche, Benoît; Paradis, Marie-Eve; Thiboutot, Hélène; Laurin, Émilie; Roy, Denis

    2011-09-01

    This randomized, placebo-controlled, double blind, parallel dose-response study investigated the impact of 4-week commercial yoghurt consumption supplemented with Bifidobacterium animalis subsp. lactis (BB-12) and Lactobacillus acidophilus (LA-5) on fecal bacterial counts of healthy adults. Fifty-eight volunteers were randomly assigned to three different groups: 1. placebo (no probiotic, no starter and no green tea extract); 2. Yoptimal (10(9)cfu/100g of BB-12 and LA-5 and 40mg of green tea extract) and 3. Yoptimal-10 (10(10)cfu/100g of BB-12, 10(9)cfu/100g of LA-5 and 40mg of green tea extract). These yoghurt products also contained Lactobacillus delbrueckii subsp. bulgaricus (10(7)cfu/100g) and Streptococcus thermophilus (10(10)cfu/100g). The quantitative PCR (qPCR) results showed that there were significant increases (P=0.02) in bifidobacteria counts with the Yoptimal treatment as compared to baseline. The fecal numbers of B. animalis subsp. lactis and LA-5 significantly increased in the two probiotic treatments compared to the placebo treatment. Viable counts of fecal lactobacilli were significantly higher (P=0.05) and those of enterococci were significantly lower (P=0.04) after the intervention when compared to placebo. No significant difference was observed between treatments in volunteers' weight, waist girth, blood pressure, fasting plasma triglyceride and HDL-C concentrations, as well as cholesterol/HDL-cholesterol ratio. However, a significant increase in plasma cholesterol levels was observed in the placebo group (P=0.0018) but the levels remained stable in the two probiotic yoghurt groups. These results show that probiotic strains supplemented in the form of yoghurt remain active during gut transit and are associated with an increase in beneficial bacteria and a reduction in potentially pathogenic bacteria. This trial was registered at clinicaltrials.gov as NCT00730626. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Quantitative PCR for the specific quantification of Lactococcus lactis and Lactobacillus paracasei and its interest for Lactococcus lactis in cheese samples.

    Science.gov (United States)

    Achilleos, Christine; Berthier, Françoise

    2013-12-01

    The first objective of this work was to develop real-time quantitative PCR (qPCR) assays to quantify two species of mesophilic lactic acid bacteria technologically active in food fermentation, including cheese making: Lactococcus lactis and Lactobacillus paracasei. The second objective was to compare qPCR and plate counts of these two species in cheese samples. Newly designed primers efficiently amplified a region of the tuf gene from the target species. Sixty-three DNA samples from twenty different bacterial species, phylogenetically related or commonly found in raw milk and dairy products, were selected as positive and negative controls. Target DNA was successfully amplified showing a single peak on the amplicon melting curve; non-target DNA was not amplified. Quantification was linear over 5 log units (R(2) > 0.990), down to 22 gene copies/μL per well for Lc. lactis and 73 gene copies/μL per well for Lb. paracasei. qPCR efficiency ranged from 82.9% to 93.7% for Lc. lactis and from 81.1% to 99.5% for Lb. paracasei. At two stages of growth, Lc. lactis was quantified in 12 soft cheeses and Lb. paracasei in 24 hard cooked cheeses. qPCR proved to be useful for quantifying Lc. lactis, but not Lb. paracasei. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Non-fusion and fusion expression of beta-galactosidase from Lactobacillus bulgaricus in Lactococcus lactis.

    Science.gov (United States)

    Wang, Chuan; Zhang, Chao-Wu; Liu, Heng-Chuan; Yu, Qian; Pei, Xiao-Fang

    2008-10-01

    To construct four recombinant Lactococcus lactis strains exhibiting high beta-galactosidase activity in fusion or non-fusion ways, and to study the influence factors for their protein expression and secretion. The gene fragments encoding beta-galactosidase from two strains of Lactobacillus bulgaricus, wch9901 isolated from yogurt and 1.1480 purchased from the Chinese Academy of Sciences, were amplified and inserted into lactococcal expression vector pMG36e. For fusion expression, the open reading frame of the beta-galactosidase gene was amplified, while for non-fusion expression, the open reading frame of the beta-galactosidase gene was amplified with its native Shine-Dalgarno sequence upstream. The start codon of the beta-galactosidase gene partially overlapped with the stop codon of vector origin open reading frame. Then, the recombinant plasmids were transformed into Escherichia coli DH5 alpha and Lactococcus lactis subsp. lactis MG1363 and confirmed by determining beta-galactosidase activities. The non-fusion expression plasmids showed a significantly higher beta-galactosidase activity in transformed strains than the fusion expression plasmids. The highest enzyme activity was observed in Lactococcus lactis transformed with the non-fusion expression plasmids which were inserted into the beta-galactosidase gene from Lactobacillus bulgaricus wch9901. The beta-galactosidase activity was 2.75 times as high as that of the native counterpart. In addition, beta-galactosidase expressed by recombinant plasmids in Lactococcus lactis could be secreted into the culture medium. The highest secretion rate (27.1%) was observed when the culture medium contained 20 g/L of lactose. Different properties of the native bacteria may have some effects on the protein expression of recombinant plasmids. Non-fusion expression shows a higher enzyme activity in host bacteria. There may be a host-related weak secretion signal peptide gene within the structure gene of Lb. bulgaricus beta

  15. Induction of heat shock proteins DnaK, GroEL, and GroES by salt stress in Lactococcus lactis

    DEFF Research Database (Denmark)

    Kilstrup, Mogens; Jacobsen, Susanne; Hammer, Karin

    1997-01-01

    The bacterium Lactococcus lactis has become a model organism in studies of growth physiology and membrane transport, as a result of its simple fermentative metabolism. It is also used as a model for studying the importance of specific genes and functions during lie in excess nutrients, by compari...... the timing during heat stress although at a lower induction level. These data indicate an overlap between the heat shock and salt stress responses in L. lactis......., by comparison of prototrophic wild-type strains and auxotrophic domesticated (daily) strains. In a study of the capacity of domesticated strains to perform directed responses toward various stress conditions, we have analyzed the heat and salt stress response in the established L,. lactis subsp. cremoris...... laboratory strain MG1363, which was originally derived from a dairy strain, After two-dimensional separation of proteins, the DnaK, GroEL, and GroES heat shock proteins, the HrcA (Orf1) heat shack repressor, and the glycolytic enzymes pyruvate kinase, glyceral-dehyde-3-phosphate dehydrogenase...

  16. Nucleotide Sequence and Analysis of an orotate transporter-containing plasmid isolated from the Lactococcus lactis ssp. lactis biovar diacetylactis strain DB0410

    DEFF Research Database (Denmark)

    Defoor, Els Marie Celine; Martinussen, Jan

    A new lactococcal plasmid, pDBORO, was isolated from the Lactococcus lactis ssp. lactis biovar diacetylactis strain DB0410 responsible for the sensitivity of DB0410 towards the pyrimidine-analog 5´-fluoroorotate. The plasmid pDBORO amounts to 16404 bp and its complete nucleotide sequence has been...

  17. Characterization, expression, and mutation of the Lactococcus lactis galPMKTE genes, involved in galactose utilization via the Leloir pathway

    NARCIS (Netherlands)

    Groossiord, B.P.; Luesink, E.J.; Vaughan, E.E.; Arnaud, A.; Vos, de W.M.

    2003-01-01

    A cluster containing five similarly oriented genes involved in the metabolism of galactose via the Leloir pathway in Lactococcus lactis subsp. cremoris MG1363 was cloned and characterized. The order of the genes is galPMKTE, and these genes encode a galactose permease (GalP), an aldose I-epimerase

  18. Engineering of the Lactococcus lactis serine proteinase by construction of hybrid enzymes

    NARCIS (Netherlands)

    Boerrigter, Ingrid J.; Buist, Girbe; Haandrikman, Alfred J.; Nijhuis, Monique; Reuver, Marjon B. de; Siezen, Roland J.; Venema, Gerhardus; Vos, Willem M. de; Kok, Jan

    Plasmids containing wild-type and hybrid proteinase genes were constructed from DNA fragments of the prtP genes of Lactococcus lactis strains Wg2 and SK11. These plasmids were introduced into the plasmid-free strain L. lactis MG1363. The serine proteinases produced by these L. lactis strains were

  19. Lactococcus lactis Metabolism and Gene Expression during Growth on Plant Tissues

    Science.gov (United States)

    Golomb, Benjamin L.

    2014-01-01

    Lactic acid bacteria have been isolated from living, harvested, and fermented plant materials; however, the adaptations these bacteria possess for growth on plant tissues are largely unknown. In this study, we investigated plant habitat-specific traits of Lactococcus lactis during growth in an Arabidopsis thaliana leaf tissue lysate (ATL). L. lactis KF147, a strain originally isolated from plants, exhibited a higher growth rate and reached 7.9-fold-greater cell densities during growth in ATL than the dairy-associated strain L. lactis IL1403. Transcriptome profiling (RNA-seq) of KF147 identified 853 induced and 264 repressed genes during growth in ATL compared to that in GM17 laboratory culture medium. Genes induced in ATL included those involved in the arginine deiminase pathway and a total of 140 carbohydrate transport and metabolism genes, many of which are involved in xylose, arabinose, cellobiose, and hemicellulose metabolism. The induction of those genes corresponded with L. lactis KF147 nutrient consumption and production of metabolic end products in ATL as measured by gas chromatography-time of flight mass spectrometry (GC-TOF/MS) untargeted metabolomic profiling. To assess the importance of specific plant-inducible genes for L. lactis growth in ATL, xylose metabolism was targeted for gene knockout mutagenesis. Wild-type L. lactis strain KF147 but not an xylA deletion mutant was able to grow using xylose as the sole carbon source. However, both strains grew to similarly high levels in ATL, indicating redundancy in L. lactis carbohydrate metabolism on plant tissues. These findings show that certain strains of L. lactis are well adapted for growth on plants and possess specific traits relevant for plant-based food, fuel, and feed fermentations. PMID:25384484

  20. Production and crystallization of α-phosphoglucomutase from Lactococcus lactis

    International Nuclear Information System (INIS)

    Nogly, Przemyslaw; Castro, Rute; Rosa, Matteo de; Neves, Ana Rute; Santos, Helena; Archer, Margarida

    2012-01-01

    α-Phosphoglucomutase from L. lactis, a homologue of human phosphomannomutase 1, was produced and crystallized. X-ray diffraction data were collected to 1.5 Å resolution. α-Phosphoglucomutase (α-PGM) is an enzyme that is essential for the growth of Lactococcus lactis. The enzyme links bacterial anabolism with sugar utilization through glycolysis by catalyzing the reversible interconversion of glucose 6-phosphate and α-glucose 1-phosphate. The gene encoding α-PGM was cloned and overexpressed in L. lactis. The purified protein was functionally active and was crystallized with ammonium sulfate as a precipitant using vapour-diffusion and seeding techniques. Optimized crystals diffracted to 1.5 Å resolution at a synchrotron source

  1. Expression of biologically active murine interleukin-18 in Lactococcus lactis.

    Science.gov (United States)

    Feizollahzadeh, Sadegh; Khanahmad, Hossein; Rahimmanesh, Ilnaz; Ganjalikhani-Hakemi, Mazdak; Andalib, Alireza; Sanei, Mohammad Hossein; Rezaei, Abbas

    2016-11-01

    The food-grade bacterium Lactococcus lactis is increasingly used for heterologous protein expression in therapeutic and industrial applications. The ability of L. lactis to secrete biologically active cytokines may be used for the generation of therapeutic cytokines. Interleukin (IL)-18 enhances the immune response, especially on mucosal surfaces, emphasizing its therapeutic potential. However, it is produced as an inactive precursor and has to be enzymatically cleaved for maturation. We genetically manipulated L. lactis to secrete murine IL-18. The mature murine IL-18 gene was inserted downstream of a nisin promoter in pNZ8149 plasmid and the construct was used to transform L. lactis NZ3900. The transformants were selected on Elliker agar and confirmed by restriction enzyme digestion and sequencing. The expression and secretion of IL-18 protein was verified by SDS-PAGE, western blotting and ELISA. The biological activity of recombinant IL-18 was determined by its ability to induce interferon (IFN)-γ production in L. lactis co-cultured with murine splenic T cells. The amounts of IL-18 in bacterial lysates and supernatants were 3-4 μg mL -1 and 0.6-0.7 ng mL -1 , respectively. The successfully generated L. lactis strain that expressed biologically active murine IL-18 can be used to evaluate the possible therapeutic effects of IL-18 on mucosal surfaces. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Statistical optimization of cell disruption techniques for releasing intracellular X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis spp. lactis.

    Science.gov (United States)

    Üstün-Aytekin, Özlem; Arısoy, Sevda; Aytekin, Ali Özhan; Yıldız, Ece

    2016-03-01

    X-prolyl dipeptidyl aminopeptidase (PepX) is an intracellular enzyme from the Gram-positive bacterium Lactococcus lactis spp. lactis NRRL B-1821, and it has commercial importance. The objective of this study was to compare the effects of several cell disruption methods on the activity of PepX. Statistical optimization methods were performed for two cavitation methods, hydrodynamic (high-pressure homogenization) and acoustic (sonication), to determine the more appropriate disruption method. Two level factorial design (2FI), with the parameters of number of cycles and pressure, and Box-Behnken design (BBD), with the parameters of cycle, sonication time, and power, were used for the optimization of the high-pressure homogenization and sonication methods, respectively. In addition, disruption methods, consisting of lysozyme, bead milling, heat treatment, freeze-thawing, liquid nitrogen, ethylenediaminetetraacetic acid (EDTA), Triton-X, sodium dodecyl sulfate (SDS), chloroform, and antibiotics, were performed and compared with the high-pressure homogenization and sonication methods. The optimized values of high-pressure homogenization were one cycle at 130 MPa providing activity of 114.47 mU ml(-1), while sonication afforded an activity of 145.09 mU ml(-1) at 28 min with 91% power and three cycles. In conclusion, sonication was the more effective disruption method, and its optimal operation parameters were manifested for the release of intracellular enzyme from a L. lactis spp. lactis strain, which is a Gram-positive bacterium. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Dual recombinant Lactococcus lactis for enhanced delivery of DNA vaccine reporter plasmid pPERDBY.

    Science.gov (United States)

    Yagnik, Bhrugu; Sharma, Drashya; Padh, Harish; Desai, Priti

    2017-04-01

    Food grade Lactococcus lactis has been widely used as an antigen and DNA delivery vehicle. We have previously reported the use of non-invasive L. lactis to deliver the newly constructed immunostimulatory DNA vaccine reporter plasmid, pPERDBY. In the present report, construction of dual recombinant L. lactis expressing internalin A of Listeria monocytogenes and harboring pPERDBY (LL InlA + pPERDBY) to enhance the efficiency of delivery of DNA by L. lactis is outlined. After confirmation and validation of LL InlA + pPERDBY, its DNA delivery potential was compared with previously developed non-invasive r- L. lactis::pPERDBY. The use of invasive L. lactis resulted in around threefold increases in the number of enhanced green fluorescent protein-expressing Caco-2 cells. These findings reinforce the prospective application of invasive strain of L. lactis for delivery of DNA/RNA and antigens. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

  4. A review on Lactococcus lactis: from food to factory.

    Science.gov (United States)

    Song, Adelene Ai-Lian; In, Lionel L A; Lim, Swee Hua Erin; Rahim, Raha Abdul

    2017-04-04

    Lactococcus lactis has progressed a long way since its discovery and initial use in dairy product fermentation, to its present biotechnological applications in genetic engineering for the production of various recombinant proteins and metabolites that transcends the heterologous species barrier. Key desirable features of this gram-positive lactic acid non-colonizing gut bacteria include its generally recognized as safe (GRAS) status, probiotic properties, the absence of inclusion bodies and endotoxins, surface display and extracellular secretion technology, and a diverse selection of cloning and inducible expression vectors. This have made L. lactis a desirable and promising host on par with other well established model bacterial or yeast systems such as Escherichia coli, Salmonella cerevisiae and Bacillus subtilis. In this article, we review recent technological advancements, challenges, future prospects and current diversified examples on the use of L. lactis as a microbial cell factory. Additionally, we will also highlight latest medical-based applications involving whole-cell L. lactis as a live delivery vector for the administration of therapeutics against both communicable and non-communicable diseases.

  5. Enhanced heterologous protein productivity by genome reduction in Lactococcus lactis NZ9000.

    Science.gov (United States)

    Zhu, Duolong; Fu, Yuxin; Liu, Fulu; Xu, Haijin; Saris, Per Erik Joakim; Qiao, Mingqiang

    2017-01-03

    The implementation of novel chassis organisms to be used as microbial cell factories in industrial applications is an intensive research field. Lactococcus lactis, which is one of the most extensively studied model organisms, exhibits superior ability to be used as engineered host for fermentation of desirable products. However, few studies have reported about genome reduction of L. lactis as a clean background for functional genomic studies and a model chassis for desirable product fermentation. Four large nonessential DNA regions accounting for 2.83% in L. lactis NZ9000 (L. lactis 9 k) genome (2,530,294 bp) were deleted using the Cre-loxP deletion system as the first steps toward a minimized genome in this study. The mutants were compared with the parental strain in several physiological traits and evaluated as microbial cell factories for heterologous protein production (intracellular and secretory expression) with the red fluorescent protein (RFP) and the bacteriocin leucocin C (LecC) as reporters. The four mutants grew faster, yielded enhanced biomass, achieved increased adenosine triphosphate content, and diminished maintenance demands compared with the wild strain in the two media tested. In particular, L. lactis 9 k-4 with the largest deletion was identified as the optimum candidate host for recombinant protein production. With nisin induction, not only the transcriptional efficiency but also the production levels of the expressed reporters were approximately three- to fourfold improved compared with the wild strain. The expression of lecC gene controlled with strong constitutive promoters P5 and P8 in L. lactis 9 k-4 was also improved significantly. The genome-streamlined L. lactis 9 k-4 outcompeted the parental strain in several physiological traits assessed. Moreover, L. lactis 9 k-4 exhibited good properties as platform organism for protein production. In future works, the genome of L. lactis will be maximally reduced by using our specific design

  6. Molecular Cloning and Nucleotide Sequence of the Gene Encoding the Major Peptidoglycan Hydrolase of Lactococcus lactis, a Muramidase Needed for Cell Separation

    NARCIS (Netherlands)

    Buist, Girbe; Kok, Jan; Leenhouts, Kees J.; Dabrowska, Magdalena; Venema, Gerhardus; Haandrikman, Alfred J.

    A gene of Lactococcus lactis subsp, cremoris MG1363 encoding a peptidoglycan hydrolase was identified in a genomic library of the strain in pUC19 by screening Escherichia coli transformants for cell wall lysis activity on a medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells,

  7. Effects of Lactococcus lactis on composition of intestinal microbiota: Role of nisin

    DEFF Research Database (Denmark)

    Bernbom, Nete; Licht, Tine Rask; Brogren, Carl-Henrik

    2006-01-01

    This study examined the ability of (i) pure nisin, (ii) nisin-producing Lactococcus lactis strain CHCC5826, and (iii) the non-nisin-producing L. lactis strain CHCH2862 to affect the composition of the intestinal microbiota of human flora-associated rats. The presence of both the nisin-producing a......This study examined the ability of (i) pure nisin, (ii) nisin-producing Lactococcus lactis strain CHCC5826, and (iii) the non-nisin-producing L. lactis strain CHCH2862 to affect the composition of the intestinal microbiota of human flora-associated rats. The presence of both the nisin...... in the rat fecal microbiota were observed after dosage with nisin. Pearson cluster analysis of denaturing gradient gel electrophoresis profiles of the 16S rRNA genes present in the fecal microbial population revealed that the microbiota of animals dosed with either of the two L. lactis strains were different...

  8. Novel phage group infecting Lactobacillus delbrueckii subsp. lactis, as revealed by genomic and proteomic analysis of bacteriophage Ldl1.

    Science.gov (United States)

    Casey, Eoghan; Mahony, Jennifer; Neve, Horst; Noben, Jean-Paul; Dal Bello, Fabio; van Sinderen, Douwe

    2015-02-01

    Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii. In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 +/- 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species.

  9. Probiotic Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp lactis Bl-04 interactions with prebiotic carbohydrates using differential proteomics and protein characterization

    DEFF Research Database (Denmark)

    Hansen, Morten Ejby

    of probiotics, primarily non-digestible carbohydrates, are termed prebiotics. The knowledge of prebiotic utilization and in particular the specificities of carbohydrate transport and metabolism are limited, hampering robust understanding for the basis of selective utilization of known prebiotics...... and the discovery and documentation of novel ones. In this project we set out to investigate the metabolism of carbohydrates that are prebiotic or potential prebiotic compounds utilized by the probiotic organisms Lactobacillus acidophilus NCFM (NCFM) and Bifidobacterium animalis subsp. lactis BL-04 (Bl-04). The aim...... of this Ph.D. thesis was the study of probiotic NCFM and Bl-04 interaction with prebiotic carbohydrates using differential proteomics and protein characterization. Proteomics is a potential omics tool to investigate probiotic bacteria and its response to prebiotic carbohydrates at the protein level...

  10. Secretory expression of a heterologous nattokinase in Lactococcus lactis.

    Science.gov (United States)

    Liang, Xiaobo; Zhang, Lixin; Zhong, Jin; Huan, Liandong

    2007-05-01

    Nattokinase has been reported as an oral health product for the prevention of atherosclerosis. We developed a novel strategy to express a nattokinase from Bacillus subtilis in a live delivery vehicle, Lactococcus lactis. Promoter P( nisZ) and signal peptide SP(Usp) were used for inducible and secretory expression of nattokinase in L. lactis. Western blotting analysis demonstrated that nattokinase was successfully expressed, and about 94% of the enzyme was secreted to the culture. The recombinant nattokinase showed potent fibrinolytic activity, equivalent to 41.7 urokinase units per milliliter culture. Expression and delivery of such a fibrinolytic enzyme in the food-grade vehicle L. lactis would facilitate the widespread application of nattokinase in the control and prevention of thrombosis diseases.

  11. Lactococcus lactis As a Versatile Vehicle for Tolerogenic Immunotherapy

    Science.gov (United States)

    Cook, Dana P.; Gysemans, Conny; Mathieu, Chantal

    2018-01-01

    Genetically modified Lactococcus lactis bacteria have been engineered as a tool to deliver bioactive proteins to mucosal tissues as a means to exert both local and systemic effects. They have an excellent safety profile, the result of years of human consumption in the food industry, as well as a lack of toxicity and immunogenicity. Also, containment strategies have been developed to promote further application as clinical protein-based therapeutics. Here, we review technological advancements made to enhanced the potential of L. lactis as live biofactories and discuss some examples of tolerogenic immunotherapies mediated by mucosal drug delivery via L. lactis. Additionally, we highlight their use to induce mucosal tolerance by targeted autoantigen delivery to the intestine as an approach to reverse autoimmune type 1 diabetes. PMID:29387056

  12. Cytoplasmic expression of a thermostable invertase from Thermotoga maritima in Lactococcus lactis.

    Science.gov (United States)

    Pek, Han Bin; Lim, Pei Yu; Liu, Chengcheng; Lee, Dong-Yup; Bi, Xuezhi; Wong, Fong Tian; Ow, Dave Siak-Wei

    2017-05-01

    To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis. The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively. Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.

  13. Genetically Modified Lactococcus lactis for Delivery of Human Interleukin-10 to Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Inge L. Huibregtse

    2012-01-01

    Full Text Available Interleukin-10 (IL-10 plays an indispensable role in mucosal tolerance by programming dendritic cells (DCs to induce suppressor Th-cells. We have tested the modulating effect of L. lactis secreting human IL-10 (L.  lactisIL-10 on DC function in vitro. Monocyte-derived DC incubated with L.  lactisIL-10 induced effector Th-cells that markedly suppressed the proliferation of allogenic Th-cells as compared to L. lactis. This suppressive effect was only seen when DC showed increased CD83 and CD86 expression. Furthermore, enhanced production of IL-10 was measured in both L.  lactisIL-10-derived DC and Th-cells compared to L. lactis-derived DC and Th-cells. Neutralizing IL-10 during DC-Th-cell interaction and coculturing L.  lactisIL-10-derived suppressor Th-cells with allogenic Th-cells in a transwell system prevented the induction of suppressor Th-cells. Only 130 pg/mL of bacterial-derived IL-10 and 40 times more exogenously added recombinant human IL-10 were needed during DC priming for the generation of suppressor Th-cells. The spatially restricted delivery of IL-10 by food-grade bacteria is a promising strategy to induce suppressor Th-cells in vivo and to treat inflammatory diseases.

  14. Modeling Lactococcus lactis using a genome-scale flux model

    Directory of Open Access Journals (Sweden)

    Nielsen Jens

    2005-06-01

    Full Text Available Abstract Background Genome-scale flux models are useful tools to represent and analyze microbial metabolism. In this work we reconstructed the metabolic network of the lactic acid bacteria Lactococcus lactis and developed a genome-scale flux model able to simulate and analyze network capabilities and whole-cell function under aerobic and anaerobic continuous cultures. Flux balance analysis (FBA and minimization of metabolic adjustment (MOMA were used as modeling frameworks. Results The metabolic network was reconstructed using the annotated genome sequence from L. lactis ssp. lactis IL1403 together with physiological and biochemical information. The established network comprised a total of 621 reactions and 509 metabolites, representing the overall metabolism of L. lactis. Experimental data reported in the literature was used to fit the model to phenotypic observations. Regulatory constraints had to be included to simulate certain metabolic features, such as the shift from homo to heterolactic fermentation. A minimal medium for in silico growth was identified, indicating the requirement of four amino acids in addition to a sugar. Remarkably, de novo biosynthesis of four other amino acids was observed even when all amino acids were supplied, which is in good agreement with experimental observations. Additionally, enhanced metabolic engineering strategies for improved diacetyl producing strains were designed. Conclusion The L. lactis metabolic network can now be used for a better understanding of lactococcal metabolic capabilities and potential, for the design of enhanced metabolic engineering strategies and for integration with other types of 'omic' data, to assist in finding new information on cellular organization and function.

  15. Growth, nisA Gene Expression, and In Situ Activity of Novel Lactococcus lactis subsp. cremoris Costarter Culture in Commercial Hard Cheese Production.

    Science.gov (United States)

    Noutsopoulos, Dimitrios; Kakouri, Athanasia; Kartezini, Eleftheria; Pappas, Dimitrios; Hatziloukas, Efstathios; Samelis, John

    2017-12-01

    This study evaluated in situ expression of the nisA gene by an indigenous, nisin A-producing (NisA+) Lactococcus lactis subsp. cremoris raw milk genotype, represented by strain M78, in traditional Greek Graviera cheeses under real factory-scale manufacturing and ripening conditions. Cheeses were produced with added a mixed thermophilic and mesophilic commercial starter culture (CSC) or with the CSC plus strain M78 (CSC+M78). Cheeses were sampled after curd cooking (day 0), fermentation of the unsalted molds for 24 h (day 1), brining (day 7), and ripening of the brined molds (14 to 15 kg each) for 30 days in a fully controlled industrial room (16.5°C; 91% relative humidity; day 37). Total RNA was directly extracted from the cheese samples, and the expression of nisA gene was evaluated by real-time reverse transcription PCR (qRT-PCR). Agar overlay and well diffusion bioassays were correspondingly used for in situ detection of the M78 NisA+ colonies in the cheese agar plates and antilisterial activity in whole-cheese slurry samples, respectively. Agar overlay assays showed good growth (>8 log CFU/g of cheese) of the NisA+ strain M78 in coculture with the CSC and vice versa. The nisA expression was detected in CSC+M78 cheese samples only, with its expression levels being the highest (16-fold increase compared with those of the control gene) on day 1, followed by significant reduction on day 7 and almost negligible expression on day 37. Based on the results, certain intrinsic and mainly implicit hurdle factors appeared to reduce growth prevalence rates and decrease nisA gene expression, as well as the nisin A-mediated antilisterial activities of the NisA+ strain M78 postfermentation. To our knowledge, this is the first report on quantitative expression of the nisA gene in a Greek cooked hard cheese during commercial manufacturing and ripening conditions by using a novel, rarely isolated, indigenous NisA+ L. lactis subsp. cremoris genotype as costarter culture.

  16. The Impact of Storage Conditions on the Stability of Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis Bb12 in Human Milk.

    Science.gov (United States)

    Mantziari, Anastasia; Aakko, Juhani; Kumar, Himanshu; Tölkkö, Satu; du Toit, Elloise; Salminen, Seppo; Isolauri, Erika; Rautava, Samuli

    2017-11-01

    Human milk is the optimal source of complete nutrition for neonates and it also guides the development of infant gut microbiota. Importantly, human milk can be supplemented with probiotics to complement the health benefits of breastfeeding. Storage of human milk for limited periods of time is often unavoidable, but little is known about the effect of different storage conditions (temperature) on the viability of the added probiotics. Therefore, in this study, we evaluated how different storage conditions affect the viability of two specific widely used probiotics, Lactobacillus rhamnosus GG (LGG) and Bifidobacterium animalis subsp. lactis (Bb12), in human milk by culturing and quantitative polymerase chain reaction. Our results indicate that LGG and Bb12 remained stable throughout the storage period. Thus, we conclude that human milk offers an appropriate matrix for probiotic supplementation.

  17. Production of recombinant peanut allergen Ara h 2 using Lactococcus lactis

    DEFF Research Database (Denmark)

    Glenting, J.; Poulsen, Lars K.; Kato, K.

    2007-01-01

    lactis is an attractive microorganism for use in the production of protein therapeutics. L. lactis is considered food grade, free of endotoxins, and is able to secrete the heterologous product together with few other native proteins. Hypersensitivity to peanut represents a serious allergic problem. Some...... of the major allergens in peanut have been described. However, for therapeutic usage more information about the individual allergenic components is needed. In this paper we report recombinant production of the Ara h 2 peanut allergen using L. lactis. Results: A synthetic ara h 2 gene was cloned into an L...

  18. Putrescine production by Lactococcus lactis subsp. cremoris CECT 8666 is reduced by NaCl via a decrease in bacterial growth and the repression of the genes involved in putrescine production.

    Science.gov (United States)

    Del Rio, Beatriz; Redruello, Begoña; Ladero, Victor; Fernandez, Maria; Martin, Maria Cruz; Alvarez, Miguel A

    2016-09-02

    The reduction of NaCl in food is a public health priority; high NaCl intakes have been associated with serious health problems. However, it is reported that reducing the NaCl content of cheeses may lead to an increase in the content of biogenic amines (BAs). The present work examines the effect of NaCl on the accumulation of putrescine (one of the BAs often detected at high concentration in cheese) in experimental Cabrales-like cheeses containing Lactococcus lactis subsp. cremoris CECT 8666, a dairy strain that catabolises agmatine to putrescine via the agmatine deiminase (AGDI) pathway. The genes responsible for this pathway are grouped in the AGDI cluster. This comprises a regulatory gene (aguR) (transcribed independently), followed by the catabolic genes that together form an operon (aguBDAC). Reducing the NaCl concentration of the cheese led to increased putrescine accumulation. In contrast, increasing the NaCl concentration of both pH-uncontrolled and pH-controlled (pH 6) cultures of L. lactis subsp. cremoris CECT 8666 significantly inhibited its growth and the production of putrescine. Such production appeared to be inhibited via a reduction in the transcription of the aguBDAC operon; no effect on the transcription of aguR was recorded. The present results suggest that low-sodium cheeses are at risk of accumulating higher concentrations of putrescine. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Membrane Protein Production in Lactococcus lactis for Functional Studies.

    Science.gov (United States)

    Seigneurin-Berny, Daphne; King, Martin S; Sautron, Emiline; Moyet, Lucas; Catty, Patrice; André, François; Rolland, Norbert; Kunji, Edmund R S; Frelet-Barrand, Annie

    2016-01-01

    Due to their unique properties, expression and study of membrane proteins in heterologous systems remains difficult. Among the bacterial systems available, the Gram-positive lactic bacterium, Lactococcus lactis, traditionally used in food fermentations, is nowadays widely used for large-scale production and functional characterization of bacterial and eukaryotic membrane proteins. The aim of this chapter is to describe the different possibilities for the functional characterization of peripheral or intrinsic membrane proteins expressed in Lactococcus lactis.

  20. Plasmid-mediated UV-protection in Streptococcus lactis

    Energy Technology Data Exchange (ETDEWEB)

    Chopin, M.C.; Rouault, A. (Institut National de la Recherche Agronomique, Rennes (France). Lab. de Recherches de Technologie Laitiere); Moillo-Batt, A. (Institut National de la Sante et de la Recherche Medicale (INSERM), Hopital de Pontchaillon, 35 - Rennes (France))

    1985-02-01

    Streptococcus lactis strain IL594 contains 9 plasmids, designated pIL1 to pIL9. On the basis of protoplast-induced curing experiments the authors showed that derivatives containing pIL7 were resistant to UV-irradiation while derivatives lacking pIL7 were sensitive. The pIL7-determined UV-protection was confirmed by co-transfer of the plasmid and of the character into a plasmid-free derivative of S. lactis IL594. Moreover, prophage induction required higher UV-fluence in this derivative carrying pIL7 than in the plasmid-free strain. This is the first report of a plasmid-mediated UV-protection in group N streptococci.

  1. Plasmid-mediated UV-protection in Streptococcus lactis

    International Nuclear Information System (INIS)

    Chopin, M.-C.; Rouault, A.

    1985-01-01

    Streptococcus lactis strain IL594 contains 9 plasmids, designated pIL1 to pIL9. On the basis of protoplast-induced curing experiments the authors showed that derivatives containing pIL7 were resistant to UV-irradiation while derivatives lacking pIL7 were sensitive. The pIL7-determined UV-protection was confirmed by cotransfer of the plasmid and of the character into a plasmid-free derivative of S. lactis IL594. Moreover, prophage induction required higher UV-fluence in this derivative carrying pIL7 than in the plasmid-free strain. This is the first report of a plasmid-mediated UV-protection in group N streptococci. (orig.)

  2. The extracellular proteome of Bifidobacterium animalis subsp. lactis BB‐12 reveals proteins with putative roles in probiotic effects

    DEFF Research Database (Denmark)

    Gilad, Ofir; Svensson, Birte; Viborg, Alexander Holm

    2011-01-01

    Probiotics are live microorganisms that exert health‐promoting effects on the human host, as demonstrated for numerous strains of the genus Bifidobacterium. To unravel the proteins involved in the interactions between the host and the extensively used and well‐studied probiotic strain Bifidobacte......Probiotics are live microorganisms that exert health‐promoting effects on the human host, as demonstrated for numerous strains of the genus Bifidobacterium. To unravel the proteins involved in the interactions between the host and the extensively used and well‐studied probiotic strain...... Bifidobacterium animalis subsp. lactis BB‐12, proteins secreted by the bacterium, i.e. belonging to the extracellular proteome present in the culture medium, were identified by 2‐DE coupled with MALDI‐TOF MS. Among the 74 distinct proteins identified, 31 are predicted to carry out their physiological role either...... functions include binding of plasminogen, formation of fimbriae, adhesion to collagen, attachment to mucin and intestinal cells as well as induction of immunomodulative response. These findings suggest a role of the proteins in colonization of the gastrointestinal tract, adhesion to host tissues...

  3. From Streptococcus lactis to Lactococcus lactis: A qualitative and quantitative analysis of the scope of research undertaken around a microbial concept

    OpenAIRE

    Yann Demarigny; Virginie Soldat; Laetitia Gemelas

    2015-01-01

    The lactic acid bacterium Lactococcus lactis, formerly named Streptococcus lactis, has been known and used for many years, even before its re-affiliation in 1985. The number of published papers featuring one of the two names, either in the title or in the key words, currently stands at more than 2,900. From 1945 to 2014, a bibliometric analysis of the evolution of this bacterium allowed us to identify three phases we have called 1, the “exploratory period” (or the “US period” if we refer to t...

  4. Vaccination against Staphylococcus aureus experimental endocarditis using recombinant Lactococcus lactis expressing ClfA or FnbpA.

    Science.gov (United States)

    Veloso, Tiago Rafael; Mancini, Stefano; Giddey, Marlyse; Vouillamoz, Jacques; Que, Yok-Ai; Moreillon, Philippe; Entenza, José Manuel

    2015-07-09

    Staphylococcus aureus is a major cause of serious infections in humans and animals and a vaccine is becoming a necessity. Lactococcus lactis is a non-pathogenic bacterium that can be used as a vector for the delivery of antigens. We investigated the ability of non-living L. lactis heterologously expressing S. aureus clumping factor A (ClfA) and fibronectin-binding protein A (FnbpA), alone or together, to elicit an immune response in rats and protect them from S. aureus experimental infective endocarditis (IE). L. lactis ClfA was used for immunization against S. aureus Newman (expressing ClfA but not FnbpA), while L. lactis ClfA, L. lactis FnbpA, as well as L. lactis ClfA/FnbpA, were used against S. aureus P8 (expressing ClfA and FnbpA). Vaccination of rats with L. lactis ClfA elicited antibodies that inhibited binding of S. aureus Newman to fibrinogen, triggered the production of IL-17A and conferred protection to 13/19 (68%) of the animals from IE (Plactis ClfA, L. lactis FnbpA or L. lactis ClfA/FnbpA also produced antibodies against the target proteins, but these did not prevent binding of S. aureus P8 to fibrinogen or fibronectin and did not protect animals against S. aureus P8 IE. Moreover, immunization with constructs containing FnbpA did not increase IL-17A production. These results indicate that L. lactis is a valuable antigen delivery system able to elicit efficient humoral and cellular responses. However, the most appropriate antigens affording protection against S. aureus IE are yet to be elucidated. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Simultaneous lactic acidification and coagulation by using recombinant Lactococcus lactis strain.

    Science.gov (United States)

    Raftari, M; Ghafourian, S; Abu Bakar, F

    2017-04-01

    This study was an attempt to create a novel milk clotting procedure using a recombinant bacterium capable of milk coagulation. The Rhizomucor pusillus proteinase (RPP) gene was sub-cloned into a pALF expression vector. The recombinant pALF-RPP vector was then electro-transferred into Lactococcus lactis. Finally, the milk coagulation ability of recombinant L. lactis carrying a RPP gene was evaluated. Nucleotide sequencing of DNA insertion from the clone revealed that the RPP activity corresponded to an open reading frame consisting of 1218 bp coding for a 43·45 kDa RPP protein. The RPP protein assay results indicated that the highest RPP enzyme expression with 870 Soxhlet units (SU) per ml and 7914 SU/OD were obtained for cultures which were incubated at pH 5·5 and 30°C. Interestingly, milk coagulation was observed after 205 min of inoculating milk with recombinant L. lactis carrying the RPP gene. The recombinant L. lactis carrying RPP gene has the ability to function as a starter culture for acidifying and subsequently coagulating milk by producing RPP as a milk coagulant agent. Creating a recombinant starter culture bacterium that is able to coagulate milk. It is significant because the recombinant L. lactis has the ability to work as a starter culture and milk coagulation agent. © 2016 The Society for Applied Microbiology.

  6. Improvement of the respiration efficiency of Lactococcus lactis by decreasing the culture pH.

    Science.gov (United States)

    Shi, Weijia; Li, Yu; Gao, Xueling; Fu, Ruiyan

    2016-03-01

    The growth characteristics and intracellular hemin concentrations of Lactococcus lactis grown under different culture pH and aeration conditions were examined to investigate the effect of culture pH on the respiration efficiency of L. lactis NZ9000 (pZN8148). Cell biomass and biomass yield of L. lactis grown with 4 μg hemin/ml and O2 were higher than those without aeration when the culture pH was controlled at 5-6.5. The culture pH affected the respiratory efficiency in the following order of pH: 5 > 5.5 > 6 > 6.5; the lag phase increased as the culture pH decreased. Hemin accumulation was sensitive to culture pH. Among the four pH conditions, pH 5.5 was optimal for hemin accumulation in the cells. The highest intracellular hemin level in L. lactis resting cells incubated at different pH saline levels (5-6.5) was at pH 5.5. The respiration efficiency of L. lactis under respiration-permissive conditions increases markedly as the culture pH decreases. These results may help develop high cell-density L. lactis cultures. Thus, this microorganism may be used for industrial applications.

  7. Expression of Helicobacter pylori hspA Gene in Lactococcus lactis NICE System and Experimental Study on Its Immunoreactivity

    Directory of Open Access Journals (Sweden)

    Xiao-Juan Zhang

    2015-01-01

    Full Text Available Aim. The aim of this study was to develop an oral Lactococcus lactis (L. lactis vaccine against Helicobacter pylori (H. pylori. Methods. After L. lactis NZ3900/pNZ8110-hspA was constructed, growth curves were plotted to study whether the growth of recombinant L. lactis was affected after hspA was cloned into L. lactis and whether the growth of empty bacteria, empty plasmid bacteria, and recombinant L. lactis was affected by different concentrations of Nisin; SDS-PAGE and Western blot were adopted, respectively, to detect the HspA expressed by recombinant L. lactis and its immunoreactivity. Results. There was no effect observed from the growth curve after exogenous gene hspA was cloned into L. lactis NZ3900; different concentrations of Nisin did not affect the growth of NZ3900 and NZ3900/pNZ8110, while different concentrations of Nisin inhibited the growth of NZ3900/pNZ8110-hspA except 10 ng/mL Nisin. No HspA strip was observed from SDS-PAGE. Western blot analysis showed that HspA expressed by recombinant bacteria had favorable immunoreactivity. Conclusion. The growth of recombinant L. lactis was suppressed even though a small amount of HspA had been induced to express. Therefore recombinant L. lactis only express HspA which was not suitable to be oral vaccine against Helicobacter pylori.

  8. Cloning in Streptococcus lactis of plasmid-mediated UV resistance and effect on prophage stability

    International Nuclear Information System (INIS)

    Chopin, M.C.; Chopin, A.; Rouault, A.; Simon, D.

    1986-01-01

    Plasmid pIL7 (33 kilobases) from Streptococcus lactis enhances UV resistance and prophage stability. A 5.4-kilobase pIL7 fragment carrying genes coding for both characters was cloned into S. lactis, using plasmid pHV1301 as the cloning vector. The recombinant plasmid was subsequently transferred to three other S. lactis strains by transformation or protoplast fusion. Cloned genes were expressed in all tested strains

  9. Genetic Variation of Lactobacillus delbrueckii subsp. lactis Bacteriophages Isolated from Cheese Processing Plants in Finland

    Science.gov (United States)

    Forsman, Päivi; Alatossava, Tapani

    1991-01-01

    The genomes of four Lactobacillus delbrueckii subsp. lactis bacteriophages were characterized by restriction endonuclease mapping, Southern hybridization, and heteroduplex analysis. The phages were isolated from different cheese processing plants in Finland between 1950 and 1972. All four phages had a small isometric head and a long noncontractile tail. Two different types of genome (double-stranded DNA) organization existed among the different phages, the pac type and the cos type, corresponding to alternative types of phage DNA packaging. Three phages belonged to the pac type, and a fourth was a cos-type phage. The pac-type phages were genetically closely related. In the genomes of the pac-type phages, three putative insertion/deletions (0.7 to 0.8 kb, 1.0 kb, and 1.5 kb) and one other region (0.9 kb) containing clustered base substitutions were discovered and localized. At the phenotype level, three main differences were observed among the pac-type phages. These concerned two minor structural proteins and the efficiency of phage DNA packaging. The genomes of the pac-type phages showed only weak homology with that of the cos-type phage. Phage-related DNA, probably a defective prophage, was located in the chromosome of the host strain sensitive to the cos-type phage. This DNA exhibited homology under stringent conditions to the pac-type phages. Images PMID:16348513

  10. Rewiring Lactococcus lactis for Ethanol Production

    DEFF Research Database (Denmark)

    Solem, Christian; Dehli, Tore Ibsen; Jensen, Peter Ruhdal

    2013-01-01

    to redirect the metabolism of LAB model organism Lactococcus lactis toward ethanol production. Codon-optimized Zymomonas mobilis pyruvate decarboxylase (PDC) was introduced and expressed from synthetic promoters in different strain backgrounds. In the wild-type L. lactis strain MG1363 growing on glucose, only...... small amounts of ethanol were obtained after introducing PDC, probably due to a low native alcohol dehydrogenase activity. When the same strains were grown on maltose, ethanol was the major product and lesser amounts of lactate, formate, and acetate were formed. Inactivating the lactate dehydrogenase...... genes ldhX, ldhB, and ldh and introducing codon-optimized Z. mobilis alcohol dehydrogenase (ADHB) in addition to PDC resulted in high-yield ethanol formation when strains were grown on glucose, with only minor amounts of by-products formed. Finally, a strain with ethanol as the sole observed...

  11. Generation of a membrane potential by Lactococcus lactis through aerobic electron transport

    NARCIS (Netherlands)

    Brooijmans, R. J. W.; Poolman, B.; Schuurman-Wolters, G. K.; de Vos, W. M.; Hugenholtz, J.

    Lactococcus lactis, a facultative anaerobic lactic acid bacterium, is known to have an increased growth yield when grown aerobically in the presence of heme. We have now established the presence of a functional, proton motive force-generating electron transfer chain (ETC) in L. lactis under these

  12. Elucidating Flux Regulation of the Fermentation Modes of Lactococcus lactis:A Mutlilevel Study

    OpenAIRE

    Chan, Siu Hung Joshua; Solem, Christian; Jensen, Peter Ruhdal

    2014-01-01

    De mange års anvendelse af mælkesyrebakterien Lactococcus lactis (L. lactis) indenfor mejeriindustrien, har været medvirkende til at L. lactis er blevet en af de mest velkarakteriserede bakterier. Denne Gram positive bakterie, som har et lavt GC indhold, har en relativt simpel metabolisme og er let at modificere genetisk. Dette har gjort den til et attraktivt mål for ”metabolic engineering”, bl.a. med henblik på produktion af non-food relaterede kemikalier. Derudover har den status som den fø...

  13. Lactococcus lactis KR-050L inhibit IL-6/STAT3 activation.

    Science.gov (United States)

    Hwang, J T; Jang, H-J; Kim, J H; Park, C S; Kim, Y; Lim, C-H; Lee, S W; Rho, M-C

    2017-05-01

    The purpose of this study was to investigate IL-6/STAT3 inhibitory activity using lactic acid bacteria (LABs) isolated from Gajuknamu kimchi. Six LABs were isolated from Gajuknamu kimchi and identified through 16S rRNA sequencing. Among them, the culture broth of Lactococcus lactis KR-050L inhibited IL-6-induced STAT3 luciferase activity. Fifteen compounds were isolated from the EtOAc extract of culture broth though column chromatography and preparative high-performance liquid chromatography, and they were identified as 2,5-diketopipperazine structures by spectroscopic analyses (MS, 1 H- and 13 C-NMR). They also showed inhibitory activities on IL-6-induced STAT3 activation, and showed the different in activity according to the presence of a phenylalanine residue, hydroxyl groups and isometric structure. The six new LABs isolated from Gajuknamu kimchi, and Lc. lactis KR-050L was selected as candidate IL-6/STAT3 inhibitors. The activity levels of 15 2,5-DKPs isolated from Lc. lactis KR-050L were verified. This study constitutes the first attempt to isolate various LABs from Gajuknamu kimchi and to discover IL-6/STAT3 inhibitors in the EtOAc extract of Lc. lactis KR-050L culture broth. Moreover, our data provide useful biochemical information regarding the commercialization of Lc. lactis isolated from Gajuknamu kimchi as an approach to use functional foods for the treatment of various diseases via IL-6/STAT3 activation. © 2017 The Society for Applied Microbiology.

  14. Generation of Dipeptidyl Peptidase-IV-Inhibiting Peptides from β-Lactoglobulin Secreted by Lactococcus lactis

    Directory of Open Access Journals (Sweden)

    Suguru Shigemori

    2014-01-01

    Full Text Available Previous studies showed that hydrolysates of β-lactoglobulin (BLG prepared using gastrointestinal proteases strongly inhibit dipeptidyl peptidase-IV (DPP-IV activity in vitro. In this study, we developed a BLG-secreting Lactococcus lactis strain as a delivery vehicle and in situ expression system. Interestingly, trypsin-digested recombinant BLG from L. lactis inhibited DPP-IV activity, suggesting that BLG-secreting L. lactis may be useful in the treatment of type 2 diabetes mellitus.

  15. Biochemical and kinetic characterisation of a novel xylooligosaccharide-upregulated GH43 β-d-xylosidase/α-l-arabinofuranosidase (BXA43) from the probiotic Bifidobacterium animalis subsp. lactis BB-12

    DEFF Research Database (Denmark)

    Viborg, Alexander Holm; Sørensen, Kim Ib; Gilad, Ofir

    2013-01-01

    The Bifidobacterium animalis subsp. lactis BB-12 gene BIF_00092, assigned to encode a β-d-xylosidase (BXA43) of glycoside hydrolase family 43 (GH43), was cloned with a C-terminal His-tag and expressed in Escherichia coli. BXA43 was purified to homogeneity from the cell lysate and found to be a dual......-specificity exo-hydrolase active on para-nitrophenyl-β-d-xylopyranoside (pNPX), para-nitrophenyl-α-L-arabinofuranoside (pNPA), β-(1 → 4)-xylopyranosyl oligomers (XOS) of degree of polymerisation (DP) 2–4, and birchwood xylan. A phylogenetic tree of the 92 characterised GH43 enzymes displayed five distinct groups...

  16. Expression of Six Peptidases from Lactobacillus helveticus in Lactococcus lactis

    OpenAIRE

    Luoma, Susanna; Peltoniemi, Kirsi; Joutsjoki, Vesa; Rantanen, Terhi; Tamminen, Marja; Heikkinen, Inka; Palva, Airi

    2001-01-01

    For development of novel starter strains with improved proteolytic properties, the ability of Lactococcus lactis to produce Lactobacillus helveticus aminopeptidase N (PepN), aminopeptidase C (PepC), X-prolyl dipeptidyl aminopeptidase (PepX), proline iminopeptidase (PepI), prolinase (PepR), and dipeptidase (PepD) was studied by introducing the genes encoding these enzymes into L. lactis MG1363 and its derivatives. According to Northern analyses and enzyme activity measurements, the L. helvetic...

  17. [The humoral immune response in mice induced by recombinant Lactococcus lactis expressing HIV-1 gag].

    Science.gov (United States)

    Zhao, Xiaofei; Zhang, Cairong; Liu, Xiaojuan; Ma, Zhenghai

    2014-11-01

    To analyze the humoral immune response induced by recombinant Lactococcus lactis expressing HIV-1 gag in mice immunized orally, intranasally, subcutaneously or in the combined way of above three. Fifty BALB/c mice were randomly divided into 5 groups, 10 mice per group. The mice were immunized consecutively three times at two week intervals with 10(9) CFU of recombinant Lactococcus lactis expressing gag through oral, intranasal, subcutaneous administration or the mix of them. The mice that were immunized orally with Lactococcus lactis containing PMG36e served as a control group. The sera of mice were collected before primary immunization and 2 weeks after each immunization to detect the gag specific IgG by ELISA. Compared with the control group, the higher titer of serum gag specific IgG was detected in the four groups immunized with recombinant Lactococcus lactis expressing gag, and it was the highest in the mixed immunization group (PLactococcus lactis expressing gag can induce humoral immune response in mice by oral, intranasal, subcutaneous injection or the mix of them, and the mixed immunization can enhance the immune effects of Lactococcus lactis vector vaccine.

  18. Oral Administration of Recombinant Lactococcus lactis Expressing the Cellulase Gene Increases Digestibility of Fiber in Geese.

    Science.gov (United States)

    Zhou, Haizhu; Gao, Yunhang; Gao, Guang; Lou, Yujie

    2015-12-01

    Enhancing cellulose digestibility in animals is important for improving the utilization of forage, which can decrease the amount of food used in animal production. The aim of the present study was to achieve recombinant expression of the cellulase gene in Lactococcus lactis and evaluate the effects of oral administration of the recombinant L. lactis on fiber digestibility in geese. Cellulase (Cell) and green fluorescent protein (GFP) genes were cloned into a L. lactis expression vector (pNZ8149) to construct the recombinant expression plasmid (pNZ8149-GFP-Cell). Then, the recombinant expression plasmid was transformed into L. lactis (NZ3900) competent cells by electroporation to obtain recombinant L. lactis (pNZ8149-GFP-Cell/NZ3900) in which protein expression was induced by Nisin. Expression of GFP and Cell by the recombinant L. lactis was confirmed using SDS-PAGE, fluorescence detection, and Congo red assays. A feeding experiment showed that oral administration of pNZ8149-GFP-Cell/NZ3900 significantly increased the digestibility of dietary fiber in geese fed either a maize stalk diet or a rice chaff diet. Therefore, oral administration of recombinant L. lactis cells expressing the cellulase gene increases fiber digestibility in geese, offering a way to increase the utilization of dietary fiber in geese.

  19. Experimental determination of control of glycolysis in Lactococcus lactis

    DEFF Research Database (Denmark)

    Købmann, Brian Jensen; Andersen, Heidi Winterberg; Solem, Christian

    2002-01-01

    ), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PYK) and lactate dehydrogenase (LDH) are shown to have no significant control on the glycolytic flux in exponentially growing cells of L. lactis MG1363. Introduction of an uncoupled ATPase activity results in uncoupling of glycolysis from biomass...... production. With MG1363 growing in defined medium supplemented with glucose, the ATP demanding processes do not have a significant control on the glycolytic flux; it appears that glycolysis is running at maximal rate. It is likely that the flux control is distributed over many enzymes in L. lactis...

  20. The prophylactic effect of probiotic Enterococcus lactis IW5 against different human cancer cells

    Directory of Open Access Journals (Sweden)

    YOUSEF eNAMI

    2015-11-01

    Full Text Available Enterococcus lactis IW5 was obtained from human gut and the potential probiotic characteristics of this organism were then evaluated. Results showed that this strain was highly resistant to low pH and high bile salt and adhered strongly to Caco-2 human epithelial colorectal cell lines. The supernatant of E. lactis IW5 strongly inhibited the growth of several pathogenic bacteria and decreased the viability of different cancer cells, such as HeLa, AGS, HT-29, and MCF-7. Conversely, E. lactis IW5 did not inhibit the viability of normal FHs-74 cells. This strain did not generate toxic enzymes, including β-glucosidase, β-glucuronidase, and N-acetyl-β-glucosaminidase and was highly susceptible to ampicillin, gentamycin, penicillin, vancomycin, clindamycin, sulfamethoxazol, and chloramphenicol but resistant to erythromycin and tetracyclin. This study provided evidence for the effect of E. lactis IW5 on cancer cells. Therefore, E. lactis IW5, as a bioactive therapeutics, should be subjected to other relevant tests to verify the therapeutic suitability of this strain for clinical applications.

  1. Cell wall anchoring of the Campylobacter antigens to Lactococcus lactis

    Directory of Open Access Journals (Sweden)

    Patrycja Anna Kobierecka

    2016-02-01

    Full Text Available Campylobacter jejuni is the most frequent cause of human food-borne gastroenteritis and chicken meat is the main source of infection. Recent studies showed that broiler chicken immunization against Campylobacter should be the most efficient way to lower the number of human infections by this pathogen. Induction of the mucosal immune system after oral antigen administration should provide protective immunity to chickens. In this work we tested the usefulness of Lactococcus lactis, the most extensively studied lactic acid bacterium, as a delivery vector for Campylobacter antigens. First we constructed hybrid protein – CjaA antigen presenting CjaD peptide epitopes on its surface. We showed that specific rabbit anti-rCjaAD serum reacted strongly with both CjaA and CjaD produced by a wild type Campylobacter jejuni strain. Next, rCjaAD and CjaA were fused to the C-terminus of the L. lactis YndF containing the LPTXG motif. The genes expressing these proteins were transcribed under control of the L. lactis Usp45 promoter and their products contain the Usp45 signal sequences. This strategy ensures a cell surface location of both analysed proteins, which was confirmed by immunofluorescence assay. In order to evaluate the impact of antigen location on vaccine prototype efficacy, a L. lactis strain producing cytoplasm-located rCjaAD was also generated. Animal experiments showed a decrease of Campylobacter cecal load in vaccinated birds as compared with the control group and showed that the L. lactis harboring the surface-exposed rCjaAD antigen afforded greater protection than the L. lactis producing cytoplasm-located rCjaAD. To the best of our knowledge, this is the first attempt to employ LAB (Lactic Acid Bacteria strains as a mucosal delivery vehicle for chicken immunization. Although the observed reduction of chicken colonization by Campylobacter resulting from vaccination was rather moderate, the experiments showed that LAB strains can be considered

  2. Lactose metabolism in Streptococcus lactis: studies with a mutant lacking glucokinase and mannose-phosphotransferase activities

    International Nuclear Information System (INIS)

    Thompson, J.; Chassy, B.M.; Egan, W.

    1985-01-01

    A mutant of Streptococcus lactis 133 has been isolated that lacks both glucokinase and phosphoenolpyruvate-dependent mannose- phosphotransferase (mannose-PTS) activities. The double mutant S. lactis 133 mannose-PTSd GK- is unable to utilize either exogenously supplied or intracellularly generated glucose for growth. Fluorographic analyses of metabolites formed during the metabolism of [ 14 C]lactose labeled specifically in the glucose or galactosyl moiety established that the cells were unable to phosphorylate intracellular glucose. However, cells of S. lactis 133 mannose-PTSd GK- readily metabolized intracellular glucose 6-phosphate, and the growth rates and cell yield of the mutant and parental strains on sucrose were the same. During growth on lactose, S. lactis 133 mannose-PTSd GK- fermented only the galactose moiety of the disaccharide, and 1 mol of glucose was generated per mol of lactose consumed. For an equivalent concentration of lactose, the cell yield of the mutant was 50% that of the wild type. The specific rate of lactose utilization by growing cells of S. lactis 133 mannose-PTSd GK- was ca. 50% greater than that of the wild type, but the cell doubling times were 70 and 47 min, respectively. High-resolution 31 P nuclear magnetic resonance studies of lactose transport by starved cells of S. lactis 133 and S. lactis 133 mannose-PTSd GK- showed that the latter cells contained elevated lactose-PTS activity. Throughout exponential growth on lactose, the mutant maintained an intracellular steady-state glucose concentration of 100 mM

  3. Lactose metabolism in Streptococcus lactis: studies with a mutant lacking glucokinase and mannose-phosphotransferase activities

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, J.; Chassy, B.M.; Egan, W.

    1985-04-01

    A mutant of Streptococcus lactis 133 has been isolated that lacks both glucokinase and phosphoenolpyruvate-dependent mannose- phosphotransferase (mannose-PTS) activities. The double mutant S. lactis 133 mannose-PTSd GK- is unable to utilize either exogenously supplied or intracellularly generated glucose for growth. Fluorographic analyses of metabolites formed during the metabolism of (/sup 14/C)lactose labeled specifically in the glucose or galactosyl moiety established that the cells were unable to phosphorylate intracellular glucose. However, cells of S. lactis 133 mannose-PTSd GK- readily metabolized intracellular glucose 6-phosphate, and the growth rates and cell yield of the mutant and parental strains on sucrose were the same. During growth on lactose, S. lactis 133 mannose-PTSd GK- fermented only the galactose moiety of the disaccharide, and 1 mol of glucose was generated per mol of lactose consumed. For an equivalent concentration of lactose, the cell yield of the mutant was 50% that of the wild type. The specific rate of lactose utilization by growing cells of S. lactis 133 mannose-PTSd GK- was ca. 50% greater than that of the wild type, but the cell doubling times were 70 and 47 min, respectively. High-resolution /sup 31/P nuclear magnetic resonance studies of lactose transport by starved cells of S. lactis 133 and S. lactis 133 mannose-PTSd GK- showed that the latter cells contained elevated lactose-PTS activity. Throughout exponential growth on lactose, the mutant maintained an intracellular steady-state glucose concentration of 100 mM.

  4. A counterselection method for Lactococcus lactis genome editing based on class IIa bacteriocin sensitivity.

    Science.gov (United States)

    Wan, Xing; Usvalampi, Anne M; Saris, Per E J; Takala, Timo M

    2016-11-01

    In this paper, we present a new counterselection method for deleting fragments from Lactococcus lactis chromosome. The method uses a non-replicating plasmid vector, which integrates into the chromosome and makes the cell sensitive to bacteriocins. The integration vector carries pUC ori functional in Escherichia coli but not in L. lactis, an erythromycin resistance gene for selecting single crossover integrants, and two fragments from L. lactis chromosome for homologous recombinations. In addition, the integration vector is equipped with the Listeria monocytogenes gene mptC encoding the mannose-phosphotransferase system component IIC, the receptor for class IIa bacteriocins. Expression of mptC from the integration vector renders the naturally resistant L. lactis sensitive to class IIa bacteriocins. This sensitivity is then used to select the double crossover colonies on bacteriocin agar. Only the cells which have regained the endogenous bacteriocin resistance through the loss of the mptC plasmid will survive. The colonies carrying the desired deletion can then be distinguished from the wild-type revertants by PCR. By using the class IIa bacteriocins leucocin A, leucocin C or pediocin AcH as the counterselective agents, we deleted 22- and 33-kb chromosomal fragments from the wild-type nisin producing L. lactis strain N8. In conclusion, this counterselection method presented here is a convenient, efficient and inexpensive technique to generate successive deletions in L. lactis chromosome.

  5. Hemin reconstitutes proton extrusion in an H+-ATPase-negative mutant of Lactococcus lactis

    DEFF Research Database (Denmark)

    Blank, L.M.; Købmann, Brian Jensen; Michelsen, Ole

    2001-01-01

    H+-ATPase is considered essential for growth of Lactococcus lactis. However, media containing hemin restored the aerobic growth of an H+-ATPase-negative mutant, suggesting that hemin complements proton extrusion. We show that inverted membrane vesicles prepared from hemin-grown L. lactis cells...

  6. Elucidating Flux Regulation of the Fermentation Modes of Lactococcus lactis

    DEFF Research Database (Denmark)

    Chan, Siu Hung Joshua

    an important subject for basic research in cellular metabolism because L. lactis exhibits an interesting metabolic shift. Under anaerobic conditions, on fast fermentable sugars, L. lactis produces lactate as the primary product, known as homolactic fermentation but on slowly fermentable sugars, significant...... amounts of formate, acetate and ethanol are formed, known as mixed-acid fermentation. This shift is termed the mixedacid shift. This type of shift between a low-yield and a high-yield metabolism has drawn a lot of research focus and has similarly been observed in other bacteria, yeast and even tumor cells....... Efforts have been put to find out the mechanism regulating the mixed-acid shift as well as to answer questions such as why L. lactis prefers such a switch. Until now, some pieces of evidence have been reported and several factors and models have been proposed as the keys to regulating the shift, including...

  7. Production of Recombinant Peanut Allergen Ara h 2 using Lactococcus lactis

    Directory of Open Access Journals (Sweden)

    Frøkiær Hanne

    2007-08-01

    Full Text Available Abstract Background Natural allergen sources can supply large quantities of authentic allergen mixtures for use as immunotherapeutics. However, such extracts are complex, difficult to define, vary from batch to batch, which may lead to unpredictable efficacy and/or unacceptable levels of side effects. The use of recombinant expression systems for allergen production can alleviate some of these issues. Several allergens have been tested in high-level expression systems and in most cases show immunereactivity comparable to their natural counterparts. The gram positive lactic acid bacterium Lactococcus lactis is an attractive microorganism for use in the production of protein therapeutics. L. lactis is considered food grade, free of endotoxins, and is able to secrete the heterologous product together with few other native proteins. Hypersensitivity to peanut represents a serious allergic problem. Some of the major allergens in peanut have been described. However, for therapeutic usage more information about the individual allergenic components is needed. In this paper we report recombinant production of the Ara h 2 peanut allergen using L. lactis. Results A synthetic ara h 2 gene was cloned into an L. lactis expression plasmid containing the P170 promoter and the SP310mut2 signal sequence. Flask cultures grown overnight showed secretion of the 17 kDa Ara h 2 protein. A batch fermentation resulted in 40 mg/L recombinant Ara h 2. Purification of Ara h 2 from the culture supernatant was done by hydrophobic exclusion and size separation. Mass spectrometry and N-terminal analysis showed a recombinant Ara h 2 of full length and correctly processed by the signal peptidase. The immunological activity of recombinant Ara h 2 was analysed by ELISA using antibodies specific for native Ara h 2. The recombinant Ara h 2 showed comparable immunereactivity to that of native Ara h 2. Conclusion Recombinant production of Ara h 2 using L. lactis can offer high yields

  8. Environmental stress responses in Lactococcus lactis

    NARCIS (Netherlands)

    Sanders, JW; Venema, G; Kok, J

    Bacteria can encounter a variety of physical conditions during their life, Bacterial cells are able to survive these (often adverse) conditions by the induction of specific or general protection mechanisms. The lactic acid bacterium Lactococcus lactis is widely used for the production of cheese.

  9. Controlles modulation of folate polyglutamyl tail length by metabolic engineering of Lactococcus lactis

    NARCIS (Netherlands)

    Sybesma, W.F.H.; Born, van den E.; Starrenburg, M.; Mierau, I.; Kleerebezem, M.; Vos, de W.M.; Hugenholtz, J.

    2003-01-01

    The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates >90% of the produced folate intracellularly, predominantly in the polyglutamyl form. Approximately 10% of the produced folate is released into the environment. Overexpression of folC in L. lactis led to an

  10. 13C based proteinogenic amino acid (PAA) and metabolic flux ratio analysis of Lactococcus lactis reveals changes in pentose phosphate (PP) pathway in response to agitation and temperature related stresses.

    Science.gov (United States)

    Azizan, Kamalrul Azlan; Ressom, Habtom W; Mendoza, Eduardo R; Baharum, Syarul Nataqain

    2017-01-01

    Lactococcus lactis subsp. cremoris MG1363 is an important starter culture for dairy fermentation. During industrial fermentations, L. lactis is constantly exposed to stresses that affect the growth and performance of the bacterium. Although the response of L. lactis to several stresses has been described, the adaptation mechanisms at the level of in vivo fluxes have seldom been described. To gain insights into cellular metabolism, 13 C metabolic flux analysis and gas chromatography mass spectrometry (GC-MS) were used to measure the flux ratios of active pathways in the central metabolism of L. lactis when subjected to three conditions varying in temperature (30°C, 37°C) and agitation (with and without agitation at 150 rpm). Collectively, the concentrations of proteinogenic amino acids (PAAs) and free fatty acids (FAAs) were compared, and Pearson correlation analysis ( r ) was calculated to measure the pairwise relationship between PAAs. Branched chain and aromatic amino acids, threonine, serine, lysine and histidine were correlated strongly, suggesting changes in flux regulation in glycolysis, the pentose phosphate (PP) pathway, malic enzyme and anaplerotic reaction catalysed by pyruvate carboxylase (pycA). Flux ratio analysis revealed that glucose was mainly converted by glycolysis, highlighting the stability of L. lactis' central carbon metabolism despite different conditions. Higher flux ratios through oxaloacetate (OAA) from pyruvate (PYR) reaction in all conditions suggested the activation of pyruvate carboxylate (pycA) in L. lactis , in response to acid stress during exponential phase. Subsequently, more significant flux ratio differences were seen through the oxidative and non-oxidative pentose phosphate (PP) pathways, malic enzyme, and serine and C1 metabolism, suggesting NADPH requirements in response to environmental stimuli. These reactions could play an important role in optimization strategies for metabolic engineering in L. lactis . Overall, the

  11. The proteolytic system of Lactococcus lactis

    NARCIS (Netherlands)

    Kunji, Edmundus Richardus Stephanus

    1997-01-01

    The bacterium Lactococcus lactis usues an extencive proteolytic system to utilize milk proteins (caseins) in orde to meet its need for amino acids. The genetic and biochemical properties of the putative components of the proteolytic pathway are well-described. However, little is known about the role

  12. Heterologous Expression of Aldehyde Dehydrogenase in Lactococcus lactis for Acetaldehyde Detoxification at Low pH.

    Science.gov (United States)

    Lyu, Yunbin; LaPointe, Gisèle; Zhong, Lei; Lu, Jing; Zhang, Chong; Lu, Zhaoxin

    2018-02-01

    Aldehyde dehydrogenase (E.C. 1.2.1.x) can catalyze detoxification of acetaldehydes. A novel acetaldehyde dehydrogenase (istALDH) from the non-Saccharomyces yeast Issatchenkia terricola strain XJ-2 has been previously characterized. In this work, Lactococcus lactis with the NIsin Controlled Expression (NICE) System was applied to express the aldehyde dehydrogenase gene (istALDH) in order to catalyze oxidation of acetaldehyde at low pH. A recombinant L. lactis NZ3900 was obtained and applied for the detoxification of acetaldehyde as whole-cell biocatalysts. The activity of IstALDH in L. lactis NZ3900 (pNZ8148-istALDH) reached 36.4 U mL -1 when the recombinant cells were induced with 50 ng mL -1 nisin at 20 °C for 2 h. The IstALDH activity of recombinant L. lactis cells showed higher stability at 37 °C and pH 4.0 compared with the crude enzyme. L. lactis NZ3900 (pNZ8148-istALDH) could convert acetaldehyde at pH 2.0 while the crude enzyme could not. Moreover, the resting cells of L. lactis NZ3900 (pNZ8148-istALDH) showed a 2.5-fold higher activity and better stability in catalyzing oxidation of acetaldehyde at pH 2.0 compared with that of Escherichia coli expressing the IstALDH. Taken together, the L. lactis cells expressing recombinant IstALDH are potential whole-cell biocatalysts that can be applied in the detoxification of aldehydes.

  13. CTP limitation increases expression of CTP synthase in Lactococcus lactis

    DEFF Research Database (Denmark)

    Jørgensen, C.M.; Hammer, Karin; Martinussen, Jan

    2003-01-01

    CTP synthase is encoded by the pyrG gene and catalyzes the conversion of UTP to CTP. A Lactococcus lactis pyrG mutant with a cytidine requirement was constructed, in which beta-galactosidase activity in a pyrG-lacLM transcriptional fusion was used to monitor gene expression of pyrG. A 10-fold...... decrease in the CTP pool induced by cytidine limitation was found to immediately increase expression of the L. lactis pyrG gene. The final level of expression of pyrG is 37-fold higher than the uninduced level. CTP limitation has pronounced effects on central cellular metabolism, and both RNA and protein...... for regulation of the pyrG gene. It is possible to fold the pyrG leader in an alternative structure that would prevent the formation of the terminator. We suggest a model for pyrG regulation in L. lactis, and probably in other gram-positive bacteria as well, in which pyrG expression is directly dependent...

  14. Development of Chemically Defined Media to Express Trp-Analog-Labeled Proteins in a Lactococcus lactis Trp Auxotroph.

    Science.gov (United States)

    Shao, Jinfeng; Marcondes, Marcelo F M; Oliveira, Vitor; Broos, Jaap

    2016-01-01

    Chemically defined media for growth of Lactococcus lactis strains contain about 50 components, making them laborious and expensive growth media. However, they are crucial for metabolism studies as well as for expression of heterologous proteins labeled with unnatural amino acids. In particular, the L. lactis Trp auxotroph PA1002, overexpressing the tryptophanyl tRNA synthetase enzyme of L. lactis, is very suitable for the biosynthetic incorporation of Trp analogs in proteins because of its most relaxed substrate specificity reported towards Trp analogs. Here we present two much simpler defined media for L. lactis, which consist of only 24 or 31 components, respectively, and with which the L. lactis Trp auxotroph shows similar growth characteristics as with a 50-component chemically defined medium. Importantly, the expression levels of two recombinant proteins used for evaluation were up to 2-3 times higher in these new media than in the 50-component medium, without affecting the Trp analog incorporation efficiency. Taken together, the simplest chemically defined media reported so far for L. lactis are presented. Since L. lactis also shows auxotrophy for Arg, His, Ile, Leu Val, and Met, our simplified media may also be useful for the biosynthetic incorporation of analogs of these five amino acids. © 2016 The Author(s) Published by S. Karger AG, Basel.

  15. Heterologous Protein Expression by Lactococcus lactis

    NARCIS (Netherlands)

    Villatoro-Hernández, J.; Kuipers, O.P.; Saucedo-Cárdenas, O.; Montes-de-Oca-Luna, R.

    2012-01-01

    This chapter describes the use of Lactococcus lactis as a safe and efficient cell factory to produce heterologous proteins of medical interest. The relevance of the use of this lactic acid bacterium (LAB) is that it is a noncolonizing, nonpathogenic microorganism that can be delivered in vivo at a

  16. The extracellular proteinase of Lactococcus lactis

    NARCIS (Netherlands)

    Laan, Harm Willem Frederik

    1991-01-01

    Lactococci are used in the production of fermentated dairy products of which cheese is one of the most important. In starter cultures used in dutch cheese manufacturing Lactococcus lactis the dominants pecies. The main functions of the Lactococci are a fast conversion of lactose into lactate and the

  17. An ecological study of lactococci isolated from raw milk in the camembert cheese registered designation of origin area.

    Science.gov (United States)

    Corroler, D; Mangin, I; Desmasures, N; Gueguen, M

    1998-12-01

    The genetic diversity of lactococci isolated from raw milk in the Camembert cheese Registered Designation of Origin area was studied. Two seasonal samples (winter and summer) of raw milk were obtained from six farms in two areas (Bessin and Bocage Falaisien) of Normandy. All of the strains analyzed had a Lactococcus lactis subsp. lactis phenotype, whereas the randomly amplified polymorphic DNA (RAPD) technique genotypically identified the strains as members of L. lactis subsp. lactis or L. lactis subsp. cremoris. The genotypes were confirmed by performing standard PCR with primers corresponding to a region of the histidine biosynthesis operon. The geographic distribution of each subspecies of L. lactis was determined; 80% of the Bocage Falaisien strains were members of L. lactis subsp. lactis, and 30.5% of the Bessin strains were members of L. lactis subsp. lactis. A dendrogram was produced from a computer analysis of the RAPD profiles in order to evaluate the diversity of the lactococci below the subspecies level. The coefficient of similarity for 117 of the 139 strains identified as members of L. lactis subsp. cremoris was as high as 66%. The L. lactis subsp. lactis strains were more heterogeneous and formed 10 separate clusters (the level of similarity among the clusters was 18%). Reference strains of L. lactis subsp. lactis fell into 2 of these 10 clusters, demonstrating that lactococcal isolates are clearly different. As determined by the RAPD profiles, some L. lactis subsp. lactis strains were specific to the farms from which they originated and were recovered throughout the year (in both summer and winter). Therefore, the typicality of L. lactis subsp. lactis strains was linked to the farm of origin rather than the area. These findings emphasize the significance of designation of origin and the specificity of "Camembert de Normandie" cheese.

  18. Probiotic Yogurt Culture Bifidobacterium Animalis Subsp. Lactis BB-12 and Lactobacillus Acidophilus LA-5 Modulate the Cytokine Secretion by Peripheral Blood Mononuclear Cells from Patients with Ulcerative Colitis.

    Science.gov (United States)

    Sheikhi, A; Shakerian, M; Giti, H; Baghaeifar, M; Jafarzadeh, A; Ghaed, V; Heibor, M R; Baharifar, N; Dadafarin, Z; Bashirpour, G

    2016-06-01

    There are some evidences for the immunomodulation disorders in the response to intestinal microbiota in inflammatory bowel disease. Yogurt is a fermented milk product made with a starter culture consisting of different probiotics which could be colonized in intestine. However, the role of probiotics in the aetiopathogenesis of ulcerative colitis (UC) has not been clarified. To determine how the immune system responds to these bacteria this study was planned. Bifidobacterium lactis BB-12 (B. lactis) and Lactobacillus acidophilus LA-5 (L. acidophilus) were cultivated on MRS broth. PBMCs of 36 UC patients were separated by Ficoll-Hypaque centrifugation and co-cultured with different concentrations of UV killed bacteria in RPMI-1 640 plus 10% FCS for 48/72 h. IL-10, TGF-β, IFN-γ and TNF-α were measured in supernatant of PBMCs by ELISA. Both bacteria significantly augmented IL-10, TGF-β, IFN-γ and TNF-α compared to control (p<0.001). The secretion levels of IL-10 and TGF-β by B. lactis- compared to L. acidophilus-stimulated PBMCs were significantly higher (p<0.05, p<0.01 respectively). The secretion levels of TNF-α and IFN-γ by PBMCs after 72 h were significantly lower compared to 48 h stimulation by B. lactis (p<0.001, p<0.035 respectively). These data show that both probiotics may trigger the pro- and anti-inflammatory immune response of UC patients. It seems that IL-10/TGF-β uprising by B. lactis could be the reason of TNF-α/IFN-γ reduction. Therefore albeit B. lactis still stimulates the effector Th cells but because of more stimulatory effect on Tregs, it could be a good potential therapeutic candidate for further investigation. © Georg Thieme Verlag KG Stuttgart · New York.

  19. Expression of PprI from Deinococcus radiodurans Improves Lactic Acid Production and Stress Tolerance in Lactococcus lactis.

    Directory of Open Access Journals (Sweden)

    Xiangrong Dong

    Full Text Available PprI is a general switch protein that regulates the expression of certain proteins involved in pathways of cellular resistance in the extremophilic bacterium Deinococcus radiodurans. In this study, we transformed pprI into Lactococcus lactis strain MG1363 using the lactococcal shuttle vector pMG36e and investigated its effects on the tolerance and lactic acid production of L. lactis while under stress. PprI was stably expressed in L. lactis as confirmed by western blot assays. L. lactis expressing PprI exhibited significantly improved resistance to oxidative stress and high osmotic pressure. This enhanced cellular tolerance to stressors might be due to the regulation of resistance-related genes (e.g., recA, recO, sodA, and nah by pprI. Moreover, transformed L. lactis demonstrated increased lactic acid production, attributed to enhanced lactate dehydrogenase activity. These results suggest that pprI can improve the tolerance of L. lactis to environmental stresses, and this transformed bacterial strain is a promising candidate for industrial applications of lactic acid production.

  20. Expression of PprI from Deinococcus radiodurans Improves Lactic Acid Production and Stress Tolerance in Lactococcus lactis.

    Science.gov (United States)

    Dong, Xiangrong; Tian, Bing; Dai, Shang; Li, Tao; Guo, Linna; Tan, Zhongfang; Jiao, Zhen; Jin, Qingsheng; Wang, Yanping; Hua, Yuejin

    2015-01-01

    PprI is a general switch protein that regulates the expression of certain proteins involved in pathways of cellular resistance in the extremophilic bacterium Deinococcus radiodurans. In this study, we transformed pprI into Lactococcus lactis strain MG1363 using the lactococcal shuttle vector pMG36e and investigated its effects on the tolerance and lactic acid production of L. lactis while under stress. PprI was stably expressed in L. lactis as confirmed by western blot assays. L. lactis expressing PprI exhibited significantly improved resistance to oxidative stress and high osmotic pressure. This enhanced cellular tolerance to stressors might be due to the regulation of resistance-related genes (e.g., recA, recO, sodA, and nah) by pprI. Moreover, transformed L. lactis demonstrated increased lactic acid production, attributed to enhanced lactate dehydrogenase activity. These results suggest that pprI can improve the tolerance of L. lactis to environmental stresses, and this transformed bacterial strain is a promising candidate for industrial applications of lactic acid production.

  1. Bioactive Films Containing Alginate-Pectin Composite Microbeads with Lactococcus lactis subsp. lactis: Physicochemical Characterization and Antilisterial Activity

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    Mariam Bekhit

    2018-02-01

    Full Text Available Novel bioactive films were developed from the incorporation of Lactococcus lactis into polysaccharide films. Two different biopolymers were tested: cellulose derivative (hydroxylpropylmethylcellulose (HPMC and corn starch. Lactic acid bacteria (LAB free or previously encapsulated in alginate-pectin composite hydrogel microbeads were added directly to the film forming solution and films were obtained by casting. In order to study the impact of the incorporation of the protective culture into the biopolymer matrix, the water vapour permeability, oxygen permeability, optical and mechanical properties of the dry films were evaluated. Furthermore, the antimicrobial effect of bioactive films against Listeria monocytogenes was studied in synthetic medium. Results showed that the addition of LAB or alginate-pectin microbeads modified slightly films optical properties. In comparison with HPMC films, starch matrix proves to be more sensitive to the addition of bacterial cells or beads. Indeed, mechanical resistance of corn starch films was lower but barrier properties were improved, certainly related to the possible establishment of interactions between alginate-pectin beads and starch. HPMC and starch films containing encapsulated bioactive culture showed a complete inhibition of listerial growth during the first five days of storage at 5 °C and a reduction of 5 logs after 12 days.

  2. Light Sensitivity of Lactococcus lactis Thioredoxin Reductase

    DEFF Research Database (Denmark)

    Skjoldager, Nicklas

    The thioredoxin system has evolved in all kingdoms of life acting as a key antioxidant system in the defense against oxidative stress. The thioredoxin system utilizes reducing equivalents from NADPH to reduce protein disulfide targets. The reducing equivalents are shuttled via a flavin and redox...... active dithiol motif in thioredoxin reductase (TrxR) to reduce the small ubiquitous thioredoxin (Trx). Trx in turn regulates the protein dithiol/disulfide balance by reduction of protein disulfide targets in e.g. ribonucleotide reductase, peroxiredoxins and methionine sulfoxide reductase. The glutathione......, thus expected to rely mainly on the Trx system for thiol-disulfide control. L. lactis is an important industrial microorganism used as starter culture in the dairy production of cheese, buttermilk etc. and known to be sensitive to oxidative stress. The L. lactis TrxR (LlTrxR) is a homodimeric...

  3. Isolation of a bacteriocin-producing Lactococcus lactis subsp. lactis and application to control Listeria monocytogenes in Moroccan jben.

    Science.gov (United States)

    Benkerroum, N; Oubel, H; Zahar, M; Dlia, S; Filali-Maltouf, A

    2000-12-01

    Use of a bacteriocin-producing lactococcal strain to control Listeria monocytogenes in jben. A Lactococcus lactis strain isolated from lben was shown, by the spot technique, to produce a bacteriocin different from nisin. Inhibitory activity of the bacteriocin-producing strain against Listeria monocytogenes was investigated in jben, made from cow's milk fermented with the producer organism and contaminated with 104 or 107 cfu ml-1. Listeria counts were monitored during manufacture, and during conservation at room and at refrigeration temperatures. Results showed that the pathogen was reduced by 2.7 logarithmic units after 30 h of jben processing when the initial inoculum of 107 cfu ml(-1) was used. For the initial inoculum of 104 cfu ml(-1), the bacterium was completely eliminated at 24 h. Furthermore, the use of the bacteriocin-producing starter culture extended the shelf-life of jben by 5 days. In situ production of the lactococcal bacteriocin is an efficient biological means of controlling L. monocytogenes in jben and of allowing shelf-life extension. The proposed technology will essentially benefit minimally processed dairy products and those made with raw milk.

  4. Construction of a new shuttle vector for DNA delivery into mammalian cells using non-invasive Lactococcus lactis.

    Science.gov (United States)

    Yagnik, Bhrugu; Padh, Harish; Desai, Priti

    2016-04-01

    Use of food grade Lactococcus lactis (L. lactis) is fast emerging as a safe alternative for delivery of DNA vaccine. To attain efficient DNA delivery, L. lactis, a non-invasive bacterium is converted to invasive strain either by expressing proteins like Internalin A (InlA) or Fibronectin binding protein A (FnBPA) or through chemical treatments. However the safety status of invasive L. lactis is questionable. In the present report, we have shown that non-invasive L. lactis efficiently delivered the newly constructed reporter plasmid pPERDBY to mammalian cells without any chemical enhancers. The salient features of the vector are; I) Ability to replicate in two different hosts; Escherichia coli (E. coli) and Lactic Acid Bacteria (LAB), II) One of the smallest reporter plasmid for DNA vaccine, III) Enhanced Green Fluorescence Protein (EGFP) linked to Multiple Cloning Site (MCS), IV) Immunostimulatory CpG motifs functioning as an adjuvant. Expression of EGFP in pPERDBY transfected CHO-K1 and Caco-2 cells demonstrates its functionality. Non-invasive r-L. lactis was found efficient in delivering pPERDBY to Caco-2 cells. The in vitro data presented in this article supports the hypothesis that in the absence of invasive proteins or relevant chemical treatment, L. lactis was found efficient in delivering DNA to mammalian cells. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  5. Control analysis of the role of triosephosphate isomerase in glucose metabolism in Lactococcus lactis

    DEFF Research Database (Denmark)

    Solem, Christian; Købmann, Brian Jensen; Jensen, Peter Ruhdal

    2008-01-01

    Triosephosphate isomerase (TPI), which catalyses the conversion of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P), was studied for its control on glycolysis and mixed acid production in L. lactis subspecies lactis IL1403 and L. lactis subspecies cremoris MG1363. Strains...... metabolites glucose-6-phosphate, fructose-1,6-bisphosphate and DHAP in the IL1403 derivatives were essentially unchanged for TPI activities from 26% to 225%. At a TPI activity of 3%, the level of DHAP increased four times. The finding that an increased level of DHAP coincides with an increase in formate...

  6. Characterization of a Lactococcus lactis promoter for heterologous protein production

    Directory of Open Access Journals (Sweden)

    Christian E. Ogaugwu

    2018-03-01

    Full Text Available Constitutively active promoter elements for heterologous protein production in Lactococcus lactis are scarce. Here, the promoter of the PTS-IIC gene cluster from L. lactis NZ3900 is described. This promoter was cloned upstream of an enhanced green fluorescent protein, GFPmut3a, and transformed into L. lactis. Transformants produced up to 13.5 μg of GFPmut3a per milliliter of log phase cells. Addition of cellobiose further increased the production of GFPmut3a by up to two-fold when compared to glucose. Analysis of mutations at two specific positions in the PTS-IIC promoter showed that a ‘T’ to ‘G’ mutation within the −35 element resulted in constitutive expression in glucose, while a ‘C’ at nucleotide 7 in the putative cre site enhanced promoter activity in cellobiose. Finally, this PTS-IIC promoter is capable of mediating protein expression in Bacillus subtilis and Escherichia coli Nissle 1917, suggesting the potential for future biotechnological applications of this element and its derivatives.

  7. Impact of bile salt adaptation of Lactobacillus delbrueckii subsp. lactis 200 on its interaction capacity with the gut.

    Science.gov (United States)

    Burns, Patricia; Reinheimer, Jorge; Vinderola, Gabriel

    2011-10-01

    In a previous work, bile-salt-resistant derivatives were obtained from non-intestinal lactobacilli. The aim of this work was to investigate the impact of bile adaptation of Lactobacillus delbrueckii subsp. lactis 200 on morphology, surface properties, in vivo interaction capacity with the gut and ability to activate the gut immune response. Electron microscopy studies, growth kinetics in the presence of bovine and porcine bile, the capacity to deconjugate bile acids, hydrophobicity, autoaggregation and co-aggregation capacities were studied for the parental strain and its bile-resistant derivative in vitro. Additionally, survival in intestinal fluid, the interaction with the gut and the immunomodulating capacities were studied in mice. Bile salt adaptation conferred upon the adapted strain a higher capacity to withstand physiological concentrations of bile salts and greater survival capacity in intestinal fluid. However, bile salt exposure reduced cell hydrophobicity, autoaggregation and adhesion capacities, resulting in reduced persistence in the intestinal lumen and delayed capacity to activate the gut immune response. Insight into the effects of bile salts upon the interaction and immunomodulating capacity of lactobacilli with the gut is provided, relating in vitro and in vivo results. Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  8. Immunization against Leishmania major infection using LACK- and IL-12-expressing Lactococcus lactis induces delay in footpad swelling.

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    Felix Hugentobler

    Full Text Available BACKGROUND: Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines. METHODOLOGY/PRINCIPAL FINDINGS: We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional T(H1 CD4(+ and CD8(+ T cells and a systemic LACK-specific T(H1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific T(H1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective T(H1 response. CONCLUSIONS/SIGNIFICANCE: This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania.

  9. Immunization against Leishmania major Infection Using LACK- and IL-12-Expressing Lactococcus lactis Induces Delay in Footpad Swelling

    Science.gov (United States)

    Hugentobler, Felix; Yam, Karen K.; Gillard, Joshua; Mahbuba, Raya; Olivier, Martin; Cousineau, Benoit

    2012-01-01

    Background Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines. Methodology/Principal findings We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional TH1 CD4+ and CD8+ T cells and a systemic LACK-specific TH1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific TH1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective TH1 response. Conclusions/Significance This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania. PMID:22348031

  10. 13C based proteinogenic amino acid (PAA and metabolic flux ratio analysis of Lactococcus lactis reveals changes in pentose phosphate (PP pathway in response to agitation and temperature related stresses

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    Kamalrul Azlan Azizan

    2017-07-01

    Full Text Available Lactococcus lactis subsp. cremoris MG1363 is an important starter culture for dairy fermentation. During industrial fermentations, L. lactis is constantly exposed to stresses that affect the growth and performance of the bacterium. Although the response of L. lactis to several stresses has been described, the adaptation mechanisms at the level of in vivo fluxes have seldom been described. To gain insights into cellular metabolism, 13C metabolic flux analysis and gas chromatography mass spectrometry (GC-MS were used to measure the flux ratios of active pathways in the central metabolism of L. lactis when subjected to three conditions varying in temperature (30°C, 37°C and agitation (with and without agitation at 150 rpm. Collectively, the concentrations of proteinogenic amino acids (PAAs and free fatty acids (FAAs were compared, and Pearson correlation analysis (r was calculated to measure the pairwise relationship between PAAs. Branched chain and aromatic amino acids, threonine, serine, lysine and histidine were correlated strongly, suggesting changes in flux regulation in glycolysis, the pentose phosphate (PP pathway, malic enzyme and anaplerotic reaction catalysed by pyruvate carboxylase (pycA. Flux ratio analysis revealed that glucose was mainly converted by glycolysis, highlighting the stability of L. lactis’ central carbon metabolism despite different conditions. Higher flux ratios through oxaloacetate (OAA from pyruvate (PYR reaction in all conditions suggested the activation of pyruvate carboxylate (pycA in L. lactis, in response to acid stress during exponential phase. Subsequently, more significant flux ratio differences were seen through the oxidative and non-oxidative pentose phosphate (PP pathways, malic enzyme, and serine and C1 metabolism, suggesting NADPH requirements in response to environmental stimuli. These reactions could play an important role in optimization strategies for metabolic engineering in L. lactis. Overall

  11. 13C based proteinogenic amino acid (PAA) and metabolic flux ratio analysis of Lactococcus lactis reveals changes in pentose phosphate (PP) pathway in response to agitation and temperature related stresses

    Science.gov (United States)

    2017-01-01

    Lactococcus lactis subsp. cremoris MG1363 is an important starter culture for dairy fermentation. During industrial fermentations, L. lactis is constantly exposed to stresses that affect the growth and performance of the bacterium. Although the response of L. lactis to several stresses has been described, the adaptation mechanisms at the level of in vivo fluxes have seldom been described. To gain insights into cellular metabolism, 13C metabolic flux analysis and gas chromatography mass spectrometry (GC-MS) were used to measure the flux ratios of active pathways in the central metabolism of L. lactis when subjected to three conditions varying in temperature (30°C, 37°C) and agitation (with and without agitation at 150 rpm). Collectively, the concentrations of proteinogenic amino acids (PAAs) and free fatty acids (FAAs) were compared, and Pearson correlation analysis (r) was calculated to measure the pairwise relationship between PAAs. Branched chain and aromatic amino acids, threonine, serine, lysine and histidine were correlated strongly, suggesting changes in flux regulation in glycolysis, the pentose phosphate (PP) pathway, malic enzyme and anaplerotic reaction catalysed by pyruvate carboxylase (pycA). Flux ratio analysis revealed that glucose was mainly converted by glycolysis, highlighting the stability of L. lactis’ central carbon metabolism despite different conditions. Higher flux ratios through oxaloacetate (OAA) from pyruvate (PYR) reaction in all conditions suggested the activation of pyruvate carboxylate (pycA) in L. lactis, in response to acid stress during exponential phase. Subsequently, more significant flux ratio differences were seen through the oxidative and non-oxidative pentose phosphate (PP) pathways, malic enzyme, and serine and C1 metabolism, suggesting NADPH requirements in response to environmental stimuli. These reactions could play an important role in optimization strategies for metabolic engineering in L. lactis. Overall, the

  12. Surface Proteins of Lactococcus lactis: Bacterial Resources for Muco-adhesion in the Gastrointestinal Tract

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    Muriel Mercier-Bonin

    2017-11-01

    Full Text Available Food and probiotic bacteria, in particular lactic acid bacteria, are ingested in large amounts by humans and are part of the transient microbiota which is increasingly considered to be able to impact the resident microbiota and thus possibly the host health. The lactic acid bacterium Lactococcus lactis is extensively used in starter cultures to produce dairy fermented food. Also because of a generally recognized as safe status, L. lactis has been considered as a possible vehicle to deliver in vivo therapeutic molecules with anti-inflammatory properties in the gastrointestinal tract. One of the key factors that may favor health effects of beneficial bacteria to the host is their capacity to colonize transiently the gut, notably through close interactions with mucus, which covers and protects the intestinal epithelium. Several L. lactis strains have been shown to exhibit mucus-binding properties and bacterial surface proteins have been identified as key determinants of such capacity. In this review, we describe the different types of surface proteins found in L. lactis, with a special focus on mucus-binding proteins and pili. We also review the different approaches used to investigate the adhesion of L. lactis to mucus, and particularly to mucins, one of its major components, and we present how these approaches allowed revealing the role of surface proteins in muco-adhesion.

  13. Transcriptome analysis of the Lactococcus lactis ArgR and AhrC regulons

    DEFF Research Database (Denmark)

    Larsen, Rasmus; van Hijum, Sacha A. F. T.; Martinussen, Jan

    2008-01-01

    In previous studies, we have shown that direct protein-protein. interaction between the two regulators ArgR and AhrC in Lactococcus lactis is required for arginine-dependent repression of the biosynthetic argC promoter and the activation of the catabolic arcA promoter. Here, we establish the global...... ArgR and AhrC regulons by transcriptome analyses and show that both regulators are dedicated to the control of arginine metabolism in L. lactis....

  14. A mouse mastitis model to study the effects of the intramammary infusion of a food-grade Lactococcus lactis strain.

    Directory of Open Access Journals (Sweden)

    Cristina Camperio

    Full Text Available Lactococcus lactis is one of the most important microorganisms in the dairy industry and has "generally recognized as safe" (GRAS status. L. lactis belongs to the group of lactic acid bacteria (LAB and is encountered in a wide range of environments. Recently, the use of the intramammary infusion of a live culture of LAB has been investigated as a new antibiotic alternative for treating mastitis in dairy ruminants. Controversial results are described in literature regarding its efficacy and safety. In this study we conducted in-depth investigation of the mammary gland immune response induced by intramammary inoculum of a live culture of L. lactis LMG 7930 using the mouse mastitis model. Overnight cultures either of L. lactis (≈ 107 CFU or of the mastitis pathogens Staphylococcus chromogenes (≈ 105 CFU or S. aureus (≈ 102 CFU/ml were injected into the mouse inguinal glands. A double injection, consisting of S. chromogenes first and then L. lactis, was also investigated. Bacterial recovery from the gland and inflammatory cell infiltration were assessed. L. lactis-treated and control glands were analysed for proinflammatory cytokine production. Microbiological results showed that L. lactis was able to survive in the mammary gland 24 h post infection, as were the mastitis pathogens S. chromogenes and S. aureus. L. lactis reduced S. chromogenes survival in the glands and increased its own survival ability by coexisting with the pathogen. Histology showed that L. lactis-treated glands presented variable histological features, ranging from undamaged tissue with no inflammatory cell infiltrate to severe PMN infiltrate with focal areas of tissue damage. S. aureus-treated glands showed the most severe histological grade of inflammation despite the fact that the inoculum size was the smallest. In contrast, most S. chromogenes-treated glands showed normal structures with no infiltration or lesions. Significant increases in IL-1β and TNF-α levels were

  15. A mouse mastitis model to study the effects of the intramammary infusion of a food-grade Lactococcus lactis strain

    Science.gov (United States)

    Biasibetti, Elena; Frassanito, Paolo; Giovannelli, Carlo; Spuria, Liliana; D’Agostino, Claudia; Tait, Sabrina; Capucchio, Maria Teresa

    2017-01-01

    Lactococcus lactis is one of the most important microorganisms in the dairy industry and has “generally recognized as safe” (GRAS) status. L. lactis belongs to the group of lactic acid bacteria (LAB) and is encountered in a wide range of environments. Recently, the use of the intramammary infusion of a live culture of LAB has been investigated as a new antibiotic alternative for treating mastitis in dairy ruminants. Controversial results are described in literature regarding its efficacy and safety. In this study we conducted in-depth investigation of the mammary gland immune response induced by intramammary inoculum of a live culture of L. lactis LMG 7930 using the mouse mastitis model. Overnight cultures either of L. lactis (≈ 107 CFU) or of the mastitis pathogens Staphylococcus chromogenes (≈ 105 CFU) or S. aureus (≈ 102 CFU/ml) were injected into the mouse inguinal glands. A double injection, consisting of S. chromogenes first and then L. lactis, was also investigated. Bacterial recovery from the gland and inflammatory cell infiltration were assessed. L. lactis-treated and control glands were analysed for proinflammatory cytokine production. Microbiological results showed that L. lactis was able to survive in the mammary gland 24 h post infection, as were the mastitis pathogens S. chromogenes and S. aureus. L. lactis reduced S. chromogenes survival in the glands and increased its own survival ability by coexisting with the pathogen. Histology showed that L. lactis-treated glands presented variable histological features, ranging from undamaged tissue with no inflammatory cell infiltrate to severe PMN infiltrate with focal areas of tissue damage. S. aureus-treated glands showed the most severe histological grade of inflammation despite the fact that the inoculum size was the smallest. In contrast, most S. chromogenes-treated glands showed normal structures with no infiltration or lesions. Significant increases in IL-1β and TNF-α levels were also found

  16. A mouse mastitis model to study the effects of the intramammary infusion of a food-grade Lactococcus lactis strain.

    Science.gov (United States)

    Camperio, Cristina; Armas, Federica; Biasibetti, Elena; Frassanito, Paolo; Giovannelli, Carlo; Spuria, Liliana; D'Agostino, Claudia; Tait, Sabrina; Capucchio, Maria Teresa; Marianelli, Cinzia

    2017-01-01

    Lactococcus lactis is one of the most important microorganisms in the dairy industry and has "generally recognized as safe" (GRAS) status. L. lactis belongs to the group of lactic acid bacteria (LAB) and is encountered in a wide range of environments. Recently, the use of the intramammary infusion of a live culture of LAB has been investigated as a new antibiotic alternative for treating mastitis in dairy ruminants. Controversial results are described in literature regarding its efficacy and safety. In this study we conducted in-depth investigation of the mammary gland immune response induced by intramammary inoculum of a live culture of L. lactis LMG 7930 using the mouse mastitis model. Overnight cultures either of L. lactis (≈ 107 CFU) or of the mastitis pathogens Staphylococcus chromogenes (≈ 105 CFU) or S. aureus (≈ 102 CFU/ml) were injected into the mouse inguinal glands. A double injection, consisting of S. chromogenes first and then L. lactis, was also investigated. Bacterial recovery from the gland and inflammatory cell infiltration were assessed. L. lactis-treated and control glands were analysed for proinflammatory cytokine production. Microbiological results showed that L. lactis was able to survive in the mammary gland 24 h post infection, as were the mastitis pathogens S. chromogenes and S. aureus. L. lactis reduced S. chromogenes survival in the glands and increased its own survival ability by coexisting with the pathogen. Histology showed that L. lactis-treated glands presented variable histological features, ranging from undamaged tissue with no inflammatory cell infiltrate to severe PMN infiltrate with focal areas of tissue damage. S. aureus-treated glands showed the most severe histological grade of inflammation despite the fact that the inoculum size was the smallest. In contrast, most S. chromogenes-treated glands showed normal structures with no infiltration or lesions. Significant increases in IL-1β and TNF-α levels were also found in

  17. Effects of metal ions on growth, β-oxidation system, and thioesterase activity of Lactococcus lactis.

    Science.gov (United States)

    Li, Liang; Ma, Ying

    2014-10-01

    The effects of divalent metal ions (Ca(2+), Mg(2+), Fe(2+), and Cu(2+)) on the growth, β-oxidation system, and thioesterase activity of Lactococcus lactis were investigated. Different metal ions significantly influenced the growth of L. lactis: Ca(2+) and Fe(2+) accelerated growth, whereas Cu(2+) inhibited growth. Furthermore, Mg(2+) inhibited growth of L. lactis at a low concentration but stimulated growth of L. lactis at a high concentration. The divalent metal ions had significant effects on activity of the 4 key enzymes of the β-oxidation system (acyl-CoA dehydrogenase, enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and thiolase) and thioesterase of L. lactis. The activity of acyl-CoA dehydrogenases increased markedly in the presence of Ca(2+) and Mg(2+), whereas it decreased with 1 mmol/L Fe(2+) or 12 mmol/L Mg(2+). All the metal ions could induce activity of enoyl-CoA hydratase. In addition, 12 mmol/L Mg(2+) significantly stimulated activity of L-3-hydroxyacyl-CoA dehydrogenase, and all metal ions could induce activity of thiolase, although thiolase activity decreased significantly when 0.05 mmol/L Cu(2+) was added into M17 broth. Inhibition of thioesterase activity by all 4 metal ions could be reversed by 2 mmol/L Ca(2+). These results help us understand the effect of metal ions on the β-oxidation system and thioesterase activity of Lactococcus lactis. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  18. Endosomal recognition of Lactococcus lactis G121 and its RNA by dendritic cells is key to its allergy-protective effects.

    Science.gov (United States)

    Stein, Karina; Brand, Stephanie; Jenckel, André; Sigmund, Anna; Chen, Zhijian James; Kirschning, Carsten J; Kauth, Marion; Heine, Holger

    2017-02-01

    Bacterial cowshed isolates are allergy protective in mice; however, the underlying mechanisms are largely unknown. We examined the ability of Lactococcus lactis G121 to prevent allergic inflammatory reactions. We sought to identify the ligands and pattern recognition receptors through which L lactis G121 confers allergy protection. L lactis G121-induced cytokine release and surface expression of costimulatory molecules by untreated or inhibitor-treated (bafilomycin and cytochalasin D) human monocyte-derived dendritic cells (moDCs), bone marrow-derived mouse dendritic cells (BMDCs), and moDC/naive CD4 + T-cell cocultures were analyzed by using ELISA and flow cytometry. The pathology of ovalbumin-induced acute allergic airway inflammation after adoptive transfer of BMDCs was examined by means of microscopy. L lactis G121-treated murine BMDCs and human moDCs released T H 1-polarizing cytokines and induced T H 1 T cells. Inhibiting phagocytosis and endosomal acidification in BMDCs or moDCs impaired the release of T H 1-polarizing cytokines, costimulatory molecule expression, and T-cell activation on L lactis G121 challenge. In vivo allergy protection mediated by L lactis G121 was dependent on endosomal acidification in dendritic cells (DCs). Toll-like receptor (Tlr) 13 -/- BMDCs showed a weak response to L lactis G121 and were unresponsive to its RNA. The T H 1-polarizing activity of L lactis G121-treated human DCs was blocked by TLR8-specific inhibitors, mediated by L lactis G121 RNA, and synergistically enhanced by activation of nucleotide-binding oligomerization domain-containing protein (NOD) 2. Bacterial RNA is the main driver of L lactis G121-mediated protection against experimentally induced allergy and requires both bacterial uptake by DCs and endosomal acidification. In mice L lactis G121 RNA signals through TLR13; however, the most likely intracellular receptor in human subjects is TLR8. Copyright © 2016 American Academy of Allergy, Asthma & Immunology

  19. Probiotic Lactococcus lactis: A Review

    OpenAIRE

    Priti Khemariya; Sudhir Singh; Gopal Nath; Anil K Gulati

    2017-01-01

    Lactococcus lactis plays a critical role in food, dairy and health sectors. In food and dairy industries, it is found in production processes of various fermented products such as sausages, pickled vegetables, beverages such as beer and wine, breads, soymilk kefir, sour milk, butter, cream, fresh cheese and different types of cheeses, like Cheddar, Colby, Cottage cheese, Camembert, cream cheese, Roquefort and Brie. Additionally, there is an increasing interest towards the possible health bene...

  20. Lactococcus lactis expressing either Staphylococcus aureus fibronectin-binding protein A or Listeria monocytogenes internalin A can efficiently internalize and deliver DNA in human epithelial cells.

    Science.gov (United States)

    Innocentin, Silvia; Guimarães, Valeria; Miyoshi, Anderson; Azevedo, Vasco; Langella, Philippe; Chatel, Jean-Marc; Lefèvre, François

    2009-07-01

    Lactococci are noninvasive bacteria frequently used as protein delivery vectors and, more recently, as in vitro and in vivo DNA delivery vehicles. We previously showed that a functional eukaryotic enhanced green fluorescent protein (eGFP) expression plasmid vector was delivered in epithelial cells by Lactococcus lactis producing Listeria monocytogenes internalin A (L. lactis InlA(+)), but this strategy is limited in vivo to transgenic mice and guinea pigs. In this study, we compare the internalization ability of L. lactis InlA(+) and L. lactis producing either the fibronectin-binding protein A of Staphylococcus aureus (L. lactis FnBPA(+)) or its fibronectin binding domains C and D (L. lactis CD(+)). L. lactis FnBPA(+) and L. lactis InlA(+) showed comparable internalization rates in Caco-2 cells, while the internalization rate observed with L. lactis CD(+) was lower. As visualized by conventional and confocal fluorescence microscopy, large clusters of L. lactis FnBPA(+), L. lactis CD(+), and L. lactis InlA(+) were present in the cytoplasm of Caco-2 cells after internalization. Moreover, the internalization rates of Lactobacillus acidophilus NCFM and of an NCFM mutant strain with the gene coding for the fibronectin-binding protein (fbpA) inactivated were also evaluated in Caco-2 cells. Similar low internalization rates were observed for both wild-type L. acidophilus NCFM and the fbpA mutant, suggesting that commensal fibronectin binding proteins have a role in adhesion but not in invasion. L. lactis FnBPA(+), L. lactis CD(+), and L. lactis InlA(+) were then used to deliver a eukaryotic eGFP expression plasmid in Caco-2 cells: flow cytometry analysis showed that the highest percentage of green fluorescent Caco-2 cells was observed after coculture with either L. lactis FnBPA(+) or L. lactis InlA(+). Analysis of the in vivo efficiency of these invasive recombinant strains is currently in progress to validate their potential as DNA vaccine delivery vehicles.

  1. Detection of bacteriophage-infected cells of Lactococcus lactis using flow cytometry

    DEFF Research Database (Denmark)

    Michelsen, Ole; Cuesta-Dominguez, Álvaro; Albrektsen, Bjarne

    2007-01-01

    Bacteriophage infection in dairy fermentation constitutes a serious problem worldwide. We have studied bacteriophage infection in Lactococcus lactis by using the flow cytometer. The first effect of the infection of the bacterium is a change from cells in chains toward single cells. We interpret...... describe a new method for detection of phage infection in Lactococcus lactis dairy cultures. The method is based on flow cytometric detection of cells with low-density cell walls. The method allows fast and early detection of phage-infected bacteria, independently of which phage has infected the culture...

  2. Versatile Cas9-Driven Subpopulation Selection Toolbox for Lactococcus lactis.

    Science.gov (United States)

    van der Els, Simon; James, Jennelle K; Kleerebezem, Michiel; Bron, Peter A

    2018-04-15

    CRISPR-Cas9 technology has been exploited for the removal or replacement of genetic elements in a wide range of prokaryotes and eukaryotes. Here, we describe the extension of the Cas9 application toolbox to the industrially important dairy species Lactococcus lactis The Cas9 expression vector pLABTarget, encoding the Streptocccus pyogenes Cas9 under the control of a constitutive promoter, was constructed, allowing plug and play introduction of short guide RNA (sgRNA) sequences to target specific genetic loci. Introduction of a pepN -targeting derivative of pLABTarget into L. lactis strain MG1363 led to a strong reduction in the number of transformants obtained, which did not occur in a pepN deletion derivative of the same strain, demonstrating the specificity and lethality of the Cas9-mediated double-strand breaks in the lactococcal chromosome. Moreover, the same pLABTarget derivative allowed the selection of a pepN deletion subpopulation from its corresponding single-crossover plasmid integrant precursor, accelerating the construction and selection of gene-specific deletion derivatives in L. lactis Finally, pLABTarget, which contained sgRNAs designed to target mobile genetic elements, allowed the effective curing of plasmids, prophages, and integrative conjugative elements (ICEs). These results establish that pLABTarget enables the effective exploitation of Cas9 targeting in L. lactis , while the broad-host-range vector used suggests that this toolbox could readily be expanded to other Gram-positive bacteria. IMPORTANCE Mobile genetic elements in Lactococcus lactis and other lactic acid bacteria (LAB) play an important role in dairy fermentation, having both positive and detrimental effects during the production of fermented dairy products. The pLABTarget vector offers an efficient cloning platform for Cas9 application in lactic acid bacteria. Targeting Cas9 toward mobile genetic elements enabled their effective curing, which is of particular interest in the

  3. Physiochemical parameters optimization for enhanced nisin production by Lactococcus lactis (MTCC 440

    Directory of Open Access Journals (Sweden)

    Puspadhwaja Mall

    2010-02-01

    Full Text Available The influence of various physiochemical parameters on the growth of Lactococcus lactis sub sp. lactis MTCC 440 was studied at shake flask level for 20 h. Media optimization (MRS broth was studied to achieve enhanced growth of the organism and also nisin production. Bioassay of nisin was done with agar diffusion method using Streptococcus agalactae NCIM 2401 as indicator strain. MRS broth (6%, w/v with 0.15μg/ml of nisin supplemented with 0.5% (v/v skimmed milk was found to be the best for nisin production as well as for growth of L lactis. The production of nisin was strongly influenced by the presence of skimmed milk and nisin in MRS broth. The production of nisin was affected by the physical parameters and maximum nisin production was at 30(0C while the optimal temperature for biomass production was 37(0C.

  4. The transcriptional and gene regulatory network of Lactococcus lactis MG1363 during growth in milk.

    Directory of Open Access Journals (Sweden)

    Anne de Jong

    Full Text Available In the present study we examine the changes in the expression of genes of Lactococcus lactis subspecies cremoris MG1363 during growth in milk. To reveal which specific classes of genes (pathways, operons, regulons, COGs are important, we performed a transcriptome time series experiment. Global analysis of gene expression over time showed that L. lactis adapted quickly to the environmental changes. Using upstream sequences of genes with correlated gene expression profiles, we uncovered a substantial number of putative DNA binding motifs that may be relevant for L. lactis fermentative growth in milk. All available novel and literature-derived data were integrated into network reconstruction building blocks, which were used to reconstruct and visualize the L. lactis gene regulatory network. This network enables easy mining in the chrono-transcriptomics data. A freely available website at http://milkts.molgenrug.nl gives full access to all transcriptome data, to the reconstructed network and to the individual network building blocks.

  5. Taxonomy, physiology and growth of Lactococcus lactis: a review

    Directory of Open Access Journals (Sweden)

    Dubravka Samaržija

    2001-01-01

    Full Text Available Lactococcus lactis species is one of the most important groups of lactic acid bacteria that are used in the dairy industry. The major functions of this species in dairy fermentation are the production of lactic acid from lactose, hydrolysis of casein and citric acid fermentation. Thus their metabolic end products and enzymes directly or indirectly have significant influence in determining the texture and flavour of the final products. In recent years, genetics and physiological properties of lactococci have considerable changed. Therefore, both for basic research and for application purposes in this paper the general view of the new taxonomic classification of Lactococcus lactis, the role of their plasmids and the physiology and nutritional requirements during growth are discussed.

  6. One-pot synthesis of GDP-l-fucose by a four-enzyme cascade expressed in Lactococcus lactis.

    Science.gov (United States)

    Li, Ling; Kim, Seul-Ah; Heo, Ji Eun; Kim, Tae-Jip; Seo, Jin-Ho; Han, Nam Soo

    2017-12-20

    GDP-l-fucose is an l-fucose donor to synthesize fucosylated compounds such as human milk oligosaccharides or Lewis antigen. In this study, we used Lactococcus lactis subsp. cremoris NZ9000 to express 4 enzymes, ManB, ManC, Gmd, and WcaG and produced GDP-l-fucose by using one-pot synthesis method with mannose-6-phosphate as substrate and the enzymes as biocatalyst. For preparation of enzyme mixture, 4 genes (manB, manC, gmd, and wcaG) cloned from Escherichia coli were transformed into L. lactis strains using pNZ8008 and the recombinant cell lysates were obtained after cultivation. When mannose-6-phosphate was used as the substrate, the consecutive reactions with ManB, ManC, Gmd, and WcaG resulted in the successful production of GDP-l-fucose (0.13mM). When GDP-d-mannose was used as the substrate, it was entirely converted to GDP-l-fucose (0.2mM; 0.12g/L) via 2 enzymatic reactions mediated by Gmd and WcaG. This is the first report of GDP-l-fucose production by using multiple enzymes expressed in lactic acid bacteria. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Use of murine models to detect the allergenicity of genetically modified Lactococcus lactis NZ9000/pNZPNK.

    Science.gov (United States)

    Chiang, Shen-Shih; Liu, Chin-Feng; Ku, Ting-Wei; Mau, Jeng-Leun; Lin, Hsin-Tang; Pan, Tzu-Ming

    2011-04-27

    By introducing aprN into Lactococcus lactis NZ9000, the genetically modified L. lactis NZ9000/pNZPNK successfully expressed the nattokinase. The safety assessment of this novel strain was based on allergenicity of pepsin digestion stability and murine model serologic identity. Subjecting to the GM strain and host to pepsin digestion, the soluble fractions and cell debris were fast degraded completely. Feeding with ovalbumin resulted in significantly higher production of IgG1 and IgE as compared to that of L. lactis NZ9000/pNZPNK or L. lactis NZ9000. Further, the serum IgG2a level increased dose-dependently at week 2 and induced immune reaction toward Th1 pathway. Secretion of cytokines IL-4 and IL-10 fed with lactococci was significantly lower than that of the OVA group. L. lactis NZ9000/pNZPNK did not increase the proliferation of type 2 helper T cells in spleen or induce allergenicity in BALB/c mice. On the basis of the results, the new GM lactic acid bacterium is regarded as safe to use.

  8. Autolysis of Lactococcus lactis is increased upon D-alanine depletion of peptidoglycan and lipoteichoic acids

    NARCIS (Netherlands)

    Steen, Anton; Palumbo, Emmanuelle; Deghorain, Marie; Cocconcelli, Pier Sandro; Delcour, Jean; Kuipers, Oscar P.; Kok, Jan; Buist, Girbe; Hols, Pascal

    Mutations in the genes encoding enzymes responsible for the incorporation of D-Ala into the cell wall of Lactococcus lactis affect autolysis. An L. lactis alanine racemase (alr) mutant is strictly dependent on an external Supply Of D-Ala to be able to synthesize peptidoglycan and to incorporate

  9. Lactic Acid Bacteria from Kefir Increase Cytotoxicity of Natural Killer Cells to Tumor Cells

    OpenAIRE

    Takuya Yamane; Tatsuji Sakamoto; Takenori Nakagaki; Yoshihisa Nakano

    2018-01-01

    The Japanese fermented beverage, homemade kefir, contains six lactic acid bacteria: Lactococcus. lactis subsp. Lactis, Lactococcus. lactis subsp. Cremoris, Lactococcus. Lactis subsp. Lactis biovar diacetylactis, Lactobacillus plantarum, Leuconostoc meseuteroides subsp. Cremoris and Lactobacillus casei. In this study, we found that a mixture of the six lactic acid bacteria from kefir increased the cytotoxicity of human natural killer KHYG-1 cells to human chronic myelogenous leukemia K562 cell...

  10. Genome‐scale diversity and niche adaptation analysis of Lactococcus lactis by comparative genome hybridization using multi‐strain arrays

    Science.gov (United States)

    Siezen, Roland J.; Bayjanov, Jumamurat R.; Felis, Giovanna E.; van der Sijde, Marijke R.; Starrenburg, Marjo; Molenaar, Douwe; Wels, Michiel; van Hijum, Sacha A. F. T.; van Hylckama Vlieg, Johan E. T.

    2011-01-01

    Summary Lactococcus lactis produces lactic acid and is widely used in the manufacturing of various fermented dairy products. However, the species is also frequently isolated from non‐dairy niches, such as fermented plant material. Recently, these non‐dairy strains have gained increasing interest, as they have been described to possess flavour‐forming activities that are rarely found in dairy isolates and have diverse metabolic properties. We performed an extensive whole‐genome diversity analysis on 39 L. lactis strains, isolated from dairy and plant sources. Comparative genome hybridization analysis with multi‐strain microarrays was used to assess presence or absence of genes and gene clusters in these strains, relative to all L. lactis sequences in public databases, whereby chromosomal and plasmid‐encoded genes were computationally analysed separately. Nearly 3900 chromosomal orthologous groups (chrOGs) were defined on basis of four sequenced chromosomes of L. lactis strains (IL1403, KF147, SK11, MG1363). Of these, 1268 chrOGs are present in at least 35 strains and represent the presently known core genome of L. lactis, and 72 chrOGs appear to be unique for L. lactis. Nearly 600 and 400 chrOGs were found to be specific for either the subspecies lactis or subspecies cremoris respectively. Strain variability was found in presence or absence of gene clusters related to growth on plant substrates, such as genes involved in the consumption of arabinose, xylan, α‐galactosides and galacturonate. Further niche‐specific differences were found in gene clusters for exopolysaccharides biosynthesis, stress response (iron transport, osmotolerance) and bacterial defence mechanisms (nisin biosynthesis). Strain variability of functions encoded on known plasmids included proteolysis, lactose fermentation, citrate uptake, metal ion resistance and exopolysaccharides biosynthesis. The present study supports the view of L. lactis as a species with a very flexible

  11. Characterization of the role of para-aminobenzoic acid biosynthesis in folate production by Lactococcus lactis

    NARCIS (Netherlands)

    Wegkamp, H.B.A.; Oorschot, van A.; Vos, de W.M.; Smid, E.J.

    2007-01-01

    The pab genes for para-aminobenzoic acid (pABA) biosynthesis in Lactococcus lactis were identified and characterized. In L. lactis NZ9000, only two of the three genes needed for pABA production were initially found. No gene coding for 4-amino-4-deoxychorismate lyase (pabC) was initially annotated,

  12. Deciphering a unique biotin scavenging pathway with redundant genes in the probiotic bacterium Lactococcus lactis.

    Science.gov (United States)

    Zhang, Huimin; Wang, Qingjing; Fisher, Derek J; Cai, Mingzhu; Chakravartty, Vandana; Ye, Huiyan; Li, Ping; Solbiati, Jose O; Feng, Youjun

    2016-05-10

    Biotin protein ligase (BPL) is widespread in the three domains of the life. The paradigm BPL is the Escherichia coli BirA protein, which also functions as a repressor for the biotin biosynthesis pathway. Here we report that Lactococcus lactis possesses two different orthologues of birA (birA1_LL and birA2_LL). Unlike the scenario in E. coli, L. lactis appears to be auxotrophic for biotin in that it lacks a full biotin biosynthesis pathway. In contrast, it retains two biotin transporter-encoding genes (bioY1_LL and bioY2_LL), suggesting the use of a scavenging strategy to obtain biotin from the environment. The in vivo function of the two L. lactis birA genes was judged by their abilities to complement the conditional lethal E. coli birA mutant. Thin-layer chromatography and mass spectroscopy assays demonstrated that these two recombinant BirA proteins catalyze the biotinylation reaction of the acceptor biotin carboxyl carrier protein (BCCP), through the expected biotinoyl-AMP intermediate. Gel shift assays were used to characterize bioY1_LL and BirA1_LL. We also determined the ability to uptake (3)H-biotin by L. lactis. Taken together, our results deciphered a unique biotin scavenging pathway with redundant genes present in the probiotic bacterium L. lactis.

  13. An engineered Lactococcus lactis strain exerts significant immune responses through efficient expression and delivery of Helicobacter pylori Lpp20 antigen.

    Science.gov (United States)

    Zhang, Rongguang; Peng, Xiaoyan; Duan, Guangcai; Shi, Qingfeng; Chen, Shuaiyin; Wang, Chen; Fan, Qingtang; Xi, Yuanlin

    2016-12-01

    To produce and deliver Helicobacter pylori lipoprotein Lpp20 via using Lactococcus lactis with aim of developing an efficient way to use this protective antigen in vaccine formulation. An engineered L. lactis strain carrying the lpp20 gene from H. pylori was constructed. The inducible expression of Lpp20 in L. lactis was detected as a 20 kDa intracellular protein by SDS-PAGE. Lpp20 constituted 10 % of the L. lactis cellular proteins. The expression product was highly immunoreactive, as demonstrated by western blot assays using mouse anti-H. pylori sera. Animal experimentation showed that oral vaccination with the engineered strain excited significantly elevated levels of serum Lpp20-specific IgG antibodies in BALB/c mice (P lactis, demonstrating an efficient utilization mode of Lpp20 in anti-H. pylori vaccination.

  14. ORF Alignment: NC_002662 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available eptidase [Lactococcus lactis subsp. lactis Il1403] ... gb|AAK04710.1| mathionine aminopeptidase [Lact...ococcus ... lactis subsp. lactis Il1403] pir||D86701 mathionine ... aminopeptidase [imported

  15. Secretion and properties of a hybrid Kluyveromyces lactis-Aspergillus niger β-galactosidase

    Directory of Open Access Journals (Sweden)

    Becerra Manuel

    2006-12-01

    Full Text Available Abstract Background The β-galactosidase from Kluyveromyces lactis is a protein of outstanding biotechnological interest in the food industry and milk whey reutilization. However, due to its intracellular nature, its industrial production is limited by the high cost associated to extraction and downstream processing. The yeast-system is an attractive method for producing many heterologous proteins. The addition of a secretory signal in the recombinant protein is the method of choice to sort it out of the cell, although biotechnological success is not guaranteed. The cell wall acting as a molecular sieve to large molecules, culture conditions and structural determinants present in the protein, all have a decisive role in the overall process. Protein engineering, combining domains of related proteins, is an alternative to take into account when the task is difficult. In this work, we have constructed and analyzed two hybrid proteins from the β-galactosidase of K. lactis, intracellular, and its Aspergillus niger homologue that is extracellular. In both, a heterologous signal peptide for secretion was also included at the N-terminus of the recombinant proteins. One of the hybrid proteins obtained has interesting properties for its biotechnological utilization. Results The highest levels of intracellular and extracellular β-galactosidase were obtained when the segment corresponding to the five domain of K. lactis β-galactosidase was replaced by the corresponding five domain of the A. niger β-galactosidase. Taking into account that this replacement may affect other parameters related to the activity or the stability of the hybrid protein, a thoroughly study was performed. Both pH (6.5 and temperature (40°C for optimum activity differ from values obtained with the native proteins. The stability was higher than the corresponding to the β-galactosidase of K. lactis and, unlike this, the activity of the hybrid protein was increased by the presence

  16. Lactobacillus delbrueckii ssp. lactis and ssp. bulgaricus: a chronicle of evolution in action.

    Science.gov (United States)

    El Kafsi, Hela; Binesse, Johan; Loux, Valentin; Buratti, Julien; Boudebbouze, Samira; Dervyn, Rozenn; Kennedy, Sean; Galleron, Nathalie; Quinquis, Benoît; Batto, Jean-Michel; Moumen, Bouziane; Maguin, Emmanuelle; van de Guchte, Maarten

    2014-05-28

    Lactobacillus delbrueckii ssp. lactis and ssp. bulgaricus are lactic acid producing bacteria that are largely used in dairy industries, notably in cheese-making and yogurt production. An earlier in-depth study of the first completely sequenced ssp. bulgaricus genome revealed the characteristics of a genome in an active phase of rapid evolution, in what appears to be an adaptation to the milk environment. Here we examine for the first time if the same conclusions apply to the ssp. lactis, and discuss intra- and inter-subspecies genomic diversity in the context of evolutionary adaptation. Both L. delbrueckii ssp. show the signs of reductive evolution through the elimination of superfluous genes, thereby limiting their carbohydrate metabolic capacities and amino acid biosynthesis potential. In the ssp. lactis this reductive evolution has gone less far than in the ssp. bulgaricus. Consequently, the ssp. lactis retained more extended carbohydrate metabolizing capabilities than the ssp. bulgaricus but, due to high intra-subspecies diversity, very few carbohydrate substrates, if any, allow a reliable distinction of the two ssp. We further show that one of the most important traits, lactose fermentation, of one of the economically most important dairy bacteria, L. delbruecki ssp. bulgaricus, relies on horizontally acquired rather than deep ancestral genes. In this sense this bacterium may thus be regarded as a natural GMO avant la lettre. The dairy lactic acid producing bacteria L. delbrueckii ssp. lactis and ssp. bulgaricus appear to represent different points on the same evolutionary track of adaptation to the milk environment through the loss of superfluous functions and the acquisition of functions that allow an optimized utilization of milk resources, where the ssp. bulgaricus has progressed further away from the common ancestor.

  17. Recombinant invasive Lactococcus lactis can transfer DNA vaccines either directly to dendritic cells or across an epithelial cell monolayer.

    Science.gov (United States)

    de Azevedo, Marcela; Meijerink, Marjolein; Taverne, Nico; Pereira, Vanessa Bastos; LeBlanc, Jean Guy; Azevedo, Vasco; Miyoshi, Anderson; Langella, Philippe; Wells, Jerry M; Chatel, Jean-Marc

    2015-09-11

    Lactococcus lactis (L. lactis), a generally regarded as safe (GRAS) bacterium has recently been investigated as a mucosal delivery vehicle for DNA vaccines. Because of its GRAS status, L. lactis represents an attractive alternative to attenuated pathogens. Previous studies showed that eukaryotic expression plasmids could be delivered into intestinal epithelial cells (IECs) by L. lactis, or recombinant invasive strains of L. lactis, leading to heterologous protein expression. Although expression of antigens in IECs might lead to vaccine responses, it would be of interest to know whether uptake of L. lactis DNA vaccines by dendritic cells (DCs) could lead to antigen expression as they are unique in their ability to induce antigen-specific T cell responses. To test this, we incubated mouse bone marrow-derived DCs (BMDCs) with invasive L. lactis strains expressing either Staphylococcus aureus Fibronectin Binding Protein A (LL-FnBPA+), or Listeria monocytogenes mutated Internalin A (LL-mInlA+), both strains carrying a plasmid DNA vaccine (pValac) encoding for the cow milk allergen β-lactoglobulin (BLG). We demonstrated that they can transfect BMDCs, inducing the secretion of the pro-inflammatory cytokine IL-12. We also measured the capacity of strains to invade a polarized monolayer of IECs, mimicking the situation encountered in the gastrointestinal tract. Gentamycin survival assay in these cells showed that LL-mInlA+ is 100 times more invasive than L. lactis. The cross-talk between differentiated IECs, BMDCs and bacteria was also evaluated using an in vitro transwell co-culture model. Co-incubation of strains in this model showed that DCs incubated with LL-mInlA+ containing pValac:BLG could express significant levels of BLG. These results suggest that DCs could sample bacteria containing the DNA vaccine across the epithelial barrier and express the antigen. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  18. Genetically engineered Lactococcus lactis protect against house dust mite allergy in a BALB/c mouse model.

    Directory of Open Access Journals (Sweden)

    Chunqing Ai

    Full Text Available Mucosal vaccine based on lactic acid bacteria is an attractive concept for the prevention and treatment of allergic diseases, but their mechanisms of action in vivo are poorly understood. Therefore, we sought to investigate how recombinant major dust mite allergen Der p2-expressing Lactococcus lactis as a mucosal vaccine induced the immune tolerance against house dust mite allergy in a mouse model.Three strains of recombinant L. lactis producing Der p2 in different cell components (extracellular, intracellular and cell wall were firstly constructed. Their prophylactic potential was evaluated in a Der p2-sensitised mouse model, and immunomodulation properties at the cellular level were determined by measuring cytokine production in vitro.Der p2 expressed in the different recombinant L. lactis strains was recognized by a polyclonal anti-Der p2 antibody. Oral treatment with the recombinant L. lactis prior sensitization significantly prevented the development of airway inflammation in the Der p2-sensitized mice, as determined by the attenuation of inflammatory cells infiltration in the lung tissues and decrease of Th2 cytokines IL-4 and IL-5 levels in bronchoalveolar lavage. In addition, the serum allergen-specific IgE levels were significantly reduced, and the levels of IL-4 in the spleen and mesenteric lymph nodes cell cultures were also markedly decreased upon allergen stimulation in the mice fed with the recombinant L. lactis strains. These protective effects correlated with a significant up-regulation of regulatory T cells in the mesenteric lymph nodes.Oral pretreatment with live recombinant L. lactis prevented the development of allergen-induced airway inflammation primarily by the induction of specific mucosal immune tolerance.

  19. Fermentation of protopanaxadiol type ginsenosides (PD) with probiotic Bifidobacterium lactis and Lactobacillus rhamnosus.

    Science.gov (United States)

    Tan, Joanne Sh; Yeo, Chia-Rou; Popovich, David G

    2017-07-01

    Ginsenosides are believed to be the principal components behind the pharmacological actions of ginseng, and their bioactive properties are closely related to the type, position, and number of sugar moieties attached to the aglycone; thus, modification of the sugar chains may markedly change their biological activities. In this study, major protopanaxadiol type ginsenosides (PD) Rb1, Rc, and Rb2 were isolated from Panax ginseng and were transformed using two probiotic strains namely Bifidobacterium lactis Bi-07 and Lactobacillus rhamnosus HN001 to obtain specific deglycosylated ginsenosides. It was demonstrated that B. lactis transformed ginsenosides Rb1, Rc, and Rb2 to Rd within 1 h of fermentation and rare ginsenoside F2 by the conversion of Rd after 12-h fermentation. The maximum Rd concentration was 147.52 ± 1.45 μg/mL after 48-h fermentation as compared to 45.85 ± 0.71 μg/mL before fermentation. In contrast, L. rhamnosus transformed Rb1, Rc, and Rb2 into Rd as the final metabolite after 72-h fermentation. B. lactis displayed significantly (p fermentation. The present study suggests that the fermentation of major PD type ginsenosides with B. lactis Bi-07 may serve as an effective means to afford bioactive deglycosylated ginsenosides and to create novel ginsenoside extracts.

  20. Heterologous expression of Streptococcus mutans Cnm in Lactococcus lactis promotes intracellular invasion, adhesion to human cardiac tissues and virulence.

    Science.gov (United States)

    Freires, Irlan A; Avilés-Reyes, Alejandro; Kitten, Todd; Simpson-Haidaris, P J; Swartz, Michael; Knight, Peter A; Rosalen, Pedro L; Lemos, José A; Abranches, Jacqueline

    2017-01-02

    In S. mutans, the expression of the surface glycoprotein Cnm mediates binding to extracellular matrix proteins, endothelial cell invasion and virulence in the Galleria mellonella invertebrate model. To further characterize Cnm as a virulence factor, the cnm gene from S. mutans strain OMZ175 was expressed in the non-pathogenic Lactococcus lactis NZ9800 using a nisin-inducible system. Despite the absence of the machinery necessary for Cnm glycosylation, Western blot and immunofluorescence microscopy analyses demonstrated that Cnm was effectively expressed and translocated to the cell wall of L. lactis. Similar to S. mutans, expression of Cnm in L. lactis enabled robust binding to collagen and laminin, invasion of human coronary artery endothelial cells and increased virulence in G. mellonella. Using an ex vivo human heart tissue colonization model, we showed that Cnm-positive strains of either S. mutans or L. lactis outcompete their Cnm-negative counterparts for tissue colonization. Finally, Cnm expression facilitated L. lactis adhesion and colonization in a rabbit model of infective endocarditis. Collectively, our results provide unequivocal evidence that binding to extracellular matrices mediated by Cnm is an important virulence attribute of S. mutans and confirm the usefulness of the L. lactis heterologous system for further characterization of bacterial virulence factors.

  1. Proposal for creation of a new genus Neomicrococcus gen. nov. to accommodate Zhihengliuella aestuarii Baik et al. 2011 and Micrococcus lactis Chittpurna et al. 2011 as Neomicrococcus aestuarii comb. nov. and Neomicrococcus lactis comb. nov.

    Science.gov (United States)

    Prakash, Om; Sharma, Avinash; Nimonkar, Yogesh; Shouche, Yogesh S

    2015-11-01

    Micrococcus lactis and Zhihengliuella aestuarii were described independently in 2011. Their type strains showed high levels of 16S rRNA gene sequence similarity (99.3%). Phylogenetic analysis revealed that M. lactis MCC 2278T and Z. aestuarii JCM 16166T formed a monophyletic group and showed distant relationships to other members of closely related genera such as Micrococcus, Zhihengliuella, Arthrobacter and Citricoccus. The presence of large proportions of iso-C14:0 and iso-C16:0 with small amounts of iso-C15:0 distinguished M. lactis MCC 2278T and Z. aestuarii JCM 16166T from other members of the genera Micrococcus and Zhihengliuella. Unlike other members of the genera Zhihengliuella and Micrococcus, M. lactis MCC 2278T and Z. aestuarii JCM 16166T showed growth at low concentrations of NaCl. Thus, based on distinctive phylogenetic, chemotaxonomic and physiological features of these two organisms in comparison with other members of the genera Micrococcus and Zhihengliuella, it is clear that they do not fit within the existing classification and deserve separate status. DNA-DNA hybridization between the two type strains was 63%, indicating that they represent separate species. In this study, we propose the creation of a novel genus, Neomicrococcus gen. nov., to accommodate the two species with Neomicrococcus aestuarii gen. nov., comb. nov. (type strain JCM 16166T = KCTC 19557T) as the type species. Neomicrococcus lactis comb. nov. (type strain MCC 2278T = DSM 23694T) is also proposed.

  2. Contribution of the CesR-regulated genes llmg0169 and llmg2164-2163 to Lactococcus lactis fitness

    NARCIS (Netherlands)

    Roces, Clara; Campelo, Ana B.; Veiga, Patrick; Pinto, Joao P. C.; Rodriguez, Ana; Martinez, Beatriz

    2009-01-01

    Lactococcus lactis is one of the main components of the starter cultures used in cheese manufacture. As starter, L lactis must tolerate harsh conditions encountered either during their production in bulk quantities or during dairy products processing. To face these hostile conditions, bacteria

  3. Subcutaneous or oral immunization of mice with Lactococcus lactis expressing F4 fimbrial adhesin FaeG.

    Science.gov (United States)

    Liu, Shujie; Li, Yongming; Xu, Ziwei; Wang, Yicheng

    2013-01-01

    Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrhea in neonatal and postweaning piglets. Fimbrial adhesion of ETEC has been considered an important colonization factor with antigenicity. To safely and effectively deliver the F4 (K88) fimbrial adhesin FaeG to the immune system, we have previously constructed the secretory expression vector pNZ8112-faeG, and FaeG was produced in cytoplasmic form in Lactococcus lactis. In this work, BALB/c mice were immunized with recombinant L. lactis to further determine the immunogenicity of recombinant FaeG (rFaeG) via the subcutaneous or oral route. Subcutaneous immunization in mice with recombinant L. lactis induced a significant increase in the F4-specific serum IgG titer and the number of antibody-secreting cells (ASCs) in the spleen. Oral immunization of mice with recombinant L. lactis induced mucosal and systemic F4-specific immune responses and increased the number of ASCs in the spleen, mesenteric lymph nodes and Peyer's patches. High-dose (2.8 × 10(11) CFU) recombinant strains and adjuvant cholera toxin B subunit enhanced specific mucosal immune responses. The results suggest the feasibility of delivering rFaeG expressed in L. lactis to the immune system in order to induce an F4-specific immune response.

  4. The major facilitator superfamily transporter Knq1p modulates boron homeostasis in Kluyveromyces lactis.

    Science.gov (United States)

    Svrbicka, Alexandra; Toth Hervay, Nora; Gbelska, Yvetta

    2016-03-01

    Boron is an essential micronutrient for living cells, yet its excess causes toxicity. To date, the mechanisms of boron toxicity are poorly understood. Recently, the ScATR1 gene has been identified encoding the main boron efflux pump in Saccharomyces cerevisiae. In this study, we analyzed the ScATR1 ortholog in Kluyveromyces lactis--the KNQ1 gene, to understand whether it participates in boron stress tolerance. We found that the KNQ1 gene, encoding a permease belonging to the major facilitator superfamily, is required for K. lactis boron tolerance. Deletion of the KNQ1 gene led to boron sensitivity and its overexpression increased K. lactis boron tolerance. The KNQ1 expression was induced by boron and the intracellular boron concentration was controlled by Knq1p. The KNQ1 promoter contains two putative binding motifs for the AP-1-like transcription factor KlYap1p playing a central role in oxidative stress defense. Our results indicate that the induction of the KNQ1 expression requires the presence of KlYap1p and that Knq1p like its ortholog ScAtr1p in S. cerevisiae functions as a boron efflux pump providing boron resistance in K. lactis.

  5. The novel sRNA s015 improves nisin yield by increasing acid tolerance of Lactococcus lactis F44.

    Science.gov (United States)

    Qi, Jiakun; Caiyin, Qinggele; Wu, Hao; Tian, Kairen; Wang, Binbin; Li, Yanni; Qiao, Jianjun

    2017-08-01

    Nisin, a polycyclic antibacterial peptide produced by Lactococcus lactis, is stable at low pH. Improving the acid tolerance of L. lactis could thus enhance nisin yield. Small non-coding RNAs (sRNAs) play essential roles in acid tolerance by regulating their target mRNAs at the post-transcriptional level. In this study, a novel sRNA, s015, was identified in L. lactis F44 via the use of RNA sequencing, qRT-PCR analysis, and Northern blotting. s015 improved the acid tolerance of L. lactis and boosted nisin yield at low pH. In silico predictions enabled us to construct a library of possible s015 target mRNAs. Statistical analysis and validation suggested that s015 contains a highly conserved region (5'-GAAAAAAAC-3') that likely encompasses the regulatory core of the sRNA. atpG, busAB, cysD, ilvB, tcsR, ung, yudD, and ywdA were verified as direct targets of s015, and the interactions between s015 and its target genes were elucidated. This work provided new insight into the adaptation mechanism of L. lactis under acid stress.

  6. An application in cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147.

    Science.gov (United States)

    Ryan, M P; Rea, M C; Hill, C; Ross, R P

    1996-01-01

    Lactococcus lactis DPC3147, a strain isolated from an Irish kefir grain, produces a bacteriocin with a broad spectrum of inhibition. The bacteriocin produced is heat stable, particularly at a low pH, and inhibits nisin-producing (Nip+) lactococci. On the basis of the observation that the nisin structural gene (nisA) does not hybridize to DPC3147 genomic DNA, the bacteriocin produced was considered novel and designated lacticin 3147. The genetic determinants which encode lacticin 3147 are contained on a 63-kb plasmid, which was conjugally mobilized to a commercial cheese starter, L. lactis subsp. cremoris DPC4268. The resultant transconjugant, DPC4275, both produces and is immune to lacticin 3147. The ability of lacticin 3147-producing lactococci to perform as cheddar cheese starters was subsequently investigated in cheesemaking trials. Bacteriocin-producing starters (which included the transconjugant strain DPC4275) produced acid at rates similar to those of commercial strains. The level of lacticin 3147 produced in cheese remained constant over 6 months of ripening and correlated with a significant reduction in the levels of nonstarter lactic acid bacteria. Such results suggest that these starters provide a means of controlling developing microflora in ripened fermented products. PMID:8593062

  7. Production and secretion of heterologous proteins by Lactococcus lactis

    NARCIS (Netherlands)

    Asseldonk, van M.

    1994-01-01

    Lactococcus lactis strains have been used for centuries in food fermentation, now appreciated as traditional biotechnology. They have been applied in the cheesemaking process and for the manufacturing of other dairy products. Years of experience with these lactic acid

  8. Factors affecting proteolytic action of Lactococcus lactis in cheese

    NARCIS (Netherlands)

    Youssef, Y.B.

    1992-01-01

    Model cheeses were developed to study the behaviour of proteolytic agents involved in cheese maturation under conditions that closely resemble those in normal cheese. The models were applied to study protein breakdown by Lactococcus lactis ssp. cremoris HP , as a

  9. Antibacterial Activity of Lactic Acid Bacteria Isolated from Healthy ...

    African Journals Online (AJOL)

    Abstract. Lactic acid bacteria (LAB), namely, Lactobacillus acidophilus 1, Lactobacillus acidophilus 2, Lactobacillus brevis 1, Lactobacillus rhamnosus 1, Lactococcus lactis subsp. lactis 1, Lactococcus lactis subsp. lactis 2, Lactococcus raffinolactis 1, Pediococcus acidilactici 1, Pediococcus pentosaceus 1, and Pediococcus ...

  10. Investigation of glycerol assimilation and cofactor metabolism in Lactococcus lactis

    DEFF Research Database (Denmark)

    Holm, Anders Koefoed

    of glycerol kinase from L. lactis, introduction of a heterologous glycerol assimilation pathway and construction of a library of NADH oxidase activity. Based on a preliminary analysis of transcription level data, an attempt was made to stimulate glycerol assimilation by overexpressing the glycerol kinase...... already present in L. lactis. The construction and verification of a strain with increased glycerol kinase activity was not fully completed and is still ongoing. Similarly the construction of mutants expressing a heterologous pathway for glycerol dissimilation is also an ongoing task. An artificial...... effects and improve the growth rate, though not completely to the level of the reference strain. The fact that this effect was predominantly observed while utilizing xylose implicates the involvement of the pentose phosphate pathway. A possible mechanism underlying the observed growth characteristics...

  11. The effects of RecO deficiency in Lactococcus lactis NZ9000 on resistance to multiple environmental stresses.

    Science.gov (United States)

    Zhang, Mengru; Chen, Jian; Zhang, Juan; Du, Guocheng

    2014-12-01

    Multiple stresses could cause damage to DNA and other macromolecules. RecO, belonging to the family of DNA repair proteins, plays an important part in homologous recombination and replication repair. In order to explore the role of RecO in overcoming multiple stresses, a mutant of recO deletion is constructed in Lactococcus lactis ssp. cremoris NZ9000. Compared with the mutant strain, the original strain L. lactis NZ9000 shows better performance in growth under multiple stresses. The survival rates of the original strain under acid, osmotic and chill stresses are 13.49-, 2.78- and 60.89-fold higher. In our deeper research on fermentation capability under osmotic stress, lactate dehydrogenase activity after 8 h fermentation, maximum lactate acid production, lactate yield and maximum lactate productivity of L. lactis NZ9000 are 1.63-, 1.28-, 1.28- and 1.5-fold higher, respectively. Results indicate that RecO has positively improved the survival of L. lactis NZ9000, protected its key enzymes and enhanced its fermentation efficiencies. Our research confirms the role of RecO in enhancing tolerances to multiple stresses of L. lactis NZ9000, and puts forward the suggestion that RecO could be used in other industrial microorganisms as a new anti-stress component to improve their resistance to various stresses. © 2014 Society of Chemical Industry.

  12. PpiA, a surface PPIase of the cyclophilin family in Lactococcus lactis.

    Directory of Open Access Journals (Sweden)

    Nicolas Trémillon

    Full Text Available BACKGROUND: Protein folding in the envelope is a crucial limiting step of protein export and secretion. In order to better understand this process in Lactococcus lactis, a lactic acid bacterium, genes encoding putative exported folding factors like Peptidyl Prolyl Isomerases (PPIases were searched for in lactococcal genomes. RESULTS: In L. lactis, a new putative membrane PPIase of the cyclophilin subfamily, PpiA, was identified and characterized. ppiA gene was found to be constitutively expressed under normal and stress (heat shock, H(2O(2 conditions. Under normal conditions, PpiA protein was synthesized and released from intact cells by an exogenously added protease, showing that it was exposed at the cell surface. No obvious phenotype could be associated to a ppiA mutant strain under several laboratory conditions including stress conditions, except a very low sensitivity to H(2O(2. Induction of a ppiA copy provided in trans had no effect i on the thermosensitivity of an mutant strain deficient for the lactococcal surface protease HtrA and ii on the secretion and stability on four exported proteins (a highly degraded hybrid protein and three heterologous secreted proteins in an otherwise wild-type strain background. However, a recombinant soluble form of PpiA that had been produced and secreted in L. lactis and purified from a culture supernatant displayed both PPIase and chaperone activities. CONCLUSIONS: Although L. lactis PpiA, a protein produced and exposed at the cell surface under normal conditions, displayed a very moderate role in vivo, it was found, as a recombinant soluble form, to be endowed with folding activities in vitro.

  13. Lactococcus lactis, an alternative system for functional expression of peripheral and intrinsic Arabidopsis membrane proteins.

    Directory of Open Access Journals (Sweden)

    Annie Frelet-Barrand

    Full Text Available BACKGROUND: Despite their functional and biotechnological importance, the study of membrane proteins remains difficult due to their hydrophobicity and their low natural abundance in cells. Furthermore, into established heterologous systems, these proteins are frequently only produced at very low levels, toxic and mis- or unfolded. Lactococcus lactis, a gram-positive lactic bacterium, has been traditionally used in food fermentations. This expression system is also widely used in biotechnology for large-scale production of heterologous proteins. Various expression vectors, based either on constitutive or inducible promoters, are available for this system. While previously used to produce bacterial and eukaryotic membrane proteins, the ability of this system to produce plant membrane proteins was until now not tested. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this work was to test the expression, in Lactococcus lactis, of either peripheral or intrinsic Arabidopsis membrane proteins that could not be produced, or in too low amount, using more classical heterologous expression systems. In an effort to easily transfer genes from Gateway-based Arabidopsis cDNA libraries to the L. lactis expression vector pNZ8148, we first established a cloning strategy compatible with Gateway entry vectors. Interestingly, the six tested Arabidopsis membrane proteins could be produced, in Lactococcus lactis, at levels compatible with further biochemical analyses. We then successfully developed solubilization and purification processes for three of these proteins. Finally, we questioned the functionality of a peripheral and an intrinsic membrane protein, and demonstrated that both proteins were active when produced in this system. CONCLUSIONS/SIGNIFICANCE: Altogether, these data suggest that Lactococcus lactis might be an attractive system for the efficient and functional production of difficult plant membrane proteins.

  14. Temperate phages TP901-1 and phi LC3, belonging to the P335 species, apparently use different pathways for DNA injection in Lactococcus lactis subsp cremoris 3107

    DEFF Research Database (Denmark)

    Breum, Solvej Østergaard; Neve, Horst; Heller, Knut J.

    2007-01-01

    Five mutants of Lactococcus lactis subsp. cremoris 3107 resistant to phage TP901-1 were obtained after treatment with ethyl methanesulfonate. Two of the mutants were also resistant to phage phi LC3. The remaining three mutants were as sensitive as 3107. Mutants E46 and E100 did not adsorb the two...... phages. Mutants E119, E121 and E126 adsorbed phage phi LC3 as well as 3107 but phage TP901-1 with significantly reduced efficiency. All, except E46, could be lysogenized with phage TP901-BC1034, a derivative of TP901-1 harboring an erythromycin-resistance marker. However, the lysogenization frequency......-1. As such impairment was not seen when infecting E119, E121 and E126 with phi LC3, we conclude that TP901-1 and phi LC3 either are differently triggered by their receptor or utilize different pathways of injection....

  15. Lactococcus lactis NCC 2287 Alleviates Food Allergic Manifestations in Sensitized Mice by Reducing IL-13 Expression Specifically in the Ileum

    Directory of Open Access Journals (Sweden)

    Adrian W. Zuercher

    2012-01-01

    Full Text Available Objective. Utilizing a food allergy murine model, we have investigated the intrinsic antiallergic potential of the Lactococcus lactis NCC 2287 strain. Methods. BALB/c mice were sensitized at weekly intervals with ovalbumin (OVA plus cholera toxin (CT by the oral route for 7 weeks. In this model, an oral challenge with a high dose of OVA at the end of the sensitization period leads to clinical symptoms. Lactococcus lactis NCC 2287 was given to mice via the drinking water during sensitization (prevention phase or after sensitization (management phase. Results. Lactococcus lactis NCC 2287 administration to sensitized mice strikingly reduced allergic manifestations in the management phase upon challenge, when compared to control mice. No preventive effect was observed with the strain. Lactococcus lactis NCC 2287 significantly decreased relative expression levels of the Th-2 cytokine, IL-13, and associated chemokines CCL11 (eotaxin-1 and CCL17 (TARC in the ileum. No effect was observed in the jejunum. Conclusion/Significance. These results taken together designate Lactococcus lactis NCC 2287 as a candidate probiotic strain appropriate in the management of allergic symptoms.

  16. Assessing the effects of exposure to environmental stress on some functional properties of Bifidobacterium animalis ssp. lactis.

    Science.gov (United States)

    Amund, O D; Ouoba, L I I; Sutherland, J P; Ghoddusi, H B

    2014-12-01

    This study assessed the effects of exposing a strain of Bifidobacterium animalis ssp. lactis to acid, bile and osmotic stresses on antagonistic properties, biofilm formation and antibiotic susceptibility/resistance profile. Exposure to each stress factor appeared to have no significant effect on the antagonism against Escherichia coli NCTC 12900 and Salmonella enterica serovar Enteritidis PT4. No suppression in biofilm formation due to exposure to stress was observed. Bile and osmotic stresses resulted in significantly higher biofilm formation. Expression of an exopolysaccharide synthesis gene, gtf 01207, was significantly higher when the B. animalis ssp. lactis strain was exposed to osmotic stress. Susceptibility of the B. animalis ssp. lactis strain to chloramphenicol, erythromycin, ampicillin and vancomycin, and resistance to tetracycline remained unchanged when exposed to each stress. The expression of a tetracycline resistance gene, tet(W), was significantly higher when exposed to each stress. These results may suggest that the potential for the B. animalis ssp. lactis strain to provide probiotic benefit, after exposure to the stressful conditions of the gastrointestinal tract, remains intact.

  17. Recombinant Lactococcus lactis Expressing Haemagglutinin from a Polish Avian H5N1 Isolate and Its Immunological Effect in Preliminary Animal Trials

    Directory of Open Access Journals (Sweden)

    Agnieszka K. Szczepankowska

    2017-01-01

    Full Text Available Lactic acid bacteria (LAB are Gram-positive, nonpathogenic microorganisms that are gaining much interest as antigen producers for development of live vaccine vectors. Heterologous proteins of different origin have been successfully expressed in various LAB species, including Lactococcus lactis. Recombinant L. lactis strains have been shown to induce specific local and systemic immune responses against various antigens. Our study aimed at constructing a L. lactis strain expressing haemagglutinin of a Polish avian H5H1 influenza isolate and examining its effect on animals. Expression of the cloned H5 gene was achieved using the nisin-controlled gene expression system. Detection of the intracellular H5 antigen produced in L. lactis was performed by Western blot analysis and confirmed using mass spectrometry. The potential of L. lactis recombinant cells to induce an immune response was examined by setting up preliminary immunization trials on chickens and mice. Obtained sera were tested for specific antibodies by ELISA assays. The results of these studies are a promising step toward developing a vaccine against the bird flu using Lactococcus lactis cells as bioreactors for efficient antigen production and delivery to the mucosal surface.

  18. Recombinant Lactococcus lactis Expressing Haemagglutinin from a Polish Avian H5N1 Isolate and Its Immunological Effect in Preliminary Animal Trials.

    Science.gov (United States)

    Szczepankowska, Agnieszka K; Szatraj, Katarzyna; Sałański, Przemysław; Rózga, Agnieszka; Górecki, Roman K; Bardowski, Jacek K

    2017-01-01

    Lactic acid bacteria (LAB) are Gram-positive, nonpathogenic microorganisms that are gaining much interest as antigen producers for development of live vaccine vectors. Heterologous proteins of different origin have been successfully expressed in various LAB species, including Lactococcus lactis . Recombinant L. lactis strains have been shown to induce specific local and systemic immune responses against various antigens. Our study aimed at constructing a L. lactis strain expressing haemagglutinin of a Polish avian H5H1 influenza isolate and examining its effect on animals. Expression of the cloned H5 gene was achieved using the nisin-controlled gene expression system. Detection of the intracellular H5 antigen produced in L. lactis was performed by Western blot analysis and confirmed using mass spectrometry. The potential of L. lactis recombinant cells to induce an immune response was examined by setting up preliminary immunization trials on chickens and mice. Obtained sera were tested for specific antibodies by ELISA assays. The results of these studies are a promising step toward developing a vaccine against the bird flu using Lactococcus lactis cells as bioreactors for efficient antigen production and delivery to the mucosal surface.

  19. Sec-mediated secretion of bacteriocin enterocin P by Lactococcus lactis

    NARCIS (Netherlands)

    Herranz, C; Driessen, AJM

    Most lactic acid bacterium bacteriocins utilize specific leader peptides and dedicated machineries for secretion. In contrast, the enterococcal bacteriocin enterocin P (EntP) contains a typical signal peptide that directs its secretion when heterologously expressed in Lactococcus lactis. Signal

  20. Statistical optimization of lactic acid production by Lactococcus lactis ...

    African Journals Online (AJOL)

    The individual and interactive effects of a total inoculums size (% v/v), fermentation temperature and skim milk dry matter added (% w/v) on the lactic acid production by Lactococcus lactis LCL strain were studied by quadratic response surface methodology. The central composite design (CCD) was employed to determine ...

  1. Multilocus sequence typing of Lactococcus lactis from naturally fermented milk foods in ethnic minority areas of China.

    Science.gov (United States)

    Xu, Haiyan; Sun, Zhihong; Liu, Wenjun; Yu, Jie; Song, Yuqin; Lv, Qiang; Zhang, Jiachao; Shao, Yuyu; Menghe, Bilige; Zhang, Heping

    2014-05-01

    To determine the genetic diversity and phylogenetic relationships among Lactococcus lactis isolates, 197 strains isolated from naturally homemade yogurt in 9 ethnic minority areas of 6 provinces of China were subjected to multilocus sequence typing (MLST). The MLST analysis was performed using internal fragment sequences of 12 housekeeping genes (carB, clpX, dnaA, groEL, murC, murE, pepN, pepX, pyrG, recA, rpoB, and pheS). Six (dnaA) to 8 (murC) different alleles were detected for these genes, which ranged from 33.62 (clpX) to 41.95% (recA) GC (guanine-cytosine) content. The nucleotide diversity (π) ranged from 0.00362 (murE) to 0.08439 (carB). Despite this limited allelic diversity, the allele combinations of each strain revealed 72 different sequence types, which denoted significant genotypic diversity. The dN/dS ratios (where dS is the number of synonymous substitutions per synonymous site, and dN is the number of nonsynonymous substitutions per nonsynonymous site) were lower than 1, suggesting potential negative selection for these genes. The standardized index of association of the alleles IA(S)=0.3038 supported the clonality of Lc. lactis, but the presence of network structure revealed by the split decomposition analysis of the concatenated sequence was strong evidence for intraspecies recombination. Therefore, this suggests that recombination contributed to the evolution of Lc. lactis. A minimum spanning tree analysis of the 197 isolates identified 14 clonal complexes and 23 singletons. Phylogenetic trees were constructed based on the sequence types, using the minimum evolution algorithm, and on the concatenated sequence (6,192 bp), using the unweighted pair-group method with arithmetic mean, and these trees indicated that the evolution of our Lc. lactis population was correlated with geographic origin. Taken together, our results demonstrated that MLST could provide a better understanding of Lc. lactis genome evolution, as well as useful information for

  2. Developing a xylanase XYNZG from Plectosphaerella cucumerina for baking by heterologously expressed in Kluyveromyces lactis.

    Science.gov (United States)

    Zhan, Fei Xiang; Wang, Qin Hong; Jiang, Si Jing; Zhou, Yu Ling; Zhang, Gui Min; Ma, Yan He

    2014-12-16

    Xylanase can replace chemical additives to improve the volume and sensory properties of bread in the baking. Suitable baking xylanase with improved yield will promote the application of xylanase in baking industry. The xylanase XYNZG from the Plectosphaerella cucumerina has been previously characterized by heterologous expression in Pichia pastoris. However, P. pastoris is not a suitable host for xylanase to be used in the baking process since P. pastoris does not have GRAS (Generally Regarded As Safe) status and requires large methanol supplement during the fermentation in most conditions, which is not allowed to be used in the food industry. Kluyveromyces lactis, as another yeast expression host, has a GRAS status, which has been successfully used in food and feed applications. No previous work has been reported concerning the heterologous expression of xylanase gene xynZG in K. lactis with an aim for application in baking. The xylanase gene xynZG from the P. cucumerina was heterologously expressed in K. lactis. The recombinant protein XYNZG in K. lactis presented an approximately 19 kDa band on SDS-PAGE and zymograms analysis. Transformant with the highest halo on the plate containing the RBB-xylan (Remazol Brilliant Blue-xylan) was selected for the flask fermentation in different media. The results indicated that the highest activity of 115 U/ml at 72 h was obtained with the YLPU medium. The mass spectrometry analysis suggested that the hydrolytic products of xylan by XYNZG were mainly xylobiose and xylotriose. The results of baking trials indicated that the addition of XYNZG could reduce the kneading time of dough, increase the volume of bread, improve the texture, and have more positive effects on the sensory properties of bread. Xylanase XYNZG is successfully expressed in K. lactis, which exhibits the highest activity among the published reports of the xylanase expression in K. lactis. The recombinant XYNZG can be used to improve the volume and sensory

  3. AbiV, a Novel Antiphage Abortive Infection Mechanism on the Chromosome of Lactococcus lactis subsp. cremoris MG1363

    DEFF Research Database (Denmark)

    Haaber, Jakob Brandt Borup; Moineau, Sylvain; Fortier, Louis-Charles

    2008-01-01

    phenotype was caused by a chromosomal gene turned on by a promoter from the inserted construct. Reverse transcription-PCR analysis confirmed that there were higher levels of transcription of a downstream open reading frame (ORF) in the phage-resistant integrants than in the phage-sensitive strain L. lactis...... searches revealed no homology to other phage resistance mechanisms, and thus, this novel Abi mechanism was designated AbiV. The mode of action of AbiV is unknown, but the activity of AbiV prevented cleavage of the replicated phage DNA of 936-like phages....

  4. Oxidative Stress at High Temperatures in Lactococcus lactis Due to an Insufficient Supply of Riboflavin

    DEFF Research Database (Denmark)

    Chen, Jun; Shen, Jing; Solem, Christian

    2013-01-01

    Lactococcus lactis MG1363 was found to be unable to grow at temperatures above 37°C in a defined medium without riboflavin, and the cause was identified to be dissolved oxygen introduced during preparation of the medium. At 30°C, growth was unaffected by dissolved oxygen and oxygen was consumed...... riboflavin to the medium, it was possible to improve growth and oxygen consumption at 37°C, and this also normalized the [ATP]-to-[ADP] ratio. A codon-optimized redox-sensitive green fluorescent protein (GFP) was introduced into L. lactis and revealed a more oxidized cytoplasm at 37°C than at 30°C....... These results indicate that L. lactis suffers from heat-induced oxidative stress at increased temperatures. A decrease in intracellular flavin adenine dinucleotide (FAD), which is derived from riboflavin, was observed with increasing growth temperature, but the presence of riboflavin made the decrease smaller...

  5. The pyrH gene of Lactococcus lactis subsp. cremoris encoding UMP kinase is transcribed as part of an operon including the frr1 gene encoding ribosomal recycling factor

    DEFF Research Database (Denmark)

    Wadskov-Hansen, Steen Lüders; Martinussen, Jan; Hammer, Karin

    2000-01-01

    establishing the ability of the encoded protein to synthesize UDP. The pyrH gene in L. lactis is flanked downstream by frr1 encoding ribosomal recycling factor 1 and upstream by an open reading frame, orfA, of unknown function. The three genes were shown to constitute an operon transcribed in the direction orf......A-pyrH-frr1 from a promoter immediately in front of orfA. This operon belongs to an evolutionary highly conserved gene cluster, since the organization of pyrH on the chromosomal level in L. lactis shows a high resemblance to that found in Bacillus subtilis as well as in Escherichia coli and several other...

  6. Proteome analysis of a Lactococcus lactis strain overexpressing gapA suggests that the gene product is an auxiliary glyceraldehyde 3-phosphate dehydrogenase

    DEFF Research Database (Denmark)

    Willemoes, Martin; Kilstrup, Mogens; Roepstorff, P.

    2002-01-01

    revealed two neighbouring protein spots, GapBI and GapBII, with amino terminal sequences identical to the product of gapA from the L. lactis subspecies cremoris strain LM0230 and that of the two IL1403 sequences. In order to assign the two protein spots to their respective genes we constructed an L. lactis...... was specific for NAD. No NADP dependent activity was detected. Proteome analysis of the gapA overexpressing strain revealed two new protein spots, GapAI and GapAII, not previously detected in proteome analysis of MG1363. Results from mass spectrometry analysis of GapA and GapB and comparison with the deduced......The sequence of the genome from the Lactococcus lactis subspecies lactis strain IL1403 shows the presence of two reading frames, gapA and gapB, putatively encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Previous proteomic analysis of the L. lactis subspecies cremoris strain MG1363 has...

  7. Lactococcus lactis as host for overproduction of functional membrane proteins

    NARCIS (Netherlands)

    Kunji, ERS; Slotboom, DJ; Poolman, B

    2003-01-01

    Lactococcus lactis has many properties that are ideal for enhanced expression of membrane proteins. The organism is easy and inexpensive to culture, has a single membrane and relatively mild proteolytic activity. Methods for genetic manipulation are fully established and a tightly controlled

  8. Nucleotide metabolism in Lactococcus lactis: Salvage pathways of exogenous pyrimidines

    DEFF Research Database (Denmark)

    Martinussen, Jan; Andersen, Paal Skytt; Hammer, Karin

    1994-01-01

    By measuring enzyme activities in crude extracts and studying the effect of toxic analogs (5-fluoropyrimidines) on cell growth, the metabolism of pyrimidines in Lactococcus lactis was analyzed. Pathways by which uracil, uridine, deoxyuridine, cytidine, and deoxycytidine are metabolized in L. lact...

  9. Two nucleoside transporters in Lactococcus lactis with different substrate specificities

    DEFF Research Database (Denmark)

    Martinussen, Jan; Sørensen, Claus; Jendresen, Christian Bille

    2010-01-01

    , and the utilization of nucleotides is dependent on exogenous phosphatases. The composition of transporters with specificity for purine and pyrimidine nucleosides and nucleobases is subject to variation. The ability of Lactococcus lactis to transport different nucleosides across the cell membrane was characterized...

  10. Development and evaluation of an efficient heterologous gene knock-in reporter system in Lactococcus lactis.

    Science.gov (United States)

    Lu, Yifei; Yan, Hongxiang; Deng, Jiezhong; Huang, Zhigang; Jin, Xurui; Yu, Yanlan; Hu, Qiwen; Hu, Fuquan; Wang, Jing

    2017-09-18

    Lactococcus lactis is a food grade probiotics and widely used to express heterologous proteins. Generally, target genes are knocked into the L. lactis genome through double-crossover recombination to express heterologous proteins stably. However, creating marker-less heterologous genes knocked-in clones is laborious. In this study, an efficient heterologous gene knock-in reporter system was developed in L. lactis NZ9000. Our knock-in reporter system consists of a temperature-sensitive plasmid pJW and a recombinant L. lactis strain named NZB. The pJW contains homologous arms, and was constructed to knock-in heterologous genes at a fixed locus of NZ9000 genome. lacZ (β-galactosidase) gene was knocked into the chromosome of NZ9000 as a counter-selective marker through the plasmid pJW to generate NZB. The engineered NZB strain formed blue colonies on X-Gal plate. The desired double-crossover mutants formed white colonies distinctive from the predominantly blue colonies (parental and plasmid-integrated clones) when the embedded lacZ was replaced with the target heterologous genes carried by pJW in NZB. By using the system, the heterologous gene knocked-in clones are screened by colony phenotype change rather than by checking colonies individually. Our new knock-in reporter system provides an efficient method to create heterologous genes knocked-in clones.

  11. Production of spent mushroom substrate hydrolysates useful for cultivation of Lactococcus lactis by dilute sulfuric acid, cellulase and xylanase treatment.

    Science.gov (United States)

    Qiao, Jian-Jun; Zhang, Yan-Fei; Sun, Li-Fan; Liu, Wei-Wei; Zhu, Hong-Ji; Zhang, Zhijun

    2011-09-01

    Spent mushroom substrate (SMS) was treated with dilute sulfuric acid followed by cellulase and xylanase treatment to produce hydrolysates that could be used as the basis for media for the production of value added products. A L9 (3(4)) orthogonal experiment was performed to optimize the acid treatment process. Pretreatment with 6% (w/w) dilute sulfuric acid at 120°C for 120 min provided the highest reducing sugar yield of 267.57 g/kg SMS. No furfural was detected in the hydrolysates. Exposure to 20PFU of cellulase and 200 XU of xylanase per gram of pretreated SMS at 40°C resulted in the release of 79.85 g/kg or reducing sugars per kg acid pretreated SMS. The dilute sulfuric acid could be recycled to process fresh SMS four times. SMS hydrolysates neutralized with ammonium hydroxide, sodium hydroxide, or calcium hydroxide could be used as the carbon source for cultivation of Lactococcus lactis subsp. lactis W28 and a cell density of 2.9×10(11)CFU/mL could be obtained. The results provide a foundation for the development of value-added products based on SMS. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. CINÉTICA, PRUEBA DE CRECIMIENTO Y EFECTO DE INHIBICIÓN DE Lactococcus lactis SOBRE Yersinia pseudotuberculosis

    Directory of Open Access Journals (Sweden)

    HENRY JURADO GÁMEZ

    2016-12-01

    Full Text Available Lactic acid bacteria have demonstrated a high ability to inhibit pathogenic microorganisms, which improve knowledge of such microorganisms is important, for this, the kinetics was determined, growth and the inhibition effect of Lactococcus lactis on Yersinia pseudotuberculosis. The research was conducted at the University of Nariño, by susceptibility testing in all strains; in vitro inhibition of Lc. lactis and supernatant on bacterial pathogen; gastrointestinal lactic strain testing (gas production and catalase, bile, bile salts and 2 temperatures, growth kinetics and HPLC determination of peptides in the supernatant. dicloxacillin resistance was found in both strains. Lactic strain and the supernatant inhibited Y. pseudotuberculosis. growths and 3,9x1010 3x1011 CFU/150 uL to 3 to 5% bile salts, 3x1011 5x1012 and CFU/150 uL 1 and 2% bovine 3x1013 and 3x1012 bile and CFU/150 uL was found 38 and 45°C. The logarithmic phase of Lc. lactis was found at 3 hours with values 6,4x1012 CFU/150 uL. The VAL-TIR-VAL peptide was found in the supernatant. It is concluded that Lc lactis shows probiotic characteristics in in vitro conditions.

  13. Oral Vaccination with the Porcine Rotavirus VP4 Outer Capsid Protein Expressed by Lactococcus lactis Induces Specific Antibody Production

    Directory of Open Access Journals (Sweden)

    Yi-jing Li

    2010-01-01

    Full Text Available The objective of this study to design a delivery system resistant to the gastrointestinal environment for oral vaccine against porcine rotavirus. Lactococcus lactis NZ9000 was transformed with segments of vP4 of the porcine rotavirus inserted into the pNZ8112 surface-expression vector, and a recombinant L. lactis expressing VP4 protein was constructed. An approximately 27 kDa VP4 protein was confirmed by SDS-PAGE , Western blot and immunostaining analysis. BALB/c mice were immunized orally with VP4-expression recombinant L. lactis and cellular, mucosal and systemic humoral immune responses were examined. Specific anti-VP4 secretory IgA and IgG were found in feces, ophthalmic and vaginal washes and in serum. The induced antibodies demonstrated neutralizing effects on porcine rotavirus infection on MA104 cells. Our findings suggest that oral immunization with VP4-expressing L. lactis induced both specific local and systemic humoral and cellular immune responses in mice.

  14. Characterization of the Fermentation Process and the Inhibition Effect of Lactobacillus lactis in Staphylococcus aureus and Staphylococcus epidermidis

    Directory of Open Access Journals (Sweden)

    Henry Jurado-Gámez

    2015-09-01

    Full Text Available The fermentative process and in vitro inhibition of L. lactis in Staphylococcus aureus and Staphylococcus epidermidis were assessed. The growth of L. lactis at three pH (2.5, 4.5 and 7, bile salts (0.5, 1 and 2 %, bovine bile (1 and 1.2 % and two temperatures (38 and 45 °C were evaluated. Peptides and organic acids in supernatant of L. lactis by HPLC were determined. Fermentation kinetics was carried out, evaluating: pH, total sugar, protein and lactic acid. An antibiogram of dicloxacilin, cefepime, penilicin and cefalotin was made. The inhibition of L. lactis and its supernatant were defined in pathogenic strains. The best growth was at a pH of 2.5 (3 × 1012 UFC/ml; of 1 × 1010 and 4 × 109 UFC/ml for 0.5 % of bile salts and 1.2 % of bovine bile, respectively; of 3.5 × 1013 and 3.4 × 1013 UFC/ml for 38 and 45 °C, respectively. The HPLC determined the peptides VAR-TIR-VAR and lactic acid (83.11 %. The fermentation kinetics determined the exponential phase at 14:24 h with a value of 77 × 1010 UFC/ ml, pH values of 4.284, 2.33 mg/ml sugar, 1.44 mg/ml protein and acidity of 0.79 %. It was found that S. aureus and S. epidermidis were sensitive to all antibiotics. The pathogenic bacteria were resistant to the lactic strain, but S. epidermidis was sensitive to the supernatant of L. lactis. The conclusion is that Lactobacillus lactis showed adequate growth capacity, good fermentation parameters and inhibitory effect in strains of S. aureus and S. epidermidis in in vitro conditions.

  15. Nisin production of Lactococcus lactis N8 with hemin-stimulated cell respiration in fed-batch fermentation system.

    Science.gov (United States)

    Kördikanlıoğlu, Burcu; Şimşek, Ömer; Saris, Per E J

    2015-01-01

    In this study, nisin production of Lactococcus lactis N8 was optimized by independent variables of glucose, hemin and oxygen concentrations in fed-batch fermentation in which respiration of cells was stimulated with hemin. Response surface model was able to explain the changes of the nisin production of L. lactis N8 in fed-batch fermentation system with high fidelity (R(2) 98%) and insignificant lack of fit. Accordingly, the equation developed indicated the optimum parameters for glucose, hemin, and dissolved oxygen were 8 g L(-1) h(-1) , 3 μg mL(-1) and 40%, respectively. While 1711 IU mL(-1) nisin was produced by L. lactis N8 in control fed-batch fermentation, 5410 IU mL(-1) nisin production was achieved within the relevant optimum parameters where the respiration of cell was stimulated with hemin. Accordingly, nisin production was enhanced 3.1 fold in fed-batch fermentation using hemin. In conclusion the nisin production of L. lactis N8 was enhanced extensively as a result of increasing the biomass by stimulating the cell respiration with adding the hemin in the fed-batch fermentation. © 2015 American Institute of Chemical Engineers.

  16. Flavins contained in yeast extract are exploited for anodic electron transfer by Lactococcus lactis.

    Science.gov (United States)

    Masuda, Masaki; Freguia, Stefano; Wang, Yung-Fu; Tsujimura, Seiya; Kano, Kenji

    2010-06-01

    Cyclic voltammograms of yeast extract-containing medium exhibit a clear redox peak around -0.4V vs. Ag|AgCl. Fermentative bacterium Lactococcus lactis was hereby shown to exploit this redox compound for extracellular electron transfer towards a graphite anode using glucose as an electron donor. High performance liquid chromatography revealed that this may be a flavin-type compound. The ability of L. lactis to exploit exogenous flavins for anodic glucose oxidation was confirmed by tests where flavin-type compounds were supplied to the bacterium in well defined media. Based on its mid-point potential, riboflavin can be regarded as a near-optimal mediator for microbially catalyzed anodic electron transfer. Riboflavin derivative flavin mononucleotide (FMN) was also exploited by L. lactis as a redox shuttle, unlike flavin adenine dinucleotide (FAD), possibly due to the absence of a specific transporter for the latter. The use of yeast extract in microbial fuel cell media is herein discouraged based on the related unwanted artificial addition of redox mediators which may distort experimental results. Copyright 2009 Elsevier B.V. All rights reserved.

  17. Crystallization and preliminary X-ray crystallographic analysis of β-galactosidase from Kluyveromyces lactis

    International Nuclear Information System (INIS)

    Pereira-Rodríguez, Ángel; Fernández-Leiro, Rafael; González Siso, M. Isabel; Cerdán, M. Esperanza; Becerra, Manuel; Sanz-Aparicio, Julia

    2010-01-01

    β-Galactosidase from K. lactis has been expressed in S. cerevisiae, purified by affinity chromatography and crystallized in its native form. β-Galactosidase from Kluyveromyces lactis catalyses the hydrolysis of the β-galactosidic linkage in lactose. Owing to its many industrial applications, the biotechnological potential of this enzyme is substantial. This protein has been expressed in yeast and purified for crystallization trials. However, optimization of the best crystallization conditions yielded crystals with poor diffraction quality that precluded further structural studies. Finally, the crystal quality was improved using the streak-seeding technique and a complete diffraction data set was collected at 2.8 Å resolution

  18. Development and Diversity of Lactococcus lactis and Leuconostoc Bacteriophages in Dairies Using Undefined Mesophilic DL-Starter Cultures

    DEFF Research Database (Denmark)

    Muhammed, Musemma Kedir

    complete loss of fermentation. Dairy phages have for long time been studied using traditional culture-dependent methods but not using metagenomic approaches. Part of this project was devoted to develop a method for dairy metavirome extraction and analysis. Several whey mixtures derived by defined......Bacteriophages (phages) attacking strains of Lactococcus (Lc.) lactis and Leuconostoc species, used as starter cultures in mesophilic dairy productions, produce huge problems through waste of ingredients, increased processing time, reduced product quality, consistency and safety, and occasionally...... in dairies using undefined starters and of mostly Lc. lactis c2 phages in dairies using defined cultures. Certain evidence indicating possible co-induction of temperate P335 phages and smaller Lc. lactis satellite phages was obtained. Also addressed was the issue of accurate and simultaneous quantification...

  19. Metabolic behavior of Lactococcus lactis MG1363 in microaerobic continuous cultivation at a low dilution rate

    DEFF Research Database (Denmark)

    Jensen, Niels B.S.; Melchiorsen, Claus Rix; Jochumsen, Kirsten Væver

    2001-01-01

    Minute amounts of oxygen were supplied to a continuous cultivation of Lactococcus lactis subsp. cremoris MG1363 grown on a defined glucose-limited medium at a dilution rate of 0.1 h(-1). More than 80% of the carbon supplied with glucose ended up in fermentation products other than lactate. Addition...... of even minute amounts of oxygen increased the yield of biomass on glucose by more than 10% compared to that obtained under anaerobic conditions and had a dramatic impact on catabolic enzyme activities and hence on the distribution of carbon at the pyruvate branch point. Increasing aeration caused carbon...... dehydrogenase while increasing the enzyme activity levels of the pyruvate dehydrogenase complex, alpha -acetolactate synthase, and the NADH oxidases. Lactate dehydrogenase and glyceraldehyde dehydrogenase enzyme activity levels were unaffected by aeration....

  20. 21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Lactase enzyme preparation from Kluyveromyces... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1388 Lactase enzyme preparation from Kluyveromyces lactis. (a) This enzyme preparation is derived from the nonpathogenic...

  1. Versatile Cas9-driven subpopulation selection toolbox for Lactococcus lactis

    NARCIS (Netherlands)

    Els, van der Simon; James, Jennelle K.; Kleerebezem, Michiel; Bron, Peter A.

    2018-01-01

    CRISPR-Cas9 technology has been exploited for the removal or replacement of genetic elements in a wide range of prokaryotes and eukaryotes. Here, we describe the extension of the Cas9 application toolbox to the industrially important dairy species Lactococcus lactis. The Cas9 expression vector

  2. Construction of an expression vector for Lactococcus lactis based on ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-11-02

    Nov 2, 2009 ... Tamez-Guerra RS, Oliveira SC, Saucedo-Cardenas O, de Oca-Luna. RM, Le Loir Y (2003). Intranasal immunization with recombinant. Lactococcus lactis secreting murine interleukin-12 enhances antigen- specific Th1 cytokine production. Infection Immunity, 71: 1887-1896. De Vos WM, Simons G (1994).

  3. Lipid-protein interactions. The leucine transport system of Lactococcus lactis.

    NARCIS (Netherlands)

    Veld, Geertruida Elisabeth in 't

    1992-01-01

    In summary, it is concluded, that a functionally reconstituted leucine transport system of L. lactis is affected by bilayer features in the following order of importance: lipid headgroup (H+-bonding) › acyl chain carbon number (thickness) › cholesterol (fluidity) › acyl chain unsaturation (indirect

  4. Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system

    Directory of Open Access Journals (Sweden)

    Langella P.

    1999-01-01

    Full Text Available Lactic acid bacteria (LAB are Gram-positive bacteria and are generally regarded as safe (GRAS organisms. Therefore, LAB could be used for heterologous protein secretion and they are good potential candidates as antigen delivery vehicles. To develop such live vaccines, a better control of protein secretion is required. We developed an efficient secretion system in the model LAB, Lactococcus lactis. Staphylococcal nuclease (Nuc was used as the reporter protein. We first observed that the quantity of secreted Nuc correlated with the copy number of the cloning vector. The nuc gene was cloned on a high-copy number cloning vector and no perturbation of the metabolism of the secreting strain was observed. Replacement of nuc native promoter by a strong lactococcal one led to a significant increase of nuc expression. Secretion efficiency (SE of Nuc in L. lactis was low, i.e., only 60% of the synthesized Nuc was secreted. Insertion of a synthetic propeptide between the signal peptide and the mature moiety of Nuc increased the SE of Nuc. On the basis of these results, we developed a secretion system and we applied it to the construction of an L. lactis strain which secretes a bovine coronavirus (BCV epitope-protein fusion (BCV-Nuc. BCV-Nuc was recognized by both anti-BCV and anti-Nuc antibodies. Secretion of this antigenic fusion is the first step towards the development of a novel antigen delivery system based on LAB-secreting strains.

  5. Absolute Enumeration of Probiotic Strains Lactobacillus acidophilus NCFM® and Bifidobacterium animalis subsp. lactis Bl-04® via Chip-Based Digital PCR

    Directory of Open Access Journals (Sweden)

    Sarah J. Z. Hansen

    2018-04-01

    Full Text Available The current standard for enumeration of probiotics to obtain colony forming units by plate counts has several drawbacks: long time to results, high variability and the inability to discern between bacterial strains. Accurate probiotic cell counts are important to confirm the delivery of a clinically documented dose for its associated health benefits. A method is described using chip-based digital PCR (cdPCR to enumerate Bifidobacterium animalis subsp. lactis Bl-04 and Lactobacillus acidophilus NCFM both as single strains and in combination. Primers and probes were designed to differentiate the target strains against other strains of the same species using known single copy, genetic differences. The assay was optimized to include propidium monoazide pre-treatment to prevent amplification of DNA associated with dead probiotic cells as well as liberation of DNA from cells with intact membranes using bead beating. The resulting assay was able to successfully enumerate each strain whether alone or in multiplex. The cdPCR method had a 4 and 5% relative standard deviation (RSD for Bl-04 and NCFM, respectively, making it more precise than plate counts with an industry accepted RSD of 15%. cdPCR has the potential to replace traditional plate counts because of its precision, strain specificity and the ability to obtain results in a matter of hours.

  6. A Lactococcus lactis expression vector set with multiple affinity tags to facilitate isolation and direct labeling of heterologous secreted proteins

    NARCIS (Netherlands)

    Pastrana, Francisco Romero; Neef, Jolanda; van Dijl, Jan Maarten; Buist, Girbe

    The gram-positive bacterium Lactococcus lactis is a useful host for extracellular protein production. A main advantage of L. lactis over other bacterial expression systems is that lactococcal cells display low levels of autolysis and proteolysis. Previously, we developed a set of vectors for

  7. Bile salt tolerance of Lactococcus lactis is enhanced by expression of bile salt hydrolase thereby producing less bile acid in the cells.

    Science.gov (United States)

    Bi, Jie; Liu, Song; Du, Guocheng; Chen, Jian

    2016-04-01

    Changes of bile salt tolerance, morphology and amount of bile acid within cells were studied to evaluate the exact effects of bile salt hydrolase (BSH) on bile salt tolerance of microorganism. The effect of BSHs on the bile salt tolerance of Lactococcus lactis was examined by expressing two BSHs (BSH1 and BSH2). Growth of L. lactis expressing BSH1 or BSH2 was better under bile salt stress compared to wild-type L. lactis. As indicated by transmission electron microscopy, bile acids released by the action of BSH induced the formation of micelles around the membrane surface of cells subject to conjugated bile salt stress. A similar micelle containing bile acid was observed in the cytoplasm by liquid chromatography-mass spectrometry. BSH1 produced fewer bile acid micelles in the cytoplasm and achieved better cell growth of L. lactis compared to BSH2. Expression of BSH improved bile salt tolerance of L. lactis but excessive production by BSH of bile acid micelles in the cytoplasm inhibited cell growth.

  8. Lactococcus lactis Thioredoxin Reductase Is Sensitive to Light Inactivation

    DEFF Research Database (Denmark)

    Björnberg, Olof; Viennet, Thibault; Skjoldager, Nicklas

    2015-01-01

    Thioredoxin, involved in numerous redox pathways, is maintained in the dithiol state by the nicotinamide adenine dinucleotide phosphate-dependent flavoprotein thioredoxin reductase (TrxR). Here, TrxR from Lactococcus lactis is compared with the well-characterized TrxR from Escherichia coli. The two...... enzymes belong to the same class of low-molecular weight thioredoxin reductases and display similar kcat values (∼25 s-1) with their cognate thioredoxin. Remarkably, however, the L. lactis enzyme is inactivated by visible light and furthermore reduces molecular oxygen 10 times faster than E. coli Trx......-resolution mass spectrometric analysis of heat-extracted FAD from light-damaged TrxR revealed a mass increment of 13.979 Da, relative to that of unmodified FAD, corresponding to the addition of one oxygen atom and the loss of two hydrogen atoms. Tandem mass spectrometry confined the increase in mass...

  9. Alternatives for biosurfactants and bacteriocins extraction from Lactococcus lactis cultures produced under different pH conditions.

    Science.gov (United States)

    Rodríguez, N; Salgado, J M; Cortés, S; Domínguez, J M

    2010-08-01

    Study of the potential of Lactococcus lactis CECT-4434 as a biosurfactants and nisin (the only bacteriocin allowed to be used in the food industry) producer for industrial applications, exploiting the possibility of recovering separately both metabolites, taking into account that L. lactis is an interesting micro-organism with several applications in the food industry because it is recognized as GRAS. The results showed the ability of this strain to produce cell-bound biosurfactants, under controlled pH, and cell-bound biosurfactants and bacteriocins, when pH was not controlled. Three extraction procedures were designed to separately recover these substances. The strain L. lactis CECT-4434 showed to be a cell-bound biosurfactants and bacterocins producer when fermentations were carried out under uncontrolled pH. Both products can be recovered separately. Development of a convenient tool for the extraction of cell-bound biosurfactants and bacteriocins from the fermentation broth.

  10. The properties of Kluyveromyces lactis for the production of D ...

    African Journals Online (AJOL)

    Production of D-arabitol from lactose by Kluyveromyces lactis NBRC 1903 was investigated to make a solution for utilization of whey. It turned out that initial concentration of yeast extract, working volume and initial cell mass concentration are important factors in D-arabitol production from lactose by this strain.

  11. Disruption of a Transcriptional Repressor by an Insertion Sequence Element Integration Leads to Activation of a Novel Silent Cellobiose Transporter in Lactococcus lactis MG1363.

    Science.gov (United States)

    Solopova, Ana; Kok, Jan; Kuipers, Oscar P

    2017-12-01

    Lactococcus lactis subsp. cremoris strains typically carry many dairy niche-specific adaptations. During adaptation to the milk environment these former plant strains have acquired various pseudogenes and insertion sequence elements indicative of ongoing genome decay and frequent transposition events in their genomes. Here we describe the reactivation of a silenced plant sugar utilization cluster in an L. lactis MG1363 derivative lacking the two main cellobiose transporters, PtcBA-CelB and PtcBAC, upon applying selection pressure to utilize cellobiose. A disruption of the transcriptional repressor gene llmg_1239 by an insertion sequence (IS) element allows expression of the otherwise silent novel cellobiose transporter Llmg_1244 and leads to growth of mutant strains on cellobiose. Llmg_1239 was labeled CclR, for c ellobiose cl uster r epressor. IMPORTANCE Insertion sequences (ISs) play an important role in the evolution of lactococci and other bacteria. They facilitate DNA rearrangements and are responsible for creation of new genetic variants with selective advantages under certain environmental conditions. L. lactis MG1363 possesses 71 copies in a total of 11 different types of IS elements. This study describes yet another example of an IS-mediated adaptive evolution. An integration of IS 981 or IS 905 into a gene coding for a transcriptional repressor led to activation of the repressed gene cluster coding for a plant sugar utilization pathway. The expression of the gene cluster allowed assembly of a novel cellobiose-specific transporter and led to cell growth on cellobiose. Copyright © 2017 American Society for Microbiology.

  12. Purification and characterization of two new cell-bound bioactive compounds produced by wild Lactococcus lactis strain.

    Science.gov (United States)

    Saraiva, Margarete Alice Fontes; Brede, Dag Anders; Nes, Ingolf Figved; Baracat-Pereira, Maria Cristina; de Queiroz, Marisa Vieira; de Moraes, Célia Alencar

    2017-07-03

    Novel compounds and innovative methods are required considering that antibiotic resistance has reached a crisis point. In the study, two cell-bound antimicrobial compounds produced by Lactococcus lactis ID1.5 were isolated and partially characterized. Following purification by cationic exchange and a solid-phase C18 column, antimicrobial activity was recovered after three runs of RPC using 60% (v/v) and 100% (v/v) of 2-propanol for elution, suggesting that more than one antimicrobial compound were produced by L. lactis ID1.5, which were in this study called compounds AI and AII. The mass spectrum of AI and AII showed major intensity ions at m/z 1070.05 and 955.9 Da, respectively. The compound AI showed a spectrum of antimicrobial activity mainly against L. lactis species, while the organisms most sensitive to compound AII were Bacillus subtilis, Listeria innocua, Streptococcus pneumoniae and Pseudomonas aeruginosa. The antimicrobial activity of both compounds was suppressed by treatment with Tween 80. Nevertheless, both compounds showed high stability to heat and proteases treatments. The isolated compounds, AI and AII, showed distinct properties from other antimicrobial substances already reported as produced by L. lactis, and have a significant inhibitory effect against two clinically important respiratory pathogens. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Immunization with r-Lactococcus lactis expressing outer membrane protein A of Shigella dysenteriae type-1: evaluation of oral and intranasal route of administration.

    Science.gov (United States)

    Yagnik, B; Sharma, D; Padh, H; Desai, P

    2017-02-01

    To evaluate the comparative immunogenic potential of food grade Lactococcus lactis expressing outer membrane protein A (OmpA) of Shigella dysenteriae type-1 (SD-1) when administered either orally or intranasally. OmpA of SD-1 was cloned and expressed first in Escherichia coli and then in L. lactis. Presence of recombinant gene was confirmed by restriction enzyme digestion and immunoblot analysis. Using immobilized metal affinity chromatography, OmpA was purified from recombinant E. coliBL21 (DE3) and subcutaneously administered in BALB/c mice. Detection of OmpA-specific IgG antibodies by enzyme-linked immunosorbent assay (ELISA) confirmed the immunogenicity of OmpA. In order to establish r-L. lactis as a mucosal delivery vehicle, it was administered orally and nasally in BALB/c mice. Serum IgG and faecal IgA were assessed through ELISA to compare the relative potential of immunization routes and immunogenic potential of r-L. lactis. Immunization via the oral route proved superior to intranasal exposure. Recombinant L. lactis expressing OmpA of SD-1 was found to be immunogenic. Oral administration of r-L. lactis elicited higher systemic and mucosal immune response when compared with the nasal route. Using food grade recombinant L. lactis has implications in the development of a prophylactic against multidrug-resistant Shigella, which can be used as a prospective vaccine candidate. Evaluating mucosal routes of immunization demonstrated that the oral route of administration elicited better immune response against OmpA of Shigella. © 2016 The Society for Applied Microbiology.

  14. Yeast Kluyveromyces lactis as host for expression of the bacterial lipase: cloning and adaptation of the new lipase gene from Serratia sp.

    Science.gov (United States)

    Šiekštelė, Rimantas; Veteikytė, Aušra; Tvaska, Bronius; Matijošytė, Inga

    2015-10-01

    Many microbial lipases have been successfully expressed in yeasts, but not in industrially attractive Kluyveromyces lactis, which among other benefits can be cultivated on a medium supplemented with whey--cheap and easily available industrial waste. A new bacterial lipase from Serratia sp. was isolated and for the first time expressed into the yeast Kluyveromyces lactis by heterologous protein expression system based on a strong promoter of Kluyveromyces marxianus triosephosphate isomerase gene and signal peptide of Kluyveromyces marxianus endopolygalacturonase gene. In addition, the bacterial lipase gene was synthesized de novo by taking into account a codon usage bias optimal for K. lactis and was expressed into the yeast K. lactis also. Both resulting strains were characterized by high output level of the target protein secreted extracellularly. Secreted lipases were characterized for activity and stability.

  15. Engineering signal peptides for enhanced protein secretion from Lactococcus lactis.

    Science.gov (United States)

    Ng, Daphne T W; Sarkar, Casim A

    2013-01-01

    Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion from L. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels of Staphylococcus aureus nuclease and further evaluated them for secretion of Bacillus subtilis α-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered L. lactis signal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts.

  16. Lactococcus lactis expressing food-grade β-galactosidase alleviates lactose intolerance symptoms in post-weaning Balb/c mice.

    Science.gov (United States)

    Li, Jingjie; Zhang, Wen; Wang, Chuan; Yu, Qian; Dai, Ruirui; Pei, Xiaofang

    2012-12-01

    The endogenous β-galactosidase expressed in intestinal microbes is demonstrated to help humans in lactose usage, and treatment associated with the promotion of beneficial microorganism in the gut is correlated with lactose tolerance. From this point, a kind of recombinant live β-galactosidase delivery system using food-grade protein expression techniques and selected probiotics as vehicle was promoted by us for the purpose of application in lactose intolerance subjects. Previously, a recombinant Lactococcus lactis MG1363 strain expressing food-grade β-galactosidase, the L. lactis MG1363/FGZW, was successfully constructed and evaluated in vitro. This study was conducted to in vivo evaluate its efficacy on alleviating lactose intolerance symptoms in post-weaning Balb/c mice, which were orally administered with 1 × 10⁶ CFU or 1 × 10⁸ CFU of L. lactis MG1363/FGZW daily for 4 weeks before lactose challenge. In comparison with naïve mice, the mice administered with L. lactis MG1363/FGZW showed significant alleviation of diarrhea symptoms in less total feces weight within 6 h post-challenge and suppressed intestinal motility after lactose challenge, although there was no significant increase of β-galactosidase activity in small intestine. The alleviation also correlated with higher species abundance, more Bifidobacterium colonization, and stronger colonization resistance in mice intestinal microflora. Therefore, this recombinant L. lactis strain effectively alleviated diarrhea symptom induced by lactose uptake in lactose intolerance model mice with the probable mechanism of promotion of lactic acid bacteria to differentiate and predominantly colonize in gut microbial community, thus making it a promising probiotic for lactose intolerance subjects.

  17. Influence of Technological Treatments on the Functionality of Bifidobacterium lactis INL1, a Breast Milk-Derived Probiotic.

    Science.gov (United States)

    Zacarías, María Florencia; Souza, Tassia Costa; Zaburlín, Natalia; Carmona Cara, Denise; Reinheimer, Jorge; Nicoli, Jacques; Vinderola, Gabriel

    2017-10-01

    The aim of this study is to evaluate the influence of the technological processing on the functionality of the human breast milk probiotic strain Bifidobacterium lactis INL1. In vitro antagonistic activity of B. lactis INL1 was detected for Gram-positive and Gram-negative pathogens. B. lactis INL1 was administered to mice as fresh (F), frozen (Z), spray-dried (S), or lyophilized (L) culture. Immune parameters (IgA, IL-10, and IFN-γ) were determined and histological analysis was performed to assess functionality and protection capacity against Salmonella. In BALB/c mice, F and S cultures induced an increase in the number of IgA-producing cells in the small intestine and IL-10 levels were increased for L culture in the large intestine. In Swiss mice, B. lactis INL1 increased secretory-IgA levels in the small intestine before and after Salmonella infection, both as F or dehydrated culture. Also, an attenuation of damage in the intestinal epithelium and less inflammatory infiltrates were observed in animals that received F and S cultures, whereas in liver only F showed some effect. The anti-inflammatory effect was confirmed in both tissues by myeloperoxidase activity and by IFN-γ levels in the intestinal content. B. lactis INL1 showed inhibitory activity against pathogens and confirmed its probiotic potential in animal models. Technological processing of the probiotic strain affected its functionality. This work provides evidence about the influence of technology on the functionality of probiotics, which may help probiotics and functional food manufacturers to take processing into consideration when assessing the functionality of new strains. © 2017 Institute of Food Technologists®.

  18. Host lysozyme-mediated lysis of Lactococcus lactis facilitates delivery of colitis-attenuating superoxide dismutase to inflamed colons

    Science.gov (United States)

    Ballal, Sonia A.; Veiga, Patrick; Fenn, Kathrin; Michaud, Monia; Kim, Jason H.; Gallini, Carey Ann; Glickman, Jonathan N.; Quéré, Gaëlle; Garault, Peggy; Béal, Chloé; Derrien, Muriel; Courtin, Pascal; Kulakauskas, Saulius; Chapot-Chartier, Marie-Pierre; van Hylckama Vlieg, Johan; Garrett, Wendy S.

    2015-01-01

    Beneficial microbes that target molecules and pathways, such as oxidative stress, which can negatively affect both host and microbiota, may hold promise as an inflammatory bowel disease therapy. Prior work showed that a five-strain fermented milk product (FMP) improved colitis in T-bet−/− Rag2−/− mice. By varying the number of strains used in the FMP, we found that Lactococcus lactis I-1631 was sufficient to ameliorate colitis. Using comparative genomic analyses, we identified genes unique to L. lactis I-1631 involved in oxygen respiration. Respiration of oxygen results in reactive oxygen species (ROS) generation. Also, ROS are produced at high levels during intestinal inflammation and cause tissue damage. L. lactis I-1631 possesses genes encoding enzymes that detoxify ROS, such as superoxide dismutase (SodA). Thus, we hypothesized that lactococcal SodA played a role in attenuating colitis. Inactivation of the sodA gene abolished L. lactis I-1631’s beneficial effect in the T-bet−/− Rag2−/− model. Similar effects were obtained in two additional colonic inflammation models, Il10−/− mice and dextran sulfate sodium-treated mice. Efforts to understand how a lipophobic superoxide anion (O2−) can be detoxified by cytoplasmic lactoccocal SodA led to the finding that host antimicrobial-mediated lysis is a prerequisite for SodA release and SodA’s extracytoplasmic O2− scavenging. L. lactis I-1631 may represent a promising vehicle to deliver antioxidant, colitis-attenuating SodA to the inflamed intestinal mucosa, and host antimicrobials may play a critical role in mediating SodA’s bioaccessibility. PMID:26056274

  19. Growth interactions and antilisterial effects of the bacteriocinogenic Lactococcus lactis subsp. cremoris M104 and Enterococcus faecium KE82 strains in thermized milk in the presence or absence of a commercial starter culture.

    Science.gov (United States)

    Lianou, Alexandra; Kakouri, Athanasia; Pappa, Eleni C; Samelis, John

    2017-06-01

    Traditional Greek cheeses are often produced from thermized milk (TM) with the use of commercial starter cultures (CSCs), which may not inhibit growth of Listeria monocytogenes completely. Therefore, this study evaluated the behavior of an artificial L. monocytogenes contamination in commercially TM (63 °C; 30 s) inoculated with a CSC plus Lactococcus lactis subsp. lactis M104 and/or Enterococcus faecium KE82, two indigenous strains producing nisin A and enterocin A and B, respectively. Inoculation treatments included TM with the CSC only, and TM without the CSC but with strain M104 alone, or combined with strain KE82. All treatments were incubated at 37 °C for 6 h followed by 66 h at 18 °C. L. monocytogenes grew by 0.66-1.24 log cfu/ml at 37 °C, whereas its further growth at 18 °C was retarded, suppressed, or accompanied by different inactivation rates, depending on each TM treatment. Strain M104 caused the greatest inactivation, whereas the CSC per se was the least effective treatment. Strain KE82 assisted the CSC in controlling pathogen growth at 37 °C, whereas both reduced the nisin A-mediated antilisterial activity of strain M104. Overall, the most 'balanced' treatment against L. monocytogenes was CSC+M104+KE82. Hence, this starter/co-starter combination may be utilized in traditional Greek cheese technologies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Lactic Acid Bacteria from Kefir Increase Cytotoxicity of Natural Killer Cells to Tumor Cells.

    Science.gov (United States)

    Yamane, Takuya; Sakamoto, Tatsuji; Nakagaki, Takenori; Nakano, Yoshihisa

    2018-03-27

    The Japanese fermented beverage, homemade kefir, contains six lactic acid bacteria: Lactococcus. lactis subsp. Lactis , Lactococcus . lactis subsp. Cremoris , Lactococcus. Lactis subsp. Lactis biovar diacetylactis , Lactobacillus plantarum , Leuconostoc meseuteroides subsp. Cremoris and Lactobacillus casei . In this study, we found that a mixture of the six lactic acid bacteria from kefir increased the cytotoxicity of human natural killer KHYG-1 cells to human chronic myelogenous leukemia K562 cells and colorectal tumor HCT116 cells. Furthermore, levels of mRNA expression and secretion of IFN-γ (interferon gamma) increased in KHYG-1 cells that had been treated with the six lactic acid bacteria mixture from kefir. The results suggest that the six lactic acid bacteria mixture from kefir has strong effects on natural immunity and tumor cell cytotoxicity.

  1. Construction of a recombinant Lactococcus lactis strain expressing a fusion protein of Omp22 and HpaA from Helicobacter pylori for oral vaccine development.

    Science.gov (United States)

    Zhang, Rongguang; Duan, Guangcai; Shi, Qingfeng; Chen, Shuaiyin; Fan, Qingtang; Sun, Nan; Xi, Yuanlin

    2016-11-01

    To develop orally administrated anti-Helicobacter pylori vaccination, a Lactococcus lactis strain was genetically constructed for fusion expression of H. pylori protective antigens HpaA and Omp22. The fusion gene of omp22 and hpaA with an adapter encoding three glycines was cloned from a plasmid pMAL-c2x-omp22-hpaA into Escherichia coli MC1061 and L. lactis NZ3900 successively using a shutter vector pNZ8110. Expression of the fusion gene in L. lactis was induced with nisin resulting in production of proteins with molecular weights of 50 and 28 kDa. Both of them were immunoreactive with mouse anti-H. pylori sera as determined via western blotting. Oral vaccination of BALB/c mice using the L. lactis strain carrying pNZ8110-omp22-hpaA elicited significant systematic humoral immune response (P lactis with immunogenicity. This is a considerable step towards H. pylori vaccines.

  2. The properties of Kluyveromyces lactis for the production of D ...

    African Journals Online (AJOL)

    USER

    2010-08-02

    Aug 2, 2010 ... Production of D-arabitol from lactose by Kluyveromyces lactis NBRC 1903 was investigated to make a solution for ... lactose and are characterized by high biological oxygen demand ... to calculate cell mass concentration (X). One unit of .... cultures, time required for the complete conversion of lactose in ...

  3. Relationships between MDR proteins, bacteriocin production and proteolysis in Lactococcus lactis

    NARCIS (Netherlands)

    Gajic, Olivera

    2003-01-01

    The Gram-positive lactic acid bacterium Lactococcus lactis can harbour a wide variety of circular extrachromosomal DNA molecules, so-called plasmids. Many of the traits that make them useful for manufacturing of fermented food products (e.g. bacteriophage resistance, bacteriocin and proteinase

  4. Preliminary X-ray crystallographic analysis of the d-xylulose 5-phosphate phosphoketolase from Lactococcus lactis

    International Nuclear Information System (INIS)

    Petrareanu, Georgiana; Balasu, Mihaela C.; Zander, Ulrich; Scheidig, Axel J.; Szedlacsek, Stefan E.

    2010-01-01

    The expression, purification, preliminary crystallization and crystallographic analysis of phosphoketolase from L. lactis ssp. lactis (strain IL 1403) are reported. Phosphoketolases are thiamine diphosphate-dependent enzymes which play a central role in the pentose-phosphate pathway of heterofermentative lactic acid bacteria. They belong to the family of aldehyde-lyases and in the presence of phosphate ion cleave the carbon–carbon bond of the specific substrate d-xylulose 5-phosphate (or d-fructose 6-phosphate) to give acetyl phosphate and d-glyceraldehyde 3-phosphate (or d-erythrose 4-phosphate). Structural information about phosphoketolases is particularly important in order to fully understand their mechanism as well as the steric course of phosphoketolase-catalyzed reactions. Here, the purification, preliminary crystallization and crystallographic characterization of d-xylulose 5-phosphate phosphoketolase from Lactococcus lactis are reported. The presence of thiamine diphosphate during purification was essential for the enzymatic activity of the purified protein. The crystals belonged to the monoclinic space group P2 1 . Diffraction data were obtained to a resolution of 2.2 Å

  5. Identification and functional characterization of the Lactococcus lactis CodY-regulated branched-chain amino acid permease BcaP (CtrA)

    NARCIS (Netherlands)

    den Hengst, CD; Groeneveld, M; Kuipers, OP; Kok, J; Hengst, Chris D. den

    Transcriptome analyses have previously revealed that a gene encoding the putative amino acid transporter CtrA (YhdG) is one of the major targets of the pleiotropic regulator CodY in Lactococcus lactis and Bacillus subtilis. The role of ctrA in L. lactis was further investigated with respect to both

  6. Acid or erythromycin stress significantly improves transformation efficiency through regulating expression of DNA binding proteins in Lactococcus lactis F44.

    Science.gov (United States)

    Wang, Binbin; Zhang, Huawei; Liang, Dongmei; Hao, Panlong; Li, Yanni; Qiao, Jianjun

    2017-12-01

    Lactococcus lactis is a gram-positive bacterium used extensively in the dairy industry and food fermentation, and its biological characteristics are usually improved through genetic manipulation. However, poor transformation efficiency was the main restriction factor for the construction of engineered strains. In this study, the transformation efficiency of L. lactis F44 showed a 56.1-fold increase in acid condition (pH 5.0); meanwhile, erythromycin stress (0.04 μg/mL) promoted the transformation efficiency more significantly (76.9-fold). Notably, the transformation efficiency of F44e (L. lactis F44 harboring empty pLEB124) increased up to 149.1-fold under the synergistic stresses of acid and erythromycin. In addition, the gene expression of some DNA binding proteins (DprA, RadA, RadC, RecA, RecQ, and SsbA) changed correspondingly. Especially for radA, 25.1-fold improvement was detected when F44e was exposed to pH 5.0. Overexpression of some DNA binding proteins could improve the transformation efficiency. The results suggested that acid or erythromycin stress could improve the transformation efficiency of L. lactis through regulating gene expression of DNA binding proteins. We have proposed a simple but promising strategy for improving the transformation efficiency of L. lactis and other hard-transformed microorganisms. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  7. Dextran sulphate sodium colitis in C57BL/6J mice is alleviated by Lactococcus lactis and worsened by the neutralization of Tumor necrosis Factor α.

    Science.gov (United States)

    Berlec, Aleš; Perše, Martina; Ravnikar, Matjaž; Lunder, Mojca; Erman, Andreja; Cerar, Anton; Štrukelj, Borut

    2017-02-01

    TNFα has a well-established role in inflammatory bowel disease that affects the gastrointestinal tract and is usually manifested as Crohn's disease or ulcerative colitis. We have compared Lactococcus lactis NZ9000 displaying TNFα-binding affibody with control Lactococcus lactis and with anti-TNFα antibody infliximab for the treatment of mice with dextran sulphate sodium (DSS)-induced colitis. L. lactis NZ9000 alleviated the colitis severity one week after colitis induction with DSS, more effectively when administered in preventive fashion prior to, during and after DSS administration. TNFα-binding L. lactis was less effective than control L. lactis, particularly when TNFα-binding L. lactis was administered in preventive fashion. Similarly, an apparently detrimental effect of TNFα neutralization was observed in mice that were intraperitoneally administered anti-TNFα monoclonal antibody infliximab prior to colitis induction. The highest concentrations of tissue TNFα were observed in groups without DSS colitis that were treated either with TNFα-binding L. lactis or infliximab. To conclude, we have confirmed that L. lactis exerts a protective effect on DSS-induced colitis in mice. Contrary to expectations, but in line with some reports, the neutralization of TNFα aggravated disease symptoms in the acute phase of colitis and increased TNFα concentration in colon tissue of healthy mice. Nevertheless, we have demonstrated that oral administration of bacteria with surface displayed TNFα-binding affibody can interfere significantly with TNFα signaling and mimic the infliximab response in the given animal model of colitis. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Cloning of nis gene and Nisin purification from Lactococcus lactis ...

    African Journals Online (AJOL)

    The purified nisin by chloroform extraction was analyzed on 20% SDS-PAGE and gave sharp band at ~ 3.4 kDa. The 3 dimension structure of the purified Nisin was studied by CPHModels as pdb with chimera program. Keywords: Lactococcus lactis, nis cloning, 16S rRNA, chloroform extraction and SDS-PAGE

  9. Construction of an expression vector for Lactococcus lactis based on ...

    African Journals Online (AJOL)

    To construct an expression vector for Lactococcus lactis, the EmPMT fragment which contained the erythromycin resistance gene, P32 promoter, multiple cloning site (MCS) and terminator (T) was subcloned into the small cryptic plasmid pAR141. The resulting vector, designated as pAR1411, was found to be stably ...

  10. Intramammary infusion of a live culture of Lactococcus lactis in ewes to treat staphylococcal mastitis.

    Science.gov (United States)

    Mignacca, Sebastian Alessandro; Dore, Simone; Spuria, Liliana; Zanghì, Pietro; Amato, Benedetta; Duprè, Ilaria; Armas, Federica; Biasibetti, Elena; Camperio, Cristina; Lollai, Stefano A; Capucchio, Maria Teresa; Cannas, Eugenia Agnese; Di Marco Lo Presti, Vincenzo; Marianelli, Cinzia

    2017-12-01

    Alternatives to antibiotic therapy for mastitis in ruminants are needed. We present an evaluation, in two trials, of the efficacy of an intramammary infusion of a live culture of Lactococcus lactis for the treatment of subclinical and clinical mastitis in ewes. In total, 67 animals were enrolled: 19 lactating ewes (study 1), including healthy (N=6) and coagulase-negative staphylococci (CNS)-infected ewes (N=13); and 48 lactating ewes (study 2) with either CNS mastitis (N=32), or Staphylococcus aureus mastitis (N=16), for a total of 123 mammary glands. Intramammary infusions were performed with either L. lactis or PBS for 3 (study 1) or 7 (study 2) consecutive days. Antibiotic-treated and untreated control glands were included. Milk samples for microbiology, somatic cell analysis and milk production were collected before and after treatment.Results/Key findings.L. lactis rapidly activated the mammary glands' innate immune response and initiated an inflammatory response as evidenced by the recruitment of polymorphonuclear neutrophils and increased somatic cell counts. But while leading to a transient clearance of CNS in the gland, this response caused mild to moderate clinical cases of mastitis characterized by abnormal milk secretions and udder inflammation. Moreover, S. aureus infections did not improve, and CNS infections tended to relapse. Under our experimental conditions, the L. lactis treatment led to a transient clearance of the pathogen in the gland, but also caused mild to moderate clinical cases of mastitis. We believe it is still early to implement bacterial formulations as alternatives in treating mastitis in ruminants and further experimentation is needed.

  11. Genome-wide transcriptional responses to carbon starvation in nongrowing Lactococcus lactis

    NARCIS (Netherlands)

    Ercan, O.; Wels, M.; Smid, E.J.; Kleerebezem, M.

    2015-01-01

    This paper describes the transcriptional adaptations of nongrowing, retentostat cultures of Lactococcus lactis to starvation. Near-zero-growth cultures (µ = 0.0001 h-1) obtained by extended retentostat cultivation were exposed to starvation by termination of the medium supply for 24 h, followed by a

  12. In Vitro characterization of Lactococcus lactis strains Isolated from Iranian Traditional Dairy Products as a Potential Probiotic

    OpenAIRE

    Fatemeh Nejati; Tobias Oelschlaeger

    2015-01-01

    Few studies have been reported regarding probiotic properties of Lactococcus lactis strains although they are extensively used as starter cultures in the production of dairy products. In this study 8 wild isolates of Lactococcus lactis were evaluated in vitro with regard to resistance to simulated gastric and intestinal juices, adherence ability to Caco-2 cells and HT29-MTX-E12 cell lines, anti-microbial activity, hydrophobicity and antibiotic susceptibility. The results revealed that all iso...

  13. Ornithine transport and exchange in Streptococcus lactis

    International Nuclear Information System (INIS)

    Thompson, J.

    1987-01-01

    Resting cells of Streptococcus lactis 133 appeared to accumulate [ 14 C]ornithine to a high concentration in the absence of an exogenous energy source. However, analysis of intracellular amino acid pool constituents and results of transport experiments revealed that the accumulation of ornithine represented a homoexchange between extracellular [ 14 C]ornithine and unlabeled ornithine in the cell. The energy-independent exchange of ornithine was not inhibited by proton-conducting uncouplers or by metabolic inhibitors. Intracellular [ 14 C]ornithine was retained by resting cells after suspension in a buffered medium. However, addition of unlabeled ornithine to the suspension elicited rapid exit of labeled amino acid. The initial rate of exist of [ 14 C]ornithine was dependent on the concentration of unlabeled ornithine in the medium, but this accelerative exchange diffusion process caused no net loss of amino acid. By contrast, the presence of a fermentable energy source caused a rapid expulsion of and new decrease in the concentration of intracellular ornithine. Kinetic analyses of amino acid transport demonstrated competitive inhibition between lysine and ornithine, and data obtained by two-dimensional thin-layer chromatography established the heteroexchange of these basic amino acids. The effects of amino acids and of ornithine analogs on both entry and exit of [ 14 C]ornithine have been examined. The data suggest that common carrier mediates the entry and exchange of lysine, arginine, and ornithine in cells of S. lactis

  14. AguR is a transmembrane transcription activator of the putrescine biosynthesis operon in Lactococcus lactis, and acts in response to agmatine concentration

    NARCIS (Netherlands)

    Linares, Daniel M; Del Rio, Beatriz; Redruello, Begoña; Ladero, Victor; Martin, Ma Cruz; de Jong, Anne; Kuipers, Oscar P; Fernandez, Maria; Alvarez, Miguel A

    2015-01-01

    Dairy industry fermentative processes mostly use Lactococcus lactis as a starter. However, some dairy L. lactis strains produce putrescine - a biogenic amine that raises food safety and spoilage concerns - via the agmatine deiminase pathway (AGDI). The enzymatic activities responsible for putrescine

  15. Lactic Acid Bacteria from Kefir Increase Cytotoxicity of Natural Killer Cells to Tumor Cells

    Directory of Open Access Journals (Sweden)

    Takuya Yamane

    2018-03-01

    Full Text Available The Japanese fermented beverage, homemade kefir, contains six lactic acid bacteria: Lactococcus. lactis subsp. Lactis, Lactococcus. lactis subsp. Cremoris, Lactococcus. Lactis subsp. Lactis biovar diacetylactis, Lactobacillus plantarum, Leuconostoc meseuteroides subsp. Cremoris and Lactobacillus casei. In this study, we found that a mixture of the six lactic acid bacteria from kefir increased the cytotoxicity of human natural killer KHYG-1 cells to human chronic myelogenous leukemia K562 cells and colorectal tumor HCT116 cells. Furthermore, levels of mRNA expression and secretion of IFN-γ (interferon gamma increased in KHYG-1 cells that had been treated with the six lactic acid bacteria mixture from kefir. The results suggest that the six lactic acid bacteria mixture from kefir has strong effects on natural immunity and tumor cell cytotoxicity.

  16. Multi-omics approach to study the growth efficiency and amino acid metabolism in Lactococcus lactis at various specific growth rates

    Directory of Open Access Journals (Sweden)

    Arike Liisa

    2011-02-01

    Full Text Available Abstract Background Lactococcus lactis is recognised as a safe (GRAS microorganism and has hence gained interest in numerous biotechnological approaches. As it is fastidious for several amino acids, optimization of processes which involve this organism requires a thorough understanding of its metabolic regulations during multisubstrate growth. Results Using glucose limited continuous cultivations, specific growth rate dependent metabolism of L. lactis including utilization of amino acids was studied based on extracellular metabolome, global transcriptome and proteome analysis. A new growth medium was designed with reduced amino acid concentrations to increase precision of measurements of consumption of amino acids. Consumption patterns were calculated for all 20 amino acids and measured carbon balance showed good fit of the data at all growth rates studied. It was observed that metabolism of L. lactis became more efficient with rising specific growth rate in the range 0.10 - 0.60 h-1, indicated by 30% increase in biomass yield based on glucose consumption, 50% increase in efficiency of nitrogen use for biomass synthesis, and 40% reduction in energy spilling. The latter was realized by decrease in the overall product formation and higher efficiency of incorporation of amino acids into biomass. L. lactis global transcriptome and proteome profiles showed good correlation supporting the general idea of transcription level control of bacterial metabolism, but the data indicated that substrate transport systems together with lower part of glycolysis in L. lactis were presumably under allosteric control. Conclusions The current study demonstrates advantages of the usage of strictly controlled continuous cultivation methods combined with multi-omics approach for quantitative understanding of amino acid and energy metabolism of L. lactis which is a valuable new knowledge for development of balanced growth media, gene manipulations for desired product

  17. Physiological Studies of Lactococcus lactis

    DEFF Research Database (Denmark)

    Hansen, Gunda

    medium and (specific) acidification activity in milk in response to the extracellular pH (pHex) during batch fermentations and in response to stress during downstream processing and storage as frozen, freeze- and spray-dried cells. In this PhD thesis, in situ flow cytometric viability assessment...... fermentation, downstream processing and storage in the absence of a protectant. However, storing freeze-dried L. lactis cells at 30 °C negatively affected the culturability and acidification activity. The reactivation of freeze-dried cells in fermentation medium prior to flow cytometric viability assessment...

  18. Oligomerized backbone pilin helps piliated Lactococcus lactis to withstand shear flow.

    Science.gov (United States)

    Castelain, Mickaël; Duviau, Marie-Pierre; Oxaran, Virginie; Schmitz, Philippe; Cocaign-Bousquet, Muriel; Loubière, Pascal; Piard, Jean-Christophe; Mercier-Bonin, Muriel

    2016-09-01

    The present work focuses on the role of pili present at the cell surface of Lactococcus lactis in bacterial adhesion to abiotic (hydrophobic polystyrene) and biotic (mucin-coated polystyrene) surfaces. Native pili-displaying strains and isogenic derivatives in which pilins or sortase C structural genes had been modified were used. Surface physico-chemistry, morphology and shear-flow-induced detachment of lactococcal cells were evaluated. The involvement of pili in L. lactis adhesion was clearly demonstrated, irrespective of the surface characteristics (hydrophobic/hydrophilic, presence or not of specific binding sites). The accessory pilin, PilC, and the backbone pilin, PilB, were revealed to play a major role in adhesion, provided that the PilB was present in its polymerized form. Within the population fraction that remained attached to the surface under increasing shear flow, different association behaviors were observed, showing that pili could serve as anchoring sites thus hampering the effect of shear flow on cell orientation and detachment.

  19. Induced mutants of Streptococcus lactis by gamma irradiation and its effect on milk acidity

    International Nuclear Information System (INIS)

    Daowd, A.H.; Sabbour, M.M.; Newigy, N.A.; Wahab, G.A.M.

    1988-01-01

    Streptococus lactis was exposed to different doses of gamma-irradiation (10, 50, 100 and 150 Kr). The results of acidity production in sterilized milk inoculated by isolates from radiation treatments and control could be summarized in the following: 1. The mean acidity produced by S. lactis isolates after irradiation at 10 Kr increased to be 0.66% than that of control isolates (0.62%). The acidity produced by the isolates of the 50 Kr treatment showed more increment to reach the peak (0.7%). Thereafter, acidity production decreased by isolates of the 100 Kr (0.53%) and 150 Kr (0.51%) treatments. Heme, the 50 Kr treatment could be considered activation dose to S. lactis starter for acid production. 2. Two mutants were selected. Acidity production by mutant I (from 10 Kr treatment) was 0.95%, and that of mutant II (from 50 Kr treatment) was 1.0%, while acid production by the parent S. lactis culture was 0.62%. Concerning the stability of the isolates, it was found that acid production by mutant I and mutant II slightly decreased by time. The mutants were re-irradiated after 37 and 60 days at doses 10, 25 and 50 Kr. Acid production in milk by isolates of the radiation treatments was determined. The following results were obtained: -The re-irradiation of the mutants decreased the ability of the isolates for acid production than that of parent mutants. -The re-irradiation of the mutants after 60 days yielded isolates which showed more reduction in acid produced than of isolates obtained from re-irridation of the mutants after 37-days. -The higher the dose of the re-irradiation of the mutants, the lower the mean of acid production by isolates of the treatment

  20. Physicochemical and functional characterization of a biosurfactant produced by Lactococcus lactis 53

    NARCIS (Netherlands)

    Rodrigues, LR; Teixeira, JA; van der Mei, HC; Oliveira, R

    2006-01-01

    Isolation and identification of key components of the crude biosurfactant produced by Lactococcus lactis 53 was studied. Fractionation was achieved by hydrophobic interaction chromatography which allowed the isolation of a fraction rich in glycoproteins. Molecular (by Fourier transform infrared

  1. Metabolism of [14C] bicarbonate by Streptococcus lactis: the synthesis, uptake and excretion of aspartate by resting cells

    International Nuclear Information System (INIS)

    Hillier, A.J.; Rice, G.H.; Jago, G.R.

    1978-01-01

    Resting cells of Streptococcus lactis C10 were able to synthesize aspartic acid de novo but could not actively transport aspartic acid into the cell. Intracellular aspartate was excreted from the cell in the presence of glucose but did not exchange with any extracellular amino acids. The results indicate that Str. lactis C10 obtains the aspartic acid it requires for growth by bicarbonate fixation instead of by the utilization of extracellular aspartic acid. (author)

  2. Bacteriocin producers from traditional food products

    Directory of Open Access Journals (Sweden)

    Thonart P.

    2007-01-01

    Full Text Available A total of 220 strains of LAB isolated from 32 samples of traditional fermented food from Senegal were screened for bacteriocin production. Two bacteriocin producers, Lactococcus lactis subsp. lactis and Enterococcus faecium, were identified from 12 bacteriocin-producing isolates on the basis of phenotypic analyses and 16S rDNA sequence. Both bacteriocins produced by new isolates show antimicrobial activity against Listeria monocytogenes and Bacillus coagulans whereas only that produced by Lactococcus lactis has an activity against Bacillus cereus. Bacteriocin-producing Lactococcus lactis strains were found in a variety of traditional foods indicating a high potential of growth of this strain in variable ecological complex environment. Partial 16S rDNA of the two bacteriocin producers obtained in this study has been registered to Genbank databases under the accession number AY971748 for Lactococcus lactis subsp. lactis (named CWBI-B1410 and AY971749 for Enterococcus faecium (named CWBI-B1411. The new bacteriocin-producing Lactococcus lactis subsp. lactis strain has been selected for identification and application of the bacteriocin to food preservation.

  3. Lactococcus lactis is an Efficient Expression System for Mammalian Membrane Proteins Involved in Liver Detoxification, CYP3A4, and MGST1.

    Science.gov (United States)

    Bakari, Sana; Lembrouk, Mehdi; Sourd, Laura; Ousalem, Fares; André, François; Orlowski, Stéphane; Delaforge, Marcel; Frelet-Barrand, Annie

    2016-04-01

    Despite the great importance of human membrane proteins involved in detoxification mechanisms, their wide use for biochemical approaches is still hampered by several technical difficulties considering eukaryotic protein expression in order to obtain the large amounts of protein required for functional and/or structural studies. Lactococcus lactis has emerged recently as an alternative heterologous expression system to Escherichia coli for proteins that are difficult to express. The aim of this work was to check its ability to express mammalian membrane proteins involved in liver detoxification, i.e., CYP3A4 and two isoforms of MGST1 (rat and human). Genes were cloned using two different strategies, i.e., classical or Gateway-compatible cloning, and we checked the possible influence of two affinity tags (6×-His-tag and Strep-tag II). Interestingly, all proteins could be successfully expressed in L. lactis at higher yields than those previously obtained for these proteins with classical expression systems (E. coli, Saccharomyces cerevisiae) or those of other eukaryotic membrane proteins expressed in L. lactis. In addition, rMGST1 was fairly active after expression in L. lactis. This study highlights L. lactis as an attractive system for efficient expression of mammalian detoxification membrane proteins at levels compatible with further functional and structural studies.

  4. Microbial domestication signatures of Lactococcus lactis can be reproduced by experimental evolution

    NARCIS (Netherlands)

    Bachmann, H.; Starrenburg, M.J.C.; Molenaar, D.; Kleerebezem, M.; Hylckama Vlieg, van J.E.T.

    2012-01-01

    Experimental evolution is a powerful approach to unravel how selective forces shape microbial genotypes and phenotypes. To this date, the available examples focus on the adaptation to conditions specific to the laboratory. The lactic acid bacterium Lactococcus lactis naturally occurs on plants and

  5. Microbial domestication signatures of Lactococcus lactis can be reproduced by experimental evolution.

    NARCIS (Netherlands)

    Bachmann, H.; Starrenburg, M.J.; Molenaar, D.; Kleerebezem, M.; van Hylckama Vlieg, J.E.T.

    2012-01-01

    Experimental evolution is a powerful approach to unravel how selective forces shape microbial genotypes and phenotypes. To this date, the available examples focus on the adaptation to conditions specific to the laboratory. The lactic acid bacterium Lactococcus lactis naturally occurs on plants and

  6. Isolation and characterisation of an enterocin P-producing Enterococcus lactis strain from a fresh shrimp (Penaeus vannamei).

    Science.gov (United States)

    Ben Braïek, Olfa; Ghomrassi, Hamdi; Cremonesi, Paola; Morandi, Stefano; Fleury, Yannick; Le Chevalier, Patrick; Hani, Khaled; Bel Hadj, Omrane; Ghrairi, Taoufik

    2017-06-01

    Screening for lactic acid bacteria (LAB) from fresh shrimp samples (Penaeus vannamei) collected from retail seafood markets in the Tunisian's coast, resulted in the isolation of an Enterococcus strain termed Q1. This strain was selected for its antagonistic activity against pathogenic bacteria such as Listeria monocytogenes, Pseudomonas aeruginosa, Lactococcus garvieae and against fungi (Aspergillus niger and Fusarium equiseti). The Q1 strain was characterised using standard morphological and biochemical tests, growth assays at different temperatures, pH and salinity. 16S rRNA, rpoA and pheS gene sequencing, as well as the 16S-23S rRNA intergenic spacer analyses, were combined to identify strain Q1 as a strain of Enterococcus lactis. The bacteriocin produced by E. lactis Q1 is thermostable, active in the pH range from 4.0 to 9.0 and has a bactericidal mode of action. The enterocin P structural gene was detected by specific PCR in strain E. lactis Q1, which is in good agreement with SDS-PAGE data of the purified bacteriocin. A lack of significant antibiotic resistance genes and virulence determinants was confirmed by specific PCRs. This work provides the first description of an enterocin P producer E. lactis strain isolated from a fresh shrimp. Based on its safety properties (absence of haemolytic activity, virulence factors and antibiotic resistance genes), this strain has the potential to be used as a natural additive or adjunct protective culture in food biopreservation and/or probiotic culture.

  7. Natural sweetening of food products by engineering Lactococcus lactis for glucose production

    NARCIS (Netherlands)

    Pool, Wietske A.; Neves, Ana Rute; Kok, Jan; Santos, Helena; Kuipers, Oscar P.

    We show that sweetening of food products by natural fermentation can be achieved by a combined metabolic engineering and transcriptome analysis approach. A Lactococcus lactis ssp. cremoris strain was constructed in which glucose metabolism was completely disrupted by deletion of the genes coding for

  8. An exoproteome approach to monitor safety of a cheese-isolated Lactococcus lactis

    DEFF Research Database (Denmark)

    Genovese, Federica; Coïsson, Jean Daniel; Majumder, Avishek

    2013-01-01

    . A DIGE comparative exoproteomic analysis was performed on the L. lactis 11D strain grown on glucose and the disaccharide trehalose, examined here due to its common use as lyophilization stabilizer, respectively. The experiment showed that chitinase biosynthesis was enhanced in presence of trehalose...

  9. Characterization of the Lactococcus lactis lactose genes and regulation of their expression

    NARCIS (Netherlands)

    Rooijen, van R.J.

    1993-01-01

    An important trait of the lactic acid bacterium Lactococcus lactis , that is used in industrial dairy fermentations, is the conversion of lactose into lactic acid. The enzymatic steps involved in the breakdown of lactose, that is transported into the cell via a

  10. Butanol is cytotoxic to Lactococcus lactis while ethanol and hexanol are cytostatic

    DEFF Research Database (Denmark)

    Hviid, Anne-Mette Meisner; Jensen, Peter Ruhdal; Kilstrup, Mogens

    2017-01-01

    resistant lactic acid bacteria. Combined results from alcohol survival rate, live/dead staining, and a novel usage of the beta-galactosidase assay, revealed that while high concentrations of ethanol and hexanol were cytostatic to L. lactis, high concentrations of butanol were cytotoxic, causing irreparable...

  11. Increased biomass yield of Lactococcus lactis during energetically limited growth and respiratory conditions

    DEFF Research Database (Denmark)

    Købmann, Brian Jensen; Blank, Lars Mathias; Solem, Christian

    2008-01-01

    (glucose/mannose-specific phosphotransferase system). Amino acid catabolism could be excluded as the source of the additional ATP. Since mutants without a functional H+-ATPase produced less ATP under sugar starvation and respiratory conditions, the additional ATP yield appears to come partly from energy......Lactococcus lactis is known to be capable of respiration under aerobic conditions in the presence of haemin. In the present study the effect of respiration on ATP production during growth on different sugars was examined. With glucose as the sole carbon source, respiratory conditions in L. lactis...... MG1363 resulted in only a minor increase, 21%, in biomass yield. Since ATP production through substrate-level phosphorylation was essentially identical with and without respiration, the increased biomass yield was a result of energy-saving under respiratory conditions estimated to be 0.4 mol of ATP...

  12. Construction of a food-grade multiple-copy integration system for Lactococcus lactis

    NARCIS (Netherlands)

    Leenhouts, K.; Bolhuis, A.; Venema, G.; Kok, J.

    A food-grade vector system was developed that allows stable integration of multiple plasmid copies in the chromosome of Lactococcus lactis. The vector consists of the plus origin of replication (Ori(+)) of the lactococcal plasmid pWV01, the sucrose genes of the lactic acid bacterium Pediococcus

  13. Molecular insights on the recognition of a Lactococcus lactis cell wall pellicle by the phage 1358 receptor binding protein.

    Science.gov (United States)

    Farenc, Carine; Spinelli, Silvia; Vinogradov, Evgeny; Tremblay, Denise; Blangy, Stéphanie; Sadovskaya, Irina; Moineau, Sylvain; Cambillau, Christian

    2014-06-01

    The Gram-positive bacterium Lactococcus lactis is used for the production of cheeses and other fermented dairy products. Accidental infection of L. lactis cells by virulent lactococcal tailed phages is one of the major risks of fermentation failures in industrial dairy factories. Lactococcal phage 1358 possesses a host range limited to a few L. lactis strains and strong genomic similarities to Listeria phages. We report here the X-ray structures of phage 1358 receptor binding protein (RBP) in complex with monosaccharides. Each monomer of its trimeric RBP is formed of two domains: a "shoulder" domain linking the RBP to the rest of the phage and a jelly roll fold "head/host recognition" domain. This domain harbors a saccharide binding crevice located in the middle of a monomer. Crystal structures identified two sites at the RBP surface, ∼8 Å from each other, one accommodating a GlcNAc monosaccharide and the other accommodating a GlcNAc or a glucose 1-phosphate (Glc1P) monosaccharide. GlcNAc and GlcNAc1P are components of the polysaccharide pellicle that we identified at the cell surface of L. lactis SMQ-388, the host of phage 1358. We therefore modeled a galactofuranose (Galf) sugar bridging the two GlcNAc saccharides, suggesting that the trisaccharidic motif GlcNAc-Galf-GlcNAc (or Glc1P) might be common to receptors of genetically distinct lactococcal phages p2, TP091-1, and 1358. Strain specificity might therefore be elicited by steric clashes induced by the remaining components of the pellicle hexasaccharide. Taken together, these results provide a first insight into the molecular mechanism of host receptor recognition by lactococcal phages. Siphophages infecting the Gram-positive bacterium Lactococcus lactis are sources of milk fermentation failures in the dairy industry. We report here the structure of the pellicle polysaccharide from L. lactis SMQ-388, the specific host strain of phage 1358. We determined the X-ray structures of the lytic lactococcal phage

  14. Improved ethanol tolerance of Saccharomyces cerevisiae in mixed cultures with Kluyveromyces lactis on high-sugar fermentation.

    Science.gov (United States)

    Yamaoka, Chizuru; Kurita, Osamu; Kubo, Tomoko

    2014-12-01

    The influence of non-Saccharomyces yeast, Kluyveromyces lactis, on metabolite formation and the ethanol tolerance of Saccharomyces cerevisiae in mixed cultures was examined on synthetic minimal medium containing 20% glucose. In the late stage of fermentation after the complete death of K. lactis, S. cerevisiae in mixed cultures was more ethanol-tolerant than that in pure culture. The chronological life span of S. cerevisiae was shorter in pure culture than mixed cultures. The yeast cells of the late stationary phase both in pure and mixed cultures had a low buoyant density with no significant difference in the non-quiescence state between both cultures. In mixed cultures, the glycerol contents increased and the alanine contents decreased when compared with the pure culture of S. cerevisiae. The distinctive intracellular amino acid pool concerning its amino acid concentrations and its amino acid composition was observed in yeast cells with different ethanol tolerance in the death phase. Co-cultivation of K. lactis seems to prompt S. cerevisiae to be ethanol tolerant by forming opportune metabolites such as glycerol and alanine and/or changing the intracellular amino acid pool. Copyright © 2014 Elsevier GmbH. All rights reserved.

  15. From field to fermentation: the origins of Lactococcus lactis and its domestication to the dairy environment.

    Science.gov (United States)

    Cavanagh, Daniel; Fitzgerald, Gerald F; McAuliffe, Olivia

    2015-05-01

    Lactococcus lactis is an organism of substantial economic importance, used extensively in the production of fermented foods and widely held to have evolved from plant strains. The domestication of this organism to the milk environment is associated with genome reduction and gene decay, and the acquisition of specific genes involved in protein and lactose utilisation by horizontal gene transfer. In recent years, numerous studies have focused on uncovering the physiology and molecular biology of lactococcal strains from the wider environment for exploitation in the dairy industry. This in turn has facilitated comparative genome analysis of lactococci from different environments and provided insight into the natural phenotypic and genetic diversity of L. lactis. This diversity may be exploited in dairy fermentations to develop products with improved quality and sensory attributes. In this review, we discuss the classification of L. lactis and the problems that arise with phenotype/genotype designation. We also discuss the adaptation of non-dairy lactococci to milk, the traits associated with this adaptation and the potential application of non-dairy lactococci to dairy fermentations. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Expression of lycopene biosynthesis genes fused in line with Shine-Dalgarno sequences improves the stress-tolerance of Lactococcus lactis.

    Science.gov (United States)

    Dong, Xiangrong; Wang, Yanping; Yang, Fengyuan; Zhao, Shanshan; Tian, Bing; Li, Tao

    2017-01-01

    Lycopene biosynthetic genes from Deinococcus radiodurans were co-expressed in Lactococcus lactis to produce lycopene and improve its tolerance to stress. Lycopene-related genes from D. radiodurans, DR1395 (crtE), DR0862 (crtB), and DR0861 (crtI), were fused in line with S hine-Dalgarno (SD) sequences and co-expressed in L. lactis. The recombinant strain produced 0.36 mg lycopene g -1  dry cell wt after 48 h fermentation. The survival rate to UV irradiation of the recombinant strain was higher than that of the non-transformed strain. The L. lactis with co-expressed genes responsible for lycopene biosynthesis from D. radiodurans produced lycopene and exhibited increased resistance to UV stress, suggesting that the recombinant strain has important application potential in food industry.

  17. Topology of a type I secretion system for bacteriocins of Lactococcus lactis

    NARCIS (Netherlands)

    Franke, Christian Marc

    1998-01-01

    This thesis describes the analysis of a number of aspects of the secretion and muturation machinery of the bacteriocin lactococcin A (LcnA) from Lactococcus lactis, whick is initially synthesized as a precursor protein (preLcnA), containing an N-terminal extension of 20 amino acids (the leader)....

  18. Transcriptome landscape of Lactococcus lactis reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and metabolism.

    Science.gov (United States)

    van der Meulen, Sjoerd B; de Jong, Anne; Kok, Jan

    2016-01-01

    RNA sequencing has revolutionized genome-wide transcriptome analyses, and the identification of non-coding regulatory RNAs in bacteria has thus increased concurrently. Here we reveal the transcriptome map of the lactic acid bacterial paradigm Lactococcus lactis MG1363 by employing differential RNA sequencing (dRNA-seq) and a combination of manual and automated transcriptome mining. This resulted in a high-resolution genome annotation of L. lactis and the identification of 60 cis-encoded antisense RNAs (asRNAs), 186 trans-encoded putative regulatory RNAs (sRNAs) and 134 novel small ORFs. Based on the putative targets of asRNAs, a novel classification is proposed. Several transcription factor DNA binding motifs were identified in the promoter sequences of (a)sRNAs, providing insight in the interplay between lactococcal regulatory RNAs and transcription factors. The presence and lengths of 14 putative sRNAs were experimentally confirmed by differential Northern hybridization, including the abundant RNA 6S that is differentially expressed depending on the available carbon source. For another sRNA, LLMGnc_147, functional analysis revealed that it is involved in carbon uptake and metabolism. L. lactis contains 13% leaderless mRNAs (lmRNAs) that, from an analysis of overrepresentation in GO classes, seem predominantly involved in nucleotide metabolism and DNA/RNA binding. Moreover, an A-rich sequence motif immediately following the start codon was uncovered, which could provide novel insight in the translation of lmRNAs. Altogether, this first experimental genome-wide assessment of the transcriptome landscape of L. lactis and subsequent sRNA studies provide an extensive basis for the investigation of regulatory RNAs in L. lactis and related lactococcal species.

  19. Oral administration of Lactococcus lactis-expressing heat shock protein 65 and tandemly repeated IA2P2 prevents type 1 diabetes in NOD mice.

    Science.gov (United States)

    Liu, Kun-Feng; Liu, Xiao-Rui; Li, Guo-Liang; Lu, Shi-Ping; Jin, Liang; Wu, Jie

    2016-06-01

    Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease characterized by the destruction of insulin-secreting β cells upon autoreactive T cell attack. Oral administration of autoantigens is an attractive approach to treating T1DM, but an effective carrier should be used in order to protect antigens. Lactococcus lactis, a safe engineering strain, was used for this task in the present study. Two recombinant L. lactis expressing protein HSP65-6IA2P2 were used and be investigated the effects and mechanisms against T1DM in NOD mice. Our findings demonstrate that recombinant L. lactis strains can successfully both deliver antigens to intestinal mucosa and maintain the epitopes for a long time in NOD mice. Oral administration of recombinant L. lactis could prevent hyperglycemia, improve glucose tolerance, and reduce insulitis by inhibiting antigen-specific proliferation of T cells, augmenting regulatory immune reactions, and balancing ratios of Th17/Tregs and Th1/Th2. These results prove that orally administrated L. lactis expressing HSP65-6IA2P2 is an effective approach for the prevention of T1DM in NOD mice. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  20. Biosorption of silver cations onto Lactococcus lactis and Lactobacillus casei isolated from dairy products.

    Directory of Open Access Journals (Sweden)

    Maciej Milanowski

    Full Text Available The current work deals with the phenomenon of silver cations uptake by two kinds of bacteria isolated from dairy products. The mechanism of sorption of silver cations by Lactococcus lactis and Lactobacillus casei bacteria was investigated. Inductively coupled plasma-mass spectrometry (ICP-MS was used for determination of silver concentration sorbed by bacteria. Analysis of charge distribution was conducted by diffraction light scattering method. Changes in the ultrastructure of Lactococcus lactis and Lactobacillus casei cells after treatment with silver cations were investigated using transmission electron microscopy observation. Molecular spectroscopy methods, namely Fourier transform-infrared spectroscopy (FT-IR and matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS were employed for description of the sorption mechanism. Moreover, an analysis of volatile organic compounds (VOCs extracted from bacterial cells was performed.

  1. Hsp65-producing Lactococcus lactis prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells

    Science.gov (United States)

    Rezende, Rafael M.; Oliveira, Rafael P.; Medeiros, Samara R.; Gomes-Santos, Ana C.; Alves, Andrea C.; Loli, Flávia G.; Guimarães, Mauro A.F.; Amaral, Sylvia S.; da Cunha, André P.; Weiner, Howard L.; Azevedo, Vasco; Miyoshi, Anderson; Faria, Ana M.C.

    2013-01-01

    Heat shock proteins (Hsps) participate in the cellular response to stress and they are hiperexpressed in inflammatory conditions. They are also known to play a major role in immune modulation, controlling, for instance, autoimmune responses. In this study, we showed that oral administration of a recombinant Lactococcus lactis strain that produces and releases LPS-free Hsp65 prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. This was confirmed by the reduced inflammatory cell infiltrate and absence of injury signs in the spinal cord. The effect was associated with reduced IL-17 and increased IL-10 production in mesenteric lymph node and spleen cell cultures. Hsp65-producing-L. lactis-fed mice had a remarkable increase in the number of natural and inducible CD4+Foxp3+ regulatory T (Treg) cells and CD4+LAP+ (Latency-associated peptide) Tregs - which express the membrane-bound TGF-β - in spleen, inguinal and mesenteric lymph nodes as well as in spinal cord. Moreover, many Tregs co-expressed Foxp3 and LAP. In vivo depletion of LAP+ cells abrogated the effect of Hsp65-producing L. lactis in EAE prevention and worsened disease in medium-fed mice. Thus, Hsp65-L.lactis seems to boost this critical regulatory circuit involved in controlling EAE development in mice. PMID:22939403

  2. Plasmid Complement of Lactococcus lactis NCDO712 Reveals a Novel Pilus Gene Cluster.

    Science.gov (United States)

    Tarazanova, Mariya; Beerthuyzen, Marke; Siezen, Roland; Fernandez-Gutierrez, Marcela M; de Jong, Anne; van der Meulen, Sjoerd; Kok, Jan; Bachmann, Herwig

    2016-01-01

    Lactococcus lactis MG1363 is an important gram-positive model organism. It is a plasmid-free and phage-cured derivative of strain NCDO712. Plasmid-cured strains facilitate studies on molecular biological aspects, but many properties which make L. lactis an important organism in the dairy industry are plasmid encoded. We sequenced the total DNA of strain NCDO712 and, contrary to earlier reports, revealed that the strain carries 6 rather than 5 plasmids. A new 50-kb plasmid, designated pNZ712, encodes functional nisin immunity (nisCIP) and copper resistance (lcoRSABC). The copper resistance could be used as a marker for the conjugation of pNZ712 to L. lactis MG1614. A genome comparison with the plasmid cured daughter strain MG1363 showed that the number of single nucleotide polymorphisms that accumulated in the laboratory since the strains diverted more than 30 years ago is limited to 11 of which only 5 lead to amino acid changes. The 16-kb plasmid pSH74 was found to contain a novel 8-kb pilus gene cluster spaCB-spaA-srtC1-srtC2, which is predicted to encode a pilin tip protein SpaC, a pilus basal subunit SpaB, and a pilus backbone protein SpaA. The sortases SrtC1/SrtC2 are most likely involved in pilus polymerization while the chromosomally encoded SrtA could act to anchor the pilus to peptidoglycan in the cell wall. Overexpression of the pilus gene cluster from a multi-copy plasmid in L. lactis MG1363 resulted in cell chaining, aggregation, rapid sedimentation and increased conjugation efficiency of the cells. Electron microscopy showed that the over-expression of the pilus gene cluster leads to appendices on the cell surfaces. A deletion of the gene encoding the putative basal protein spaB, by truncating spaCB, led to more pilus-like structures on the cell surface, but cell aggregation and cell chaining were no longer observed. This is consistent with the prediction that spaB is involved in the anchoring of the pili to the cell.

  3. Phosphoglycerate Mutase Is a Highly Efficient Enzyme without Flux Control in Lactococcus lactis

    DEFF Research Database (Denmark)

    Solem, Christian; Petranovic, D.; Købmann, Brian

    2010-01-01

    The glycolytic enzyme phosphoglycerate mutase (PGM), which catalyzes the conversion of 3-phosphoglycerate to 2-phosphoglycerate, was examined in Lactococcus lactis with respect to its function, kinetics and glycolytic flux control. A library of strains with PGM activities ranging between 15-465% ...

  4. Spray drying of starter cultures: Diverse solutions within Lactococcus lactis to improve robustness

    NARCIS (Netherlands)

    Dijkstra, A.R.

    2015-01-01

    This thesis describes the assessment and the possible exploitation of the natural diversity within Lactococcus lactis strains with respect to robustness. Special focus was on survival during heat and oxidative stress, which are both important parameters for optimal performance and survival during

  5. Molecular characterization and exploitation of the temperate Lactococcus lactis bacteriophage r1t

    NARCIS (Netherlands)

    Nauta, Arjen

    1997-01-01

    When comprehending the scale at wich the Gram-positive lactic acid bacterium Lactococcus lactis is used in the dairy-industry, one can imagine the consequences of fermentation failures. To date, the most serious threat to these large scale fermentation processes. ... Zie Summary

  6. Biofortification of riboflavin and folate in idli batter, based on fermented cereal and pulse, by Lactococcus lactis N8 and Saccharomyces boulardii SAA655.

    Science.gov (United States)

    Chandrasekar Rajendran, S C; Chamlagain, B; Kariluoto, S; Piironen, V; Saris, P E J

    2017-06-01

    Lactococcus lactis N8 and Saccharomyces boulardii SAA655 were investigated for their ability to synthesize B-vitamins (riboflavin and folate) and their functional role as microbial starters in idli fermentation. In this study, ultra-high performance liquid chromatography and microbiological assay were used to determine the total riboflavin and folate content respectively. Increased levels of folate were evident in both L. lactis N8 and S. boulardii SAA655 cultivated medium. Enhanced riboflavin levels were found only in S. boulardii SAA655 grown medium, whereas decreased riboflavin level was found in L. lactis N8 cultivated medium. To evaluate the functional role of microbial starter strains, L. lactis N8 and S. boulardii SAA655 were incorporated individually and in combination into idli batter, composed of wet grounded rice and black gram. For the experiments, naturally fermented idli batter was considered as control. The results indicated that natural idli fermentation did not enhance the riboflavin level and depleted folate levels by half. In comparison with control, L. lactis N8 and S. boulardii SAA655 incorporated idli batter (individually and in combination) increased riboflavin and folate levels by 40-90%. Apart from compensating the folate loss caused by natural fermentation, S. boulardii SAA655 fermented idli batter individually and in combination with L. lactis N8 also showed the highest leavening character. Moreover, the microbial starter incorporation did not significantly influence the pH of idli batter. Incorporation of L. lactis N8 and S. boulardii SAA655 can evidently enhance the functional and technological characteristics of idli batter. UN General Assembly declared 2016 the International Year of pulses emphasizing the importance of legumes as staple food. Furthermore, this is the first experimental report of in situ biofortifcation of riboflavin and folate using microbes in pulse based fermented staple food. The current study suggests possible

  7. Efficient Overproduction of Membrane Proteins in Lactococcus lactis Requires the Cell Envelope Stress Sensor/Regulator Couple CesSR

    Science.gov (United States)

    Pinto, Joao P. C.; Kuipers, Oscar P.; Marreddy, Ravi K. R.; Poolman, Bert; Kok, Jan

    2011-01-01

    Background Membrane proteins comprise an important class of molecules whose study is largely frustrated by several intrinsic constraints, such as their hydrophobicity and added requirements for correct folding. Additionally, the complexity of the cellular mechanisms that are required to insert membrane proteins functionally in the membrane and to monitor their folding state makes it difficult to foresee the yields at which one can obtain them or to predict which would be the optimal production host for a given protein. Methods and Findings We describe a rational design approach to improve the lactic acid bacterium Lactococcus lactis as a producer of membrane proteins. Our transcriptome data shows that the two-component system CesSR, which senses cell envelope stresses of different origins, is one of the major players when L. lactis is forced to overproduce the endogenous membrane protein BcaP, a branched-chain amino acid permease. Growth of the BcaP-producing L. lactis strain and its capability to produce membrane proteins are severely hampered when the CesSR system itself or particular members of the CesSR regulon are knocked out, notably the genes ftsH, oxaA2, llmg_2163 and rmaB. Overexpressing cesSR reduced the growth defect, thus directly improving the production yield of BcaP. Applying this rationale to eukaryotic proteins, some of which are notoriously more difficult to produce, such as the medically-important presenilin complex, we were able to significantly diminish the growth defect seen in the wild-type strain and improve the production yield of the presenilin variant PS1Δ9-H6 more than 4-fold. Conclusions The results shed light into a key, and perhaps central, membrane protein quality control mechanism in L. lactis. Modulating the expression of CesSR benefited the production yields of membrane proteins from different origins. These findings reinforce L. lactis as a legitimate alternative host for the production of membrane proteins. PMID:21818275

  8. Perdeuteration and methyl-selective 1H, 13C-labeling by using a Kluyveromyces lactis expression system

    International Nuclear Information System (INIS)

    Miyazawa-Onami, Mayumi; Takeuchi, Koh; Takano, Toshiaki; Sugiki, Toshihiko; Shimada, Ichio; Takahashi, Hideo

    2013-01-01

    The production of stable isotope-labeled proteins is critical in structural analyses of large molecular weight proteins using NMR. Although prokaryotic expression systems using Escherichia coli have been widely used for this purpose, yeast strains have also been useful for the expression of functional eukaryotic proteins. Recently, we reported a cost-effective stable isotope-labeled protein expression using the hemiascomycete yeast Kluyveromyces lactis (K. lactis), which allow us to express exogenous proteins at costs comparable to prokaryotic expression systems. Here, we report the successful production of highly deuterated (>90 %) protein in the K. lactis system. We also examined the methyl-selective 1 H, 13 C-labeling of Ile, Leu, and Val residues using commonly used amino acid precursors. The efficiency of 1 H- 13 C-incorporation varied significantly based on the amino acid. Although a high level of 1 H- 13 C-incorporation was observed for the Ile δ1 position, 1 H, 13 C-labeling rates of Val and Leu methyl groups were limited due to the mitochondrial localization of enzymes involved in amino acid biosynthesis and the lack of transporters for α-ketoisovalerate in the mitochondrial membrane. In line with this notion, the co-expression with branched-chain-amino-acid aminotransferase in the cytosol significantly improved the incorporation rates of amino acid precursors. Although it would be less cost-effective, addition of 13 C-labeled valine can circumvent problems associated with precursors and achieve high level 1 H, 13 C-labeling of Val and Leu. Taken together, the K. lactis system would be a good alternative for expressing large eukaryotic proteins that need deuteration and/or the methyl-selective 1 H, 13 C-labeling for the sensitive detection of NMR resonances

  9. Carbon catabolite repression and global control of the carbohydrate metabolism in Lactococcus lactis

    NARCIS (Netherlands)

    Luesink, E.J.

    1998-01-01

    In view of the economic importance of fermented dairy products considerable scientific attention has been given to various steps of fermentation processes, including the L-lactate formation of lactic acid bacteria (de Vos, 1996). In particular, the carbohydrate metabolism of L. lactis has

  10. Characterization of Probiotic Fermented Milk Prepared by Different Inoculation Size of Mesophilic and Thermophilic Lactic Acid Bacteria

    Directory of Open Access Journals (Sweden)

    Sara Nasiri Boosjin

    2016-10-01

    Full Text Available Background and Objectives: Importance of development of novel probiotic fermented milk and challenge made for its acceptability is well known. In this research, the impact of different inoculation sizes of yogurt and DL-type starter culture (mesophilic and thermophilic LAB on titratable acidity, viscosity, sensorial and microbial properties of fermented milk was investigated; and finally, probiotic Langfil was produced.Materials and Methods: Fermented milk produced by 1, 2 and 3% v v-1 inocula consisting thermophilic: mesophilic starter cultures 10:90 (Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc mesenteroides subsp. cremoris. Streptococcus thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus were analyzed for determination of titratable acidity, viscosity, viability of mesophilic starter cultures and sensory properties on days 5, 10, and 15 of storage at 4°C. Then, the most suitable treatments were selected for the producing probiotic Langfil, containing probiotic starter culture (2% v v-1 inoculums with equal ratio of Lactobacillus acidophilus and Bifidobacterium bifidum. Lactococcus lactis and L. cremoris were counted on M17 agar, while Leuconostoc and Lactobacillus were counted aerobically on tomato juice agar and MRS bile agar, respectively. Bifidobacterium was cultured anaerobically on MRS bile agar. Sensory evaluation was carried out by ten trained panelists, based on a nine-point hedonic scale during the cold storage.Results and Conclusion: According to results, the best organoleptic properties were achieved in the product prepared with 2% the mesophilic and thermophilic starter cultures and 2% probiotic. This product had a high viscosity. An Iranian probiotic Langfil with desired properties was produced using the best treatment prepared.Conflict of interests: The authors declare no conflict of

  11. The Lactococcus lactis Thioredoxin System

    DEFF Research Database (Denmark)

    Efler, Petr

    -dependent thioredoxin reductase (NTR) in order to complete its catalytic cycle. Glutathione-dependent glutaredoxin complements Trx in many organisms. This thesis focuses on disulfide reduction pathways in Lactococcus lactis, an important industrial microorganism used traditionally for cheese and buttermilk production...... caused about 30% growth inhibition at non-stressed conditions and significantly increased sensitivity to oxidants (e.g. H2O2, diamide), while deletion of trxD displayed an effect predominantly in the ΔtrxAΔtrxD mutant. The ΔtrxD mutant exhibited a significantly higher sensitivity only in case of exposure......D mutants by difference gel electrophoresis (DIGE) revealed significant changes between ΔtrxA and wt. Higher levels of several oxidative stress-related proteins (e.g. glutathione peroxidase) were observed in the ΔtrxA mutant. Proteomic analysis (pulse labeling by [35S]-L-methionine) of the ΔtrxD mutant vs...

  12. Antimicrobial effect of selected lactic acid bacteria against microorganisms with decarboxylase activity

    Directory of Open Access Journals (Sweden)

    Khatantuul Purevdorj

    2017-01-01

    Full Text Available The main purpose of this study was to evaluate the antimicrobial activity of twenty-one bacteriocinogenic lactic acid bacteria (12 strains of Lactococcus lactis subsp. lactis, 4 strains of Lactobacillus gasseri, 3 strains of Lb. helveticus and 2 strains of Lb. acidophilus, LAB against 28 Staphylococcus and 33 Enterococcus strains able to produce tyramine, putrescine, 2-phenylethylamine and cadaverine. The antimicrobial activity of cell-free supernatants (CFS from tested LAB was examined by an agar-well diffusion assay. Nine out of twenty-one strains (33% showed the inhibitory effect on tested enterococci and staphylococci, namely 9 strains of Lactococcus lactis subsp. lactis. The diameters of inhibition zones ranged between 7 mm and 14 mm. The biggest diameter of 14 mm inhibition was obtained with the CFS's from strains CCDM 670 and CCDM 731 on Enterococcus sp. E16 and E28. The cell-free supernatants from Lactococcus lactis subsp. lactis CCDM 71 and from Lactococcus lactis subsp. lactis CCDM 731 displayed the broadest antibacterial activity (52% inhibition of all tested strains. On the other hand, the cell-free supernatants from the screened Lactobacillus strains did not show any inhibitory effect on the tested Staphylococcus and Enterococcus strains. Nowadays, the great attention is given to the antibacterial substances produced by lactic acid bacteria. With the ability to produce a variety of metabolites displaying inhibitory effect, the LAB have great potential in biopreservation of food.

  13. Unleashing Natural Competence in Lactococcus lactis by Induction of the Competence Regulator ComX

    NARCIS (Netherlands)

    Mulder, Joyce; Wels, Michiel; Kuipers, Oscar P; Kleerebezem, Michiel; Bron, Peter A

    2017-01-01

    In biotechnological workhorses like Streptococcus thermophilus and Bacillus subtilis, natural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starter Lactococcus lactis, natural competence has not been established to

  14. Probiotic assessment of Enterococcus durans 6HL and Lactococcus lactis 2HL isolated from vaginal microflora.

    Science.gov (United States)

    Nami, Yousef; Abdullah, Norhafizah; Haghshenas, Babak; Radiah, Dayang; Rosli, Rozita; Khosroushahi, Ahmad Yari

    2014-08-01

    Forty-five lactic acid bacteria (LAB) were isolated from the vaginal specimens of healthy fertile women, and the identities of the bacteria were confirmed by sequencing of their 16S rDNA genes. Among these bacteria, only four isolates were able to resist and survive in low pH, bile salts and simulated in vitro digestion conditions. Lactococcus lactis 2HL, Enterococcus durans 6HL, Lactobacillus acidophilus 36YL and Lactobacillus plantarum 5BL showed the best resistance to these conditions. These strains were evaluated further to assess their ability to adhere to human intestinal Caco-2 cells. Lactococcus lactis 2HL and E. durans 6HL were the most adherent strains. In vitro tests under neutralized pH proved the antimicrobial activity of both strains. Results revealed that the growth of Escherichia coli O26, Staphylococcus aureus and Shigella flexneri was suppressed by both LAB strains. The antibiotic susceptibility tests showed that these strains were sensitive to all nine antibiotics: vancomycin, tetracycline, ampicillin, penicillin, gentamicin, erythromycin, clindamycin, sulfamethoxazole and chloramphenicol. These data suggest that E. durans 6HL and Lactococcus lactis 2HL could be examined further for their useful properties and could be developed as new probiotics. © 2014 The Authors.

  15. The structure of Lactococcus lactis thioredoxin reductase reveals molecular features of photo-oxidative damage

    DEFF Research Database (Denmark)

    Skjoldager, Nicklas; Bang, Maria Blanner; Rykær, Martin

    2017-01-01

    The NADPH-dependent homodimeric flavoenzyme thioredoxin reductase (TrxR) provides reducing equivalents to thioredoxin, a key regulator of various cellular redox processes. Crystal structures of photo-inactivated thioredoxin reductase (TrxR) from the Gram-positive bacterium Lactococcus lactis have...

  16. Growth Phase-Dependent Proteomes of the Malaysian Isolated Lactococcus lactis Dairy Strain M4 Using Label-Free Qualitative Shotgun Proteomics Analysis

    Directory of Open Access Journals (Sweden)

    Theresa Wan Chen Yap

    2014-01-01

    Full Text Available Lactococcus lactis is the most studied mesophilic fermentative lactic acid bacterium. It is used extensively in the food industry and plays a pivotal role as a cell factory and also as vaccine delivery platforms. The proteome of the Malaysian isolated L. lactis M4 dairy strain, obtained from the milk of locally bred cows, was studied to elucidate the physiological changes occurring between the growth phases of this bacterium. In this study, ultraperformance liquid chromatography nanoflow electrospray ionization tandem mass spectrometry (UPLC- nano-ESI-MSE approach was used for qualitative proteomic analysis. A total of 100 and 121 proteins were identified from the midexponential and early stationary growth phases, respectively, of the L. lactis strain M4. During the exponential phase, the most important reaction was the generation of sufficient energy, whereas, in the early stationary phase, the metabolic energy pathways decreased and the biosynthesis of proteins became more important. Thus, the metabolism of the cells shifted from energy production in the exponential phase to the synthesis of macromolecules in the stationary phase. The resultant proteomes are essential in providing an improved view of the cellular machinery of L. lactis during the transition of growth phases and hence provide insight into various biotechnological applications.

  17. Growth phase-dependent proteomes of the Malaysian isolated Lactococcus lactis dairy strain M4 using label-free qualitative shotgun proteomics analysis.

    Science.gov (United States)

    Yap, Theresa Wan Chen; Rabu, Amir; Abu Bakar, Farah Diba; Rahim, Raha Abdul; Mahadi, Nor Muhammad; Illias, Rosli Md; Murad, Abdul Munir Abdul

    2014-01-01

    Lactococcus lactis is the most studied mesophilic fermentative lactic acid bacterium. It is used extensively in the food industry and plays a pivotal role as a cell factory and also as vaccine delivery platforms. The proteome of the Malaysian isolated L. lactis M4 dairy strain, obtained from the milk of locally bred cows, was studied to elucidate the physiological changes occurring between the growth phases of this bacterium. In this study, ultraperformance liquid chromatography nanoflow electrospray ionization tandem mass spectrometry (UPLC- nano-ESI-MS(E)) approach was used for qualitative proteomic analysis. A total of 100 and 121 proteins were identified from the midexponential and early stationary growth phases, respectively, of the L. lactis strain M4. During the exponential phase, the most important reaction was the generation of sufficient energy, whereas, in the early stationary phase, the metabolic energy pathways decreased and the biosynthesis of proteins became more important. Thus, the metabolism of the cells shifted from energy production in the exponential phase to the synthesis of macromolecules in the stationary phase. The resultant proteomes are essential in providing an improved view of the cellular machinery of L. lactis during the transition of growth phases and hence provide insight into various biotechnological applications.

  18. Growth Phase-Dependent Proteomes of the Malaysian Isolated Lactococcus lactis Dairy Strain M4 Using Label-Free Qualitative Shotgun Proteomics Analysis

    Science.gov (United States)

    Yap, Theresa Wan Chen; Rabu, Amir; Abu Bakar, Farah Diba; Abdul Rahim, Raha; Mahadi, Nor Muhammad; Illias, Rosli Md.

    2014-01-01

    Lactococcus lactis is the most studied mesophilic fermentative lactic acid bacterium. It is used extensively in the food industry and plays a pivotal role as a cell factory and also as vaccine delivery platforms. The proteome of the Malaysian isolated L. lactis M4 dairy strain, obtained from the milk of locally bred cows, was studied to elucidate the physiological changes occurring between the growth phases of this bacterium. In this study, ultraperformance liquid chromatography nanoflow electrospray ionization tandem mass spectrometry (UPLC- nano-ESI-MSE) approach was used for qualitative proteomic analysis. A total of 100 and 121 proteins were identified from the midexponential and early stationary growth phases, respectively, of the L. lactis strain M4. During the exponential phase, the most important reaction was the generation of sufficient energy, whereas, in the early stationary phase, the metabolic energy pathways decreased and the biosynthesis of proteins became more important. Thus, the metabolism of the cells shifted from energy production in the exponential phase to the synthesis of macromolecules in the stationary phase. The resultant proteomes are essential in providing an improved view of the cellular machinery of L. lactis during the transition of growth phases and hence provide insight into various biotechnological applications. PMID:24982972

  19. Immunogenicity of a malaria parasite antigen displayed by Lactococcus lactis in oral immunisations

    NARCIS (Netherlands)

    Ramasamy, R; Yasawardena, S; Zomer, A; Venema, G; Kok, J; Leenhouts, K

    2006-01-01

    A putative protective protein from Plasmodium falciparum merozoites, MSA2, was expressed in two different ways on the cell surface of the Gram-positive food-grade bacterium, Lactococcus lactis. The first display format exploits an LPXTG-type anchoring motif of the lactococcal proteinase PrtP to

  20. Targeting diseases with genetically engineered Lactococcus lactis and its course towards medical translation

    NARCIS (Netherlands)

    Villatoro-Hernandez, Julio; Montes-de-Oca-Luna, Roberto; Kuipers, Oscar P.

    The use of the lactic acid bacterium Lactococcus lactis, primarily used in food fermentations, as therapeutic agent is no longer speculative but an imminent reality. After the successful completion of Phase I and II clinical trials in humans for the treatment of inflammatory bowel disease, an

  1. A novel multi-locus sequence typing (MLST) protocol for Leuconostoc lactis isolates from traditional dairy products in China and Mongolia.

    Science.gov (United States)

    Dan, Tong; Liu, Wenjun; Sun, Zhihong; Lv, Qiang; Xu, Haiyan; Song, Yuqin; Zhang, Heping

    2014-06-09

    Economically, Leuconostoc lactis is one of the most important species in the genus Leuconostoc. It plays an important role in the food industry including the production of dextrans and bacteriocins. Currently, traditional molecular typing approaches for characterisation of this species at the isolate level are either unavailable or are not sufficiently reliable for practical use. Multilocus sequence typing (MLST) is a robust and reliable method for characterising bacterial and fungal species at the molecular level. In this study, a novel MLST protocol was developed for 50 L. lactis isolates from Mongolia and China. Sequences from eight targeted genes (groEL, carB, recA, pheS, murC, pyrG, rpoB and uvrC) were obtained. Sequence analysis indicated 20 different sequence types (STs), with 13 of them being represented by a single isolate. Phylogenetic analysis based on the sequences of eight MLST loci indicated that the isolates belonged to two major groups, A (34 isolates) and B (16 isolates). Linkage disequilibrium analyses indicated that recombination occurred at a low frequency in L. lactis, indicating a clonal population structure. Split-decomposition analysis indicated that intraspecies recombination played a role in generating genotypic diversity amongst isolates. Our results indicated that MLST is a valuable tool for typing L. lactis isolates that can be used for further monitoring of evolutionary changes and population genetics.

  2. Exogenous transglutaminase improves multiple-stress tolerance in Lactococcus lactis and other lactic acid bacteria with glutamine and lysine in the cell wall.

    Science.gov (United States)

    Li, Yu; Kan, Zhipeng; You, Yuanli; Gao, Xueling; Wang, Zhigeng; Fu, Ruiyan

    2015-12-01

    To increase the resistance of ingested bacteria to multiple environmental stresses, the role of transglutaminase in Lactococcus lactis and possible mechanisms of action were explored. L. lactis grown with transglutaminase exhibited significantly higher resistance to bile salts, stimulated gastric juice, antibiotics, NaCl, and cold stress compared to the control (cultured without transglutaminase), with no negative influence on cell growth. Transmission electron microscopy revealed that the cell walls of L. lactis cultured with 9 U transglutaminase/ml were approx. 1.9-times thicker than the control. Further analysis demonstrated that the multi-resistant phenotype was strain-specific; that is, it occurred in bacteria with the presence of glutamine and lysine in the peptidoglycan. Supplementation of culture media with transglutaminase is an effective, simple, and inexpensive strategy to protect specific ingested bacteria against multiple environmental challenges.

  3. Effect of different NADH oxidase levels on glucose metabolism by Lactococus lactis : kinetics of intracellular metabolite pools determined by in vivo nuclear magnetic resonance

    NARCIS (Netherlands)

    Neves, A.R.; Ramos, A.; Costa, H.; Swam, van I.I.; Hugenholtz, J.; Kleerebezem, M.; Vos, de W.M.; Santos, H.

    2002-01-01

    Three isogenic strains of Lactococcus lactis with different levels of H2O-forming NADH oxidase activity were used to study the effect of oxygen on glucose metabolism: the parent strain L. lactis MG1363, a NOX- strain harboring a deletion of the gene coding for H2O-forming NADH oxidase, and a NOX

  4. Relation of Growth of Streptococcus lactis and Streptococcus cremoris to Amino Acid Transport

    NARCIS (Netherlands)

    Poolman, Bert; Konings, Wil N.

    The maximum specific growth rate of Streptococcus lactis and Streptococcus cremoris on synthetic medium containing glutamate but no glutamine decreases rapidly above pH 7. Growth of these organisms is extended to pH values in excess of 8 in the presence of glutamine. These results can be explained

  5. Fine tuning of the lactate and diacetyl production through promoter engineering in Lactococcus lactis.

    Directory of Open Access Journals (Sweden)

    Tingting Guo

    Full Text Available Lactococcus lactis is a well-studied bacterium widely used in dairy fermentation and capable of producing metabolites with organoleptic and nutritional characteristics. For fine tuning of the distribution of glycolytic flux at the pyruvate branch from lactate to diacetyl and balancing the production of the two metabolites under aerobic conditions, a constitutive promoter library was constructed by randomizing the promoter sequence of the H(2O-forming NADH oxidase gene in L. lactis. The library consisted of 30 promoters covering a wide range of activities from 7,000 to 380,000 relative fluorescence units using a green fluorescent protein as reporter. Eleven typical promoters of the library were selected for the constitutive expression of the H(2O-forming NADH oxidase gene in L. lactis, and the NADH oxidase activity increased from 9.43 to 58.17-fold of the wild-type strain in small steps of activity change under aerobic conditions. Meanwhile, the lactate yield decreased from 21.15 ± 0.08 mM to 9.94 ± 0.07 mM, and the corresponding diacetyl production increased from 1.07 ± 0.03 mM to 4.16 ± 0.06 mM with the intracellular NADH/NAD(+ ratios varying from 0.711 ± 0.005 to 0.383 ± 0.003. The results indicated that the reduced pyruvate to lactate flux was rerouted to the diacetyl with an almost linear flux variation via altered NADH/NAD(+ ratios. Therefore, we provided a novel strategy to precisely control the pyruvate distribution for fine tuning of the lactate and diacetyl production through promoter engineering in L. lactis. Interestingly, the increased H(2O-forming NADH oxidase activity led to 76.95% lower H(2O(2 concentration in the recombinant strain than that of the wild-type strain after 24 h of aerated cultivation. The viable cells were significantly elevated by four orders of magnitude within 28 days of storage at 4°C, suggesting that the increased enzyme activity could eliminate H(2O(2 accumulation and prolong cell survival.

  6. Microencapsulation of probiotics in hydrogel particles: enhancing Lactococcus lactis subsp. cremoris LM0230 viability using calcium alginate beads.

    Science.gov (United States)

    Yeung, Timothy W; Arroyo-Maya, Izlia J; McClements, David J; Sela, David A

    2016-04-01

    Probiotics are beneficial microbes often added to food products to enhance the health and wellness of consumers. A major limitation to producing efficacious functional foods containing probiotic cells is their tendency to lose viability during storage and gastrointestinal transit. In this study, the impact of encapsulating probiotics within food-grade hydrogel particles to mitigate sensitivity to environmental stresses was examined. Confocal fluorescence microscopy confirmed that Lactococcus lactis were trapped within calcium alginate beads formed by dripping a probiotic-alginate mixture into a calcium solution. Encapsulation improved the viability of the probiotics during aerobic storage: after seven days, less than a two-log reduction was observed in encapsulated cells stored at room temperature, demonstrating that a high concentration of cells survived relative to non-encapsulated bacteria. These hydrogel beads may have applications for improving the stability and efficacy of probiotics in functional foods.

  7. Micrococcus lactis sp. nov., isolated from dairy industry waste.

    Science.gov (United States)

    Chittpurna; Singh, Pradip K; Verma, Dipti; Pinnaka, Anil Kumar; Mayilraj, Shanmugam; Korpole, Suresh

    2011-12-01

    A Gram-positive, yellow-pigmented, actinobacterial strain, DW152(T), was isolated from a dairy industry effluent treatment plant. 16S rRNA gene sequence analysis indicated that strain DW152(T) exhibited low similarity with many species with validly published names belonging to the genera Micrococcus and Arthrobacter. However, phenotypic properties including chemotaxonomic markers affiliated strain DW152(T) to the genus Micrococcus. Strain DW152(T) had ai-C(15:0) and i-C(15:0) as major cellular fatty acids, and MK-8(H(2)) as the major menaquinone. The cell-wall peptidoglycan of strain DW152(T) had l-lysine as the diagnostic amino acid and the type was A4α. The DNA G+C content of strain DW152(T) was 68.0 mol%. In 16S rRNA gene sequence analysis, strain DW152(T) exhibited significant similarity with Micrococcus terreus NBRC 104258(T), but the mean value of DNA-DNA relatedness between these strains was only 42.3%. Moreover, strain DW152(T) differed in biochemical and chemotaxonomic characteristics from M. terreus and other species of the genus Micrococcus. Based on the above differences, we conclude that strain DW152(T) should be treated as a novel species of the genus Micrococcus, for which the name Micrococcus lactis sp. nov. is proposed. The type strain of Micrococcus lactis sp. nov. is DW152(T) (=MTCC10523(T) =DSM 23694(T)).

  8. Implementation of the agmatine-controlled expression system for inducible gene expression in Lactococcus lactis.

    Science.gov (United States)

    Linares, Daniel M; Alvarez-Sieiro, Patricia; del Rio, Beatriz; Ladero, Victor; Redruello, Begoña; Martin, Ma Cruz; Fernandez, Maria; Alvarez, Miguel A

    2015-12-30

    Lactococcus lactis has been safely consumed in fermented foods for millennia. This Gram-positive bacterium has now become of industrial importance as an expression host for the overproduction of lipopolysaccharide-free recombinant proteins used as food ingredients, therapeutic proteins and biotechnological enzymes. This paper reports an agmatine-controlled expression (ACE) system for L. lactis, comprising the lactococcal agmatine-sensor/transcriptional activator AguR and its target promoter P(aguB). The usefulness and efficiency of this system was checked via the reporter gene gfp and by producing PEP (Myxococcus xanthus prolyl-endopeptidase), an enzyme of biomedical interest able to degrade the immunotoxic peptides produced during the gastrointestinal breakdown of gluten. The ACE system developed in this work was suitable for the efficient expression of the functional recombinant proteins GFP and PEP. The expression system was tightly regulated by the agmatine concentration and allowed high protein production without leakiness.

  9. Transcriptional activation of the glycolytic las operon and catabolite repression of the gal operon in Lactococcus lactis are mediated by the catabolite control protein CcpA

    NARCIS (Netherlands)

    Luesink, Evert J.; Herpen, René E.M.A. van; Grossiord, Benoît P.; Kuipers, Oscar P.; Vos, Willem M. de

    1998-01-01

    The Lactococcus lactis ccpA gene, encoding the global regulatory protein CcpA, was identified and characterized. Northern blot and primer extension analyses showed that the L. lactis ccpA gene is constitutively transcribed from a promoter that does not contain a cre sequence. Inactivation of the

  10. Bifidobacterium animalis ssp. lactis CNCM-I2494 Restores Gut Barrier Permeability in Chronically Low-Grade Inflamed Mice.

    Science.gov (United States)

    Martín, Rebeca; Laval, Laure; Chain, Florian; Miquel, Sylvie; Natividad, Jane; Cherbuy, Claire; Sokol, Harry; Verdu, Elena F; van Hylckama Vlieg, Johan; Bermudez-Humaran, Luis G; Smokvina, Tamara; Langella, Philippe

    2016-01-01

    Growing evidence supports the efficacy of many probiotic strains in the management of gastrointestinal disorders associated with deregulated intestinal barrier function and/or structure. In particular, bifidobacteria have been studied for their efficacy to both prevent and treat a broad spectrum of animal and/or human gut disorders. The aim of the current work was thus to evaluate effects on intestinal barrier function of Bifidobacterium animalis ssp. lactis CNCM-I2494, a strain used in fermented dairy products. A chronic dinitrobenzene sulfonic acid (DNBS)-induced low-grade inflammation model causing gut dysfunction in mice was used in order to study markers of inflammation, intestinal permeability, and immune function in the presence of the bacterial strain. In this chronic low-grade inflammation mice model several parameters pointed out the absence of an over active inflammation process. However, gut permeability, lymphocyte populations, and colonic cytokines were found to be altered. B. animalis ssp. lactis CNCM-I2494 was able to protect barrier functions by restoring intestinal permeability, colonic goblet cell populations, and cytokine levels. Furthermore, tight junction (TJ) proteins levels were also measured by qRT-PCR showing the ability of this strain to specifically normalize the level of several TJ proteins, in particular for claudin-4. Finally, B. lactis strain counterbalanced CD4(+) lymphocyte alterations in both spleen and mesenteric lymphoid nodes. It restores the Th1/Th2 ratio altered by the DNBS challenge (which locally augments CD4(+) Th1 cells) by increasing the Th2 response as measured by the increase in the production of major representative Th2 cytokines (IL-4, IL-5, and IL-10). Altogether, these data suggest that B. animalis ssp. lactis CNCM-I2494 may efficiently prevent disorders associated with increased barrier permeability.

  11. ClpE from Lactococcus lactis promotes repression of CtsR-dependent gene expression

    DEFF Research Database (Denmark)

    Varmanen, P.; Vogensen, F.K.; Hammer, Karin

    2003-01-01

    ATPase (ClpE) in Lactococcus lactis is required for such a decrease in expression of a gene negatively regulated by the heat shock regulator (CtsR). Northern blot analysis showed that while a shift to a high temperature in wild-type cells resulted in a temporal increase followed by a decrease...

  12. Production of Ginsenoside F2 by Using Lactococcus lactis with Enhanced Expression of β-Glucosidase Gene from Paenibacillus mucilaginosus.

    Science.gov (United States)

    Li, Ling; Shin, So-Yeon; Lee, Soo Jin; Moon, Jin Seok; Im, Wan Taek; Han, Nam Soo

    2016-03-30

    This study aimed to produce a pharmacologically active minor ginsenoside F2 from the major ginsenosides Rb1 and Rd by using a recombinant Lactococcus lactis strain expressing a heterologous β-glucosidase gene. The nucleotide sequence of the gene (BglPm) was derived from Paenibacillus mucilaginosus and synthesized after codon optimization, and the two genes (unoptimized and optimized) were expressed in L. lactis NZ9000. Codon optimization resulted in reduction of unfavorable codons by 50% and a considerable increase in the expression levels (total activities) of β-glucosidases (0.002 unit/mL, unoptimized; 0.022 unit/mL, optimized). The molecular weight of the enzyme was 52 kDa, and the purified forms of the enzymes could successfully convert Rb1 and Rd into F2. The permeabilized L. lactis expressing BglPm resulted in a high conversion yield (74%) of F2 from the ginseng extract. Utilization of this microbial cell to produce F2 may provide an alternative method to increase the health benefits of Panax ginseng.

  13. Cloning and nucleotide sequence analysis of pepV, a carnosinase gene from Lactobacillus delbrueckii subsp. lactis DSM 7290, and partial characterization of the enzyme.

    Science.gov (United States)

    Vongerichten, K F; Klein, J R; Matern, H; Plapp, R

    1994-10-01

    Cell extracts of Lactobacillus delbrueckii subsp. lactis DSM 7290 were found to exhibit unique peptolytic ability against unusual beta-alanyl-dipeptides. In order to clone the gene encoding this activity, designated pepV, a gene library of strain DSM 7290 genomic DNA, prepared in the low-copy-number plasmid pLG339, was screened for heterologous expression in Escherichia coli. Recombinant clones harbouring pepV were identified by their ability to allow the utilization of carnosine (beta-alanyl-histidine) as a source of histidine by the E. coli mutant strain UK197 (pepD, hisG). Complementation was observed in a colony harbouring a recombinant plasmid (pKV101), carrying pepV. A 2.4 kb fragment containing pepV was subcloned and its nucleotide sequence revealed an open reading frame (ORF) of 1413 nucleotides, corresponding to a protein with predicted molecular mass of 51998 Da. A single transcription initiation site 71 bp upstream of the ATG translational start codon was identified by primer extension. No significant homology was detected between pepV or its deduced amino acid sequence with any entry in the databases. The only similarity was found in a region conserved in the ArgE/DapE/CPG2/YscS family of proteins. This observation, and protease inhibitor studies, indicated that pepV is of the metalloprotease type. A second ORF present in the sequenced fragment showed extensive homology to a variety of amino acid permeases from E. coli and Saccharomyces cerevisiae.

  14. Development, molecular characterisation and exploitation of the nisin controlled expression system in Lactococcus lactis

    NARCIS (Netherlands)

    Ruyter, de P.G.G.A.

    1998-01-01

    Lactic acid bacteria are gram-positive bacteria that are widely used in a variety of dairy fermentation processes. Notably, strains of the lactic acid starter bacterium Lactococcus lactis are of great economic importance because of their world-wide use in cheese making.

  15. Insertion-Sequence-Mediated Mutations Isolated During Adaptation to Growth and Starvation in Lactococcus lactis.

    NARCIS (Netherlands)

    Visser, de J.A.G.M.; Akkermans, A.D.L.; Hoekstra, R.F.; Vos, de W.M.

    2004-01-01

    We studied the activity of three multicopy insertion sequence (IS) elements in 12 populations of Lactococcus lactis IL1403 that evolved in the laboratory for 1000 generations under various environmental conditions (growth or starvation and shaken or stationary). Using RFLP analysis of single-clone

  16. Glycosylation in secreted proteins from yeast Kluyveromyces lactis

    Energy Technology Data Exchange (ETDEWEB)

    Santos, A.V.; Passos, F.M.L. [Universidade Federal de Vicosa (UFV), MG (Brazil). Dept. de Microbiologia. Lab. de Fisiologia de Microrganismos; Azevedo, B.R.; Pimenta, A.M.C.; Santoro, M.M. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Bioquimica e Imunologia. Lab. de Enzimologia e Fisico-Quimica de Proteina

    2008-07-01

    Full text: The nutritional status of a cell culture affects either the expression or the traffic of a number of proteins. The identification of the physiological conditions which favor protein secretion has important biotechnological consequences in designing systems for recombinant extracellular protein industrial production. Yeast Kluyvromyces lactis has been cultured in a continuous stirring tank bioreactor (CSTR) under nitrogen limitation at growth rates (0.03 h{sup -1} and 0.09 h{sup -1}) close to either exponential or stationary batch growth phases, respectively the objective was to investigate the extracellular glycoproteins at these two level of nitrogen limitation. Proteins from free cell extracts were separated by gradient SDS-PAGE (5-15%) and two-dimensional chromatography, and were analyzed by mass spectrometry (MALDI-TOF-TOF-MS). In SDS-PAGE analysis, differences in extracellular proteome were visualized: different proteins profiles at these two growth rates. The 0.09 h-1 growth rate showed larger number of bands using colloidal Coma ssie Blue staining. Different bands were detected at these two growth rates when the PAS assay for glycoprotein detection in polyacrylamide gel was used. The two-dimensional chromatogram profiles were comparatively distinguished between the 0.03 h{sup -1} and 0.09 h{sup -1} growth rate samples. Protein peaks from the second dimension, were subjected to mass spectrometry. The mass spectrums visualized showed glycosylated proteins with N-acetylglucosamine molecules and 8, 9 or 15 hexoses molecules. Comparisons between the proteins averaged mass values with the deduced proteins masses from K. lactis secreted proteins database indicated possible post-translational modifications, such as post-translational proteolysis, acetylation, deamidation and myristoylation.

  17. Glycosylation in secreted proteins from yeast Kluyveromyces lactis

    International Nuclear Information System (INIS)

    Santos, A.V.; Passos, F.M.L.; Azevedo, B.R.; Pimenta, A.M.C.; Santoro, M.M.

    2008-01-01

    Full text: The nutritional status of a cell culture affects either the expression or the traffic of a number of proteins. The identification of the physiological conditions which favor protein secretion has important biotechnological consequences in designing systems for recombinant extracellular protein industrial production. Yeast Kluyvromyces lactis has been cultured in a continuous stirring tank bioreactor (CSTR) under nitrogen limitation at growth rates (0.03 h -1 and 0.09 h -1 ) close to either exponential or stationary batch growth phases, respectively the objective was to investigate the extracellular glycoproteins at these two level of nitrogen limitation. Proteins from free cell extracts were separated by gradient SDS-PAGE (5-15%) and two-dimensional chromatography, and were analyzed by mass spectrometry (MALDI-TOF-TOF-MS). In SDS-PAGE analysis, differences in extracellular proteome were visualized: different proteins profiles at these two growth rates. The 0.09 h-1 growth rate showed larger number of bands using colloidal Coma ssie Blue staining. Different bands were detected at these two growth rates when the PAS assay for glycoprotein detection in polyacrylamide gel was used. The two-dimensional chromatogram profiles were comparatively distinguished between the 0.03 h -1 and 0.09 h -1 growth rate samples. Protein peaks from the second dimension, were subjected to mass spectrometry. The mass spectrums visualized showed glycosylated proteins with N-acetylglucosamine molecules and 8, 9 or 15 hexoses molecules. Comparisons between the proteins averaged mass values with the deduced proteins masses from K. lactis secreted proteins database indicated possible post-translational modifications, such as post-translational proteolysis, acetylation, deamidation and myristoylation

  18. Glutathione attenuates uranyl toxicity in Lactococcus lactis

    International Nuclear Information System (INIS)

    Fahmy, Karim; Oertel, Jana; Solioz, M.

    2017-01-01

    We investigated the role of intracellular glutathione (GSH), which in a large number of taxa plays a role in the protection against the toxicity of heavy metals. Anaerobically grown Lactococcus lactis containing an inducible GSH synthesis pathway was used as a model organism allowing the study of GSH-dependent uranyl detoxification without interference from additional reactive oxygen species. Microcalorimetric measurements of the metabolic heat showed that intracellular GSH attenuates the toxicity of uranium at a concentration in the range of 10-150 μM. Isothermal titration calorimetry revealed the endothermic binding of U(VI) to the carboxyl group(s) of GSH. The data indicate that the primary detoxifying mechanism is the intracellular sequestration of carboxyl-coordinated U(VI) into an insoluble complex with GSH.

  19. Glutathione attenuates uranyl toxicity in Lactococcus lactis

    Energy Technology Data Exchange (ETDEWEB)

    Fahmy, Karim; Oertel, Jana [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). Biophysics; Obeid, M. [Technische Univ. Dresden (Germany); Solioz, M. [Bern Univ. (Switzerland)

    2017-06-01

    We investigated the role of intracellular glutathione (GSH), which in a large number of taxa plays a role in the protection against the toxicity of heavy metals. Anaerobically grown Lactococcus lactis containing an inducible GSH synthesis pathway was used as a model organism allowing the study of GSH-dependent uranyl detoxification without interference from additional reactive oxygen species. Microcalorimetric measurements of the metabolic heat showed that intracellular GSH attenuates the toxicity of uranium at a concentration in the range of 10-150 μM. Isothermal titration calorimetry revealed the endothermic binding of U(VI) to the carboxyl group(s) of GSH. The data indicate that the primary detoxifying mechanism is the intracellular sequestration of carboxyl-coordinated U(VI) into an insoluble complex with GSH.

  20. A Food-Grade Cloning System for Industrial Strains of Lactococcus lactis

    Science.gov (United States)

    Sørensen, Kim I.; Larsen, Rasmus; Kibenich, Annette; Junge, Mette P.; Johansen, Eric

    2000-01-01

    We have previously reported the construction of a food-grade cloning vector for Lactococcus using the ochre suppressor, supB, as the selective marker. This vector, pFG1, causes only a slight growth inhibition in the laboratory strain MG1363 but is unstable in the industrial strains tested. As supB suppresses both amber and ochre stop codons, which are present in 82% of all known lactococcal genes, this undesirable finding may result from the accumulation of elongated mistranslated polypeptides. Here, we report the development of a new food-grade cloning vector, pFG200, which is suitable for overexpressing a variety of genes in industrial strains of Lactococcus lactis. The vector uses an amber suppressor, supD, as selectable marker and consists entirely of Lactococcus DNA, with the exception of a small polylinker region. Using suppressible pyrimidine auxotrophs, selection and maintenance are efficient in any pyrimidine-free medium including milk. Importantly, the presence of this vector in a variety of industrial strains has no significant effect on the growth rate or the rate of acidification in milk, making this an ideal system for food-grade modification of industrially relevant L. lactis strains. The usefulness of this system is demonstrated by overexpressing the pepN gene in a number of industrial backgrounds. PMID:10742196

  1. Isolasi dan Karakterisasi Bakteriosin yang Dihasilkan Oleh Lactobacillus lactis dari Sedimen Laut

    Directory of Open Access Journals (Sweden)

    Rofiq Sunaryanto

    2015-04-01

    Full Text Available Telah dilakukan isolasi dan karakterisasi bakteriosin yang dihasilkan oleh Lactobacillus lactis yang berasal dari sedimen laut. Karakterisasi bakteriosin meliputi uji aktivitas antimikroba, stabilitas terhadap suhu, pH, penambahan enzim, surfaktan, dan stabilitas bakteriosin terhadap penyinaran lampu UV. Aktivitas antimikroba bakteriosin diuji melawan bakteri uji Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 66923, Staphyllococcus aureus ATCC 25923, Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus casei, dan Candida albican. Hasil penelitian menunjukkan bahwa bakteriosin mampu menghambat pertumbuhan E.coli ATCC 25922, E. faecalis ATCC 29212, S. aureus ATCC 25923 dan B. subtilis ATCC 66923, namun demikian tidak mampu menghambat pertumbuhan L. plantarum, L. bulgaricus, L. casei, dan C. albican. Bakteriosin yang dihasilkan oleh Lactobacillus lactis stabil terhadap pemanasan sampai dengan suhu 70 °C dan stabil pada rentang pH 3 sampai dengan 7. Aktivitas bakteriosin hilang dengan penambahan tripsin, pepsin, dan proteinase-K, namun aktivitas bakteriosin stabil terhadap penambahan a-amilase. Penambahan tween 20, tween 80, dan EDTA mampu meningkatkan aktivitas bakteriosin sebesar 1,1 sampai dengan 1,2 kali dibandingkan dengan tanpa penambahan surfaktan. Penyinaran lampu UV selama 15 menit tidak berpengaruh terhadap aktivitas bakteriosin.

  2. Study on Batch Culture Growth Model for Lactococcus lactis IO-1

    OpenAIRE

    Ishizaki, Ayaaki; Ohta, Tomomi; Kobayashi, Genta; 石崎, 文彬; 太田, 智美; 小林, 元太

    1991-01-01

    L-lactate fermentation employing Lactncoccus lactis IO-1 demonstrated a typical end product inhibition. By numerical analysis of fermentation results of the batch culture of this microorganism, the specific rates for cell growth, substrate consumption and product formation were clearly expressed by the end product inhibition formulae. All constants for those formulae were determined by the fermentation results. A mathematical model for batch culture growth of this microorganism in which the n...

  3. CONTINUOUS MEASUREMENT OF THE CYTOPLASMIC PH IN LACTOCOCCUS-LACTIS WITH A FLUORESCENT PH INDICATOR

    NARCIS (Netherlands)

    MOLENAAR, D; ABEE, T; KONINGS, WN

    1991-01-01

    The cytoplasmic pH of Lactococcus lactis was studied with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF). A novel method was applied for loading bacterial cells with BCECF, which consists of briefly treating a dense cell suspension with acid in the

  4. A possible contribution of mRNA secondary structure to translation initiation efficiency in Lactococcus lactis

    NARCIS (Netherlands)

    Guchte, Maarten van de; Lende, Ted van der; Kok, Jan; Venema, Gerard

    1991-01-01

    Gene expression signals derived from Lactococcus lactis were linked to lacZ-fused genes with different 5'-nucleotide sequences. Computer predictions of mRNA secondary structure were combined with lacZ expression studies to direct base-substitutions that could possibly influence gene expression.

  5. Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans

    Science.gov (United States)

    We expressed a glucansucrase, DsrI, from Leuconostoc mesenteroides that catalyzes formation of water-insoluble glucans from sucrose in Lactococcus lactis using a nisin-controlled gene expression system. Production of DsrI was optimized using several different background vectors, signal peptides, str...

  6. Phenotypic and genotypic characterization of lactic acid bacteria isolated from raw goat milk and effect of farming practices on the dominant species of lactic acid bacteria.

    Science.gov (United States)

    Tormo, Hélène; Ali Haimoud Lekhal, Djamila; Roques, C

    2015-10-01

    Lactic acid bacteria, in particular Lactococcus lactis, play a decisive role in the cheese making process and more particularly in lactic cheeses which are primarily produced on goat dairy farms. The objective of this study was therefore to identify the main lactic acid bacteria found in raw goats' milk from three different regions in France and evaluate if certain farming practices have an effect on the distribution of species of lactic acid bacteria in the various milk samples. Identification at genus or species level was carried out using phenotypic tests and genotypic methods including repetitive element REP-PCR, species-specific PCR and 16S rRNA gene sequencing. The distribution of the main bacterial species in the milk samples varied depending on farms and their characteristics. Out of the 146 strains identified, L. lactis was the dominant species (60% of strains), followed by Enterococcus (38%) of which Enterococcus faecalis and Enterococcus faecium. Within the species L. lactis, L. lactis subsp lactis was detected more frequently than L. lactis subsp cremoris (74% vs. 26%). The predominance of L. lactis subsp cremoris was linked to geographical area studied. It appears that the animals' environment plays a role in the balance between the dominance of L. lactis and enterococci in raw goats' milk. The separation between the milking parlor and the goat shed (vs no separation) and only straw in the bedding (vs straw and hay) seems to promote L. lactis in the milk (vs enterococci). Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Study of the transgalactosylation activity of ß-galactosidase from a new strain Kluyveromyces lactis 3

    Directory of Open Access Journals (Sweden)

    ILIA ILIEV

    2012-01-01

    Full Text Available Beta-galactosidase (EC.3.2.1.23 is an important enzyme industrially used for the hydrolysis of lactose from milk and milk whey for several applications. Lately, the importance of this enzyme was enhanced by its galactosyltransferase activity, which is responsible for synthesis of transgalactosylated oligosaccharides that act as prebiotics with several beneficial effects on the consumers. ß-Galactosidase production by Kluyveromyces lactis 3 was studied in shake flask culture. The highest enzymatic activity was obtained at 10-th hour of the fermentation. The optimum temperature for transferase activity was 50°C. When incubated with 30% lactose in 50 mM phosphate buffer (pH 6.0 the enzyme can synthesize up to 41% galacto-oligosaccharides (GalOS. β-Galactosidase from strain Kluyveromyces lactis 3 produces mainly oligosaccharides with degree of polymerization (DP 6 at 40°C and with DP 3 at 50°C.

  8. Construction of upp deletion mutant strains of Lactobacillus casei and Lactococcus lactis based on counterselective system using temperature-sensitive plasmid.

    Science.gov (United States)

    Song, Li; Cui, Hongyu; Tang, Lijie; Qiao, Xinyuan; Liu, Min; Jiang, Yanping; Cui, Wen; Li, Yijing

    2014-07-01

    Integration plasmids are often used in constructing chromosomal mutations, as it enables the alternation of genes at any location by integration or replacement. Food-grade integration vectors can integrate into the host genome without introducing any selectable markers or residual bases, and the recombination often happens in non-coding region. In this study we used the temperature-sensitive pWV01 replicon to construct 2 chloramphenicol-resistant integration plasmids (pGBHC32-upp) containing the uracil phosphoribosyl transferase (upp) gene as a counterselective marker for Lactobacillus casei (L. casei) ATCC393 and Lactococcus lactis (L. lactis) MG1363. We then ligated the designed homologous arms to the pGBHC32-upp plasmids to allow their integration to the bacterial chromosome, and selected upp deletion mutants of L. casei ATCC393 and L. lactis MG1363 in the presence of 5-fluorouracil (5-FU). Analysis of genetic stability, growth curve, carbon utilization and scanning electronic microscopy showed that, except for 5-FU resistance, there were no significant differences between the wild type and mutant lactic acid bacteria. The integration system and the upp deletion strains could be used in the insertion or deletion of genes at any location of the chromosome of both L. casei ATCC 393 and L. lactis MG1363, and the homologous recombination would not introduce any selectable markers or residual bases. These mutant strains can be further investigated for heterologous protein expression and construction of a live mucosal vaccine carrier. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. The extracellular phage-host interactions involved in the bacteriophage LL-H infection of Lactobacillus delbrueckii ssp. lactis ATCC 15808.

    Science.gov (United States)

    Munsch-Alatossava, Patricia; Alatossava, Tapani

    2013-12-24

    The complete genome sequence of Lactobacillus bacteriophage LL-H was determined in 1996. Accordingly, LL-H has been used as a model phage for the infection of dairy Lactobacillus, specifically for thermophilic Lactobacillus delbrueckii ssp. lactis host strains, such as ATCC 15808. One of the major goals of phage LL-H research consisted of the characterization of the first phage-host interactions at the level of phage adsorption and phage DNA injection steps to determine effective and practical methods to minimize the risks associated with the appearance and attack of phages in the manufacture of yogurt, and Swiss or Italian hard type cheeses, which typically use thermophilic lactic acid bacteria starter cultures containing L. delbrueckii strains among others. This mini review article summarizes the present data concerning (i) the special features, particle structure, and components of phage LL-H and (ii) the structure and properties of lipoteichoic acids (LTAs), which are the phage LL-H receptor components of L. delbrueckii ssp. lactis host strains. Moreover, a model of the first, extracellular, phage-host interactions for the infection of L. delbrueckii ssp. lactis ATCC 15808 by phage LL-H is presented and further discussed.

  10. The extracellular phage-host interactions involved in the bacteriophage LL-H infection of Lactobacillus delbrueckii ssp. lactis ATCC 15808

    Directory of Open Access Journals (Sweden)

    Patricia eMunsch-Alatossava

    2013-12-01

    Full Text Available The complete genome sequence of Lactobacillus bacteriophage LL-H was determined in 1996. Accordingly, LL-H has been used as a model phage for the infection of dairy Lactobacillus, specifically for thermophilic Lb. delbrueckii ssp. lactis host strains, such as ATCC 15808. One of the major goals of phage LL-H research consisted of the characterization of the the first phage-host interactions at the level of phage adsorption and phage DNA injection steps to determine effective and practical methods to minimise the risks associated with the appearance and attack of phages in the manufacture of yoghurt, and Swiss or Italian type hard cheeses, which typically use thermophilic LAB starter cultures containing Lb. delbrueckii strains among others. This mini review article summarises the present data concerning (i the special features, particle structure and components of phage LL-H and (ii the structure and properties of lipoteichoic acids (LTAs, which are the phage LL-H receptor components of Lb. delbrueckii ssp. lactis host strains. Moreover, a model of the first, extracellular, phage-host interactions for the infection of Lb. delbrueckii ssp. lactis ATCC 15808 by phage LL-H is presented and further discussed.

  11. Oxidative Stress at High Temperatures in Lactococcus lactis Due to an Insufficient Supply of Riboflavin

    Science.gov (United States)

    Chen, Jun; Shen, Jing

    2013-01-01

    Lactococcus lactis MG1363 was found to be unable to grow at temperatures above 37°C in a defined medium without riboflavin, and the cause was identified to be dissolved oxygen introduced during preparation of the medium. At 30°C, growth was unaffected by dissolved oxygen and oxygen was consumed quickly. Raising the temperature to 37°C resulted in severe growth inhibition and only slow removal of dissolved oxygen. Under these conditions, an abnormally low intracellular ratio of [ATP] to [ADP] (1.4) was found (normally around 5), which indicates that the cells are energy limited. By adding riboflavin to the medium, it was possible to improve growth and oxygen consumption at 37°C, and this also normalized the [ATP]-to-[ADP] ratio. A codon-optimized redox-sensitive green fluorescent protein (GFP) was introduced into L. lactis and revealed a more oxidized cytoplasm at 37°C than at 30°C. These results indicate that L. lactis suffers from heat-induced oxidative stress at increased temperatures. A decrease in intracellular flavin adenine dinucleotide (FAD), which is derived from riboflavin, was observed with increasing growth temperature, but the presence of riboflavin made the decrease smaller. The drop was accompanied by a decrease in NADH oxidase and pyruvate dehydrogenase activities, both of which depend on FAD as a cofactor. By overexpressing the riboflavin transporter, it was possible to improve FAD biosynthesis, which resulted in increased NADH oxidase and pyruvate dehydrogenase activities and improved fitness at high temperatures in the presence of oxygen. PMID:23913422

  12. Autolysis of Lactococcus lactis caused by induced overproduction of its major autolysin, AcmA

    NARCIS (Netherlands)

    Buist, Girbe; Karsens, H; Nauta, A; van Sinderen, D; Venema, G; Kok, J

    The optical density of a culture of Lactococcus lactis MG1363 was reduced more than 60% during prolonged stationary phase, Reduction in optical density (autolysis) was almost absent in a culture of an isogenic mutant containing a deletion in the major autolysin gene, acmA. An acmA mutant carrying

  13. In ovo injection of prebiotics and synbiotics affects the digestive potency of the pancreas in growing chickens.

    Science.gov (United States)

    Pruszynska-Oszmalek, E; Kolodziejski, P A; Stadnicka, K; Sassek, M; Chalupka, D; Kuston, B; Nogowski, L; Mackowiak, P; Maiorano, G; Jankowski, J; Bednarczyk, M

    2015-08-01

    The purpose of the study was to examine the effect of 2 prebiotics and 2 synbiotics on the digestive potency of pancreas in 1-, 3-, 7-, 14-, 21-, and 34-day-old cockerels. Prebiotics (inulin and Bi²tos) and synbiotics (inulin + Lactococcus lactis subsp. lactis and Bi²tos + Lactococcus lactis subsp. cremoris) were injected in ovo into the air cell on the 12th d embryonic development. Their application increased the activity of amylase, lipase, and trypsin in the pancreas. The most pronounced changes were observed at the end of the investigated rearing period (d 34). The strongest stimulative effects on amylase were shown by both synbiotics, on lipase synbiotic Bi²tos + Lactococcus lactis subsp. cremoris, and on trypsin all the used prebiotics and synbiotics. Simultaneously, neither the absolute nor the relative mass of the pancreas in comparison to control group were changed. Also, the injected in ovo compounds did not cause a deterioration in the posthatching condition of the chicken liver, as determined by measurement of the activity of marker enzymes in the blood (alanine aminotransferase and aspartate aminotransferase). Treatment with the prebiotics and synbiotics did not change the feed conversion ratio but Bi²tos (galacto-oligosaccharide) and inulin (fructan) + Lactococcus lactis subsp. lactis significantly increased final BW. © 2015 Poultry Science Association Inc.

  14. The Transcriptional and Gene Regulatory Network of Lactococcus lactis MG1363 during Growth in Milk

    NARCIS (Netherlands)

    de Jong, Anne; Hansen, Morten E.; Kuipers, Oscar P.; Kilstrup, Mogens; Kok, Jan

    2013-01-01

    In the present study we examine the changes in the expression of genes of Lactococcus lactis subspecies cremoris MG1363 during growth in milk. To reveal which specific classes of genes (pathways, operons, regulons, COGs) are important, we performed a transcriptome time series experiment. Global

  15. Use of synthetic genes for cloning, production and functional expression of the bacteriocins enterocin A and bacteriocin E 50-52 by Pichia pastoris and Kluyveromyces lactis.

    Science.gov (United States)

    Jiménez, Juan J; Borrero, Juan; Gútiez, Loreto; Arbulu, Sara; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

    2014-06-01

    The use of synthetic genes may constitute a successful approach for the heterologous production and functional expression of bacterial antimicrobial peptides (bacteriocins) by recombinant yeasts. In this work, synthetic genes with adapted codon usage designed from the mature amino acid sequence of the bacteriocin enterocin A (EntA), produced by Enterococcus faecium T136, and the mature bacteriocin E 50-52 (BacE50-52), produced by E. faecium NRRL B-32746, were synthesized. The synthetic entA and bacE50-52 were cloned into the protein expression vectors pPICZαA and pKLAC2 for transformation of derived vectors into Pichia pastoris X-33 and Kluyveromyces lactis GG799, respectively. The recombinant vectors were linearized and transformed into competent cells selecting for P. pastoris X-33EAS (entA), P. pastoris X-33BE50-52S (bacE50-52), K. lactis GG799EAS (entA), and K. lactis GG799BE50-52S (bacE50-52). P. pastoris X-33EAS and K. lactis GG799EAS, but not P. pastoris X-33BE50-52S and K. lactis GG799BE50-52S, showed antimicrobial activity in their supernatants. However, purification of the supernatants of the producer yeasts permitted recovery of the bacteriocins EntA and BacE50-52. Both purified bacteriocins were active against Gram-positive bacteria such as Listeria monocytogenes but not against Gram-negative bacteria, including Campylobacter jejuni.

  16. Enzyme activity of β-galactosidase from Kluyveromyces lactis and Aspergillus oryzae on simulated conditions of human gastrointestinal system

    Directory of Open Access Journals (Sweden)

    Alessandra Bosso

    2015-09-01

    Full Text Available An alternative to relieve the symptoms of lactose intolerance is the intake of the enzyme β-galactosidase in pharmaceutical dosage forms. The ability of β-galactosidase produced by Kluyveromyces lactis and Aspergillus oryzae to hydrolyze lactose in simulated conditions of the human gastrointestinal tract was investigated. The experiment was carried out in the optimum temperature for each enzyme activity, 40 and 55°C, respectively, and at the normal human body temperature (37°C at concentrations of 1.5, 3.0, and 5.0 g/L (enzyme from A. oryzae or mL/L (enzyme from K. lactis. Both enzymes were completely inactivated under simulated gastric conditions (pH 2. When the enzymes were subjected to simulated small intestine conditions (pH 7.4, lactose hydrolysis has occurred, but at 37°C the percentage was lower than that under the optimal temperatures. At concentrations of 1.5, 3.0, and 5.0 mL/L the enzyme from K. lactis hydrolyzed 76.63%, 88.91% and 94.80% of lactose at 40°C, and 55.99%, 80.91% and 81.53% at 37°C, respectively. In contrast, the enzyme from A. oryzae hydrolyzed 7.11%, 16.18% and 21.29% at 55°C, and 8.4%, 11.85% and 16.43% at 37°C. It was observed that under simulated intestinal conditions, the enzyme from K. lactis was more effective on lactose hydrolysis as compared to the enzyme from A. oryzae. Considering the findings of this study, it is extremely necessary to use an enteric coating on β-galactosidase capsules so that this enzyme is released only in the small intestine, which is its site of action, thus not suffering the action of the stomach pH.Keywords: Lactase. Hydrolysis. Lactose intolerance. Gastrointestinal tract. RESUMOAtividade de β-galactosidase de Kluyveromyces lactis e Aspergillus oryzae, em condições simuladas do sistema gastrintestinal humanoUma das alternativas para amenizar os sintomas da intolerância à lactose é a ingestão de β-galactosidase em formas farmacêuticas. Neste trabalho avaliou-se a

  17. Secretion of biologically active pancreatitis-associated protein I (PAP) by genetically modified dairy Lactococcus lactis NZ9000 in the prevention of intestinal mucositis.

    Science.gov (United States)

    Carvalho, Rodrigo D; Breyner, Natalia; Menezes-Garcia, Zelia; Rodrigues, Nubia M; Lemos, Luisa; Maioli, Tatiane U; da Gloria Souza, Danielle; Carmona, Denise; de Faria, Ana M C; Langella, Philippe; Chatel, Jean-Marc; Bermúdez-Humarán, Luis G; Figueiredo, Henrique C P; Azevedo, Vasco; de Azevedo, Marcela S

    2017-02-13

    Mucositis is one of the most relevant gastrointestinal inflammatory conditions in humans, generated by the use of chemotherapy drugs, such as 5-fluoracil (5-FU). 5-FU-induced mucositis affects 80% of patients undergoing oncological treatment causing mucosal gut dysfunctions and great discomfort. As current therapy drugs presents limitations in alleviating mucositis symptoms, alternative strategies are being pursued. Recent studies have shown that the antimicrobial pancreatitis-associated protein (PAP) has a protective role in intestinal inflammatory processes. Indeed, it was demonstrated that a recombinant strain of Lactococcus lactis expressing human PAP (LL-PAP) could prevent and improve murine DNBS-induced colitis, an inflammatory bowel disease (IBD) that causes severe inflammation of the colon. Hence, in this study we sought to evaluate the protective effects of LL-PAP on 5-FU-induced experimental mucositis in BALB/c mice as a novel approach to treat the disease. Our results show that non-recombinant L. lactis NZ9000 have antagonistic activity, in vitro, against the enteroinvasive gastrointestinal pathogen L. monocytogenes and confirmed PAP inhibitory effect against Opportunistic E. faecalis. Moreover, L. lactis was able to prevent histological damage, reduce neutrophil and eosinophil infiltration and secretory Immunoglobulin-A in mice injected with 5-FU. Recombinant lactococci carrying antimicrobial PAP did not improve those markers of inflammation, although its expression was associated with villous architecture preservation and increased secretory granules density inside Paneth cells in response to 5-FU inflammation. We have demonstrated for the first time that L. lactis NZ9000 by itself, is able to prevent 5-FU-induced intestinal inflammation in BALB/c mice. Moreover, PAP delivered by recombinant L. lactis strain showed additional protective effects in mice epithelium, revealing to be a promising strategy to treat intestinal mucositis.

  18. Lactococcus lactis carrying a DNA vaccine coding for the ESAT-6 antigen increases IL-17 cytokine secretion and boosts the BCG vaccine immune response.

    Science.gov (United States)

    Pereira, V B; da Cunha, V P; Preisser, T M; Souza, B M; Turk, M Z; De Castro, C P; Azevedo, M S P; Miyoshi, A

    2017-06-01

    A regimen utilizing Bacille Calmette-Guerin (BCG) and another vaccine system as a booster may represent a promising strategy for the development of an efficient tuberculosis vaccine for adults. In a previous work, we confirmed the ability of Lactococcus lactis fibronectin-binding protein A (FnBPA+) (pValac:ESAT-6), a live mucosal DNA vaccine, to produce a specific immune response in mice after oral immunization. In this study, we examined the immunogenicity of this strain as a booster for the BCG vaccine in mice. After immunization, cytokine and immunoglobulin profiles were measured. The BCG prime L. lactis FnBPA+ (pValac:ESAT-6) boost group was the most responsive group, with a significant increase in splenic pro-inflammatory cytokines IL-17, IFN-γ, IL-6 and TNF-α compared with the negative control. Based on the results obtained here, we demonstrated that L. lactis FnBPA+ (pValac:ESAT-6) was able to increase the BCG vaccine general immune response. This work is of great scientific and social importance because it represents the first step towards the development of a booster to the BCG vaccine using L. lactis as a DNA delivery system. © 2017 The Society for Applied Microbiology.

  19. Unity in organisation and regulation of catabolic operons in Lactobacillus plantarum, Lactococcus lactis and Listeria monocytogenes

    NARCIS (Netherlands)

    Andersson, U.; Molenaar, D.; Radstrom, P.; Vos, de W.M.

    2005-01-01

    Global regulatory circuits together with more specific local regulators play a notable role when cells are adapting to environmental changes. Lactococcus lactis is a lactic acid bacterium abundant in nature fermenting most mono- and disaccharides. Comparative genomics analysis of the operons

  20. Ecosystème fromager : de l'étude du métabolisme du soufre chez Kluyveromyces lactis et Yarrowia lipolytica à l'interaction entre Kluyveromyces lactis et Brevibacterium aurantiacum

    OpenAIRE

    Hébert , Agnès

    2010-01-01

    Sulphur metabolism, which has a central role in the cell, is also important during the manufacturing of smear ripened cheeses. The cheese ecosystem degrades sulphur aminoacids, producing volatile sulphur compounds (VSCs) indispensable for the flavour of these products. We studied sulphur metabolism in two cheese-ripening microorganisms, the hemiascomycetous yeasts Kluyveromyces lactis and Yarrowia lipolytica. The in silico analysis of the phylum of hemiascomycetes gave us for the first time a...

  1. Ecosystème fromager : de l'étude du métabolisme du soufre chez Kluyveromyces lactis et Yarrowia lipolytica à l'interaction entre Kluyveromyces lactis et Brevibacterium aurantiacum

    OpenAIRE

    Hebert , Agnès

    2010-01-01

    Sulphur metabolism, which has a central role in the cell, is also important during the manufacturing of smear ripened cheeses. The cheese ecosystem degrades sulphur amino acids, producing volatile sulphur compounds (VSCs) indispensable for the flavour of these products. We studied sulphur metabolism in two cheese-ripening microorganisms, the hemiascomycetous yeasts Kluyveromyces lactis and Yarrowia lipolytica. The in silico analysis of the phylum of hemiascomycetes gave us for the first time ...

  2. Assessment of probiotic properties of lactic acid bacteria isolated from Indonesian naturally fermented milk

    Science.gov (United States)

    Jatmiko, Yoga Dwi; Howarth, Gordon S.; Barton, Mary D.

    2017-11-01

    This study aimed to characterize the probiotic properties of lactic acid bacteria from the naturally fermented milk of Indonesia, namely dangke and dadih. Fifty-one representative lactic acid bacteria belonging to the species Lactobacillus Plantarum, Lactococcus lactis subsp. lactis and Enterococcus faecium were evaluated in vitro for potential probiotic properties based on their bile salt resistance, low pH tolerance, antimicrobial activity, antibiotic susceptibility and adherence to Caco-2 colon cancer cells. In addition, bacteriocin related gene (plantaricin A), bile salt hydrolase (bsh) and mannose-specific adhesin (msa) genes in the genome of lactobacilli were also examined. None of the dangke isolates, which belonged to the species L. lactis subsp. lactis tolerated low pH. However, eight of the isolates were able to grow in the presence of bile salts. It was observed that L. plantarum strain S1.30 and SL2.7 from dadih tolerated low pH, survived bile salt concentrations and were resistant to vancomycin. Furthermore, these strains also contained bacteriocin regulating gene (plantaricin A) and msa and bsh genes in their genome. However, only the strain S1.30 exhibited optimal antimicrobial activity against the selected pathogens and was able to adhere to Caco-2 cells by up to 82.24±0.14%. Antagonistic activity of L. lactis subsp. lactis from dadih and dangke was not detected. However, 73.94±1.26% adherence to Caco-2 cells was demonstrated by L. lactis subsp. lactis strain SL3.34 sourced from dangke. These results suggest that Lactobacillus plantarum strain S1.30 associated with dadih fulfilled the in vitro probiotic criteria and could be exploited for further in vivo evaluation. In addition, dadih was an effective probiotic carrier compared to dangke.

  3. EVIDENCE-BASED DATA ON EFFECTIVENESS OF LACTOBACILLUS RHAMNOSUS GG AND BIFIDOBACTERIUM LACTIS ВB-12 IN PEDIATRIC PRACTICE

    Directory of Open Access Journals (Sweden)

    I. V. Andreyeva

    2011-01-01

    Full Text Available Prophylactic and therapeutic administration of prebiotics in treatment of different disorders is used very often nowadays. However, this kind of a treatment confirmed its efficacy in only several diseases. The review presents the data on efficacy of two probiotic microorganisms (L. rhamnosus GG and B. lactis Вb-12 in pediatric practice. Author summarizes and analyzes existing evidence-based data on efficacy of probiotics in treatment of acute diarrhea, prophylaxis of antibiotic-associated diarrhea and nosocomial infections. L. rhamnosus GG and B. lactis Вb-12 have their own place in prophylaxis of infections of airways and gastrointestinal tract. Administration of probiotics for treatment and prophylaxis of allergic and other diseases is reviewed. Safety of probiotics is described as well.

  4. Cloning, Characterization, Controlled Overexpression, and Inactivation of the Major Tributyrin Esterase Gene of Lactococcus lactis

    NARCIS (Netherlands)

    Fernández, Leonides; Beerthuyzen, Marke M.; Brown, Julie; Siezen, Roland J.; Coolbear, Tim; Holland, Ross; Kuipers, Oscar P.

    2000-01-01

    The gene encoding the major intracellular tributyrin esterase of Lactococcus lactis was cloned using degenerate DNA probes based on 19 known N-terminal amino acid residues of the purified enzyme. The gene, named estA, was sequenced and found to encode a protein of 258 amino acid residues. The

  5. The Deletion of the Succinate Dehydrogenase Gene KlSDH1 in Kluyveromyces lactis Does Not Lead to Respiratory Deficiency

    Science.gov (United States)

    Saliola, Michele; Bartoccioni, Paola Chiara; De Maria, Ilaria; Lodi, Tiziana; Falcone, Claudio

    2004-01-01

    We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose. PMID:15189981

  6. The effects of Bifidobacterium animalis ssp. lactis B94 on gastrointestinal wellness in adults with Prader-Willi syndrome: study protocol for a randomized controlled trial.

    Science.gov (United States)

    Alyousif, Zainab; Miller, Jennifer L; Sandoval, Mariana Y; MacPherson, Chad W; Nagulesapillai, Varuni; Dahl, Wendy J

    2018-04-27

    Constipation is a frequent problem in adults with Prader-Willi syndrome. Certain probiotics have been shown to improve transit and gastrointestinal symptoms of adults with functional constipation. The aim of this study is to determine the effect of daily consumption of Bifidobacterium animalis ssp. lactis B94 (B. lactis B94) on stool frequency, stool form, and gastrointestinal symptoms in adults with Prader-Willi syndrome. Adults with Prader-Willi syndrome (18-75 years old, n = 36) will be recruited and enrolled in a 20-week, randomized, double-blind, placebo-controlled, crossover study. Study subjects will be randomized to B. lactis B94 or placebo each for a 4-week period, preceded by a 4-week baseline and followed by 4-week washouts. Subjects will complete daily records of stool frequency and stool form (a proxy of transit time). Dietary intake data also will be collected. Stools, one in each period, will be collected for exploratory microbiota analyses. To our knowledge, this is the first randomized controlled trial evaluating the effectiveness of B. lactis in adults with Prader-Willi syndrome. The results of this study will provide evidence of efficacy for future clinical trials in patient populations with constipation. ClinicalTrials.gov ( NCT03277157 ). Registered on 08 September 2017.

  7. Transcriptome analysis shows activation of the arginine deiminase pathway in Lactococcus lactis as a response to ethanol stress.

    Science.gov (United States)

    Díez, Lorena; Solopova, Ana; Fernández-Pérez, Rocío; González, Miriam; Tenorio, Carmen; Kuipers, Oscar P; Ruiz-Larrea, Fernanda

    2017-09-18

    This paper describes the molecular response of Lactococcus lactis NZ9700 to ethanol. This strain is a well-known nisin producer and a lactic acid bacteria (LAB) model strain. Global transcriptome profiling using DNA microarrays demonstrated a bacterial adaptive response to the presence of 2% ethanol in the culture broth and differential expression of 67 genes. The highest up-regulation was detected for those genes involved in arginine degradation through the arginine deiminase (ADI) pathway (20-40 fold up-regulation). The metabolic responses to ethanol of wild type L. lactis strains were studied and compared to those of regulator-deletion mutants MG∆argR and MG∆ahrC. The results showed that in the presence of 2% ethanol those strains with an active ADI pathway reached higher growth rates when arginine was available in the culture broth than in absence of arginine. In a chemically defined medium strains with an active ADI pathway consumed arginine and produced ornithine in the presence of 2% ethanol, hence corroborating that arginine catabolism is involved in the bacterial response to ethanol. This is the first study of the L. lactis response to ethanol stress to demonstrate the relevance of arginine catabolism for bacterial adaptation and survival in an ethanol containing medium. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Characterization of lactococci isolated from milk produced in the Camembert region of Normandy.

    Science.gov (United States)

    Desmasures, N; Mangin, I; Corroler, D; Guéguen, M

    1998-12-01

    Thirty-eight Lactococcus strains, isolated from raw milk produced in two dairy areas in Normandy, were identified at the phenotypic level. Only Lactococcus lactis strains with the lactis phenotype were found in the milk samples. Most strains fermented lactose (97%) and showed proteinase activity (76%). Isolates were characterized by RAPD technique and rRNA gene restriction analysis. More L. lactis strains with the lactis genotype were found in the first area, while L. lactis strains with the cremoris genotype predominated in the second area. RAPD was more efficient than rRNA gene restriction analysis in differentiating between strains with the subsp. lactis genotype. For L. lactis with the subsp. cremoris genotype, the second method gave a better result but there was poor discrimination between strains. Plasmid profiles were determined. Patterns ranged in size from 1.3 to 16.5 kbp, and 29 different profiles were found. Six groups of strains were determined, five of which were specific for the area of origin. It is suggested that the region of manufacture could influence organoleptic properties of cheeses because of different Lactococcus strains in the raw milk used for cheese making.

  9. The carB gene encoding the large subunit of carbamoylphosphate synthetase from Lactococcus lactis is transcribed monocistronically

    DEFF Research Database (Denmark)

    Martinussen, Jan; Hammer, Karin

    1998-01-01

    The biosynthesis of carbamoylphosphate is catalysed by the heterodimeric enzyme carbamoylphosphate synthetase (CPSase). The genes encoding the two subunits in procaryotes are normally transcribed as an operon, whereas in Lactococcus lactis, the gene encoding the large subunit (carB) is shown...

  10. Discovery of proteins involved in the interaction between prebiotics carbohydrates and probiotics & whole proteome analysis of the probiotic strain Bifidobacterium animalis susp. lactis BB-12

    DEFF Research Database (Denmark)

    Gilad, Ofir

    Probiotic bacteria, which primarily belong to the genera Lactobascillus and Bifidobacterium, are live microorganisms that have been related to a variety of health-promoting effects. Prebiotics are indigestible food components that specifically stimulate the growth of probiotic organisms...... in the human gastrointestinal tract. Despite an increased scientific focus within this field, the mechanisms behind the beneficial effects exerted by pre- and probiotics are still far from fully understood. The purpose of the present industrial-PhD project was to identify proteins involved in interactions...... between the widely-used, extensively-studied probiotic strain Bifidobacterium animalis subsp. lactis BB-12 and potentially-prebiotic carbohydrates. The project was initiated with a screening phase in which more than 40 carbohydrates were tested for their ability to promote the growth of the bacterium...

  11. Overview on sugar metabolism and its control in Lactococcus lactis - The input from in vivo NMR

    NARCIS (Netherlands)

    Neves, AR; Pool, WA; Kok, J; Kuipers, OP; Santos, H; Neves, Ana Rute; Pool, Wietske A.

    The wide application of lactic acid bacteria in the production of fermented foods depends to a great extent on the unique features of sugar metabolism in these organisms. The relative metabolic simplicity and the availability of genetic tools made Lactococcus lactis the organism of choice to gain

  12. Intracellular and Extracellular Expression of Bacillus thuringiensis Crystal Protein Cry5B in Lactococcus lactis for Use as an Anthelminthic

    Science.gov (United States)

    Durmaz, Evelyn; Hu, Yan; Aroian, Raffi V.

    2015-01-01

    The Bacillus thuringiensis crystal (Cry) protein Cry5B (140 kDa) and a truncated version of the protein, tCry5B (79 kDa), are lethal to nematodes. Genes encoding the two proteins were separately cloned into a high-copy-number vector with a strong constitutive promoter (pTRK593) in Lactococcus lactis for potential oral delivery against parasitic nematode infections. Western blots using a Cry5B-specific antibody revealed that constitutively expressed Cry5B and tCry5B were present in both cells and supernatants. To increase production, cry5B was cloned into the high-copy-number plasmid pMSP3535H3, carrying a nisin-inducible promoter. Immunoblotting revealed that 3 h after nisin induction, intracellular Cry5B was strongly induced at 200 ng/ml nisin, without adversely affecting cell viability or cell membrane integrity. Both Cry5B genes were also cloned into plasmid pTRK1061, carrying a promoter and encoding a transcriptional activator that invoke low-level expression of prophage holin and lysin genes in Lactococcus lysogens, resulting in a leaky phenotype. Cry5B and tCry5B were actively expressed in the lysogenic strain L. lactis KP1 and released into cell supernatants without affecting culture growth. Lactate dehydrogenase (LDH) assays indicated that Cry5B, but not LDH, leaked from the bacteria. Lastly, using intracellular lysates from L. lactis cultures expressing both Cry5B and tCry5B, in vivo challenges of Caenorhabditis elegans worms demonstrated that the Cry proteins were biologically active. Taken together, these results indicate that active Cry5B proteins can be expressed intracellularly in and released extracellularly from L. lactis, showing potential for future use as an anthelminthic that could be delivered orally in a food-grade microbe. PMID:26682852

  13. Lactococcus lactis is capable of improving the riboflavin status in deficient rats

    OpenAIRE

    LeBlanc, Jean Guy; Burgess, Catherine M.; Sesma, Fernando; de Giori, Graciela Savoy; van Sinderen, Douwe

    2005-01-01

    Lactococcus lactis is a commonly used starter strain that can be converted from a vitamin B2 consumer into a vitamin B2 'factory' by over-expressing its riboflavin biosynthesis genes. The present study was conducted to assess in a rat bioassay the response of riboflavin produced by GM or native lactic acid bacteria (LAB). The riboflavin-producing strains were able to eliminate most physiological manifestations of ariboflavinosis such as stunted growth, elevated erythrocyte glutathione reducta...

  14. Casein Hydrolysates by Lactobacillus brevis and Lactococcus lactis Proteases: Peptide Profile Discriminates Strain-Dependent Enzyme Specificity.

    Science.gov (United States)

    Bounouala, Fatima Zohra; Roudj, Salima; Karam, Nour-Eddine; Recio, Isidra; Miralles, Beatriz

    2017-10-25

    Casein from ovine and bovine milk were hydrolyzed with two extracellular protease preparations from Lactobacillus brevis and Lactococcus lactis. The hydrolysates were analyzed by HPLC-MS/MS for peptide identification. A strain-dependent peptide profile could be observed, regardless of the casein origin, and the specificity of these two proteases could be computationally ascribed. The cleavage pattern yielding phenylalanine, leucine, or tyrosine at C-terminal appeared both at L. lactis and Lb. brevis hydrolysates. However, the cleavage C-terminal to lysine was favored with Lb. brevis protease. The hydrolysates showed ACE-inhibitory activity with IC 50 in the 16-70 μg/mL range. Ovine casein hydrolysates yielded greater ACE-inhibitory activity. Previously described antihypertensive and opioid peptides were found in these ovine and bovine casein hydrolysates and prediction of the antihypertensive activity of the sequences based on quantitative structure and activity relationship (QSAR) was performed. This approach might represent a useful classification tool regarding health-related properties prior to further purification.

  15. Cloning and functional expression of the mitochondrial alternative oxidase gene (aox1) of Aspergillus niger in Lactococcus lactis and its induction by oxidizing conditions.

    Science.gov (United States)

    Papagianni, Maria; Avramidis, Nicholaos

    2012-01-05

    Lactococcus lactis is a widely used food bacterium mainly known for its fermentation metabolism. An important, and for long time overlooked, trait of this species is its ability to perform respiratory metabolism in the presence of heme and under aerobic conditions. There is no evidence however for the presence of an alternative respiration pathway and AOX activity. In this study, a cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for alternative respiration, from a citric acid producing Aspergillus niger strain was cloned and expressed in L. lactis as a host strain. Expression of aox1 conferred on this organism cyanide-resistant and salicylhydroxamate-sensitive growth. Bioreactor cultures under fully aerobic conditions of the transformed L. lactis showed that the alternative respiratory pathway operates and improves significantly the microorganism's response to oxidizing stress conditions as it enhances biomass production, suppresses lactate formation, and leads to accumulation of large amounts of nisin. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. ORF Alignment: NC_002662 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available lactis subsp. lactis Il1403] ... Length = 134 ... Query: 6 ... LDNLYRAVILDHSSNPRHAGELQTGCMVDLNNPTCGDVIRLTVEFENDVI...SNIAFSGHGC 65 ... LDNLYRAVILDHSSNPRHAGELQTGCMVDLNNPTCGDVIRLTVEFENDVISNIAFSGHGC Sbjct: 1 ... LDN...LYRAVILDHSSNPRHAGELQTGCMVDLNNPTCGDVIRLTVEFENDVISNIAFSGHGC 60 ... Query: 126 KCSTLAWNALKKAI 139 ... KCSTLAWNALKKAI Sbjct: 121 KCSTLAWNALKKAI 134

  17. Use of the KlADH3 promoter for the quantitative production of the murine PDE5A isoforms in the yeast Kluyveromyces lactis.

    Science.gov (United States)

    Cardarelli, Silvia; Giorgi, Mauro; Naro, Fabio; Malatesta, Francesco; Biagioni, Stefano; Saliola, Michele

    2017-09-22

    Phosphodiesterases (PDE) are a superfamily of enzymes that hydrolyse cyclic nucleotides (cAMP/cGMP), signal molecules in transduction pathways regulating crucial aspects of cell life. PDEs regulate the intensity and duration of the cyclic nucleotides signal modulating the downstream biological effect. Due to this critical role associated with the extensive distribution and multiplicity of isozymes, the 11 mammalian families (PDE1 to PDE11) constitute key therapeutic targets. PDE5, one of these cGMP-specific hydrolysing families, is the molecular target of several well known drugs used to treat erectile dysfunction and pulmonary hypertension. Kluyveromyces lactis, one of the few yeasts capable of utilizing lactose, is an attractive host alternative to Saccharomyces cerevisiae for heterologous protein production. Here we established K. lactis as a powerful host for the quantitative production of the murine PDE5 isoforms. Using the promoter of the highly expressed KlADH3 gene, multicopy plasmids were engineered to produce the native and recombinant Mus musculus PDE5 in K. lactis. Yeast cells produced large amounts of the purified A1, A2 and A3 isoforms displaying K m , V max and Sildenafil inhibition values similar to those of the native murine enzymes. PDE5 whose yield was nearly 1 mg/g wet weight biomass for all three isozymes (30 mg/L culture), is well tolerated by K. lactis cells without major growth deficiencies and interferences with the endogenous cAMP/cGMP signal transduction pathways. To our knowledge, this is the first time that the entire PDE5 isozymes family containing both regulatory and catalytic domains has been produced at high levels in a heterologous eukaryotic organism. K. lactis has been shown to be a very promising host platform for large scale production of mammalian PDEs for biochemical and structural studies and for the development of new specific PDE inhibitors for therapeutic applications in many pathologies.

  18. Fermented Milk Containing Bifidobacterium lactis DN-173 010 in Childhood Constipation: A Randomized, Double-Blind, Controlled Trial

    NARCIS (Netherlands)

    Tabbers, Merit M.; Chmielewska, Ania; Roseboom, Maaike G.; Crastes, Nolwenn; Perrin, Catherine; Reitsma, Johannes B.; Norbruis, Obbe; Szajewska, Hania; Benninga, Marc A.

    2011-01-01

    BACKGROUND: Constipation is a frustrating symptom affecting 3% of children worldwide. A fermented dairy product containing Bifidobacterium lactis strain DN-173 010 was effective in increasing stool frequency in constipated women. Our aim was to assess the effects of this product in constipated

  19. Characterization of Lactococcus lactis response to ampicillin and ciprofloxacin using surface-enhanced Raman spectroscopy.

    Science.gov (United States)

    Wang, Panxue; Pang, Shintaro; Zhang, Hua; Fan, Mingtao; He, Lili

    2016-01-01

    Decades of antibiotic use or misuse has resulted in antibiotic resistance in lactic acid bacteria, a group of common culture starters and probiotic microorganisms. This has urged researchers to study how lactic acid bacteria respond to antibiotics, so as to have a better strategy to identify and predict the antibiotic-resistant bacteria. This study aimed to characterize the biochemical profiles of Lactococcus lactis responding to antibiotics using surface-enhanced Raman spectroscopy (SERS). Lactococcus lactis exposed to antibiotics was mixed with 50-nm gold nanoparticles for subsequent SERS measurements. The SERS spectra analyzed by principal component analysis showed no significant change after 30 min of antibiotic treatment, whereas distinct changes were clearly observed after 60 and 90 min of antibiotic treatment. Different antibiotics induced different spectral changes, and these changes revealed the detailed biochemical information of cellular responses. This study demonstrates that the SERS method developed not only senses the changes in the bacterial cell wall, but also reveals details of the biochemical profiles, which help us to understand how lactic acid bacteria respond to antibiotics, as well as to set a base for the detection of antibiotic susceptibility of bacteria by SERS.

  20. Effect of a fermented milk containing Bifidobacterium lactis DN-173010 on Chinese constipated women.

    Science.gov (United States)

    Yang, Yue-Xin; He, Mei; Hu, Gang; Wei, Jie; Pages, Philippe; Yang, Xian-Hua; Bourdu-Naturel, Sophie

    2008-10-28

    To investigate the effect of a fermented milk containing Bifidobacterium lactis DN-173010 and yogurt strains (BIO(R)) on adult women with constipation in Beijing. A total of 135 adult females with constipation were randomly allocated to consume for 2 wk either 100 g of the test fermented milk or 100 g of an acidified milk containing non-living bacteria (control). Stool frequency, defecation condition scores, stool consistency and food intake were recorded at baseline and after 1 and 2 wk in an intention-to-treat population of 126 subjects. In parallel, safety evaluation parameters were performed. At baseline, no differences were found between groups. Following consumption of test product, stool frequency was significantly increased after 1 wk (3.5 +/- 1.5 vs 2.4 +/- 0.6, P food intake did not change between the two groups, and safety parameters of the subjects were within normal ranges. This study suggests a beneficial effect of a fermented milk containing B. lactis DN-173010 on stool frequency, defecation condition and stool consistency in adult women with constipation constipated women after 1 and 2 wk of consumption.

  1. Statistical investigation of Kluyveromyces lactis cells permeabilization with ethanol by response surface methodology.

    Science.gov (United States)

    de Faria, Janaína T; Rocha, Pollyana F; Converti, Attilio; Passos, Flávia M L; Minim, Luis A; Sampaio, Fábio C

    2013-12-01

    The aim of our study was to select the optimal operating conditions to permeabilize Kluyveromyces lactis cells using ethanol as a solvent as an alternative to cell disruption and extraction. Cell permeabilization was carried out by a non-mechanical method consisting of chemical treatment with ethanol, and the results were expressed as β-galactosidase activity. Experiments were conducted under different conditions of ethanol concentration, treatment time and temperature according to a central composite rotatable design (CCRD), and the collected results were then worked out by response surface methodology (RSM). Cell permeabilization was improved by an increase in ethanol concentration and simultaneous decreases in the incubation temperature and treatment time. Such an approach allowed us to identify an optimal range of the independent variables within which the β-galactosidase activity was optimized. A maximum permeabilization of 2,816 mmol L(-1) oNP min(-1) g(-1) was obtained by treating cells with 75.0% v/v of ethanol at 20.0 °C for 15.0 min. The proposed methodology resulted to be effective and suited for K. lactis cells permeabilization at a lab-scale and promises to be of possible interest for future applications mainly in the food industry.

  2. Molecular Characterization of a Recombinant Manganese Superoxide Dismutase from Lactococcus lactis M4

    Directory of Open Access Journals (Sweden)

    Boon Hooi Tan

    2014-01-01

    Full Text Available A superoxide dismutase (SOD gene of Lactococcus lactis M4 was cloned and expressed in a prokaryotic system. Sequence analysis revealed an open reading frame of 621 bp which codes for 206 amino acid residues. Expression of sodA under T7 promoter exhibited a specific activity of 4967 U/mg when induced with 1 mM of isopropyl-β-D-thiogalactopyranoside. The recombinant SOD was purified to homogeneity by immobilised metal affinity chromatography and Superose 12 gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses of the recombinant SOD detected a molecular mass of approximately 27 kDa. However, the SOD was in dimer form as revealed by gel filtration chromatography. The purified recombinant enzyme had a pI of 4.5 and exhibited maximal activity at 25°C and pH 7.2. It was stable up to 45°C. The insensitivity of this lactococcal SOD to cyanide and hydrogen peroxide established that it was a MnSOD. Although it has 98% homology to SOD of L. lactis IL1403, this is the first elucidated structure of lactococcal SOD revealing active sites containing the catalytic manganese coordinated by four ligands (H-27, H-82, D-168, and H-172.

  3. A copper-induced quinone degradation pathway provides protection against combined copper/quinone stress in Lactococcus lactis IL1403.

    Science.gov (United States)

    Mancini, Stefano; Abicht, Helge K; Gonskikh, Yulia; Solioz, Marc

    2015-02-01

    Quinones are ubiquitous in the environment. They occur naturally but are also in widespread use in human and industrial activities. Quinones alone are relatively benign to bacteria, but in combination with copper, they become toxic by a mechanism that leads to intracellular thiol depletion. Here, it was shown that the yahCD-yaiAB operon of Lactococcus lactis IL1403 provides resistance to combined copper/quinone stress. The operon is under the control of CopR, which also regulates expression of the copRZA copper resistance operon as well as other L. lactis genes. Expression of the yahCD-yaiAB operon is induced by copper but not by quinones. Two of the proteins encoded by the operon appear to play key roles in alleviating quinone/copper stress: YaiB is a flavoprotein that converts p-benzoquinones to less toxic hydroquinones, using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as reductant; YaiA is a hydroquinone dioxygenase that converts hydroquinone putatively to 4-hydroxymuconic semialdehyde in an oxygen-consuming reaction. Hydroquinone and methylhydroquinone are both substrates of YaiA. Deletion of yaiB causes increased sensitivity of L. lactis to quinones and complete growth arrest under combined quinone and copper stress. Copper induction of the yahCD-yaiAB operon offers protection to copper/quinone toxicity and could provide a growth advantage to L. lactis in some environments. © 2014 John Wiley & Sons Ltd.

  4. LnqR, a TetR-family transcriptional regulator, positively regulates lacticin Q production in Lactococcus lactis QU 5.

    Science.gov (United States)

    Iwatani, Shun; Ishibashi, Naoki; Flores, Floirendo P; Zendo, Takeshi; Nakayama, Jiro; Sonomoto, Kenji

    2016-09-01

    Lacticin Q is an unmodified leaderless bacteriocin produced by Lactococcus lactis QU 5. It has been revealed that the production and self-immunity of lacticin Q are facilitated by a gene cluster lnqQBCDEF The gene for a putative TetR-family transcriptional regulator, termed lnqR, was found nearby the lnqQBCDEF cluster, but its involvement in lacticin Q biosynthesis remained unknown. In this study, we created an LnqR-overexpressing QU 5 recombinant by using lactococcal constitutive promoter P32 The recombinant QU 5 showed enhanced production of and self-immunity to lacticin Q. RT-PCR analysis has revealed that an overexpression of LnqR increases the amounts of lnqQBCDEF transcripts, and these six genes are transcribed as an operon in a single transcriptional unit. Interestingly, LnqR expression and thus lacticin Q production by L. lactis QU 5 was found temperature dependent, while LnzR, an LnqR-homologue, in L. lactis QU 14 was expressed in a similar but not identical manner to LnqR, resulting in dissimilar bacteriocin productivities by these strains. This report demonstrates LnqR as the first TetR-family transcriptional regulator involved in LAB bacteriocin biosynthesis and that, as an exceptional case of TetR-family regulators, LnqR positively regulates the transcription of these biosynthetic genes. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Metabolic characterization and transformation of the non-dairy Lactococcus lactis strain KF147, for production of ethanol from xylose

    DEFF Research Database (Denmark)

    Petersen, Kia Vest; Liu, Jianming; Chen, Jun

    2017-01-01

    producing ethanol as the sole fermentation product with a high yield corresponding to 83% of the theoretical maximum. The results clearly indicate the great potential of using the more metabolically diverse non-dairy L. lactis strains for bio-production based on xylose containing feedstocks.......The non-dairy lactic acid bacterium Lactococcus lactis KF147 can utilize xylose as the sole energy source. To assess whether KF147 could serve as a platform organism for converting second generation sugars into useful chemicals, we characterized growth and product formation for KF147 when grown...... the arcA gene encoding the arginine deiminase. The fermentation product profile suggested two routes for xylose degradation, the phosphoketolase pathway and the pentose phosphate pathway. Inactivation of the phosphoketolase pathway redirected the entire flux through the pentose phosphate pathway whereas...

  6. Volatile Organic Compounds in Naturally Fermented Milk and Milk Fermented Using Yeasts, Lactic Acid Bacteria and Their Combinations As Starter Cultures

    Directory of Open Access Journals (Sweden)

    Bennie C. Viljoen

    2007-01-01

    Full Text Available The volatile organic compounds present in 18 Zimbabwean naturally fermented milk (amasi samples and those produced by various yeasts, lactic acid bacteria (LAB and yeast/ LAB combinations were determined using headspace gas chromatography. The yeast strains used were: Candida kefyr 23, C. lipolytica 57, Saccharomyces cerevisiae 71, C. lusitaniae 68, C. tropicalis 78, C. lusitaniae 63, C. colliculosa 41, S. dairenensis 32, and Dekkera bruxellensis 43, and were coded Y1 to Y9, respectively. The LAB strains used were Lactococcus lactis subsp. lactis Lc39, L. lactis subsp. lactis Lc261, Lactobacillus paracasei Lb11, and L. lactis subsp. lactis biovar. diacetylactis C1, and were coded B1 to B4, respectively. Some of the volatile organic compounds found in amasi were acetaldehyde, ethanol, acetone, 2-methyl propanal, 2-methyl-1-propanol and 3-methyl-1-butanol. However, the levels of volatile organic compounds in the naturally fermented milk (NFM samples varied from one sample to another, with acetaldehyde ranging from 0.1–18.4 ppm, 3-methyl butanal from <0.1–0.47 ppm and ethanol from 39.3–656 ppm. The LAB/C. kefyr 23 (B/Y1 co-cultures produced significantly (p<0.05 higher levels of acetaldehyde and ethanol than the levels found in the NFM. The acetaldehyde levels in the B/Y1 samples ranged from 26.7–87.7 ppm, with L. lactis subsp. lactis biovar. diacetylactis C1 (B4 producing the highest level of acetaldehyde in combination with C. kefyr 23 (Y1. Using principal component analysis (PCA, most of the NFM samples were grouped together with single and co-cultures of Lc261, Lb11 and the non-lactose fermenting yeasts, mainly because of the low levels of ethanol and similar levels of 3-methyl butanal. Chromatograms of amasi showed prominent peak of methyl aldehydes and their alcohols including 3-methyl-butanal and 3-methyl-butanol, suggesting that these compounds are important attributes of Zimbabwean naturally fermented milk.

  7. Metabolic engineering of mannitol production in Lactococcus lactis: influence of overexpression of mannitol 1-phosphate dehydrogenase in different genetic backgrounds.

    Science.gov (United States)

    Wisselink, H Wouter; Mars, Astrid E; van der Meer, Pieter; Eggink, Gerrit; Hugenholtz, Jeroen

    2004-07-01

    To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and (13)C nuclear magnetic resonance analysis revealed that small amounts (<1%) of mannitol were formed by growing cells of mtlD-overexpressing LDH-deficient and phosphofructokinase-reduced strains, whereas resting cells of the LDH-deficient transformant converted 25% of glucose into mannitol. Moreover, the formed mannitol was not reutilized upon glucose depletion. Of the metabolic-engineering strategies investigated in this work, mtlD-overexpressing LDH-deficient L. lactis seemed to be the most promising strain for mannitol production.

  8. Safety characterisation and inhibition of fungi and bacteria by a novel multiple enterocin-producing Enterococcus lactis 4CP3 strain.

    Science.gov (United States)

    Ben Braïek, Olfa; Cremonesi, Paola; Morandi, Stefano; Smaoui, Slim; Hani, Khaled; Ghrairi, Taoufik

    2018-03-07

    This study aims to characterise a potential bacteriocinogenic lactic acid bacterial strain isolated from a raw pink shrimp (Palaemon serratus) and evaluate its safety aspect. The strain designated as 4CP3 was noted to display antibacterial activities (P enterocins A, B and P. To our knowledge, this feature is newly described for E. lactis strain isolated from raw shrimps. Regarding safety aspect of E. lactis 4CP3, it has been demonstrated that this strain was not haemolytic, gelatinase negative, sensitive to vancomycin, and free of common antibiotic resistance genes and virulence factors. Therefore, it may be useful as a safe natural agent in preservation of foods or as a new probiotic strain in food and feed. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. Characterization of non-starter lactic acid bacteria in traditionally produced home-made Radan cheese during ripening

    Directory of Open Access Journals (Sweden)

    Jokovic Natasa

    2011-01-01

    Full Text Available Two hundred thirteen non-starter lactic acid bacteria isolated from Radan cheese during ripening were identified with both a classical biochemical test and rep-PCR with (GTG5 primer. For most isolates, which belong to the Lactococcus lactis subsp. lactis, Leuconostoc mesenteroides, Lactobacillus plantarum, Lactobacillus paraplantarum and Enterococcus faecium, a phenotypic identification was in good agreement with rep-PCR identification. Lactococeus lactis subsp. lactis, Enterococcus faecium and subspecies from the Lenconostoc mesenteroides group were the dominant population of lactic acid bacteria in cheese until 10 days of ripening and only one Streptococcus thermophilus strain was isolated from the 5-day-old cheese sample. As ripening progressed, Lactobacillus plantarum became the predominant species together with the group of heterofermentative species of lactobacilli that could not be precisely identified with rep-PCR.

  10. A high-throughput qPCR system for simultaneous quantitative detection of dairy Lactococcus lactis and Leuconostoc bacteriophages

    DEFF Research Database (Denmark)

    Muhammed, Musemma Kedir; Krych, Lukasz; Nielsen, Dennis Sandris

    2017-01-01

    simultaneous quantitative detection of Lc. lactis 936 (now SK1virus), P335, c2 (now C2virus) and Leuconostoc phage groups. Component assays are designed to have high efficiencies and nearly the same dynamic detection ranges, i.e., from 1.1 x 105 to 1.1 x 101 phage genomes per reaction, which corresponds to 9 x......Simultaneous quantitative detection of Lactococcus (Lc.) lactis and Leuconostoc species bacteriophages (phages) has not been reported in dairies using undefined mixed-strain DL-starters, probably due to the lack of applicable methods. We optimized a high-throughput qPCR system that allows...... 107 to 9 x 103 phage particles mL-1 without any additional up-concentrating steps. The amplification efficiencies of the corresponding assays were 100.1±2.6, 98.7±2.3, 101.0±2.3 and 96.2±6.2. The qPCR system was tested on samples obtained from a dairy plant that employed traditional mother...

  11. Influence of extracellular pH on growth, viability, cell size, acidification activity, and intracellular pH of Lactococcus lactis in batch fermentations.

    Science.gov (United States)

    Hansen, Gunda; Johansen, Claus Lindvald; Marten, Gunvor; Wilmes, Jacqueline; Jespersen, Lene; Arneborg, Nils

    2016-07-01

    In this study, we investigated the influence of three extracellular pH (pHex) values (i.e., 5.5, 6.5, and 7.5) on the growth, viability, cell size, acidification activity in milk, and intracellular pH (pHi) of Lactococcus lactis subsp. lactis DGCC1212 during pH-controlled batch fermentations. A universal parameter (e.g., linked to pHi) for the description or prediction of viability, specific acidification activity, or growth behavior at a given pHex was not identified. We found viability as determined by flow cytometry to remain high during all growth phases and irrespectively of the pH set point. Furthermore, regardless of the pHex, the acidification activity per cell decreased over time which seemed to be linked to cell shrinkage. Flow cytometric pHi determination demonstrated an increase of the averaged pHi level for higher pH set points, while the pH gradient (pHi-pHex) and the extent of pHi heterogeneity decreased. Cells maintained positive pH gradients at a low pHex of 5.5 and even during substrate limitation at the more widely used pHex 6.5. Moreover, the strain proved able to grow despite small negative or even absent pH gradients at a high pHex of 7.5. The larger pHi heterogeneity at pHex 5.5 and 6.5 was associated with more stressful conditions resulting, e.g., from higher concentrations of non-dissociated lactic acid, while the low pHi heterogeneity at pHex 7.5 most probably corresponded to lower concentrations of non-dissociated lactic acid which facilitated the cells to reach the highest maximum active cell counts of the three pH set points.

  12. Contribution of plasmid-encoded peptidase S8 (PrtP) to adhesion and transit in the gut of Lactococcus lactis IBB477 strain.

    Science.gov (United States)

    Radziwill-Bienkowska, Joanna Maria; Robert, Véronique; Drabot, Karolina; Chain, Florian; Cherbuy, Claire; Langella, Philippe; Thomas, Muriel; Bardowski, Jacek Karol; Mercier-Bonin, Muriel; Kowalczyk, Magdalena

    2017-07-01

    The ability of Lactococcus lactis to adhere to the intestinal mucosa can potentially prolong the contact with the host, and therefore favour its persistence in the gut. In the present study, the contribution of plasmid-encoded factors to the adhesive and transit properties of the L. lactis subsp. cremoris IBB477 strain was investigated. Plasmid-cured derivatives as well as deletion mutants were obtained and analysed. Adhesion tests were performed using non-coated polystyrene plates, plates coated with mucin or fibronectin and mucus-secreting HT29-MTX intestinal epithelial cells. The results indicate that two plasmids, pIBB477a and b, are involved in adhesion of the IBB477 strain. One of the genes localised on plasmid pIBB477b (AJ89_14230), which encodes cell wall-associated peptidase S8 (PrtP), mediates adhesion of the IBB477 strain to bare, mucin- and fibronectin-coated polystyrene, as well as to HT29-MTX cells. Interactions between bacteria and mucus secreted by HT29-MTX cells were further investigated by fluorescent staining and confocal microscopy. Confocal images showed that IBB477 forms dense clusters embedded in secreted mucus. Finally, the ability of IBB477 strain and its ΔprtP deletion mutant to colonise the gastrointestinal tract of conventional C57Bl/6 mice was determined. Both strains were present in the gut for up to 72 h. In summary, adhesion and persistence of IBB477 were analysed by in vitro and in vivo approaches, respectively. Our studies revealed that plasmidic genes encoding cell surface proteins are more involved in the adhesion of IBB477 strain than in the ability to confer a selective advantage in the gut.

  13. Early Transcriptome Response of Lactococcus lactis to Environmental Stresses Reveals Differentially Expressed Small Regulatory RNAs and tRNAs

    NARCIS (Netherlands)

    van der Meulen, Sjoerd B.; de Jong, Anne; Kok, Jan

    2017-01-01

    Bacteria can deploy various mechanisms to combat environmental stresses. Many genes have previously been identified in Lactococcus lactis that are involved in sensing the stressors and those that are involved in regulating and mounting a defense against the stressful conditions. However, the

  14. Does the scientific evidence support the advertising claims made for products containing Lactobacillus casei and Bifidobacterium lactis? A systematic review.

    Science.gov (United States)

    Meléndez-Illanes, Lorena; González-Díaz, Cristina; Chilet-Rosell, Elisa; Álvarez-Dardet, Carlos

    2016-09-01

    To analyse the scientific evidence that exists for the advertising claims made for two products containing Lactobacillus casei and Bifidobacterium lactis and to conduct a comparison between the published literature and what is presented in the corporate website. Systematic review, using Medline through Pubmed and Embase. We included human clinical trials that exclusively measured the effect of Lactobacillus casei or Bifidobacterium lactis on a healthy population, and where the objective was related to the health claims made for certain products in advertising. We assessed the levels of evidence and the strength of the recommendation according to the classification criteria established by the Oxford Centre for Evidence Based Medicine (CEBM). We also assessed the outcomes of the studies published on the website that did not appear in the search. Of the 440 articles identified, 16 met the inclusion criteria. Only four (25%) of these presented a level of evidence of 1b and a recommendation grade of A, all corresponding to studies on product containing Bifidobacterium lactis, and only 12 of the 16 studies were published on the corporate website (47). There is insufficient scientific evidence to support the health claims made for these products, especially in the case of product containing Lactobacillus casei. © The Author 2015. Published by Oxford University Press on behalf of Faculty of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. Specificity of the second binding protein of the peptide ABC-transporter (Dpp) of Lactococcus lactis IL1403

    NARCIS (Netherlands)

    Sanz, Y; Toldra, F; Renault, P; Poolman, B

    2003-01-01

    The genome sequence of Lactococcus lactis IL1403 revealed the presence of a putative peptide-binding protein-dependent ABC-transporter (Dpp). The genes for two peptide-binding proteins (dppA and dppP) precede the membrane components, which include two transmembrane protein genes (dppB and dppC) and

  16. Consumption of Dairy Yogurt Containing Lactobacillus paracasei ssp. paracasei, Bifidobacterium animalis ssp. lactis and Heat-Treated Lactobacillus plantarum Improves Immune Function Including Natural Killer Cell Activity

    Science.gov (United States)

    Lee, Ayoung; Lee, Young Ju; Yoo, Hye Jin; Kim, Minkyung; Chang, Yeeun; Lee, Dong Seog; Lee, Jong Ho

    2017-01-01

    The aim of this study was to investigate the impact of consuming dairy yogurt containing Lactobacillus paracasei ssp. paracasei (L. paracasei), Bifidobacterium animalis ssp. lactis (B. lactis) and heat-treated Lactobacillus plantarum (L. plantarum) on immune function. A randomized, open-label, placebo-controlled study was conducted on 200 nondiabetic subjects. Over a twelve-week period, the test group consumed dairy yogurt containing probiotics each day, whereas the placebo group consumed milk. Natural killer (NK) cell activity, interleukin (IL)-12 and immunoglobulin (Ig) G1 levels were significantly increased in the test group at twelve weeks compared to baseline. Additionally, the test group had significantly greater increases in serum NK cell activity and interferon (IFN)-γ and IgG1 than placebo group. Daily consumption of dairy yogurt containing L. paracasei, B. lactis and heat-treated L. plantarum could be an effective option to improve immune function by enhancing NK cell function and IFN-γ concentration (ClinicalTrials.gov: NCT03051425). PMID:28561762

  17. Consumption of Dairy Yogurt Containing Lactobacillus paracasei ssp. paracasei, Bifidobacterium animalis ssp. lactis and Heat-Treated Lactobacillus plantarum Improves Immune Function Including Natural Killer Cell Activity

    Directory of Open Access Journals (Sweden)

    Ayoung Lee

    2017-05-01

    Full Text Available The aim of this study was to investigate the impact of consuming dairy yogurt containing Lactobacillus paracasei ssp. paracasei (L. paracasei, Bifidobacterium animalis ssp. lactis (B. lactis and heat-treated Lactobacillus plantarum (L. plantarum on immune function. A randomized, open-label, placebo-controlled study was conducted on 200 nondiabetic subjects. Over a twelve-week period, the test group consumed dairy yogurt containing probiotics each day, whereas the placebo group consumed milk. Natural killer (NK cell activity, interleukin (IL-12 and immunoglobulin (Ig G1 levels were significantly increased in the test group at twelve weeks compared to baseline. Additionally, the test group had significantly greater increases in serum NK cell activity and interferon (IFN-γ and IgG1 than placebo group. Daily consumption of dairy yogurt containing L. paracasei, B. lactis and heat-treated L. plantarum could be an effective option to improve immune function by enhancing NK cell function and IFN-γ concentration (ClinicalTrials.gov: NCT03051425.

  18. Consumption of Dairy Yogurt Containing Lactobacillus paracasei ssp. paracasei, Bifidobacterium animalis ssp. lactis and Heat-Treated Lactobacillus plantarum Improves Immune Function Including Natural Killer Cell Activity.

    Science.gov (United States)

    Lee, Ayoung; Lee, Young Ju; Yoo, Hye Jin; Kim, Minkyung; Chang, Yeeun; Lee, Dong Seog; Lee, Jong Ho

    2017-05-31

    The aim of this study was to investigate the impact of consuming dairy yogurt containing Lactobacillus paracasei ssp. paracasei ( L. paracasei ), Bifidobacterium animalis ssp. lactis ( B. lactis ) and heat-treated Lactobacillus plantarum ( L. plantarum ) on immune function. A randomized, open-label, placebo-controlled study was conducted on 200 nondiabetic subjects. Over a twelve-week period, the test group consumed dairy yogurt containing probiotics each day, whereas the placebo group consumed milk. Natural killer (NK) cell activity, interleukin (IL)-12 and immunoglobulin (Ig) G1 levels were significantly increased in the test group at twelve weeks compared to baseline. Additionally, the test group had significantly greater increases in serum NK cell activity and interferon (IFN)-γ and IgG1 than placebo group. Daily consumption of dairy yogurt containing L. paracasei , B. lactis and heat-treated L. plantarum could be an effective option to improve immune function by enhancing NK cell function and IFN-γ concentration (ClinicalTrials.gov: NCT03051425).

  19. Impact of High-Level Expression of Heterologous Protein on Lactococcus lactis Host.

    Science.gov (United States)

    Kim, Mina; Jin, Yerin; An, Hyun-Joo; Kim, Jaehan

    2017-07-28

    The impact of overproduction of a heterologous protein on the metabolic system of host Lactococcus lactis was investigated. The protein expression profiles of L. lactis IL1403 containing two near-identical plasmids that expressed high- and low-level of the green fluorescent protein (GFP) were examined via shotgun proteomics. Analysis of the two strains via high-throughput LC-MS/MS proteomics identified the expression of 294 proteins. The relative amount of each protein in the proteome of both strains was determined by label-free quantification using the spectral counting method. Although expression level of most proteins were similar, several significant alterations in metabolic network were identified in the high GFP-producing strain. These changes include alterations in the pyruvate fermentation pathway, oxidative pentose phosphate pathway, and de novo synthesis pathway for pyrimidine RNA. Expression of enzymes for the synthesis of dTDP-rhamnose and N -acetylglucosamine from glucose was suppressed in the high GFP strain. In addition, enzymes involved in the amino acid synthesis or interconversion pathway were downregulated. The most noticeable changes in the high GFP-producing strain were a 3.4-fold increase in the expression of stress response and chaperone proteins and increase of caseinolytic peptidase family proteins. Characterization of these host expression changes witnessed during overexpression of GFP was might suggested the metabolic requirements and networks that may limit protein expression, and will aid in the future development of lactococcal hosts to produce more heterologous protein.

  20. Genome-scale diversity and niche adaptation analysis of Lactococcus lactis by comparative genome hybridization using multi-strain arrays.

    NARCIS (Netherlands)

    Siezen, R.J.; Bayjanov, J.R.; Felis, G.E.; van der Sijde, M.R.; Starrenburg, M.; Molenaar, D.; Wels, M.; Hijum, S.A.; van Hylckama Vlieg, J.E.T.

    2011-01-01

    Lactococcus lactis produces lactic acid and is widely used in the manufacturing of various fermented dairy products. However, the species is also frequently isolated from non-dairy niches, such as fermented plant material. Recently, these non-dairy strains have gained increasing interest, as they

  1. African Journal of Biotechnology - Vol 13, No 53 (2014)

    African Journals Online (AJOL)

    Cloning of nis gene and Nisin purification from Lactococcus lactis subsp. lactis Fc2 · EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT. DE El-hadedy, EW El-gammal ...

  2. Regulation and Adaptive Evolution of Lactose Operon Expression in Lactobacillus delbrueckii

    Science.gov (United States)

    Lapierre, Luciane; Mollet, Beat; Germond, Jacques-Edouard

    2002-01-01

    Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis are both used in the dairy industry as homofermentative lactic acid bacteria in the production of fermented milk products. After selective pressure for the fast fermentation of milk in the manufacture of yogurts, L. delbrueckii subsp. bulgaricus loses its ability to regulate lac operon expression. A series of mutations led to the constitutive expression of the lac genes. A complex of insertion sequence (IS) elements (ISL4 inside ISL5), inserted at the border of the lac promoter, induced the loss of the palindromic structure of one of the operators likely involved in the binding of regulatory factors. A lac repressor gene was discovered downstream of the β-galactosidase gene of L. delbrueckii subsp. lactis and was shown to be inactivated by several mutations in L. delbrueckii subsp. bulgaricus. Regulatory mechanisms of the lac gene expression of L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis were compared by heterologous expression in Lactococcus lactis of the two lac promoters in front of a reporter gene (β-glucuronidase) in the presence or absence of the lac repressor gene. Insertion of the complex of IS elements in the lac promoter of L. delbrueckii subsp. bulgaricus increased the promoter's activity but did not prevent repressor binding; rather, it increased the affinity of the repressor for the promoter. Inactivation of the lac repressor by mutations was then necessary to induce the constitutive expression of the lac genes in L. delbrueckii subsp. bulgaricus. PMID:11807052

  3. Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with Various Plasmids

    OpenAIRE

    Serror, Pascale; Sasaki, Takashi; Ehrlich, S. Dusko; Maguin, Emmanuelle

    2002-01-01

    We describe, for the first time, a detailed electroporation procedure for Lactobacillus delbrueckii. Three L. delbrueckii strains were successfully transformed. Under optimal conditions, the transformation efficiency was 104 transformants per μg of DNA. Using this procedure, we identified several plasmids able to replicate in L. delbrueckii and integrated an integrative vector based on phage integrative elements into the L. delbrueckii subsp. bulgaricus chromosome. These vectors provide a goo...

  4. Cloning, production, and functional expression of the bacteriocin enterocin A, produced by Enterococcus faecium T136, by the yeasts Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, and Arxula adeninivorans.

    Science.gov (United States)

    Borrero, Juan; Kunze, Gotthard; Jiménez, Juan J; Böer, Erik; Gútiez, Loreto; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

    2012-08-01

    The bacteriocin enterocin A (EntA) produced by Enterococcus faecium T136 has been successfully cloned and produced by the yeasts Pichia pastoris X-33EA, Kluyveromyces lactis GG799EA, Hansenula polymorpha KL8-1EA, and Arxula adeninivorans G1212EA. Moreover, P. pastoris X-33EA and K. lactis GG799EA produced EntA in larger amounts and with higher antimicrobial and specific antimicrobial activities than the EntA produced by E. faecium T136.

  5. Regulation of Cell Wall Plasticity by Nucleotide Metabolism in Lactococcus lactis*

    Science.gov (United States)

    Solopova, Ana; Formosa-Dague, Cécile; Courtin, Pascal; Furlan, Sylviane; Veiga, Patrick; Péchoux, Christine; Armalyte, Julija; Sadauskas, Mikas; Kok, Jan; Hols, Pascal; Dufrêne, Yves F.; Kuipers, Oscar P.; Chapot-Chartier, Marie-Pierre; Kulakauskas, Saulius

    2016-01-01

    To ensure optimal cell growth and separation and to adapt to environmental parameters, bacteria have to maintain a balance between cell wall (CW) rigidity and flexibility. This can be achieved by a concerted action of peptidoglycan (PG) hydrolases and PG-synthesizing/modifying enzymes. In a search for new regulatory mechanisms responsible for the maintenance of this equilibrium in Lactococcus lactis, we isolated mutants that are resistant to the PG hydrolase lysozyme. We found that 14% of the causative mutations were mapped in the guaA gene, the product of which is involved in purine metabolism. Genetic and transcriptional analyses combined with PG structure determination of the guaA mutant enabled us to reveal the pivotal role of the pyrB gene in the regulation of CW rigidity. Our results indicate that conversion of l-aspartate (l-Asp) to N-carbamoyl-l-aspartate by PyrB may reduce the amount of l-Asp available for PG synthesis and thus cause the appearance of Asp/Asn-less stem peptides in PG. Such stem peptides do not form PG cross-bridges, resulting in a decrease in PG cross-linking and, consequently, reduced PG thickness and rigidity. We hypothesize that the concurrent utilization of l-Asp for pyrimidine and PG synthesis may be part of the regulatory scheme, ensuring CW flexibility during exponential growth and rigidity in stationary phase. The fact that l-Asp availability is dependent on nucleotide metabolism, which is tightly regulated in accordance with the growth rate, provides L. lactis cells the means to ensure optimal CW plasticity without the need to control the expression of PG synthesis genes. PMID:27022026

  6. Investigation of the bacteriophage community in induced lysates of undefined mesophilic mixed-strain DL-cultures using classical and metagenomic approaches

    DEFF Research Database (Denmark)

    Muhammed, Musemma K.; Olsen, Mette L.; Kot, Witold

    2018-01-01

    To investigate the notion that starter cultures can be a reservoir of bacteriophages (phages) in the dairy environment, strains of three DL-starters (undefined mesophilic mixed-strain starters containing Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc species) were selected...

  7. Effects of dietary supplementation of Lactobacillus rhamnosus or/and Lactococcus lactis on the growth, gut microbiota and immune responses of red sea bream, Pagrus major.

    Science.gov (United States)

    Dawood, Mahmoud A O; Koshio, Shunsuke; Ishikawa, Manabu; Yokoyama, Saichiro; El Basuini, Mohammed F; Hossain, Md Sakhawat; Nhu, Truong H; Dossou, Serge; Moss, Amina S

    2016-02-01

    Pagrus major fingerlings (3·29 ± 0·02 g) were fed with basal diet (control) supplemented with Lactobacillus rhamnosus (LR), Lactococcus lactis (LL), and L. rhamnosus + L. lactis (LR + LL) at 10(6) cell g(-1) feed for 56 days. Feeding a mixture of LR and LL significantly increased feed utilization (FER and PER), intestine lactic acid bacteria (LAB) count, plasma total protein, alternative complement pathway (ACP), peroxidase, and mucus secretion compared with the other groups (P red sea bream aquaculture. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Structure-guided engineering of Lactococcus lactis alcohol dehydrogenase LlAdhA for improved conversion of isobutyraldehyde to isobutanol

    KAUST Repository

    Liu, Xiang; Bastian, Sabine; Snow, Christopher D.; Brustad, Eric M.; Saleski, Tatyana E.; Xu, Jian-He; Meinhold, Peter; Arnold, Frances H.

    2013-01-01

    We have determined the X-ray crystal structures of the NADH-dependent alcohol dehydrogenase LlAdhA from Lactococcus lactis and its laboratory-evolved variant LlAdhA(RE1) at 1.9Å and 2.5Å resolution, respectively. LlAdhA(RE1), which contains three

  9. Effect of Co-overexpression of Nisin Key Genes on Nisin Production Improvement in Lactococcus lactis LS01.

    Science.gov (United States)

    Ni, Zhi-Jian; Zhang, Xiao-Yuan; Liu, Fei; Wang, Miao; Hao, Rong-Hua; Ling, Pei-Xue; Zhu, Xi-Qiang

    2017-06-01

    Nisin is a small antimicrobial peptide produced by several subset strains of Lactococcus lactis. To improve nisin yield in the producer L. lactis LS01, we proposed a successive fusion of nisA with nisRK and nisFEG into a single shuttle expression vector pMG36e under the control of the native strong constitutive promoter p32. Subsequently, the recombinant vectors were transplanted into the producer cell through electroporation. Nisin productivity was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and bioactivity assays. Expression of nisin peptide was detected by agar diffusion bioassay, and the transcriptional levels of the target genes involved in nisin biosynthesis were investigated via semi-quantitative reverse transcription PCR expression analysis using 16S ribosomal RNA (rRNA) as an internal control. Results suggested that the introduction of empty plasmid did not affect nisin production of L. lactis LS01, whereas by our rational construction and screening, the engineered strain co-overexpressing nisA, nisRK, and nisFEG achieved a maximum increment in bioactive nisin production with a yield of 2470 IU/ml in shake flasks and 4857 IU/ml in 1.0-l fermenters, which increased by approximately 66.3 and 52.6% (P < 0.05), respectively, compared with that of the original strain under the given fermentation conditions. Meanwhile, the transcriptional analysis revealed that the expression of most of these multicopy genes except nisE at transcriptional level were upregulated in the two recombinant strains (LS01/pAR and LS01/pARF), possibly contributing to the improved nisin production. Therefore, this study would provide a potential strategy to improve the economic benefits of nisin manufacture for large-scale industrial production.

  10. Recombinant Lactococcus lactis NZ9000 secretes a bioactive kisspeptin that inhibits proliferation and migration of human colon carcinoma HT-29 cells.

    Science.gov (United States)

    Zhang, Bo; Li, Angdi; Zuo, Fanglei; Yu, Rui; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

    2016-06-10

    Proteinaceous bioactive substances and pharmaceuticals are most conveniently administered orally. However, the facing problems are the side effects of proteolytic degradation and denaturation in the gastrointestinal tract. In recent years, lactic acid bacteria (LAB) have been verified to be a promising delivery vector for susceptible functional proteins and drugs. KiSS-1 peptide, a cancer suppressor, plays a critical role in inhibiting cancer metastasis and its activity has been confirmed by direct administration. However, whether this peptide can be functionally expressed in LAB and exert activity on cancer cells, thus providing a potential alternative administration manner in the future, has not been demonstrated. A recombinant Lactococcus lactis strain NZ9000-401-kiss1 harboring a plasmid containing the gene of the tumor metastasis-inhibiting peptide KiSS1 was constructed. After optimization of the nisin induction conditions, the recombinant strain efficiently secreted KiSS1 with a maximum detectable amount of 27.9 μg/ml in Dulbecco's Modified Eagle medium. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide and would healing assays, respectively, indicated that the secreted KiSS1 peptide remarkably inhibited HT-29 cell proliferation and migration. Furthermore, the expressed KiSS1 was shown to induce HT-29 cell morphological changes, apoptosis and reduce the expression of matrix metalloproteinase 9 (MMP-9) at both mRNA and protein levels. A recombinant L. lactis NZ9000-401-kiss1 successfully expressing the human kiss1 was constructed. The secreted KiSS1 peptide inhibited human HT-29 cells' proliferation and migration probably by invoking, or mediating the cell-apoptosis pathway and by down regulating MMP-9 expression, respectively. Our results suggest that L. lactis is an ideal cell factory for secretory expression of tumor metastasis-inhibiting peptide KiSS1, and the KiSS1-producing L. lactis strain may serve as a new tool for cancer therapy in

  11. Potential aquaculture probiont Lactococcus lactis TW34 produces nisin Z and inhibits the fish pathogen Lactococcus garvieae.

    Science.gov (United States)

    Sequeiros, Cynthia; Garcés, Marisa E; Vallejo, Marisol; Marguet, Emilio R; Olivera, Nelda L

    2015-04-01

    Bacteriocin-producing Lactococcus lactis TW34 was isolated from marine fish. TW34 bacteriocin inhibited the growth of the fish pathogen Lactococcus garvieae at 5 AU/ml (minimum inhibitory concentration), whereas the minimum bactericidal concentration was 10 AU/ml. Addition of TW34 bacteriocin to L. garvieae cultures resulted in a decrease of six orders of magnitude of viable cells counts demonstrating a bactericidal mode of action. The direct detection of the bacteriocin activity by Tricine-SDS-PAGE showed an active peptide with a molecular mass ca. 4.5 kDa. The analysis by MALDI-TOF-MS detected a strong signal at m/z 2,351.2 that corresponded to the nisin leader peptide mass without the initiating methionine, whose sequence STKDFNLDLVSVSKKDSGASPR was confirmed by MS/MS. Sequence analysis of nisin structural gene confirmed that L. lactis TW34 was a nisin Z producer. This nisin Z-producing strain with probiotic properties might be considered as an alternative in the prevention of lactococcosis, a global disease in aquaculture systems.

  12. Toxicity of nalidixic acid on candida albicans, Saccharomyces cerevisiae, and Kluyveromyces lactis.

    Science.gov (United States)

    Sobieski, R J; Brewer, A R

    1976-03-01

    The antibacterial drug nalidixic acid (Nal) can suppress the growth of Candida albicans at levels of the drug normally found in urine. Growth suppression increases as drug levels are increased, and Nal also causes a similar proportional inhibition of the synthesis of all cellular macromolecules. However, growth temperature (25 versus 37 C) and the divalent cations Mg(2+) and Mn(2+) can increase C. albicans resistance to Nal. Also, nitrogen depletion of Candida shows that Nal-treated and untreated cells exhibit no difference in leucine uptake during readaptation to nitrogen. In Nal-treated, nitrogen-starved cells, ribonucleic acid and deoxyribonucleic acid (DNA) biosynthesis are less affected than in unstarved Nal-treated cells, but of the two nucleic acids DNA synthesis is the most affected. Nal-resistant strains of C. albicans exhibit a slight toxicity for macromolecular synthesis. Nal treatment of a synchronized population of Saccharomyces cerevisiae results in an increase in the culture mean doubling time of, at most, 20%, but Nal causes the loss of synchronous cell division. With a synchronized population of Kluyveromyces lactis, Nal causes an increase in the mean doubling time of upwards of 300%, with synchrony of cell division being maintained. It is known that S. cerevisiae asynchronously synthesizes mitochondrial DNA during the cell cycle, whereas with K. lactis it is synchronous. Thus, with C. albicans Nal toxicity is dependent both on the dose and the physiological state of the cell. Furthermore, Nal inhibits growth of yeast with synchronous mitochondrial DNA synthesis more adversely than yeast with asynchronous mitochondrial DNA synthesis.

  13. Rapid genomic fingerprinting of Lactococcus lactis strains by arbitrarily primed polymerase chain reaction with 32P and fluorescent labels.

    OpenAIRE

    Cancilla, M R; Powell, I B; Hillier, A J; Davidson, B E

    1992-01-01

    Arbitrarily primed polymerase chain reaction, with incorporation of either radioactive or fluorescent labels, was used as a rapid and sensitive method for obtaining genomic fingerprints of strains of Lactococcus lactis. Closely related strains produced almost identical fingerprints. Fingerprints of other strains showed only some similarities.

  14. Ameliorated effects of Lactobacillus delbrueckii subsp. lactis DSM 20076 and Pediococcus acidilactici NNRL B-5627 on Fumonisin B1-induced Hepatotoxicity and Nephrotoxicity in rats

    Directory of Open Access Journals (Sweden)

    Amira A. Abdellatef

    2016-04-01

    Full Text Available Oxidative stress has been implicated in a number of human regeneration and disease processes including atherosclerosis, pulmonary fibrosis, cancer, and different neurodegenerative diseases. The aim of this study was to evaluate the protective effects of Lactobacillus delbrueckii subsp. lactis DSM 20076 (LL-DSM and Pediococcus acidilactici NNRL B-5627 (PA-NNRL against the hepatic- and nephro-toxicity of fumonisin B1 (FB1 in FB1-treated rats for an experimental period of 4-weeks. Eighty mature male Sprague-Dawley rats were divided to 12 groups: 1 untreated group; 3 groups fed by a FB1-contaminated diet (50, 100 and 200 mg FB1/kg diet, respectively; 1 group fed orally by LL-DSM (1 ml/d; 1 group fed orally by PA-NNRL (1 ml/d; 3 groups co-administered by FB1-contaminated diet and LL-DSM (1 ml/d, and 3 groups co-administered by FB1-contaminated diet and PA-NNRL (1 ml/d. Malonaldehyde (MDA nitric oxide, glutathione content, SOD activity, total antioxidant capacity (TAC, total oxidant status (TOS and oxidative stress index (OSI were determined. DPA assay was used to assess apoptosis in liver and kidney tissues. The animals fed with FB1-contaminated diet showed a significant increase in oxidative stress markers and DNA fragmentation accompanied with significant decrease in GSH content, SOD activity, and TAC in liver and kidney tissues, especially at high-dosage of FB1 (T200. Probiotics antioxidant strains (LL-DSM and PA-NNRL relatively succeeded to restore almost all parameters investigated as well as to reduce DNA fragmentation in liver and kidney tissues. As a conclusion, probiotics may induce its protective role via increasing the antioxidant capacity, inhibition of lipid peroxidation, scavenging of free radicals and decreasing DNA lesions in liver and kidney of experimental animals tested.

  15. Estudo da viabilidade de Bifidobacterium animalis ssp. lactis em suco de Yacon

    OpenAIRE

    Watanabe, Felipe Miguel Farion

    2013-01-01

    Resumo: A maioria dos produtos probióticos são elaborados à base de leite, com desvantagens aos consumidores intolerantes à lactose, tornando assim produtos probióticos não lácteos vantajosos. O yacon é uma planta que acumula em suas raízes compostos prebióticos conhecidos como frutooligossacarídeos. O objetivo deste trabalho foi produzir o suco de yacon e utilizá-lo como matriz não láctea para a bactéria probiótica Bifidobacterium animalis ssp. lactis. O yacon apresentou rápido escurecimento...

  16. Identification of Lactobacillus spp. from broiler litter in Brazil Identificação de Lactobacillus spp. de cama de frango no Brasil

    Directory of Open Access Journals (Sweden)

    Ronaldo S. Paço

    2003-07-01

    Full Text Available Lactobacillus spp. were identified in 100 broiler litter samples collected from different poultry-rearing regions in Brazil. Ten different Lactobacillus species were identified: L. plantarum, L.casei subsp. pesudoplantarum, L. delbrueckii subsp. delbrueckii, L. reuteri, L. murinus, L. agilis, L. delbrueckii subsp. lactis, L. salivarus subsp. salicinus, L. viridenscens and L. amylophilus.Foram identificadas cepas de Lactobacillus spp. de 100 amostras de camas de frango coletadas de diferentes regiões de produção avícola do Brasil. Foram isoladas dez espécies diferentes de Lactobacillus: L. plantarum, L. casei subsp. pseudoplantarum, L. delbrueckii subsp. delbrueckii, L. reuteri, L. murinus, L. agilis, L.delbrueckii subsp. lactis, L. salivarus subsp. salicinus, L. viridenscens, L. amylophilus.

  17. Metabolic Engineering of Mannitol Production in Lactococcus lactis: Influence of Overexpression of Mannitol 1-Phosphate Dehydrogenase in Different Genetic Backgrounds

    NARCIS (Netherlands)

    Wisselink, H.W.; Mars, A.E.; Meer, van der P.; Eggink, G.; Hugenholtz, J.

    2004-01-01

    To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance

  18. Cell Surface Properties of Lactococcus lactis Reveal Milk Protein Binding Specifically Evolved in Dairy Isolates

    Directory of Open Access Journals (Sweden)

    Mariya Tarazanova

    2017-09-01

    Full Text Available Surface properties of bacteria are determined by the molecular composition of the cell wall and they are important for interactions of cells with their environment. Well-known examples of bacterial interactions with surfaces are biofilm formation and the fermentation of solid materials like food and feed. Lactococcus lactis is broadly used for the fermentation of cheese and buttermilk and it is primarily isolated from either plant material or the dairy environment. In this study, we characterized surface hydrophobicity, charge, emulsification properties, and the attachment to milk proteins of 55 L. lactis strains in stationary and exponential growth phases. The attachment to milk protein was assessed through a newly developed flow cytometry-based protocol. Besides finding a high degree of biodiversity, phenotype-genotype matching allowed the identification of candidate genes involved in the modification of the cell surface. Overexpression and gene deletion analysis allowed to verify the predictions for three identified proteins that altered surface hydrophobicity and attachment of milk proteins. The data also showed that lactococci isolated from a dairy environment bind higher amounts of milk proteins when compared to plant isolates. It remains to be determined whether the alteration of surface properties also has potential to alter starter culture functionalities.

  19. Cell Surface Properties of Lactococcus lactis Reveal Milk Protein Binding Specifically Evolved in Dairy Isolates

    Science.gov (United States)

    Tarazanova, Mariya; Huppertz, Thom; Beerthuyzen, Marke; van Schalkwijk, Saskia; Janssen, Patrick; Wels, Michiel; Kok, Jan; Bachmann, Herwig

    2017-01-01

    Surface properties of bacteria are determined by the molecular composition of the cell wall and they are important for interactions of cells with their environment. Well-known examples of bacterial interactions with surfaces are biofilm formation and the fermentation of solid materials like food and feed. Lactococcus lactis is broadly used for the fermentation of cheese and buttermilk and it is primarily isolated from either plant material or the dairy environment. In this study, we characterized surface hydrophobicity, charge, emulsification properties, and the attachment to milk proteins of 55 L. lactis strains in stationary and exponential growth phases. The attachment to milk protein was assessed through a newly developed flow cytometry-based protocol. Besides finding a high degree of biodiversity, phenotype-genotype matching allowed the identification of candidate genes involved in the modification of the cell surface. Overexpression and gene deletion analysis allowed to verify the predictions for three identified proteins that altered surface hydrophobicity and attachment of milk proteins. The data also showed that lactococci isolated from a dairy environment bind higher amounts of milk proteins when compared to plant isolates. It remains to be determined whether the alteration of surface properties also has potential to alter starter culture functionalities. PMID:28936202

  20. Construction of Lactococcus lactis expressing secreted and anchored Eimeria tenella 3-1E protein and comparison of protective immunity against homologous challenge.

    Science.gov (United States)

    Ma, Chunli; Zhang, Lili; Gao, Mingyang; Ma, Dexing

    2017-07-01

    Two novel plasmids pTX8048-SP-Δ3-1E and pTX8048-SP-NAΔ3-1E-CWA were constructed. The plasmids were respectively electrotransformed into L. lactis NZ9000 to generate strain of L. lactis/pTX8048-SP-Δ3-1E in which 3-1E protein was expressed in secretion, and L. lactis/pTX8048-SP-NAΔ3-1E-CWA on which 3-1E protein was covalently anchored to the surface of bacteria cells. The expression of target proteins were examined by Western blot. The live lactococci expressing secreted 3-1E protein, anchored 3-1E protein, and cytoplasmic 3-1E protein was administered orally to chickens respectively, and the protective immunity and efficacy were compared by animal experiment. The results showed oral immunization to chickens with recombinant lactococci expressing anchored 3-1E protein elicited high 3-1E-specific serum IgG, increased high proportion of CD4 + and CD8α + cells in spleen, alleviated average lesion score in cecum, decreased the oocyst output per chicken compared to lactococci expressing cytoplasmic or secreted 3-1E protein. Taken together, these findings indicated the surface anchored Eimeria protein displayed by L. lacits can induce protective immunity and partial protection against homologous infection. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. D-Tagatose production in the presence of borate by resting Lactococcus lactis cells harboring Bifidobacterium longum L-arabinose isomerase.

    Science.gov (United States)

    Salonen, Noora; Salonen, Kalle; Leisola, Matti; Nyyssölä, Antti

    2013-04-01

    Bifidobacterium longum NRRL B-41409 L-arabinose isomerase (L-AI) was overexpressed in Lactococcus lactis using a phosphate depletion inducible expression system. The resting L. lactis cells harboring the B. longum L-AI were used for production of D-tagatose from D-galactose in the presence of borate buffer. Multivariable analysis suggested that high pH, temperature and borate concentration favoured the conversion of D-galactose to D-tagatose. Almost quantitative conversion (92 %) was achieved at 20 g L⁻¹ substrate and at 37.5 °C after 5 days. The D-tagatose production rate of 185 g L⁻¹ day ⁻¹ was obtained at 300 g L⁻¹ galactose, at 1.15 M borate, and at 41 °C during 10 days when the production medium was changed every 24 h. There was no significant loss in productivity during ten sequential 24 h batches. The initial D-tagatose production rate was 290 g L⁻¹ day⁻¹ under these conditions.

  2. Transcriptome landscape of Lactococcus lactis reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and metabolism

    NARCIS (Netherlands)

    van der Meulen, Sjoerd B; de Jong, Anne; Kok, Jan

    2016-01-01

    RNA sequencing has revolutionized genome-wide transcriptome analyses, and the identification of non-coding regulatory RNAs in bacteria has thus increased concurrently. Here we reveal the transcriptome map of the lactic acid bacterial paradigm Lactococcus lactis MG1363 by employing differential RNA

  3. Esterase activities of intracellular extracts of wild strains of lactic acid bacteria isolated from Serra da Estrela cheese

    OpenAIRE

    Macedo, Angela C.; Tavares, Tânia G.; Malcata, F. Xavier

    2003-01-01

    Lactococcus lactis subsp. lactis strain ESB110019 and Lactobacillus plantarum strain ESB5004, novel strains that were previously isolated from the wild adventitious microflora of certified Serra da Estrela cheeses, were assayed for esterase activity using, as substrates, ortho- and para-nitrophenyl derivatives of fatty acids. Both strains preferentially hydrolyzed short-chain fatty acids; L. lactis ESB110019 exhibited a stronger esterase activity than Lb. plantarum ESB5004 and cleaved the p-n...

  4. Relative rates of amino acid import via the ABC transporter GlnPQ determine the growth performance of Lactococcus lactis

    NARCIS (Netherlands)

    Fulyani, Faizah; Schuurman-Wolters, Geesina; Slotboom, Dirk-Jan; Poolman, Bert

    The GlnPQ transporter from Lactococcus lactis has the remarkable feature of having two substrate-binding domains (SBD) fused to the N-terminus of the transmembrane domain (TMD), and thus four SBDs are present in the homodimeric complex. Although X-ray structures and ligand binding data are available

  5. Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with Various Plasmids

    Science.gov (United States)

    Serror, Pascale; Sasaki, Takashi; Ehrlich, S. Dusko; Maguin, Emmanuelle

    2002-01-01

    We describe, for the first time, a detailed electroporation procedure for Lactobacillus delbrueckii. Three L. delbrueckii strains were successfully transformed. Under optimal conditions, the transformation efficiency was 104 transformants per μg of DNA. Using this procedure, we identified several plasmids able to replicate in L. delbrueckii and integrated an integrative vector based on phage integrative elements into the L. delbrueckii subsp. bulgaricus chromosome. These vectors provide a good basis for developing molecular tools for L. delbrueckii and open the field of genetic studies in L. delbrueckii. PMID:11772607

  6. Reaction kinetics and galactooligosaccharide product profiles of the β-galactosidases from Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae.

    Science.gov (United States)

    Yin, Huifang; Bultema, Jelle B; Dijkhuizen, Lubbert; van Leeuwen, Sander S

    2017-06-15

    β-Galactosidase enzymes are used in the dairy industry to convert lactose into galactooligosaccharides (GOS) that are added to infant formula to mimic the molecular sizes and prebiotic functions of human milk oligosaccharides. Here we report a detailed analysis of the clearly different GOS profiles of the commercial β-galactosidases from Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae. Also the GOS yields of these enzymes differed, varying from 48.3% (B. circulans) to 34.9% (K. lactis), and 19.5% (A. oryzae). Their incubation with lactose plus the monosaccharides Gal or Glc resulted in altered GOS profiles. Experiments with 13 C 6 labelled Gal and Glc showed that both monosaccharides act as acceptor substrates in the transgalactosylation reactions. The data shows that the lactose isomers β-d-Galp-(1→2)-d-Glcp, β-d-Galp-(1→3)-d-Glcp and β-d-Galp-(1→6)-d-Glcp are formed from acceptor reactions with free Glc and not by rearrangement of Glc in the active site. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Mucosal and systemic immune responses elicited by recombinant Lactococcus lactis expressing a fusion protein composed of pertussis toxin and filamentous hemagglutinin from Bordetella pertussis.

    Science.gov (United States)

    Torkashvand, Ali; Bahrami, Fariborz; Adib, Minoo; Ajdary, Soheila

    2018-05-05

    We constructed a food-grade expression system harboring a F1S1 fusion protein of Bordetella pertussis to be produced in Lactococcus lactis NZ3900 as a new oral vaccine model against whooping cough, caused by B. pertussis. F1S1 was composed of N-terminally truncated S1 subunit of pertussis toxin and type I immunodominant domain of filamentous hemagglutinin which are both known as protective immunogens against pertussis. The recombinant L. lactis was administered via oral or intranasal routes to BALB/c mice and the related specific systemic and mucosal immune responses were then evaluated. The results indicated significantly higher levels of specific IgA in the lung extracts and IgG in sera of mucosally-immunized mice, compared to their controls. It was revealed that higher levels of IgG2a, compared to IgG1, were produced in all mucosally-immunized mice. Moreover, immunized mice developed Th1 responses with high levels of IFN-γ production by the spleen cells. These findings provide evidence for L. lactis to be used as a suitable vehicle for expression and delivery of F1S1 fusion protein to mucosa and induction of appropriate systemic and mucosal immune responses against pertussis. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. Cloning and Expression of Plantaricin W Produced by Lactobacillus plantarum U10 Isolate from "Tempoyak" Indonesian Fermented Food as Immunity Protein in Lactococcus lactis.

    Science.gov (United States)

    Lages, Aksar Chair; Mustopa, Apon Zaenal; Sukmarini, Linda; Suharsono

    2015-10-01

    Plantaricins, one of bacteriocin produced by Lactobacillus plantarum, are already known to have activities against several pathogenic bacterium. L. plantarum U10 isolated from "tempoyak," an Indonesian fermented food, produced one kind of plantaricin designated as plantaricin W (plnW). The plnW is suggested as a putative membrane location of protein and has similar conserved motif which is important as immunity to bacteriocin itself. Thus, due to study about this plantaricin, several constructs have been cloned and protein was analyzed in Lactococcus lactis. In this study, plnW gene was successfully cloned into vector NICE system pNZ8148 and created the transformant named L. lactis NZ3900 pNZ8148-WU10. PlnW protein was 25.3 kDa in size. The concentration of expressed protein was significantly increased by 10 ng/mL nisin induction. Furthermore, PlnW exhibited protease activity with value of 2.22 ± 0.05 U/mL and specific activity about 1.65 ± 0.03 U/mg protein with 50 ng/mL nisin induction. Immunity study showed that the PlnW had immunity activity especially against plantaricin and rendered L. lactis recombinant an immunity broadly to other bacteriocins such as pediocin, fermentcin, and acidocin.

  9. Cloning, Expression, and Chromosomal Stabilization of the Propionibacterium shermanii Proline Iminopeptidase Gene (pip) for Food-Grade Application in Lactococcus lactis

    NARCIS (Netherlands)

    Leenhouts, Kees; Bolhuis, Albert; Boot, Johan; Deutz, Inge; Toonen, Marjolein; Venema, Gerard; Kok, Jan; Ledeboer, Aat

    1998-01-01

    Proline iminopeptidase produced by Propionibacterium shermanii plays an essential role in the flavor development of Swiss-type cheeses. The enzyme (Pip) was purified and characterized, and the gene (pip) was cloned and expressed in Escherichia coli and Lactococcus lactis, the latter species being an

  10. Nisin-induced Expression of Pediocin in Dairy Lactic Acid Bacteria

    Science.gov (United States)

    To test if a single vector, nisin-controlled expression (NICE) system could be used to regulate expression of the pediocin operon in Streptococcus thermophilus, Lactococcus lactis subsp. lactis and Lactobacillus casei, the intact pediocin operon was cloned into pMSP3535 immediately down stream of th...

  11. Recombinant pediocin in Lactococcus lactis : increased production by propeptide fusion and improved potency by co-production with PedC

    NARCIS (Netherlands)

    Back, Alexandre; Borges, Frederic; Mangavel, Cecile; Paris, Cedric; Rondags, Emmanuel; Kapel, Romain; Aymes, Arnaud; Rogniaux, Helene; Pavlovic, Marija; van Heel, Auke J.; Kuipers, Oscar P.; Revol-Junelles, Anne-Marie; Cailliez-Grimal, Catherine

    We describe the impact of two propeptides and PedC on the production yield and the potency of recombinant pediocins produced in Lactococcus lactis. On the one hand, the sequences encoding the propeptides SD or LEISSTCDA were inserted between the sequence encoding the signal peptide of Usp45 and the

  12. Metabolism of [14C]bicarbonate by Streptococcus lactis: identification and distribution of labelled compounds

    International Nuclear Information System (INIS)

    Hillier, A.J.; Jago, G.R.

    1978-01-01

    Streptococcus lactis C10, grown in tryptone-yeast extract-lactose broth containing [ 14 C] bicarbonate, incorporated radioactivity into the protein and nucleic acid fractions of the cell as well as into compounds which were excreted by the organism into the growth medium. Aspartic acid was the first compound to be labelled and was the only amino acid labelled in the cell protein. All 4 bases were labelled in the cell RNA. Aspartic, succunuc and lactic acids were the radioactive compounds excreted into the growth medium. (U.K.)

  13. In Vitro characterization of Lactococcus lactis strains Isolated from Iranian Traditional Dairy Products as a Potential Probiotic

    Directory of Open Access Journals (Sweden)

    Fatemeh Nejati

    2015-12-01

    Full Text Available Few studies have been reported regarding probiotic properties of Lactococcus lactis strains although they are extensively used as starter cultures in the production of dairy products. In this study 8 wild isolates of Lactococcus lactis were evaluated in vitro with regard to resistance to simulated gastric and intestinal juices, adherence ability to Caco-2 cells and HT29-MTX-E12 cell lines, anti-microbial activity, hydrophobicity and antibiotic susceptibility. The results revealed that all isolates had better survival after exposure to simulated gastrointestinal tract stresses in comparison to control probiotic Lactobacillus rhamnosus GG. Regarding adherence efficiency, almost all isolates exhibited similar adherence with control. Three isolates showed antibacterial activity against Gram-positive pathogens (Staphylococcus aureus and Listeria monocytogenes through spot-agar method. Almost all isolates (seven out of eight showed similar hydrophobicity to control probiotic. Regarding to antibiotic resistance, all isolates were susceptible to gentamicin, ampicillin, ciprofloxacin, erythromycin, tetracycline, penicillin, kanamycin and nitrofurantoin. Although, further investigations are necessary, it was concluded that strains derived from raw milk and home-made dairy products could be a remarkable reservoir for identification of new potential probiotic strains.

  14. Dynamics in copy numbers of five plasmids of a dairy Lactococcus lactis in dairy-related conditions including near-zero growth rates.

    Science.gov (United States)

    van Mastrigt, Oscar; Lommers, Marcel M A N; de Vries, Yorick C; Abee, Tjakko; Smid, Eddy J

    2018-03-23

    Lactic acid bacteria can carry multiple plasmids affecting their performance in dairy fermentations. The expression of plasmid-encoded genes and the activity of the corresponding proteins is severely affected by changes in the number of plasmid copies. We studied the impact of growth rate on dynamics of plasmid copy numbers at high growth rates in chemostat cultures and down to near-zero growth rates in retentostat cultures. Five plasmids of the dairy strain Lactococcus lactis FM03-V1 were selected which varied in size (3 to 39 kb), in replication mechanism (theta or rolling-circle) and in putative (dairy-associated) functions. Copy numbers ranged from 1.5 to 40.5 and the copy number of theta-type replicating plasmids were negatively correlated to the plasmid size. Despite the extremely wide range of growth rates (0.0003 h -1 to 0.6 h -1 ), copy numbers of the five plasmids were stable and only slightly increased at near-zero growth rates showing that the plasmid replication rate was strictly controlled. One low-copy number plasmid, carrying a large exopolysaccharide gene cluster, was segregationally unstable during retentostat cultivations reflected in complete loss of the plasmid in one of the retentostat cultures. The copy number of the five plasmids was also hardly affected by varying the pH value, nutrient limitation or presence of citrate (maximum 2.2-fold) signifying the stability in copy number of the plasmids. Importance Lactococcus lactis is extensively used in starter cultures for dairy fermentations. Important traits for growth and survival of L. lactis in dairy fermentations are encoded by genes located on plasmids, such as genes involved in lactose and citrate metabolism, protein degradation and oligopeptide uptake and bacteriophage resistance. Because the number of plasmid copies could affect the expression of plasmid-encoded genes, it is important to know the factors that influence the plasmid copy numbers. We monitored plasmid copy numbers of L

  15. The level of pyruvate-formate lyase controls the shift from homolactic to mixed-acid product formation in Lactococcus lactis

    DEFF Research Database (Denmark)

    Melchiorsen, C.R.; Jokumsen, K.V.; Villadsen, John

    2002-01-01

    promoters in L. lactis MG1363 and in the PFL-deficient strain CRM40. Strains with five different PFL levels were obtained. Variation in the PFL level markedly affected the resulting end-product formation in these strains. During growth on galactose, the flux towards mixed-acid products was to a great extent...

  16. Exploring optimization parameters to increase ssDNA recombineering in Lactococcus lactis and Lactobacillus reuteri.

    Science.gov (United States)

    Van Pijkeren, Jan-Peter; Neoh, Kar Mun; Sirias, Denise; Findley, Anthony S; Britton, Robert A

    2012-01-01

    Single-stranded DNA (ssDNA) recombineering is a technology which is used to make subtle changes in the chromosome of several bacterial genera. Cells which express a single-stranded DNA binding protein (RecT or Bet) are transformed with an oligonucleotide which is incorporated via an annealing and replication-dependent mechanism. By in silico analysis we identified ssDNA binding protein homologs in the genus Lactobacillus and Lactococcus lactis. To assess whether we could further improve the recombineering efficiency in Lactobacillus reuteri ATCC PTA 6475 we expressed several RecT homologs in this strain. RecT derived from Enterococcus faecalis CRMEN 19 yielded comparable efficiencies compared with a native RecT protein, but none of the other proteins further increased the recombineering efficiency. We successfully improved recombineering efficiency 10-fold in L. lactis by increasing oligonucleotide concentration combined with the use of oligonucleotides containing phosphorothioate-linkages (PTOs). Surprisingly, neither increased oligonucleotide concentration nor PTO linkages enhanced recombineering in L. reuteri 6475. To emphasize the utility of this technology in improving probiotic features we modified six bases in a transcriptional regulatory element region of the pdu-operon of L. reuteri 6475, yielding a 3-fold increase in the production of the antimicrobial compound reuterin. Directed genetic modification of lactic acid bacteria through ssDNA recombineering will simplify strain improvement in a way that, when mutating a single base, is genetically indistinguishable from strains obtained through directed evolution.

  17. Effect of non-nutritional factors on nisin production | Tafreshi | African ...

    African Journals Online (AJOL)

    order to assess some of non-nutritional factors and how they influence the nisin production in batch cultivation, a laboratory scale study was performed. Lactococcus lactis subsp. lactis ATCC 11454 produced nisin and Micrococcus luteus ATCC 10240 was used in bioassay measurement as the nisinsensitive strain. The age ...

  18. Use of DNA quantification to measure growth and autolysis of Lactococcus and Propionibacterium spp. in mixed populations.

    Science.gov (United States)

    Treimo, Janneke; Vegarud, Gerd; Langsrud, Thor; Rudi, Knut

    2006-09-01

    Autolysis is self-degradation of the bacterial cell wall that results in the release of enzymes and DNA. Autolysis of starter bacteria, such as lactococci and propionibacteria, is essential for cheese ripening, but our understanding of this important process is limited. This is mainly because the current tools for measuring autolysis cannot readily be used for analysis of bacteria in mixed populations. We have now addressed this problem by species-specific detection and quantification of free DNA released during autolysis. This was done by use of 16S rRNA gene single-nucleotide extension probes in combination with competitive PCR. We analyzed pure and mixed populations of Lactococcus lactis subsp. lactis and three different species of Propionibacterium. Results showed that L. lactis subsp. lactis INF L2 autolyzed first, followed by Propionibacterium acidipropionici ATCC 4965, Propionibacterium freudenreichii ISU P59, and then Propionibacterium jensenii INF P303. We also investigated the autolytic effect of rennet (commonly used in cheese production). We found that the effect was highly strain specific, with all the strains responding differently. Finally, autolysis of L. lactis subsp. lactis INF L2 and P. freudenreichii ISU P59 was analyzed in a liquid cheese model. Autolysis was detected later in this cheese model system than in broth media. A challenge with DNA, however, is DNA degradation. We addressed this challenge by using a DNA degradation marker. We obtained a good correlation between the degradation of the marker and the target in a model experiment. We conclude that our DNA approach will be a valuable tool for use in future analyses and for understanding autolysis in mixed bacterial populations.

  19. Nisin Z Production by Lactococcus lactis subsp. cremoris WA2-67 of Aquatic Origin as a Defense Mechanism to Protect Rainbow Trout (Oncorhynchus mykiss, Walbaum) Against Lactococcus garvieae.

    Science.gov (United States)

    Araújo, Carlos; Muñoz-Atienza, Estefanía; Pérez-Sánchez, Tania; Poeta, Patrícia; Igrejas, Gilberto; Hernández, Pablo E; Herranz, Carmen; Ruiz-Zarzuela, Imanol; Cintas, Luis M

    2015-12-01

    Probiotics represent an alternative to chemotherapy and vaccination to control fish diseases, including lactococcosis caused by Lactococcus garvieae. The aims of this study were (i) to determine the in vitro probiotic properties of three bacteriocinogenic Lactococcus lactis subsp. cremoris of aquatic origin, (ii) to evaluate in vivo the ability of L. cremoris WA2-67 to protect rainbow trout (Oncorhynchus mykiss, Walbaum) against infection by L. garvieae, and (iii) to demonstrate the role of nisin Z (NisZ) production as an anti-infective mechanism. The three L. cremoris strains survived in freshwater at 18 °C for 7 days, withstood exposure to pH 3.0 and 10 % (v/v) rainbow trout bile, and showed different cell surface hydrophobicity (37.93-58.52 %). The wild-type NisZ-producer L. cremoris WA2-67 and its non-bacteriocinogenic mutant L. cremoris WA2-67 ∆nisZ were administered orally (10(6) CFU/g) to rainbow trout for 21 days and, subsequently, fish were challenged with L. garvieae CLG4 by the cohabitation method. The fish fed with the bacteriocinogenic strain L. cremoris WA2-67 reduced significantly (p trout against infection with the invasive pathogen L. garvieae and the relevance of NisZ production as an anti-infective mechanism. This is the first report demonstrating the effective in vivo role of LAB bacteriocin (NisZ) production as a mechanism to protect fish against bacterial infection. Our results suggest that the wild-type NisZ-producer strain L. cremoris WA2-67 could be used in fish farming to prevent lactococcosis in rainbow trout.

  20. Functional analysis of the Lactococcus lactis galU and galE genes and their impact on sugar nucleotide and exopolysaccharide biosynthesis

    NARCIS (Netherlands)

    Boels, I.C.; Ramos, A.; Kleerebezem, M.; Vos, de W.M.

    2001-01-01

    We studied the UDP-glucose pyrophosphorylase (galU) and UDP-galactose epimerase (galE) genes of Lactococcus lactis MG1363 to investigate their involvement in biosynthesis of UDP-glucose and UDP-galactose, which are precursors of glucose- and galactose-containing exopolysaccharides (EPS) in L.