Effect of enzymes on anaerobic digestion of primary sludge and septic tank performance.

Science.gov (United States)

Diak, James; Örmeci, Banu; Kennedy, Kevin J

2012-11-01

Enzyme additives are believed to improve septic tank performance by increasing the hydrolysis and digestion rates and maintaining a healthy microbial population. Previous studies reported mixed results on the effectiveness of enzymes on mesophilic and thermophilic digestion, and it is not clear whether enzymes would be effective under septic tank conditions where there is no heating or mixing, quantities of enzymes added are small, and they can be washed out quickly. In this study, batch reactors and continuous-flow reactors designed and operated as septic tanks were used to evaluate whether enzymatic treatment would increase the hydrolysis and digestion rates in primary sludge. Total solids, volatile solids, total suspended solids, total and soluble chemical oxygen demand, concentrations of protein, carbohydrate, ammonia and volatile acids in sludge and effluent samples were measured to determine the differences in digestion rates in the presence and absence of enzymes. Overall, no significant improvement was observed in enzyme-treated reactors compared with the control reactors.

Digestive enzyme activities and gastrointestinal fermentation in wood-eating catfishes.

Science.gov (United States)

German, Donovan P; Bittong, Rosalie A

2009-11-01

To determine what capabilities wood-eating and detritivorous catfishes have for the digestion of refractory polysaccharides with the aid of an endosymbiotic microbial community, the pH, redox potentials, concentrations of short-chain fatty acids (SCFAs), and the activity levels of 14 digestive enzymes were measured along the gastrointestinal (GI) tracts of three wood-eating taxa (Panaque cf. nigrolineatus "Marañon", Panaque nocturnus, and Hypostomus pyrineusi) and one detritivorous species (Pterygoplichthys disjunctivus) from the family Loricariidae. Negative redox potentials (-600 mV) were observed in the intestinal fluids of the fish, suggesting that fermentative digestion was possible. However, SCFA concentrations were low (<3 mM in any intestinal region), indicating that little GI fermentation occurs in the fishes' GI tracts. Cellulase and xylanase activities were low (<0.03 U g(-1)), and generally decreased distally in the intestine, whereas amylolytic and laminarinase activities were five and two orders of magnitude greater, respectively, than cellulase and xylanase activities, suggesting that the fish more readily digest soluble polysaccharides. Furthermore, the Michaelis-Menten constants (K(m)) of the fishes' beta-glucosidase and N-acetyl-beta-D-glucosaminidase enzymes were significantly lower than the K(m) values of microbial enzymes ingested with their food, further suggesting that the fish efficiently digest soluble components of their detrital diet rather than refractory polysaccharides. Coupled with rapid gut transit and poor cellulose digestibility, the wood-eating catfishes appear to be detritivores reliant on endogenous digestive mechanisms, as are other loricariid catfishes. This stands in contrast to truly "xylivorous" taxa (e.g., beavers, termites), which are reliant on an endosymbiotic community of microorganisms to digest refractory polysaccharides.

An enzyme complex increases in vitro dry matter digestibility of corn and wheat in pigs.

Science.gov (United States)

Park, Kyu Ree; Park, Chan Sol; Kim, Beob Gyun

2016-01-01

Two experiments were conducted to determine the effects of enzyme complex on in vitro dry matter (DM) digestibility for feed ingredients. The objective of experiment 1 was to screen feed ingredients that can be effective substrates for an enzyme complex, mainly consisted of β-pentosanase, β-glucanase and α-amylase, using in vitro digestibility methods. In experiment 1, the test ingredients were three grain sources (barley, corn and wheat) and six protein supplements (canola meal, copra expellers, cottonseed meal, distillers dried grains with solubles, palm kernel expellers and soybean meal). In vitro ileal and total tract digestibility (IVID and IVTTD, respectively) of DM for test ingredients were determined. In vitro digestibility methods consisted of two- or three-step procedure simulating in vivo digestion in the pig gastrointestinal tracts with or without enzyme complex. As the enzyme complex added, the IVID of DM for corn and wheat increased (p digestibility, corn grains were selected to determine the in vitro digestibility of the fractions (starch, germ, hull and gluten) that maximally respond to the enzyme complex in experiment 2. The IVID of DM for corn starch, germ and hull increased (p digestibility of corn and wheat, and the digestibility increments of corn are mainly attributed to the increased digestibility of corn starch.

Digestive enzymes in the alimentary canal of Clarias anguillaris ...

African Journals Online (AJOL)

Three groups of digestive enzymes were observed in the alimentary canals of the fingerlings and adults of Clarias anguillaris. The enzymes were carbohydrases including amylase, sucrase, lactase and maltase, proteases including peptase, tryptase and peptidase, and lipase. The activities of the protease were found to be ...

Genomic-based restriction enzyme selection for specific detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP

Directory of Open Access Journals (Sweden)

Dinka eMandakovic

2016-05-01

Full Text Available The gram negative facultative bacterium P. salmonis is the etiological agent of Salmonid Rickettsial Septicaemia (SRS, a severe disease that causes important economic losses in the global salmon farmer industry. Despite efforts to control this disease, the high frequency of new epizootic events indicate that the vaccine and antibiotics treatments have limited effectiveness, therefore the preventive and diagnostic approaches must be improved. A comparison of several methodologies for SRS diagnostic indicate differences in their specificity and its capacity to detect other bacteria coexisting with P. salmonis in culture media (contamination and fish samples (coinfection, aspects relevant for research, vaccine development and clinical diagnostic. By computer-simulation analyses, we identified a group of restriction enzymes that generate unique P. salmonis 16S rDNA band patterns, distinguishable from all other bacteria. From this information, we designed and developed a PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism assay, which was validated using 16S rDNA universal primers and restriction enzyme PmaCI for the amplification and digestion, respectively. Experimental validation was performed by comparing the restriction pattern of P. salmonis with the restriction patterns generated by bacteria that cohabit with P. salmonis (fish bacterial isolates and culture media contaminants. Our results indicate that the restriction enzyme selection pipeline was suitable to design a more specific, sensible, faster and cheaper assay than the currently used P. salmonis detection methodologies.

Digestive enzyme activities in the guts of bonnethead sharks (Sphyrna tiburo) provide insight into their digestive strategy and evidence for microbial digestion in their hindguts.

Science.gov (United States)

Jhaveri, Parth; Papastamatiou, Yannis P; German, Donovan P

2015-11-01

Few investigations have studied digestive enzyme activities in the alimentary tracts of sharks to gain insight into how these organisms digest their meals. In this study, we examined the activity levels of proteases, carbohydrases, and lipase in the pancreas, and along the anterior intestine, spiral intestine, and colon of the bonnethead shark, Sphyrna tiburo. We then interpreted our data in the context of a rate-yield continuum to discern this shark's digestive strategy. Our data show anticipated decreasing patterns in the activities of pancreatic enzymes moving posteriorly along the gut, but also show mid spiral intestine peaks in aminopeptidase and lipase activities, which support the spiral intestine as the main site of absorption in bonnetheads. Interestingly, we observed spikes in the activity levels of N-acetyl-β-D-glucosaminidase and β-glucosidase in the bonnethead colon, and these chitin- and cellulose-degrading enzymes, respectively, are likely of microbial origin in this distal gut region. Taken in the context of intake and relatively long transit times of food through the gut, the colonic spikes in N-acetyl-β-D-glucosaminidase and β-glucosidase activities suggest that bonnetheads take a yield-maximizing strategy to the digestive process, with some reliance on microbial digestion in their hindguts. This is one of the first studies to examine digestive enzyme activities along the gut of any shark, and importantly, the data match with previous observations that sharks take an extended time to digest their meals (consistent with a yield-maximizing digestive strategy) and that the spiral intestine is the primary site of absorption in sharks. Copyright © 2015 Elsevier Inc. All rights reserved.

Analysis of mutation/rearrangement frequencies and methylation patterns at a given DNA locus using restriction fragment length polymorphism.

Science.gov (United States)

Boyko, Alex; Kovalchuk, Igor

2010-01-01

Restriction fragment length polymorphism (RFLP) is a difference in DNA sequences of organisms belonging to the same species. RFLPs are typically detected as DNA fragments of different lengths after digestion with various restriction endonucleases. The comparison of RFLPs allows investigators to analyze the frequency of occurrence of mutations, such as point mutations, deletions, insertions, and gross chromosomal rearrangements, in the progeny of stressed plants. The assay involves restriction enzyme digestion of DNA followed by hybridization of digested DNA using a radioactively or enzymatically labeled probe. Since DNA can be digested with methylation sensitive enzymes, the assay can also be used to analyze a methylation pattern of a particular locus. Here, we describe RFLP analysis using methylation-insensitive and methylation-sensitive enzymes.

Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers.

Directory of Open Access Journals (Sweden)

Lin Yuan

Full Text Available Three hundred one-day-old male broiler chickens (Ross-308 were fed corn-soybean basal diets containing non-starch polysaccharide (NSP enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI and average daily gain (ADG were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05. Feed-to-gain ratio (FGR was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05. Apparent digestibility of crude protein (ADCP was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05. Cholecystokinin (CCK level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05, but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05, respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05. However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05. The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05. Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased

Analytical workflow of double-digest restriction site-associated DNA sequencing based on empirical and in silico optimization in tomato.

Science.gov (United States)

Shirasawa, Kenta; Hirakawa, Hideki; Isobe, Sachiko

2016-04-01

Double-digest restriction site-associated DNA sequencing (ddRAD-Seq) enables high-throughput genome-wide genotyping with next-generation sequencing technology. Consequently, this method has become popular in plant genetics and breeding. Although computational in silico prediction of restriction sites from the genome sequence is recognized as an effective approach for choosing the restriction enzymes to be used, few reports have evaluated the in silico predictions in actual experimental data. In this study, we designed and demonstrated a workflow for in silico and empirical ddRAD-Seq analysis in tomato, as follows: (i)in silico prediction of optimum restriction enzymes from the reference genome, (ii) verification of the prediction by actual ddRAD-Seq data of four restriction enzyme combinations, (iii) establishment of a computational data processing pipeline for high-confidence single nucleotide polymorphism (SNP) calling, and (iv) validation of SNP accuracy by construction of genetic linkage maps. The quality of SNPs based on de novo assembly reference of the ddRAD-Seq reads was comparable with that of SNPs obtained using the published reference genome of tomato. Comparisons of SNP calls in diverse tomato lines revealed that SNP density in the genome influenced the detectability of SNPs by ddRAD-Seq. In silico prediction prior to actual analysis contributed to optimization of the experimental conditions for ddRAD-Seq, e.g. choices of enzymes and plant materials. Following optimization, this ddRAD-Seq pipeline could help accelerate genetics, genomics, and molecular breeding in both model and non-model plants, including crops. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Depolymerization of chitosan by enzymes from the digestive tract of ...

African Journals Online (AJOL)

A complex of enzymes was isolated in a preparation derived from the digestive tract of sea cucumber, Stichopus japonicus. Hydrolysis of chitosan using this enzyme preparation decreased its molecular weight (Mw), increased its water solubility and produced water-soluble chitosan (WSC). The conditions for hydrolysis were ...

Digestive enzymes of the Californian two-spot octopus, Octopus bimaculoides (Pickford and McConnaughey, 1949).

Science.gov (United States)

Ibarra-García, Laura Elizabeth; Tovar-Ramírez, Dariel; Rosas, Carlos; Campa-Córdova, Ángel Isidro; Mazón-Suástegui, José Manuel

2018-01-01

Octopus bimaculoides is an important commercially fished species in the California Peninsula with aquaculture potential; however, to date limited information is available regarding its digestive physiology. The objective of this study was focused on biochemically characterizing the main enzymes involved in the digestion of O. bimaculoides. Optimum pH, temperature and thermostability were determined for amylases, lipases, trypsin and chymotrypsin; optimum pH and protease inhibitor effect were assessed for acidic and alkaline proteases, and the effect of divalent ions on trypsin and chymotrypsin activity was evaluated in enzymatic extracts from the digestive (DG) and salivary glands (SG) and crop gastric juices (GJ). High amylase activity was detected in GD and GJ whereas this activity is very low in other cephalopods. Salivary glands had the greatest activity in most of the enzyme groups, showing the importance of this organ in digestion. Optimum pH was different depending on the organ and enzyme analyzed. The optimum pH in DG was 3 showing the predominance of acidic proteases in the digestion process. All enzymes were resistant and stable at high temperatures in contrast with other marine species. Trypsin and chymotrypsin activity were highly incremented with the presence of Mg 2+ , Co 2+ , Cu 2+ and Zn 2+ in some tissues. The inhibitor assay showed the importance of serine proteases, metalloproteases and aspartic proteases in the digestive process of this species. This study is the first in assessing the main digestive enzymes of O. bimaculoides and in remarking the importance of other digestive enzyme groups besides proteases in octopuses. Copyright © 2017 Elsevier Inc. All rights reserved.

Vitellin- and hemoglobin-digesting enzymes in Rhipicephalus (Boophilus) microplus larvae and females.

Science.gov (United States)

Estrela, Andréia Bergamo; Seixas, Adriana; Teixeira, Vivian de Oliveira Nunes; Pinto, Antônio Frederico Michel; Termignoni, Carlos

2010-12-01

The aim of the present study was to address the involvement of Rhipicephalus microplus larval cysteine endopeptidase (RmLCE) in protein digestion in R. microplus larvae and adult females. In this work, an improved purification protocol for native RmLCE was developed. Partial amino acid sequence of the purified enzyme indicates that it is the same enzyme as Boophilus microplus cathepsin-L1 (BmCL1). When vitellin (Vt) degradation by egg and larval enzymes was analyzed, stage-specific differences for RmLCE activity in comparison to vitellin-degrading cysteine endopeptidase (VTDCE) were observed. RmLCE is also able to degrade host hemoglobin (Hb). In agreement, an acidic cysteine endopeptidase activity was detected in larval gut. It was shown that cysteine and aspartic endopeptidases are involved in Vt and Hb digestion in R. microplus larvae and females. Interestingly, we observed that the aspartic endopeptidase Boophilus yolk cathepsin (BYC) is associated with a cysteine endopeptidase activity, in larvae. Synergic hemoglobin digestion by BYC and RmLCE was observed and indicates the presence of an Hb-degrading enzymatic cascade involving these enzymes. Our results suggest that RmLCE/BmCL1 has a continued role in vitellin and hemoglobin digestion during tick development. Copyright © 2010 Elsevier Inc. All rights reserved.

Improved Starch Digestion of Sucrase-deficient Shrews Treated With Oral Glucoamylase Enzyme Supplements.

Science.gov (United States)

Nichols, Buford L; Avery, Stephen E; Quezada-Calvillo, Roberto; Kilani, Shadi B; Lin, Amy Hui-Mei; Burrin, Douglas G; Hodges, Benjamin E; Chacko, Shaji K; Opekun, Antone R; Hindawy, Marwa El; Hamaker, Bruce R; Oda, Sen-Ichi

2017-08-01

Although named because of its sucrose hydrolytic activity, this mucosal enzyme plays a leading role in starch digestion because of its maltase and glucoamylase activities. Sucrase-deficient mutant shrews, Suncus murinus, were used as a model to investigate starch digestion in patients with congenital sucrase-isomaltase deficiency.Starch digestion is much more complex than sucrose digestion. Six enzyme activities, 2 α-amylases (Amy), and 4 mucosal α-glucosidases (maltases), including maltase-glucoamylase (Mgam) and sucrase-isomaltase (Si) subunit activities, are needed to digest starch to absorbable free glucose. Amy breaks down insoluble starch to soluble dextrins; mucosal Mgam and Si can either directly digest starch to glucose or convert the post-α-amylolytic dextrins to glucose. Starch digestion is reduced because of sucrase deficiency and oral glucoamylase enzyme supplement can correct the starch maldigestion. The aim of the present study was to measure glucogenesis in suc/suc shrews after feeding of starch and improvement of glucogenesis by oral glucoamylase supplements. Sucrase mutant (suc/suc) and heterozygous (+/suc) shrews were fed with C-enriched starch diets. Glucogenesis derived from starch was measured as blood C-glucose enrichment and oral recombinant C-terminal Mgam glucoamylase (M20) was supplemented to improve starch digestion. After feedings, suc/suc and +/suc shrews had different starch digestions as shown by blood glucose enrichment and the suc/suc had lower total glucose concentrations. Oral supplements of glucoamylase increased suc/suc total blood glucose and quantitative starch digestion to glucose. Sucrase deficiency, in this model of congenital sucrase-isomaltase deficiency, reduces blood glucose response to starch feeding. Supplementing the diet with oral recombinant glucoamylase significantly improved starch digestion in the sucrase-deficient shrew.

Development of Digestive Enzyme of Patin Pangasius hypohthalmus Larvae

Directory of Open Access Journals (Sweden)

I. Effendi

2007-01-01

Full Text Available Culture of patin Pangasius hypophthalmus especially larval rearing  very depends on the supply of natural food as energy source.  Artemia is the main natural food for fish larvae as a starter food, but its price is high.  To reduce production cost, farmers tend to reduce the feeding frequency and shorten  the Artemia feeding period.  Altering feeding regime however may reduce fry quality. This relate to the availability of digestive enzymes.  The objective of this study was  to examine digestive enzymes activity in patin larvae fed with  different feeding regime.  By shorten feeding period with Artemia to 2-4 days and Tubifex,substitution, the enzymes activity of protease, lipase and amylase were revealed similar pattern   The enzymes activity tends to increase and reach the peak at day 7 , and decrease later on until day 15 after hatching.  Survival rate of fish were varied for each treatment, and the highest survival rate was obtained when larvae were fed by Artemia for 8 days.  Blood worm were not fully digested by patin larvae at early stage. Keywords: enzyme, digestion, patin, Pangasius hypophthalmus   ABSTRAK Proses budidaya ikan patin, Pangasius hypophthalmus terutama pembenihan sangat tergantung oleh ketersediaan pakan alami sebagai sumber energinya. Artemia merupakan pakan alami yang banyak diberikan pada saat larva ikan mulai makan, namun harganya relatif tinggi. Untuk menekan biaya produksi, petani ikan patin cenderung mengurangi frekuensi pemberian Artemia dan mempersingkat waktu pemberiannya. Penggeseran jadwal ini diduga mengakibatkan penurunan kualitas benih ikan patin yang dihasilkan yang berhubungan dengan kesiapan enzim pencernaannya. Penelitian ini dilakukan untuk mengetahui aktivitas enzim pada larva ikan patin dengan jadwal pemberian pakan yang berbeda. Dengan memotong waktu pemberian Artemia 2 - 4 hari dan disubstitusi dengan Tubifex, aktifitas enzim protease, lipase dan amilase pada larva ikan patin, memiliki

Induction of DNA double-strand breaks by restriction enzymes in X-ray-sensitive mutant Chinese hamster ovary cells measured by pulsed-field gel electrophoresis

International Nuclear Information System (INIS)

Kinashi, Yuko; Nagasawa, Hatsumi; Little, J.B.; Okayasu, Ryuichi; Iliakis, G.E.

1995-01-01

This investigation was designed to determine whether the cytotoxic effects of different restriction endonucleases are related to the number and type of DNA double-strand breaks (DSBs) they produce. Chinese hamster ovary (CHO) K1 and xrs-5 cells, a radiosensitive mutant of CHO K1, were exposed to restriction endonucleases HaeIII, HinfI, PvuII and BamHI by electroporation. These enzymes represent both blunt and sticky end cutters with differing recognition sequence lengths. The number of DSBs was measured by pulsed-field gel electrophoresis (PFGE). Two forms of PFGE were employed: asymmetric field-inversion gel electrophoresis (AFIGE) for measuring the kinetics of DNA breaks by enzyme digestion and clamped homogeneous gel electrophoresis (CHEF) for examining the size distributions of damaged DNA. The amount of DNA damage induced by exposure to all four restriction enzymes was significantly greater in xrs-5 compared to CHO K1 cells, consistent with the reported DSB repair deficiency in these cells. Since restriction endonucleases produce DSBs alone as opposed to the various types of DNA damage induced by X rays, these results confirm that the repair defect in this mutant involves the rejoining of DSBs. Although the cutting frequency was directly related to the length of the recognition sequence for four restriction enzymes, there was no simple correlation between the cytotoxic effect and the amount of DNA damage produced by each enzyme in either cell line. This finding suggests that the type or nature of the cutting sequence itself may play a role in restriction enzyme-induced cell killing. 32 refs., 6 figs., 3 tabs

Effect of water quality and confounding factors on digestive enzyme activities in Gammarus fossarum.

Science.gov (United States)

Charron, L; Geffard, O; Chaumot, A; Coulaud, R; Queau, H; Geffard, A; Dedourge-Geffard, O

2013-12-01

The feeding activity and subsequent assimilation of the products resulting from food digestion allow organisms to obtain energy for growth, maintenance and reproduction. Among these biological parameters, we studied digestive enzymes (amylase, cellulase and trypsin) in Gammarus fossarum to assess the impact of contaminants on their access to energy resources. However, to enable objective assessment of a toxic effect of decreased water quality on an organisms' digestive capacity, it is necessary to establish reference values based on its natural variability as a function of changing biotic and abiotic factors. To limit the confounding influence of biotic factors, a caging approach with calibrated male organisms from the same population was used. This study applied an in situ deployment at 23 sites of the Rhone basin rivers, complemented by a laboratory experiment assessing the influence of two abiotic factors (temperature and conductivity). The results showed a small effect of conductivity on cellulase activity and a significant effect of temperature on digestive enzyme activity but only at the lowest temperature (7 °C). The experimental conditions allowed us to define an environmental reference value for digestive enzyme activities to select sites where the quality of the water impacted the digestive capacity of the organisms. In addition to the feeding rate, this study showed the relevance of digestive enzymes as biomarkers to be used as an early warning tool to reflect organisms' health and the chemical quality of aquatic ecosystems.

Nucleic acids digestion by enzymes in the stomach of snakehead (Channa argus) and banded grouper (Epinephelus awoara).

Science.gov (United States)

Liu, Yu; Zhang, Yanfang; Jiang, Wei; Wang, Jing; Pan, Xiaoming; Wu, Wei; Cao, Minjie; Dong, Ping; Liang, Xingguo

2017-02-01

Dietary nucleic acids (NAs) were important nutrients. However, the digestion of NAs in stomach has not been studied. In this study, the digestion of NAs by enzymes from fish stomach was investigated. The snakehead pepsins (SP) which were the main enzymes in stomach were extracted and purified. The purity of SP was evaluated by SDS-PAGE and HPLC. The snakehead pepsin 2 (SP2) which was the main component in the extracts was used for investigating the protein and NAs digestion activity. SP2 could digest NAs, including λ DNA and salmon sperm DNA. Interestingly, the digestion could be inhibited by treatment of alkaline solution at pH 8.0 and pepstatin A, and the digestion could happen either in the presence or absence of hemoglobin (Hb) and BSA as the protein substrates. Similarly, the stomach enzymes of banded grouper also showed the NAs digestion activity. NAs could be digested by the stomach enzymes of snakehead and banded grouper. It may be helpful for understanding both animal nutrition and NAs metabolic pathway.

Sequence specific inhibition of DNA restriction enzyme cleavage by PNA

DEFF Research Database (Denmark)

Nielsen, P.E.; Egholm, M.; Berg, R.H.

1993-01-01

Plasmids containing double-stranded 10-mer PNA (peptide nucleic acid chimera) targets proximally flanked by two restriction enzyme sites were challenged with the complementary PNA or PNAs having one or two mismatches, and the effect on the restriction enzyme cleavage of the flanking sites was ass...

Changes in growth, survival and digestive enzyme activities of Asian ...

African Journals Online (AJOL)

A study was conducted to determine the effects of different dietary treatments on the growth, survival and digestive enzyme activities of Mystus nemurus larvae. Newly hatched larvae were reared for 14 days in twelve 15 L glass aquaria (for growth and survival) and eight 300 L fiberglass tanks (for enzyme samples) at a ...

Restriction enzyme cleavage of ultraviolet-damaged Simian virus 40 and pBR322 DNA

International Nuclear Information System (INIS)

Cleaver, J.E.

1983-01-01

Cleavage of specific DNA sequences by the restriction enzymes EcoRI, HindIII and TaqI was prevented when the DNA was irradiated with ultraviolet light. Most of the effects were attributed to cyclobutane pyrimidine dimers in the recognition sequences; the effectiveness of irradiation was directly proportional to the number of potential dimer sites in the DNA. Combining EcoRI with dimer-specific endonuclease digestion revealed that pyrimidine dimers blocked cleavage within one base-pair on the strand opposite to the dimer but did not block cleavage three to four base-pairs away on the same strand. These are the probable limits for the range of influence of pyrimidine dimers along the DNA, at least for this enzyme. The effect of irradiation on cleavage by TaqI seemed far greater than expected for the cyclobutane dimer yield, possibly because of effects from photoproducts flanking the tetranucleotide recognition sequence and the effect of non-cyclobutane (6-4)pyrimidine photoproducts involving adjacent T and C bases. (author)

The complexities of hydrolytic enzymes from the termite digestive system.

Science.gov (United States)

Saadeddin, Anas

2014-06-01

The main challenge in second generation bioethanol production is the efficient breakdown of cellulose to sugar monomers (hydrolysis). Due to the recalcitrant character of cellulose, feedstock pretreatment and adapted hydrolysis steps are needed to obtain fermentable sugar monomers. The conventional industrial production process of second-generation bioethanol from biomass comprises several steps: thermochemical pretreatment, enzymatic hydrolysis and sugar fermentation. This process is undergoing continuous optimization in order to increase the bioethanol yield and reduce the economic cost. Therefore, the discovery of new enzymes with high lignocellulytic activity or new strategies is extremely important. In nature, wood-feeding termites have developed a sophisticated and efficient cellulose degrading system in terms of the rate and extent of cellulose hydrolysis and exploitation. This system, which represents a model for digestive symbiosis has attracted the attention of biofuel researchers. This review describes the termite digestive system, gut symbionts, termite enzyme resources, in vitro studies of isolated enzymes and lignin degradation in termites.

Cellulose digestion in Monochamus marmorator Kby. (coleoptera: Cerambycidae): role of acquired fungal enzymes

Energy Technology Data Exchange (ETDEWEB)

Kukol, J.J.; Martin, M.M.

1986-05-01

Larvae of the balsam fir sawyer, Monochamus marmorator Kby. (Coleoptera, Cerambycidae), contain midgut digestive enzymes active against hemicellulose and cellulose. Cellulases from larvae fed on balsam fir wood infected with the fungus, Trichoderma harzianum Rifai (Deuteromycetes, Moniliales, Moniliaceae), were found to be identical to those of the cellulase complex produced by this fungus when compared using chromatography, electrophoresis, and isofocusing. When larvae are maintained on a fungusfree diet, their midgut fluids lack cellulolytic activity, and they are unable to digest cellulose. Cellulolytic capacity can be restored by feeding the larvae wood permeated by fungi. We conclude that the enzymes which enable M. marmorator larvae to digest cellulose are not produced by the larvae. Instead, the larvae acquire the capacity to digest cellulose by ingesting active fungal cellulases while feeding in fungus-infected wood.

Cellulose digestion in Monochamus marmorator Kby. (coleoptera: Cerambycidae): role of acquired fungal enzymes

International Nuclear Information System (INIS)

Kukol, J.J.; Martin, M.M.

1986-01-01

Larvae of the balsam fir sawyer, Monochamus marmorator Kby. (Coleoptera, Cerambycidae), contain midgut digestive enzymes active against hemicellulose and cellulose. Cellulases from larvae fed on balsam fir wood infected with the fungus, Trichoderma harzianum Rifai (Deuteromycetes, Moniliales, Moniliaceae), were found to be identical to those of the cellulase complex produced by this fungus when compared using chromatography, electrophoresis, and isofocusing. When larvae are maintained on a fungusfree diet, their midgut fluids lack cellulolytic activity, and they are unable to digest cellulose. Cellulolytic capacity can be restored by feeding the larvae wood permeated by fungi. We conclude that the enzymes which enable M. marmorator larvae to digest cellulose are not produced by the larvae. Instead, the larvae acquire the capacity to digest cellulose by ingesting active fungal cellulases while feeding in fungus-infected wood

Transforming Growth Factor β/Activin Signaling Functions as a Sugar-Sensing Feedback Loop to Regulate Digestive Enzyme Expression

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Wen-bin Alfred Chng

2014-10-01

Full Text Available Summary: Organisms need to assess their nutritional state and adapt their digestive capacity to the demands for various nutrients. Modulation of digestive enzyme production represents a rational step to regulate nutriment uptake. However, the role of digestion in nutrient homeostasis has been largely neglected. In this study, we analyzed the mechanism underlying glucose repression of digestive enzymes in the adult Drosophila midgut. We demonstrate that glucose represses the expression of many carbohydrases and lipases. Our data reveal that the consumption of nutritious sugars stimulates the secretion of the transforming growth factor β (TGF-β ligand, Dawdle, from the fat body. Dawdle then acts via circulation to activate TGF-β/Activin signaling in the midgut, culminating in the repression of digestive enzymes that are highly expressed during starvation. Thus, our study not only identifies a mechanism that couples sugar sensing with digestive enzyme expression but points to an important role of TGF-β/Activin signaling in sugar metabolism. : Organisms modulate their digestive processes to reflect their nutritional state. In this study, Chng et al. demonstrate that the TGF-β/Activin pathway functions as a carbohydrate-sensing mechanism in the adult Drosophila midgut to regulate digestive enzyme expression. They show that the TGF-β ligand, Dawdle, and the canonical TGF-β/Activin signaling are essential to couple carbohydrate sensing with digestive enzyme expression. Thus, their study highlights an unexpected function of TGF-β/Activin signaling that is beyond their established roles in development and immunity.

Breakdown of mucin as barrier to digestive enzymes in the ischemic rat small intestine.

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Marisol Chang

Full Text Available Loss of integrity of the epithelial/mucosal barrier in the small intestine has been associated with different pathologies that originate and/or develop in the gastrointestinal tract. We showed recently that mucin, the main protein in the mucus layer, is disrupted during early periods of intestinal ischemia. This event is accompanied by entry of pancreatic digestive enzymes into the intestinal wall. We hypothesize that the mucin-containing mucus layer is the main barrier preventing digestive enzymes from contacting the epithelium. Mucin breakdown may render the epithelium accessible to pancreatic enzymes, causing its disruption and increased permeability. The objective of this study was to investigate the role of mucin as a protection for epithelial integrity and function. A rat model of 30 min splanchnic arterial occlusion (SAO was used to study the degradation of two mucin isoforms (mucin 2 and 13 and two epithelial membrane proteins (E-cadherin and toll-like receptor 4, TLR4. In addition, the role of digestive enzymes in mucin breakdown was assessed in this model by luminal inhibition with acarbose, tranexamic acid, or nafamostat mesilate. Furthermore, the protective effect of the mucin layer against trypsin-mediated disruption of the intestinal epithelium was studied in vitro. Rats after SAO showed degradation of mucin 2 and fragmentation of mucin 13, which was not prevented by protease inhibition. Mucin breakdown was accompanied by increased intestinal permeability to FITC-dextran as well as degradation of E-cadherin and TLR4. Addition of mucin to intestinal epithelial cells in vitro protected against trypsin-mediated degradation of E-cadherin and TLR4 and reduced permeability of FITC-dextran across the monolayer. These results indicate that mucin plays an important role in the preservation of the mucosal barrier and that ischemia but not digestive enzymes disturbs mucin integrity, while digestive enzymes actively mediate epithelial cell

Development of digestive enzymes in larvae of Mayan cichlid Cichlasoma urophthalmus.

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López-Ramírez, G; Cuenca-Soria, C A; Alvarez-González, C A; Tovar-Ramírez, D; Ortiz-Galindo, J L; Perales-García, N; Márquez-Couturier, G; Arias-Rodríguez, L; Indy, J R; Contreras-Sánchez, W M; Gisbert, E; Moyano, F J

2011-03-01

The development of digestive enzymes during the early ontogeny of the Mayan cichlid (Cichlasoma urophthalmus) was studied using biochemical and electrophoretic techniques. From yolk absorption (6 days after hatching: dah), larvae were fed Artemia nauplii until 15 dah, afterward they were fed with commercial microparticulated trout food (45% protein and 16% lipids) from 16 to 60 dah. Several samples were collected including yolk-sac larvae (considered as day 1 after hatching) and specimens up to 60 dah. Most digestive enzymes were present from yolk absorption (5-6 dah), except for the specific acid proteases activity (pepsin-like), which increase rapidly from 8 dah up to 20 dah. Three alkaline proteases isoforms (24.0, 24.8, 84.5 kDa) were detected at 8 dah using SDS-PAGE zymogram, corresponding to trypsin, chymotrypsin and probably leucine aminopeptidase enzymes, and only one isoform was detected (relative electromobility, Rf = 0.54) for acid proteases (pepsin-like) from 3 dah onwards using PAGE zymogram. We concluded that C. urophthamus is a precocious fish with a great capacity to digest all kinds of food items, including artificial diets provided from 13 dah.

Conservation of the egg envelope digestion mechanism of hatching enzyme in euteleostean fishes.

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Kawaguchi, Mari; Yasumasu, Shigeki; Shimizu, Akio; Sano, Kaori; Iuchi, Ichiro; Nishida, Mutsumi

2010-12-01

We purified two hatching enzymes, namely high choriolytic enzyme (HCE; EC 3.4.24.67) and low choriolytic enzyme (LCE; EC 3.4.24.66), from the hatching liquid of Fundulus heteroclitus, which were named Fundulus HCE (FHCE) and Fundulus LCE (FLCE). FHCE swelled the inner layer of egg envelope, and FLCE completely digested the FHCE-swollen envelope. In addition, we cloned three Fundulus cDNAs orthologous to cDNAs for the medaka precursors of egg envelope subunit proteins (i.e. choriogenins H, H minor and L) from the female liver. Cleavage sites of FHCE and FLCE on egg envelope subunit proteins were determined by comparing the N-terminal amino acid sequences of digests with the sequences deduced from the cDNAs for egg envelope subunit proteins. FHCE and FLCE cleaved different sites of the subunit proteins. FHCE efficiently cleaved the Pro-X-Y repeat regions into tripeptides to dodecapeptides to swell the envelope, whereas FLCE cleaved the inside of the zona pellucida domain, the core structure of egg envelope subunit protein, to completely digest the FHCE-swollen envelope. A comparison showed that the positions of hatching enzyme cleavage sites on egg envelope subunit proteins were strictly conserved between Fundulus and medaka. Finally, we extended such a comparison to three other euteleosts (i.e. three-spined stickleback, spotted halibut and rainbow trout) and found that the egg envelope digestion mechanism was well conserved among them. During evolution, the egg envelope digestion by HCE and LCE orthologs was established in the lineage of euteleosts, and the mechanism is suggested to be conserved. © 2010 The Authors Journal compilation © 2010 FEBS.

Effects of enzyme Additive on Nutrient intake, Digestibility and ...

African Journals Online (AJOL)

Nutrient intake, digestibility and rumen metabolites were determined in sixteen yearling cattle fed Panicum maximum hay supplemented with concentrate diet in which an exogenous fibrolytic enzyme, ROXAZYME G2® (which consist of Cellulase, hemicellulase and beta glucanase) was included at 0, 50, 100 and 150mg/kg.

Improved starch digestion of sucrase deficient shrews treated with oral glucoamylase enzyme supplements

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Although named because of its sucrose hydrolytic activity, this mucosal enzyme plays a leading role in starch digestion because of its maltase and glucoamylase activities. Sucrase deficient mutant shrews, Suncus murinus, were used as a model to investigate starch digestion in patients with Congenita...

Effect of starvation and refeeding on digestive enzyme activities in sturgeon (Acipenser naccarii) and trout (Oncorhynchus mykiss).

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Furné, Miriam; García-Gallego, Manuel; Hidalgo, M Carmen; Morales, Amalia E; Domezain, Alberto; Domezain, Julio; Sanz, Ana

2008-04-01

The digestive enzyme activities were determined in Adriatic sturgeon and rainbow trout during starvation and refeeding period. Overall, the digestive enzyme activities are affected in the same sense in both species. The protease and lipase activities were decreased later than amylase activity. Even after 1 month of starvation, both species would be prepared to digest protein and lipids in an effective way. After 72 days of starvation, the digestive machinery of the sturgeon and of the trout shows an altered capacity to digest macronutrients. The capacity to digest proteins and lipids, after 60 days of refeeding, begins to become re-established in sturgeon and trout. In contrast, in this period, the capacity to digest carbohydrates remains depressed in both species.

distribution, abundance and properties of restriction enzymes

African Journals Online (AJOL)

DNA of granule-bound starch synthase (GBSS) I and II with a view to ... properties for manipulation of the genes for production of modified starch. .... procurement, storage and handling of the ..... been made on restriction enzymes of potato,.

Effect of Alchornea cordifolia leaf meal inclusion and enzyme supplementation on performance and digestibility of rabbits

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S.O. Ayodele

2016-09-01

Full Text Available A feeding trial was conducted to study the performance, digestibility and health status of weaner rabbits fed diets including Alchornea cordifolia leaf meal (ALM: 18% crude protein [CP] and 12.9% crude fibre and supplemented with a multi-enzyme additive (cellulase, xylanase, β-glucanase, α-amylase, protease, lipase. Six experimental diets were arranged factorially: 3 levels of ALM (0, 5 and 10% substituting palm kernel cake: 16.3% CP and 39.1% neutral detergent fibre combined with 2 levels of enzyme supplementation (0 and 0.35 g/kg. One hundred and eighty healthy, 5-wk-old weaner rabbits of cross-breeds were randomly allotted to 6 dietary treatments (30 rabbits/treatment, 3 rabbits/replicate. Growth rate was not affected (P>0.05 by the main factors (exogenous enzyme and ALM inclusion and their interactions (13.5 g/d on av.. Daily feed intake and feed conversion ratio decreased (P=0.01 with the ALM inclusion by 8%, but did not affect faecal digestibility. However, enzyme supplementation improved crude protein and crude fibre digestibility (P<0.001 by 6%. In conclusion, ALM inclusion and enzyme supplementation had no adverse effect on the performance and digestibility of rabbits.

New stable isotope method to measure protein digestibility and response to pancreatic enzyme intake in cystic fibrosis.

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Engelen, M P K J; Com, G; Anderson, P J; Deutz, N E P

2014-12-01

Adequate protein intake and digestion are necessary to prevent muscle wasting in cystic fibrosis (CF). Accurate and easy-to-use methodology to quantify protein maldigestion is lacking in CF. To measure protein digestibility and the response to pancreatic enzyme intake in CF by using a new stable isotope methodology. In 19 CF and 8 healthy subjects, protein digestibility was quantified during continuous (sip) feeding for 6 h by adding (15)N-labeled spirulina protein and L-[ring-(2)H5]phenylalanine (PHE) to the nutrition and measuring plasma ratio [(15)N]PHE to [(2)H5]PHE. Pancreatic enzymes were ingested after 2 h in CF and the response in protein digestibility was assessed. To exclude difference in mucosal function, postabsorptive whole-body citrulline (CIT) production rate was measured by L-[5-(13)C-5,5-(2)H2]-CIT pulse and blood samples were taken to analyze tracer-tracee ratios. Protein digestibility was severely reduced in the CF group (47% of healthy subjects; P digestibility in CF until 90% of values obtained by healthy subjects. Maximal digestibility was reached at 100 min and maintained for 80 min. Stratification into CF children (n = 10) and adults showed comparable values for protein digestibility and similar kinetic responses to pancreatic enzyme intake. Whole-body citrulline production was elevated in CF indicating preserved mucosal function. Protein digestibility is severely compromised in patients with CF as measured by this novel and easy-to-use stable isotope approach. Pancreatic enzymes are able to normalize protein digestibility in CF, albeit with a severe delay. Registration ClinicalTrials.gov = NCT01494909. Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

High-pressure tolerance of earthworm fibrinolytic and digestive enzymes.

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Akazawa, Shin-Ichi; Tokuyama, Haruka; Sato, Shunsuke; Watanabe, Toshinori; Shida, Yosuke; Ogasawara, Wataru

2018-02-01

Earthworms contain several digestive and therapeutic enzymes that are beneficial to our health and useful for biomass utilization. Specifically, earthworms contain potent fibrinolytic enzymes called lumbrokinases, which are highly stable even at room temperature and remain active in dried earthworm powder. However, the high-temperature sterilization method leads to the inactivation of enzymes. Therefore, we investigated the effect of high-pressure treatment (HPT) (from 0.1 MPa to 500 MPa at 25°C and 50°C) on the enzymatic activity of lumbrokinase (LK), α-amylase (AMY), endoglucanase (EG), β-glucosidase (BGL), and lipase (LP) of the earthworm Eisenia fetida, Waki strain, and its sterilization ability in producing dietary supplement. LK showed thermo- and high-pressure tolerance. In addition, HPT may have resulted in pressure-induced stabilization and activation of LK. Although AMY activity was maintained up to 400 MPa at 25°C, the apparent activity decreased slightly at 50°C with HPT. EG showed almost the same pattern as AMY. However, it is possible that the effects of temperature and pressure compensated each other under 100 MPa at 50°C. BGL was shown to be a pressure- and temperature-sensitive enzyme, and LP showed a thermo- and high-pressure tolerance. The slight decrease in apparent activity occurred under 200 MPa at both temperatures. Furthermore, the low-temperature and pressure treatment completely sterilized the samples. These results provide a basis for the development of a novel earthworm dietary supplement with fibrinolytic and digestive activity and of high-pressure-tolerant enzymes to be used for biomass pretreatment. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

The Effects of Enzyme Complex on Performance, Intestinal Health and Nutrient Digestibility of Weaned Pigs

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J. Q. Yi

2013-08-01

Full Text Available Two experiments were conducted to evaluate the effect of supplementing a corn-soybean meal-based diet with an enzyme complex containing amylase, protease and xylanase on the performance, intestinal health, apparent ileal digestibility of amino acids and nutrient digestibility of weaned pigs. In Exp. 1, 108 piglets weaned at 28 d of age were fed one of three diets containing 0 (control, 100, or 150 ppm enzyme complex for 4 wks, based on a two-phase feeding program namely 1 to 7 d (phase 1 and 8 to 28 d (phase 2. At the end of the experiment, six pigs from the control group and the group supplemented with 150 ppm enzyme complex were chosen to collect digesta samples from intestine to measure viscosity and pH in the stomach, ileum, and cecum, as well as volatile fatty acid concentrations and composition of the microflora in the cecum and colon. There were linear increases (p<0.01 in weight gain, gain: feed ratio and digestibility of gross energy with the increasing dose rate of enzyme supplementation during the whole experiment. Supplementation with enzyme complex increased the digesta viscosity in the stomach (p<0.05 and significantly increased (p<0.01 the concentrations of acetic, propionic and butyric acid in the cecum and colon. Enzyme supplementation also significantly increased the population of Lactobacilli (p<0.01 in the cecum and decreased the population of E. coli (p<0.05 in the colon. In Exp. 2, six crossbred barrows (initial body weight: 18.26±1.21 kg, fitted with a simple T-cannula at the distal ileum, were assigned to three dietary treatments according to a replicated 3×3 Latin Square design. The experimental diets were the same as the diets used in phase 2 in Exp. 1. Apparent ileal digestibility of isoleucine (p<0.01, valine (p<0.05 and aspartic acid (p<0.05 linearly increased with the increasing dose rate of enzyme supplementation. In conclusion, supplementation of the diet with an enzyme complex containing amylase, protease and

Quantitative assessment of the association between the angiotensin-converting enzyme gene insertion/deletion polymorphism and digestive system cancer risk.

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Wang, J; Yang, S; Guo, F H; Mao, X; Zhou, H; Dong, Y Q; Wang, Z M; Luo, F

2015-11-13

The angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism has been reported to be associated with digestive system cancer; however, the results from previous studies have been conflicting. The present study aimed to investigate the association between the ACE I/D polymorphism and the risk of digestive system cancer using a meta-analysis of previously published studies. Databases were systematically searched to identify relevant studies published prior to December 2014. We estimated the pooled OR with its 95%CI to assess the association. The meta-analysis consisted of thirteen case-control studies that included 2557 patients and 4356 healthy controls. Meta-analysis results based on all the studies showed no significant association between the ACE I/D polymorphism and the risk of digestive system cancer (DD vs II: OR = 0.85, 95%CI = 0.59-1.24; DI vs II: OR = 0.94, 95%CI = 0.78-1.15; dominant model: OR = 0.96, 95%CI = 0.81- 1.15; recessive model: OR = 1.06, 95%CI = 0.76-1.48). Subgroup analyses by race and cancer type did not detect an association between the ACE I/D polymorphism and digestive system cancer risk. However, when the analyses were restricted to smaller studies (N digestive system cancer. Further large and well-designed studies are needed to confirm these conclusions.

Pst I restriction fragment length polymorphism of the human placental alkaline phosphatase gene in normal placentae and tumors

International Nuclear Information System (INIS)

Tsavaler, L.; Penhallow, R.C.; Kam, W.; Sussman, H.H.

1987-01-01

The structure of the human placental alkaline phosphatase gene from normal term placentae was studied by restriction enzyme digestion and Southern blot analysis using a cDNA probe to the gene for the placental enzyme. The DNA digests fall into three distinct patterns based on the presence and intensity of an extra 1.1-kilobase Pst I Band. The extra 1.1-kilobase band is present in 9 of 27 placenta samples, and in 1 of these samples the extra band is present at double intensity. No polymorphism was revealed by digestion with restriction enzymes EcoRI, Sma I, BamHI, or Sac I. The extra Pst I-digestion site may lie in a noncoding region of the gene because no correlation was observed between the restriction fragment length polymorphism and the common placental alkaline phosphatase alleles identified by starch gel electrophoresis. In addition, because placental alkaline phosphatase is frequently re-expressed in neoplasms, the authors examined tissue from ovarian, testicular, and endometrial tumors and from BeWo choriocarcinoma cells in culture. The Pst I-DNA digestion patterns from these cells and tissues were identical to those seen in the normal ovary and term placentae. The consistent reproducible digestion patterns seen in DNA from normal and tumor tissue indicate that a major gene rearrangement is not the basis for the ectopic expression of placental alkaline phosphatase in neoplasia

Expression pattern of glycoside hydrolase genes in Lutzomyia longipalpis reveals key enzymes involved in larval digestion

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Caroline da Silva Moraes

2014-08-01

Full Text Available The sand fly Lutzomyia longipalpis is the most important vector of American Visceral Leishmaniasis. Adults are phytophagous (males and females or blood feeders (females only, and larvae feed on solid detritus. Digestion in sand fly larvae has scarcely been studied, but some glycosidase activities putatively involved in microorganism digestion were already described. Nevertheless, the molecular nature of these enzymes, as the corresponding genes and transcripts, were not explored yet. Catabolism of microbial carbohydrates in insects generally involves β-1,3-glucanases, chitinases and digestive lysozymes. In this work, the transcripts of digestive β-1,3-glucanase and chitinases were identified in the L. longipalpis larvae throughout analysis of sequences and expression patterns of glycoside hydrolases families 16, 18 and 22. The activity of one i-type lysozyme was also registered. Interestingly, this lysozyme seems to play a role in immunity, rather than digestion. This is the first attempt to identify the molecular nature of sand fly larval digestive enzymes.

Expression pattern of glycoside hydrolase genes in Lutzomyia longipalpis reveals key enzymes involved in larval digestion

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Moraes, Caroline da Silva; Diaz-Albiter, Hector M.; Faria, Maiara do Valle; Sant'Anna, Maurício R. V.; Dillon, Rod J.; Genta, Fernando A.

2014-01-01

The sand fly Lutzomyia longipalpis is the most important vector of American Visceral Leishmaniasis. Adults are phytophagous (males and females) or blood feeders (females only), and larvae feed on solid detritus. Digestion in sand fly larvae has scarcely been studied, but some glycosidase activities putatively involved in microorganism digestion were already described. Nevertheless, the molecular nature of these enzymes, as the corresponding genes and transcripts, were not explored yet. Catabolism of microbial carbohydrates in insects generally involves β-1,3-glucanases, chitinases, and digestive lysozymes. In this work, the transcripts of digestive β-1,3-glucanase and chitinases were identified in the L. longipalpis larvae throughout analysis of sequences and expression patterns of glycoside hydrolases families 16, 18, and 22. The activity of one i-type lysozyme was also registered. Interestingly, this lysozyme seems to play a role in immunity, rather than digestion. This is the first attempt to identify the molecular nature of sand fly larval digestive enzymes. PMID:25140153

msgbsR: An R package for analysing methylation-sensitive restriction enzyme sequencing data.

Science.gov (United States)

Mayne, Benjamin T; Leemaqz, Shalem Y; Buckberry, Sam; Rodriguez Lopez, Carlos M; Roberts, Claire T; Bianco-Miotto, Tina; Breen, James

2018-02-01

Genotyping-by-sequencing (GBS) or restriction-site associated DNA marker sequencing (RAD-seq) is a practical and cost-effective method for analysing large genomes from high diversity species. This method of sequencing, coupled with methylation-sensitive enzymes (often referred to as methylation-sensitive restriction enzyme sequencing or MRE-seq), is an effective tool to study DNA methylation in parts of the genome that are inaccessible in other sequencing techniques or are not annotated in microarray technologies. Current software tools do not fulfil all methylation-sensitive restriction sequencing assays for determining differences in DNA methylation between samples. To fill this computational need, we present msgbsR, an R package that contains tools for the analysis of methylation-sensitive restriction enzyme sequencing experiments. msgbsR can be used to identify and quantify read counts at methylated sites directly from alignment files (BAM files) and enables verification of restriction enzyme cut sites with the correct recognition sequence of the individual enzyme. In addition, msgbsR assesses DNA methylation based on read coverage, similar to RNA sequencing experiments, rather than methylation proportion and is a useful tool in analysing differential methylation on large populations. The package is fully documented and available freely online as a Bioconductor package ( https://bioconductor.org/packages/release/bioc/html/msgbsR.html ).

Digestive enzymes and gut morphometric parameters of threespine stickleback (Gasterosteus aculeatus): Influence of body size and temperature.

Science.gov (United States)

Hani, Younes Mohamed Ismail; Marchand, Adrien; Turies, Cyril; Kerambrun, Elodie; Palluel, Olivier; Bado-Nilles, Anne; Beaudouin, Rémy; Porcher, Jean-Marc; Geffard, Alain; Dedourge-Geffard, Odile

2018-01-01

Determining digestive enzyme activity is of potential interest to obtain and understand valuable information about fish digestive physiology, since digestion is an elementary process of fish metabolism. We described for the first time (i) three digestive enzymes: amylase, trypsin and intestinal alkaline phosphatase (IAP), and (ii) three gut morphometric parameters: relative gut length (RGL), relative gut mass (RGM) and Zihler's index (ZI) in threespine stickleback (Gasterosteus aculeatus), and we studied the effect of temperature and body size on these parameters. When mimicking seasonal variation in temperature, body size had no effect on digestive enzyme activity. The highest levels of amylase and trypsin activity were observed at 18°C, while the highest IAP activity was recorded at 20°C. When sticklebacks were exposed to three constant temperatures (16, 18 and 21°C), a temporal effect correlated to fish growth was observed with inverse evolution patterns between amylase activity and the activities of trypsin and IAP. Temperature (in both experiments) had no effect on morphometric parameters. However, a temporal variation was recorded for both RGM (in the second experiment) and ZI (in both experiments), and the later was correlated to fish body mass.

Digestive enzymes and gut morphometric parameters of threespine stickleback (Gasterosteus aculeatus): Influence of body size and temperature

Science.gov (United States)

Marchand, Adrien; Turies, Cyril; Kerambrun, Elodie; Palluel, Olivier; Bado-Nilles, Anne; Beaudouin, Rémy; Porcher, Jean-Marc; Geffard, Alain; Dedourge-Geffard, Odile

2018-01-01

Determining digestive enzyme activity is of potential interest to obtain and understand valuable information about fish digestive physiology, since digestion is an elementary process of fish metabolism. We described for the first time (i) three digestive enzymes: amylase, trypsin and intestinal alkaline phosphatase (IAP), and (ii) three gut morphometric parameters: relative gut length (RGL), relative gut mass (RGM) and Zihler’s index (ZI) in threespine stickleback (Gasterosteus aculeatus), and we studied the effect of temperature and body size on these parameters. When mimicking seasonal variation in temperature, body size had no effect on digestive enzyme activity. The highest levels of amylase and trypsin activity were observed at 18°C, while the highest IAP activity was recorded at 20°C. When sticklebacks were exposed to three constant temperatures (16, 18 and 21°C), a temporal effect correlated to fish growth was observed with inverse evolution patterns between amylase activity and the activities of trypsin and IAP. Temperature (in both experiments) had no effect on morphometric parameters. However, a temporal variation was recorded for both RGM (in the second experiment) and ZI (in both experiments), and the later was correlated to fish body mass. PMID:29614133

Digestive enzymes in Rhinolophus euryale (Rhinolophidae, Chiroptera are active also during hibernation

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Maxinová Edita

2017-11-01

Full Text Available During the winter, bats use hibernation as a means of surviving the period of low prey offer. However, the Mediterranean horseshoe bat (Rhinolophus euryale arouses from torpor quite frequently. Based on the actual climatic conditions, it can profit from occasional foraging oportunities, when they occur. We analysed faeces collected on four nights during the period from November 2012 to February 2013 from the Domica-Baradla cave system (Slovakia and Hungary. In mid-November, the largest proportion of faecal contents were from Lepidoptera. Later on, the proportion of non-consumptive mass in the faeces increased and prey remnants disappeared. We analysed the activity of digestive enzymes (amylase, chitobiase, endochitinase and glukosaminidase in faeces. The activity of these enzymes was detected in fresh faeces throughout the whole winter. The faecal activity of the chitinases was relatively stable during the monitored period, whilst the activity of amylase was highest during late November and December. Some level of active digestive enzymes during the winter could be an adaptation to occasional winter foraging.

Inhibition of raw starch digestion by one glucoamylase preparation from black Aspergillus at high enzyme concentration

Energy Technology Data Exchange (ETDEWEB)

Saka, B C; Veda, S

1981-09-01

Raw starch digestion by glucoamylase I (Ab. G-I) preparation from black Aspergillus was inhibited significantly at relatively high concentration of the enzyme. The properties of this enzyme were studied together with those of another glucoamylase I (Nor. G-I), also from black Aspergillus. The two glucoamylases do not differ so much in their physico-chemical properties such as molecular weight, pH and thermal stability, pH and temperature optimum, substrate specificity, debranching activity, isoelectric point etc. The adsorption rate of both enzymes on raw starch increased by the increase of enzyme concentration. The raw starch digestion rate by adsorbed Ab. G-I, however, was decreased with the increase of concentration of enzyme whereas the same was increased in case of Nor. G-I. The inhibitory effect was weaker at 60 deg. Celcius or above. (Refs. 11).

RNA-Seq reveals the dynamic and diverse features of digestive enzymes during early development of Pacific white shrimp Litopenaeus vannamei.

Science.gov (United States)

Wei, Jiankai; Zhang, Xiaojun; Yu, Yang; Li, Fuhua; Xiang, Jianhai

2014-09-01

The Pacific white shrimp (Litopenaeus vannamei), with high commercial value, has a typical metamorphosis pattern by going through embryo, nauplius, zoea, mysis and postlarvae during early development. Its diets change continually in this period, and a high mortality of larvae also occurs in this period. Since there is a close relationship between diets and digestive enzymes, a comprehensive investigation about the types and expression patterns of all digestive enzyme genes during early development of L. vannamei is of considerable significance for shrimp diets and larvae culture. Using RNA-Seq data, the types and expression characteristics of the digestive enzyme genes were analyzed during five different development stages (embryo, nauplius, zoea, mysis and postlarvae) in L. vannamei. Among the obtained 66,815 unigenes, 296 were annotated as 16 different digestive enzymes including five types of carbohydrase, seven types of peptidase and four types of lipase. Such a diverse suite of enzymes illustrated the capacity of L. vannamei to exploit varied diets to fit their nutritional requirements. The analysis of their dynamic expression patterns during development also indicated the importance of transcriptional regulation to adapt to the diet transition. Our study revealed the diverse and dynamic features of digestive enzymes during early development of L. vannamei. These results would provide support to better understand the physiological changes during diet transition. Copyright © 2014 Elsevier Inc. All rights reserved.

Digestive enzyme ratios are good indicators of hatchling yolk reserve and digestive gland maturation in early life stages of cuttlefish Sepia officinalis L.: application of these new tools in ecology and aquaculture.

Science.gov (United States)

Safi, Georges; Martinez, A S; Le Pabic, C; Le Bihan, E; Robin, J P; Koueta, N

2018-01-01

In Sepia officinalis (Linnaeus, 1758), the digestive gland matures during the first month post-hatching, while a shift from intracellular acid to extracellular alkaline digestion occurs. The purpose of this study was to investigate the possibility of using enzymatic ratios for the description of digestive system maturation in early life stages of S. officinalis. Second, it is intended to apply these new tools as eco-physiological indicators for understanding the impact of cuttlefish eggs' life history from different spawning sites of the English Channel on digestive performance of juveniles. An experimental rearing was performed over 35 days after hatching (DAH) on juveniles from wild collected eggs in 2010 and 2011. Four digestive enzyme activities and their ratios [i.e., trypsin, cathepsin, acid (ACP), and alkaline (ALP) phosphatase, ALP/ACP, and trypsin/cathepsin] were studied along with histological features (e.g., internal yolk surface and digestive gland development). The two enzyme ratios were good indicators of digestive system maturation allowing the study of the digestive gland's development. They were highly correlated to juveniles' weight increase and histological features of the gland in early DAH. These ratios described more accurately the shift occurring between the intracellular acid and the extracellular alkaline modes of digestion in S. officinalis and were more specific than separated enzyme activities. Their application as eco-physiological tools revealed that enzyme ratios reflected yolk content and digestive gland development in new hatching juveniles. Finally, ALP/ACP ratio was shown to be a powerful tool to describe growth performance of S. officinalis which is useful for aquaculture optimization.

Bacteria and digestive enzymes in the alimentary tract of the giant ...

African Journals Online (AJOL)

A study was carried out to investigate the bacteria flora in the gut of the Giant African Land Snails (GALS), Archarchatina marginata and Achatina achatina. Microflora cultures from snail gut contents were prepared to isolate and identify microorganisms within the snail digestive tract. Enzyme assays were carried out on a few ...

Highlights of the DNA cutters: a short history of the restriction enzymes.

Science.gov (United States)

Loenen, Wil A M; Dryden, David T F; Raleigh, Elisabeth A; Wilson, Geoffrey G; Murray, Noreen E

2014-01-01

In the early 1950's, 'host-controlled variation in bacterial viruses' was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of restriction enzymes and their continuing impact on molecular biology and medicine.

Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

International Nuclear Information System (INIS)

Chen, Kai; Roberts, Gareth A.; Stephanou, Augoustinos S.; Cooper, Laurie P.; White, John H.; Dryden, David T.F.

2010-01-01

Research highlights: → Successful fusion of GFP to M.EcoKI DNA methyltransferase. → GFP located at C-terminal of sequence specificity subunit does not later enzyme activity. → FRET confirms structural model of M.EcoKI bound to DNA. -- Abstract: We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Foerster resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.

Expression of digestive enzymes and nutrient transporters in Eimeria-challenged broilers.

Science.gov (United States)

Su, S; Miska, K B; Fetterer, R H; Jenkins, M C; Wong, E A

2015-03-01

Avian coccidiosis is a disease caused by the intestinal protozoa Eimeria. The site of invasion and lesions in the intestine is species-specific, for example E. acervulina affects the duodenum, E. maxima the jejunum, and E. tenella the ceca. Lesions in the intestinal mucosa cause reduced feed efficiency and body weight gain. The growth reduction may be due to changes in expression of digestive enzymes and nutrient transporters in the intestine. The objective of this study was to compare the expression of digestive enzymes, nutrient transporters and an antimicrobial peptide in broilers challenged with either E. acervulina, E. maxima or E. tenella. The genes examined included digestive enzymes (APN and SI), peptide and amino acid transporters (PepT1, ASCT1, b(0,+)AT/rBAT, B(0)AT, CAT1, CAT2, EAAT3, LAT1, y(+)LAT1 and y(+)LAT2), sugar transporters (GLUT1, GLUT2, GLUT5 and SGLT1), zinc transporter (ZnT1) and an antimicrobial peptide (LEAP2). Duodenum, jejunum, ileum and ceca were collected 7 days post challenge. E. acervulina challenge resulted in downregulation of various nutrient transporters or LEAP2 in the duodenum and ceca, but not the jejunum or ileum. E. maxima challenge produced both downregulation and upregulation of nutrient transporters and LEAP2 in all three segments of the small intestine and ceca. E. tenella challenge resulted in the downregulation and upregulation of nutrient transporters and LEAP2 in the jejunum, ileum and ceca, but not the duodenum. At the respective target tissue, E. acervulina, E. maxima and E. tenella infection caused common downregulation of APN, b(0,+)AT, rBAT, EAAT3, SI, GLUT2, GLUT5, ZnT1 and LEAP2. The downregulation of nutrient transporters would result in a decrease in the efficiency of protein and polysaccharide digestion and uptake, which may partially explain the weight loss. The downregulation of nutrient transporters may also be a cellular response to reduced expression of the host defense protein LEAP2, which would

Site-specific DNA transesterification catalyzed by a restriction enzyme

OpenAIRE

Sasnauskas, Giedrius; Connolly, Bernard A.; Halford, Stephen E.; Siksnys, Virginijus

2007-01-01

Most restriction endonucleases use Mg2+ to hydrolyze phosphodiester bonds at specific DNA sites. We show here that BfiI, a metal-independent restriction enzyme from the phospholipase D superfamily, catalyzes both DNA hydrolysis and transesterification reactions at its recognition site. In the presence of alcohols such as ethanol or glycerol, it attaches the alcohol covalently to the 5′ terminus of the cleaved DNA. Under certain conditions, the terminal 3′-OH of one DNA strand can attack the t...

Induced and constitutive responses of digestive enzymes to plant toxins in an herbivorous mammal.

Science.gov (United States)

Kohl, Kevin D; Dearing, M Denise

2011-12-15

Many plants produce plant secondary compounds (PSCs) that bind and inhibit the digestive enzymes of herbivores, thus limiting digestibility for the herbivore. Herbivorous insects employ several physiological responses to overcome the anti-nutritive effects of PSCs. However, studies in vertebrates have not shown such responses, perhaps stemming from the fact that previously studied vertebrates were not herbivorous. The responses of the digestive system to dietary PSCs in populations of Bryant's woodrat (Neotoma bryanti) that vary in their ecological and evolutionary experience with the PSCs in creosote bush (Larrea tridentata) were compared. Individuals from naïve and experienced populations were fed diets with and without added creosote resin. Animals fed diets with creosote resin had higher activities of pancreatic amylase, as well as luminal amylase and chymotrypsin, regardless of prior experience with creosote. The experienced population showed constitutively higher activities of intestinal maltase and sucrase. Additionally, the naïve population produced an aminopeptidase-N enzyme that was less inhibited by creosote resin when feeding on the creosote resin diet, whereas the experienced population constitutively expressed this form of aminopeptidase-N. Thus, the digestive system of an herbivorous vertebrate responds significantly to dietary PSCs, which may be important for allowing herbivorous vertebrates to feed on PSC-rich diets.

Fibrolytic enzyme and ammonia application effects on the nutritive value, intake, and digestion kinetics of bermudagrass hay in beef cattle.

Science.gov (United States)

Romero, J J; Zarate, M A; Queiroz, O C M; Han, J H; Shin, J H; Staples, C R; Brown, W F; Adesogan, A T

2013-09-01

The objectives were to compare the effect of exogenous fibrolytic enzyme (Biocellulase A20) or anhydrous ammonia (4% DM) treatment on the nutritive value, voluntary intake, and digestion kinetics of bermudagrass (Cynodon dactylon cultivar Coastal) hay harvested after 2 maturities (5- and 13-wk regrowths). Six individually housed, ruminally cannulated Brangus steers (BW 325 ± 10 kg) were used in an experiment with a 6 × 6 Latin square design with a 3 (additives) × 2 (maturities) factorial arrangement of treatments. Each period consisted of 14 d of adaptation and 7, 4, 1, 1, and 4 d for measuring in vivo digestibility, in situ degradability, no measurements, rumen liquid fermentation and passage indices, and rate of solid passage, respectively. Steers were fed hay for ad libitum intake and supplemented with sugarcane molasses and distillers grain (supplement total of 2.88 kg DM/d). Enzyme did not affect the nutritional composition of hay but ammonia treatment decreased hay NDF, hemicellulose, and ADL concentrations and increased the CP concentration particularly for the mature lignified 13-wk hay. The enzyme increased NDF and hemicellulose digestibility of the 5-wk hay but decreased those of the 13-wk hay. Ammoniation decreased intake of hay but increased digestibility of DM, OM, NDF, hemicellulose, ADF, and cellulose and increased the ruminal in situ soluble and potentially digestible fractions and the rate of DM degradation of the 13-wk hay. Also, ammoniation increased the concentrations of ruminal NH3, total VFA, acetate, and butyrate but enzyme treatment did not. Neither enzyme addition nor ammoniation affected rate of liquid and solid passage. In conclusion, ammoniation decreased the concentration of most fiber fractions, decreased the intake of hays, and increased their CP concentration, in vivo digestibility, and in situ degradability at both maturities whereas enzyme application increased fiber digestibility of the 5-wk hay but decreased it in the case of

Influence of probiotics on the growth and digestive enzyme activity of white Pacific shrimp ( Litopenaeus vannamei)

Science.gov (United States)

Gómez, R. Geovanny D.; Shen, M. A.

2008-05-01

The influence of Bacillus probiotics on the digestive enzyme activity and the growth of Litopenaeus vannamei were determined in this study. The shrimp was treated with five percentages (1.5, 3.0, 4.5, 6.0 and 7.5) of probiotics ( Bacillus spp.) supplemented to the feed and cultured for 45d. The growth measured as the weight gain at the end of culturing was significantly ( Pshrimps than that of the control (without receiving probiotics). Activities of protease and amylase, two digestive enzymes of the midgut gland and the intestine were significantly ( Pshrimp than in the control.

Effects of sources of protein and enzyme supplementation on protein digestibility and chyme characteristics in broilers.

Science.gov (United States)

Yu, B; Lee, T T T; Chiou, P W S

2002-07-01

1. The purpose of this study was to evaluate the effects of protein source and enzyme supplementation on protein digestibility and chyme characteristics in broilers. 2. One hundred and twenty growing (13 d old) and 60 finishing (34 d old) Arbor Acre strain commercial male broilers were selected and placed into individual metabolic cages. 3. The experiment was a 5 x 2 factorial arrangement with 5 different sources of protein: casein, fish meal, soybean meal (SBM), soy protein concentrate (SPC), maize gluten meal (MGM) and two levels of protease (bromelain), 0 and 65 CDU/kg diets. 4. The diets were iso-nitrogenous and semi-purified, with Cr2O3 as an indicator for determination of ileal digestibility and chyme characteristics. 5. Apparent ileal protein digestibility (AIPD) in both growing and finishing chickens was highest on the casein diet, followed by fish meal, SBM, SPC and MGM. 6. Enzyme inclusion did not improve protein digestibility, but significantly decreased the digesta pH value in the gizzard and increased pH in the ileum in the 3-week-old broilers. 7. The digesta pH values in the gizzard and duodenum were significantly lower in the SBM and fish meal groups compared with the other protein groups. The molecular weight distribution pattern of the soluble protein in the chyme of the gastrointestinal (GI) segments showed a similar trend, regardless of the enzyme inclusion or the stage of growth. 8. The molecular weight profile of soluble protein changed dynamically in the casein fed broilers from the gizzard to ileum and the low molecular weight proteins, < 7 kDa, reached maximum levels at the ileum. The molecular weight profile of the soluble protein in the SBM and SPC changed between the jejunum and the ileum and in the intermediate molecular soluble protein weight (7 to 10 kDa) was significantly decreased. This indicated that the hydrolysis process began from the middle to the posterior end of the small intestine. 9. Similar profiles were also shown with

Digestibility, productive performance, and egg quality of laying hens as affected by dried cassava pulp replacement with corn and enzyme supplementation.

Science.gov (United States)

Khempaka, Sutisa; Maliwan, Prapot; Okrathok, Supattra; Molee, Wittawat

2018-02-24

Two experiments were conducted to investigate the potential use of dried cassava pulp (DCP) supplemented with enzymes as an alternative feed ingredient in laying hen diets. In experiment 1, 45 laying hens (Isa Brown) aged 45 weeks were placed in individual cages to measure nutrient digestibility for 10 days. Nine dietary treatments were control and DCP as a replacement for corn at 20, 25, 30, and 35% supplemented with mixed enzymes (cellulase, glucanase, and xylanase) at 0.10 and 0.15%. Results showed that the use of DCP at 20-35% added with mixed enzymes had no negative effects on dry matter digestibility, while organic matter digestibility and nitrogen retention decreased with increased DCP up to 30-35% in diets. Both enzyme levels (0.10 and 0.15%) showed similar results on nutrient digestibility and retention. In experiment 2, a total of 336 laying hens aged 32 weeks were randomly allocated to seven dietary treatments (control and DCP-substituted diets at 20, 25, and 30%) supplemented with mixed enzymes (0.10 and 0.15%). Diets incorporated with 20-30% of DCP and supplemented with mixed enzymes at both levels had no significant effects on egg production, egg weight, feed intake, egg mass, feed conversion ratio, protein efficiency ratio, or egg quality, except for egg yolk color being decreased with an increase of DCP in diets (P digestibility, productive performance, or egg quality.

Development of digestive enzymes in larvae of Mayancichlid Cichlasoma urophthalmus

OpenAIRE

López Ramírez, G.; Cuenca Soria, C.A.; Álvarez González, C.A.; Tovar Ramírez, D.; Ortiz Galindo, José Luis; Perales García, N.; Márquez Couturier, G.; Arias Rodríguez, L.; Indy, J.R.; Contreras Sánchez, W.M.; Gisbert, E.; Moyano, F.J.

2011-01-01

The development of digestive enzymes during the early ontogeny of the Mayan cichlid (Cichlasoma urophthalmus) was studied using biochemical and electrophoretic techniques. From yolk absorption (6 days after hatching: dah), larvae were fed Artemia nauplii until 15 dah, afterward they were fed with commercial microparticulated trout food (45% protein and 16% lipids) from 16 to 60 dah. Several samples were collected including yolk-sac larvae (considered as day 1 after hatching) and specimens up ...

Characterization and identification of enzyme-producing microflora isolated from the gut of sea cucumber Apostichopus japonicus

Science.gov (United States)

Li, Fenghui; Gao, Fei; Tan, Jie; Fan, Chaojing; Sun, Huiling; Yan, Jingping; Chen, Siqing; Wang, Xiaojun

2016-01-01

Gut microorganisms play an important role in the digestion of their host animals. The purpose of this research was to isolate and assess the enzyme-producing microbes from the Apostichopus japonicus gut. Thirty-nine strains that can produce at least one of the three digestive enzymes (protease, amylase, and cellulase) were qualitatively screened based on their extracellular enzyme-producing abilities. The enzyme-producing strains clustered into eight groups at the genetic similarity level of 100% by analyzing the restriction patterns of 16S rDNA amplified with Mbo I. Phylogenetic analysis revealed that 37 strains belonged to the genus Bacillus and two were members of the genus Virgibacillus. Enzyme-producing capability results indicate that the main enzyme-producing microflora in the A. japonicus gut was Bacillus, which can produce protease, amylase, and cellulase. Virgibacillus, however, can only produce protease. The high enzyme-producing capability of the isolates suggests that the gut microbiota play an important role in the sea cucumber digestive process.

A fast exact sequential algorithm for the partial digest problem.

Science.gov (United States)

Abbas, Mostafa M; Bahig, Hazem M

2016-12-22

Restriction site analysis involves determining the locations of restriction sites after the process of digestion by reconstructing their positions based on the lengths of the cut DNA. Using different reaction times with a single enzyme to cut DNA is a technique known as a partial digestion. Determining the exact locations of restriction sites following a partial digestion is challenging due to the computational time required even with the best known practical algorithm. In this paper, we introduce an efficient algorithm to find the exact solution for the partial digest problem. The algorithm is able to find all possible solutions for the input and works by traversing the solution tree with a breadth-first search in two stages and deleting all repeated subproblems. Two types of simulated data, random and Zhang, are used to measure the efficiency of the algorithm. We also apply the algorithm to real data for the Luciferase gene and the E. coli K12 genome. Our algorithm is a fast tool to find the exact solution for the partial digest problem. The percentage of improvement is more than 75% over the best known practical algorithm for the worst case. For large numbers of inputs, our algorithm is able to solve the problem in a suitable time, while the best known practical algorithm is unable.

Functional and transcriptomic analysis of leaf litter digestion by millipedes: Role of microorganisms and digestive enzymes. An introduction of a starting project

Czech Academy of Sciences Publication Activity Database

Tajovský, Karel; Šustr, Vladimír; Chroňáková, Alica; Chrudimský, Tomáš; Macková, Jana; Koubová, Anna; Šimek, Miloslav

-, Suppl. 5 (2017), s. 61 ISSN 1513-9700. [International Congress of Myriapodology /17./. 23.07.2017-26.07.2017, Krabi] Institutional support: RVO:60077344 Keywords : millipedes * digestive enzymes * gut microbial communities * cellulose degradation * methane release Subject RIV: EG - Zoology OBOR OECD: Zoology

Prolidase is a critical enzyme for complete gliadin digestion in Tenebrio molitor larvae.

Science.gov (United States)

Tereshchenkova, Valeriia F; Goptar, Irina A; Zhuzhikov, Dmitry P; Belozersky, Mikhail A; Dunaevsky, Yakov E; Oppert, Brenda; Filippova, Irina Yu; Elpidina, Elena N

2017-08-01

Prolidase is a proline-specific metallopeptidase that cleaves imidodipeptides with C-terminal Pro residue. Prolidase was purified and characterized from the Tenebrio molitor larval midgut. The enzyme was localized in the soluble fraction of posterior midgut tissues, corresponding to a predicted cytoplasmic localization of prolidase according to the structure of the mRNA transcript. Expression of genes encoding prolidase and the major digestive proline-specific peptidase (PSP)-dipeptidyl peptidase 4-were similar. The pH optimum of T. molitor prolidase was 7.5, and the enzyme was inhibited by Z-Pro, indicating that it belongs to type I prolidases. In mammals, prolidase is particularly important in the catabolism of a proline-rich protein-collagen. We propose that T. molitor larval prolidase is a critical enzyme for the final stages of digestion of dietary proline-rich gliadins, providing hydrolysis of imidodipeptides in the cytoplasm of midgut epithelial cells. We propose that the products of hydrolysis are absorbed from the luminal contents by peptide transporters, which we have annotated in the T. molitor larval gut transcriptome. The origin of prolidase substrates in the insect midgut is discussed in the context of overall success of grain feeding insects. © 2017 Wiley Periodicals, Inc.

Digestive Enzyme Supplementation for Autism Spectrum Disorders: A Double-Blind Randomized Controlled Trial

Science.gov (United States)

Munasinghe, Sujeeva A.; Oliff, Carolyn; Finn, Judith; Wray, John A.

2010-01-01

To examine the effects of a digestive enzyme supplement in improving expressive language, behaviour and other symptoms in children with Autism Spectrum Disorder. Randomized, double-blind placebo-controlled trial using crossover design over 6 months for 43 children, aged 3-8 years. Outcome measurement tools included monthly Global Behaviour Rating…

Relationship between digestive enzymes and food habit of Lutzomyia longipalpis (Diptera: Psychodidae) larvae: Characterization of carbohydrases and digestion of microorganisms.

Science.gov (United States)

Moraes, C S; Lucena, S A; Moreira, B H S; Brazil, R P; Gontijo, N F; Genta, F A

2012-08-01

The sandfly Lutzomyia longipalpis (Lutz and Neiva, 1912) is the main vector of American Visceral Leishmaniasis. In spite of its medical importance and several studies concerning adult digestive physiology, biochemistry and molecular biology, very few studies have been carried out to elucidate the digestion in sandfly larvae. Even the breeding sites and food sources of these animals in the field are largely uncharacterized. In this paper, we describe and characterize several carbohydrases from the gut of L. longipalpis larvae, and show that they are probably not acquired from food. The enzyme profile of this insect is consistent with the digestion of fungal and bacterial cells, which were proved to be ingested by larvae under laboratory conditions. In this respect, sandfly larvae might have a detritivore habit in nature, being able to exploit microorganisms usually encountered in the detritus as a food source. Copyright © 2012 Elsevier Ltd. All rights reserved.

The effect of water temperature on food transit time and digestive enzymes activity in Caspian kutum (Rutilus kutum larvae

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Nahid Ghysvandi

2014-07-01

Full Text Available The present study investigates the effects of water temperature on digestive enzymes activity and food transit time in Caspian kutum (Rutilus kutum larvae. Caspian kutum larvae (532 ± 0.05 and 543 ± 0.02 mg were divided into two groups with three replicates and reared at different water temperature i.e. 25.6 ± 0.4°C (T1 and 18.4 ± 0.1°C (T2. At the end of the experiment, sampling of intestine was performed at 0, 1, 3, 5, 8, 16, 24 and 30 h after feeding from each treatment. In T2, food was observed until 24 h after feeding and the intestine was empty 29 h after feeding, while in T1 19 h after feeding the intestine was empty. Digestive enzymes activities were higher in T2 treatment. The peaks of trypsin and alkaline phosphatase enzymes activity were found 8 h after feeding in T1, while occurred 16 h after feeding in T2. The highest chymotrypsin and alpha-amylase enzymes activity were observed 5 and 8h after feeding in T1 and T2, respectively. These results confirmed remarkable effects of temperature on food transit time and digestive enzymes activity of Caspian kutum.

Effects of limited concentrate feeding on growth and blood and serum variables, and on nutrient digestibility and gene expression of hepatic gluconeogenic enzymes in dairy calves.

Science.gov (United States)

Lohakare, J D; van de Sand, H; Gerlach, K; Hosseini, A; Mielenz, M; Sauerwein, H; Pries, M; Südekum, K-H

2012-02-01

This study elucidated the effects of limited concentrate feeding on growth, nutrient digestibility, blood profile and gene expression of gluconeogenic enzymes in the liver of dairy calves. The study utilized 36 German Holstein dairy calves (5-7 days of age) divided into two groups of 18 calves each for 150 days. Control group calves received 2 kg/(calf × day) of concentrate, whereas calves in the restricted group received only 1 kg/(calf × day). Good quality forage (mixture of maize and grass silages) was available for ad libitum consumption to both groups. The intake of milk replacer before weaning, and of concentrate were recorded daily per calf; however, the consumption of forages was quantified as daily average of the group. Body weights (BW) were recorded at start and on days 35, 70, 112 and 150. Blood and serum samples and spot urinary and faecal samples were also collected at similar time points. On days 70 and 150, liver biopsies were collected from seven animals in each group. The BW was not different between the groups at all times. Total BW gain in the control group was 124 kg as opposed to 111 kg in restricted group that led to average BW gain of 827 g/day and 739 g/day in respective groups, and the differences were significant (p = 0.018). As planned, the control group had higher concentrate and lower forage intake than the restricted group. The blood haemoglobin, haematocrit and serum variables (glucose, total protein, albumin and urea) were within the normal range in both groups, but serum glucose was higher (p < 0.05) in control than in restricted group at 70 days. There was no difference between groups in organic matter (OM) digestibility which declined (p < 0.001) with increasing age in both groups. Microbial crude protein (MCP) synthesis estimated from urinary allantoin excretion increased (p < 0.001) in both groups with increasing age but was not different between groups. The mRNA expressions for the gluconeogenic enzymes, cytosolic and

[Restriction endonuclease digest - melting curve analysis: a new SNP genotyping and its application in traditional Chinese medicine authentication].

Science.gov (United States)

Jiang, Chao; Huang, Lu-Qi; Yuan, Yuan; Chen, Min; Hou, Jing-Yi; Wu, Zhi-Gang; Lin, Shu-Fang

2014-04-01

Single nucleotide polymorphisms (SNP) is an important molecular marker in traditional Chinese medicine research, and it is widely used in TCM authentication. The present study created a new genotyping method by combining restriction endonuclease digesting with melting curve analysis, which is a stable, rapid and easy doing SNP genotyping method. The new method analyzed SNP genotyping of two chloroplast SNP which was located in or out of the endonuclease recognition site, the results showed that when attaching a 14 bp GC-clamp (cggcgggagggcgg) to 5' end of the primer and selecting suited endonuclease to digest the amplification products, the melting curve of Lonicera japonica and Atractylodes macrocephala were all of double peaks and the adulterants Shan-yin-hua and A. lancea were of single peaks. The results indicated that the method had good stability and reproducibility for identifying authentic medicines from its adulterants. It is a potential SNP genotyping method and named restriction endonuclease digest - melting curve analysis.

Symbiotic Chlorella variabilis incubated under constant dark conditions for 24 hours loses the ability to avoid digestion by host lysosomal enzymes in digestive vacuoles of host ciliate Paramecium bursaria.

Science.gov (United States)

Kodama, Yuuki; Fujishima, Masahiro

2014-12-01

Endosymbiosis between symbiotic Chlorella and alga-free Paramecium bursaria cells can be induced by mixing them. To establish the endosymbiosis, algae must acquire temporary resistance to the host lysosomal enzymes in the digestive vacuoles (DVs). When symbiotic algae isolated from the alga-bearing paramecia are kept under a constant dark conditions for 24 h before mixing with the alga-free paramecia, almost all algae are digested in the host DVs. To examine the cause of algal acquisition to the host lysosomal enzymes, the isolated algae were kept under a constant light conditions with or without a photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea for 24 h, and were mixed with alga-free paramecia. Unexpectedly, most of the algae were not digested in the DVs irrespective of the presence of the inhibitor. Addition of 1 mM maltose, a main photosynthetic product of the symbiotic algae or of a supernatant of the isolated algae kept for 24 h under a constant light conditions, did not rescue the algal digestion in the DVs. These observations reveal that unknown factors induced by light are a prerequisite for algal resistance to the host lysosomal enzymes. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Digestibility of the cottonseed meal with or without addition of protease and phytase enzymes in swine diet - doi: 10.4025/actascianimsci.v34i3.12360

Directory of Open Access Journals (Sweden)

Maria do Carmo Mouhaupt Marques Ludke

2012-05-01

Full Text Available This study evaluated the digestibility of cottonseed meal with or without addition of enzymes (phytase and protease for growing pigs. It was used 18 barrows, housed in metabolism cages, distributed in a completely randomized design, standardizing body weight (bw with average of 25.8 ± 3.6 kg, with three treatments and six repetitions. The treatments consisted of a reference diet based on corn and soybean meal, the second treatment with replacement of 30% of the reference diet by cottonseed meal without enzymes, and the third with 30% of the reference diet replaced by cottonseed meal with added enzymes. Was determined the digestible protein, digestible energy, digestibility of dry matter, energy and protein. It was also registered the balance of nitrogen and phosphorus. The use of cottonseed meal with the addition of enzymes in diets for growing pigs has no effect on the digestibility of dry matter, gross energy and crude protein, but improved the absorption of phosphorus, consequently reducing its excretion in the feces. There was no improvement in nitrogen balance in the diets containing cottonseed meal with enzymes.

Effects of treating sorghum wet distillers grains with solubles with fibrolytic enzymes on nutrient digestibility and performance in finishing beef steers

Science.gov (United States)

Two experiments were conducted to determine the effects of treating sorghum WDG with solubles (SWDG) with an enzyme, or enzyme-buffer combination on diet digestibility and feedlot performance. Experimental treatments are; 1) untreated SWDG (control), 2) addition of an enzyme complex to SWDG (enzyme...

Two-stage in vitro digestibility assay, a tool for formulating non-starch polysaccharide degrading enzyme combinations for commonly used feed ingredients of poultry rations

Directory of Open Access Journals (Sweden)

Y. Ramana Reddy

2013-05-01

Full Text Available Aim: An attempt was made to assess the effect of pure enzyme combinations with the objective of formulating customized enzyme mixtures based on sugar release when subjected to two-stage in vitro digestion assay. Materials and Methods: A two-stage in vitro digestibility assay was carried out for commonly used feed ingredients for poultry viz., maize, soy bean meal, sunflower cake, and de-oiled rice bran supplemented with three concentrations of xylanase (5000; 7500 and 10000 IU/kg, cellulase (50; 100 and 400 IU/kg and â-D-glucanase (100; 200 and 400 IU/kg were used to formulate various NSP enzymes combinations. In total 27 NSP enzyme combinations (3x3x3 were formulated and the sugar released due to NSP digestion was quantified by phenol sulphuric acid method. Results: The total sugar release was significantly (P<0.05 higher with supplementation of various enzymes combinations for maize, sunflower cake and de-oiled rice bran where as no significant (P<0.05 interaction of various NSP enzymes combinations was observed for soy bean meal. The NSP digestibility was highest in combination (xylanase-5000, cellulase-50 and â-D-glucanase-400 IU/kg, (xylanase-10000, cellulase-50 and â-D-glucanase-200 IU/kg and (xylanase-7500, cellulase- 100 and â-D-glucanase-100 IU/kg for maize, sunflower cake and de-oiled rice bran respectively. In case of sunflower cake, significant (P<0.01 three way interaction was observed among the xylanase, cellulose, and â-D-glucanase enzymes and the two-way interactions between the enzymes were also significant (P<0.01. Conclusion: It is concluded that 'n' number of non-starch Polysaccharide enzymes combinations can be screened for their efficiency to digest non-starch Polysaccharides present in various feed ingredients commonly used in poultry rations by employing two-stage in vitro digestibility assay as a tool. [Vet World 2013; 6(8.000: 525-529

Expression of digestive enzymes and nutrient transporters in Eimeria acervulina-challenged layers and broilers

Science.gov (United States)

Avian coccidiosis is a disease caused by the intestinal protozoa Eimeria. Eimeria-infected chickens develop lesions in the intestinal mucosa, which result in reduced feed efficiency and body weight gain. This growth reduction may be due to changes in expression of digestive enzymes and nutrient tran...

In vitro digestion of purified β-casein variants A(1), A(2), B, and I: effects on antioxidant and angiotensin-converting enzyme inhibitory capacity.

Science.gov (United States)

Petrat-Melin, B; Andersen, P; Rasmussen, J T; Poulsen, N A; Larsen, L B; Young, J F

2015-01-01

Genetic polymorphisms of bovine milk proteins affect the protein profile of the milk and, hence, certain technological properties, such as casein (CN) number and cheese yield. However, reports show that such polymorphisms may also affect the health-related properties of milk. Therefore, to gain insight into their digestion pattern and bioactive potential, β-CN was purified from bovine milk originating from cows homozygous for the variants A(1), A(2), B, and I by a combination of cold storage, ultracentrifugation, and acid precipitation. The purity of the isolated β-CN was determined by HPLC, variants were verified by mass spectrometry, and molar extinction coefficients at λ=280nm were determined. β-Casein from each of the variants was subjected to in vitro digestion using pepsin and pancreatic enzymes. Antioxidant and angiotensin-converting enzyme (ACE) inhibitory capacities of the hydrolysates were assessed at 3 stages of digestion and related to that of the undigested samples. Neither molar extinction coefficients nor overall digestibility varied significantly between these 4 variants; however, clear differences in digestion pattern were indicated by gel electrophoresis. In particular, after 60min of pepsin followed by 5min of pancreatic enzyme digestion, one ≈4kDa peptide with the N-terminal sequence (106)H-K-E-M-P-F-P-K- was absent from β-CN variant B. This is likely a result of the (122)Ser to (122)Arg substitution in variant B introducing a novel trypsin cleavage site, leading to the changed digestion pattern. All investigated β-CN variants exhibited a significant increase in antioxidant capacity upon digestion, as measured by the Trolox-equivalent antioxidant capacity assay. After 60min of pepsin + 120min of pancreatic enzyme digestion, the accumulated increase in antioxidant capacity was ≈1.7-fold for the 4 β-CN variants. The ACE inhibitory capacity was also significantly increased by digestion, with the B variant reaching the highest inhibitory

The effect of 1-week feed restriction on performance, digestibility of nutrients and digestive system development in the growing rabbit.

Science.gov (United States)

Tůmová, E; Volek, Z; Chodová, D; Härtlová, H; Makovický, P; Svobodová, J; Ebeid, T A; Uhlířová, L

2016-01-01

A 3 to 4 week feed restriction of about 20% to 25% of the free intake is widely applied in rabbit breeding systems to reduce post-weaning digestive disorders. However, a short intensive feed restriction is described in few studies and can be beneficial for growing rabbits due to a longer re-alimentation period. The aim of this study was to evaluate the effect of ad libitum (AL) and two restriction levels of feeding (50 and 65 g/rabbit per day) applied for 1 week on performance, gastrointestinal morphology and physiological parameters during the restriction and during the re-alimentation period. Rabbits were divided into three experimental groups: AL rabbits were fed AL, R1 rabbits were restricted from 42 to 49 days of age and received 50 g daily (29% of AL) and R2 rabbits were restricted at the same age and were fed 65 g of feed daily (37% of AL). In the 1(st) week after weaning and in the weeks after restriction, all the groups were fed AL. During the restriction period, daily weight gain (DWG) in R1 significantly dropped to 11% (experiment 1) and 5% (experiment 2) compared with rabbits in the AL group, although they were fed 29% of AL, whereas in the R2 group it decreased to 20% (experiment 1) and 10% (experiment 2). In the week following feed restriction, DWG in the restricted groups increased (Pdepth of crypts, which might be involved in the compensatory growth and defence mechanism.

Characterization of Biomphalaria orbignyi, Biomphalaria peregrina and Biomphalaria oligoza by polymerase chain reaction and restriction enzyme digestion of the internal transcribed spacer region of the RNA ribosomal gene

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Spatz Linus

2000-01-01

Full Text Available The correct identification of Biomphalaria oligoza, B. orbignyi and B. peregrina species is difficult due to the morphological similarities among them. B. peregrina is widely distributed in South America and is considered a potential intermediate host of Schistosoma mansoni. We have reported the use of the polymerase chain reaction and restriction fragment length polymorphism analysis of the internal transcribed spacer region of the ribosomal DNA for the molecular identification of these snails. The snails were obtained from different localities of Argentina, Brazil and Uruguay. The restriction patterns obtained with MvaI enzyme presented the best profile to identify the three species. The profiles obtained with all enzymes were used to estimate genetic similarities among B. oligoza, B. peregrina and B. orbignyi. This is also the first report of B. orbignyi in Uruguay.

The Choice of Enzyme for Human Pancreas Digestion is a Critical Factor for Increasing the Success of Islet Isolation.

Science.gov (United States)

Qi, Meirigeng; Valiente, Luis; McFadden, Brian; Omori, Keiko; Bilbao, Shiela; Juan, Jemily; Rawson, Jeffrey; Scott, Stephen; Ferreri, Kevin; Mullen, Yoko; El-Shahawy, Mohamed; Dafoe, Donald; Kandeel, Fouad; Al-Abdullah, Ismail H

2015-05-01

We evaluated three commercially available enzymes for pancreatic digestion by comparing key parameters during the islet isolation process, as well as islet quality post-isolation. Retrospectively compared and analyzed islet isolations from pancreata using three different enzyme groups: Liberase HI (n=63), Collagenase NB1/Neutral Protease (NP) (n=43), and Liberase Mammalian Tissue Free Collagenase/Thermolysin (MTF C/T) (n=115). A standardized islet isolation and purification method was used. Islet quality assessment was carried out using islet count, viability, in vitro glucose-stimulated insulin secretion (GSIS), glucose-stimulated oxygen consumption rate (ΔOCR), and in vivo transplantation model in mice. Donor characteristics were not significantly different among the three enzyme groups used in terms of age, sex, hospital stay duration, cause of death, body mass index (BMI), hemoglobin A1c (HbA1c), cold ischemia time (CIT), and pancreas weight. Digestion efficacy (percentage of digested tissue by weight) was significantly higher in the Liberase MTF C/T group (73.5 ± 1.5 %) when compared to the Liberase HI group (63.6 ± 2.3 %) (psuccess rate of transplantation in diabetic NOD Scid mice (65%), which was significantly higher than the Liberase HI (42%, p=0.001) and the Collagenase NB1/NP enzymes (41%, psuccess rate of transplantation in diabetic NOD Scid mice compared to Liberase HI and Collagenase NB1/NP enzymes.

Antioxidant properties of digestive enzyme-treated fibre-rich fractions from wheat, finger millet, pearl millet and sorghum: A comparative evaluation

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Aisha Siddiq A.

2015-12-01

Full Text Available Whole grains are rich in antioxidant components (AC, most of which are bound to fibre fraction and released during digestion. The study investigated the effect of digestive enzymes on the antioxidant properties of fibre-rich fractions from wheat (Triticum aestivum, finger millet (Eleusine coracana, pearl millet (Pennisetum typhoides and sorghum (Sorghum bicolor. Coarse (CF and fine fractions (FF of milled flour were separated using a standard sieve and analysed for nutritional composition, AC extractable in different solvents and antioxidant activity (AA in untreated and enzyme-treated fractions. The CF had a higher range of insoluble dietary fibre (17.26–20.93% than FF (10.65–17.29%. The highest amount of polyphenols and flavonoids was extractable in different solvents from finger millet and pearl millet, respectively. FF of pearl millet showed higher total AA in all solvents. Enzyme-treated samples had a much higher content of AC as well as higher total AA. Free radical scavenging assay revealed that enzyme-treated millet flours had higher activity in comparison to wheat. Between fractions, wheat exhibited variable results. Among millets, CF of finger millet and FF of pearl millet and sorghum had higher AA. In conclusion, digestive enzyme treatment released more AC from grains, and exhibited a higher AA.

Examination of digestive enzyme distribution in gut tract and functions of intestinal caecum, in megascolecid earthworms (Oligochaeta: Megascolecidae) in Japan.

Science.gov (United States)

Nozaki, Mana; Ito, Katsutoshi; Miura, Chiemi; Miura, Takeshi

2013-09-01

Earthworms ingest various materials in addition to food items, such as soil particles. Most earthworms of the family Megascolecidae, a dominant family in Japan, have intestinal caeca connected directly to the intestinal tract. The function of the caeca has not been demonstrated, although it is thought to be associated with digestion. We investigated the activity of the digestive enzymes amylase, phosphatase, cellulase, and protease in different regions of the gut, including the intestinal caeca, in three species of megascolecid earthworms, Pheretima heteropoda, Pheretima hilgendorfi, and Pheretima sieboldi. Activities of several enzymes were high in the intestinal caeca; in particular, protease activity was higher in the caeca than that in the anterior gut, foregut, midgut, and hindgut in all three species. Moreover, the ratio of enzyme activities in the intestinal caeca to whole-gut tended to be higher in manicate intestinal caeca than in simple intestinal caeca. These results suggest that the digestive system of earthworms relies on the intestinal caeca.

Consequences of lower food intake on the digestive enzymes activities, the energy reserves and the reproductive outcome in Gammarus fossarum.

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Laetitia Charron

Full Text Available Digestive enzyme activity is often used as a sensitive response to environmental pollution. However, only little is known about the negative effects of stress on digestive capacities and their consequences on energy reserves and reproduction, although these parameters are important for the maintenance of populations. To highlight if changes in biochemical responses (digestive enzymes and reserves led to impairments at an individual level (fertility, Gammarus fossarum were submitted to a lower food intake throughout a complete female reproductive cycle (i.e. from ovogenesis to offspring production. For both males and females, amylase activity was inhibited by the diet stress, whereas trypsin activity was not influenced. These results underline similar sensitivity of males and females concerning their digestive capacity. Energy reserves decreased with food starvation in females, and remained stable in males. The number of embryos per female decreased with food starvation. Lower digestive activity in males and females therefore appears as an early response. These results underline the ecological relevance of digestive markers, as they make it possible to anticipate upcoming consequences on reproduction in females, a key biological variable for population dynamics.

Andrographolide powder treatment as antifeedant decreased digestive enzyme activity from Plutella xylostella (L.) larvae midgut

Science.gov (United States)

Madihah, Malini, Desak Made; Roviani, Hana; Rani, Nessa Vidya; Hermawan, Wawan

2018-02-01

Andrographolide, an active compound of Andrographis paniculata, has shown antifeedant activity against Plutella xylostella larvae by disrupting the midgut histological structures. This study aims to determine the activity of andrographolide in crystallized powder form against several digestive enzymes from the midgut of 4th instar P. xylostella larvae. The concentrations used were 0 (control), 1000, 1600, 2500, 4000 and 6500 ppm with four replications each. No-choice antifeedant test with leaf disc method is used in a bioassay for 24 hours. The midgut was dissected from 2nd until 6th segment of 4th instar larvae and was homogenized in iced-buffer solution. Furthermore, larvae's midgut samples were centrifuged at 10,000 rpm, 4°C for 20 min and the supernatant is used as enzyme source. The results showed that andrographolide significantly reduces the amylase, invertase, protease and trypsin activity, as well as total protein concentration compared with control (p<0.05) in a dose-dependent manner. This study provides information about the mode of action of andrographolide in inhibiting feed activity by the reduced digestive enzyme activity of 4th instar P. xylostella larvae.

Sequence dependent DNA conformations: Raman spectroscopic studies and a model of action of restriction enzymes

International Nuclear Information System (INIS)

Nishimura, Y.

1985-01-01

Raman spectra have been examined to clarify the polymorphic forms of DNA, A, B, and Z forms. From an analysis the authors found that the guanine ring breathing vibration is sensitive to its local conformation. Examination of nine crystals of guanosine residues in which the local conformations are well established revealed that a guanosine residue with a C3'endo-anti gives a strong line at 666+-2 cm/sup -1/, O4'endo-anti at 682 cm/sup -1/, C1'exo-anti at 673 cm/sup -1/, C2'endo-anti at 677 cm/sup -1/ and syn-forms around 625 cm/sup -1/. Using this characteristic line, they were able to obtain the local conformations of guanosine moieties in poly(dG-dC). Such a sequence derived variation is suggested to be recognized by sequence specific proteins such as restriction enzymes. The authors found a correlation between sequence dependent DNA conformation and a mode of action of restriction enzymes. The cutting mode of restriction enzymes is classified into three groups. The classification of whether the products have blunt ends, two-base-long cohesive ends, or four-base-long cohesive ends depends primarily on the substrate, not on the enzyme. It is suggested that sequence dependent DNA conformation causes such a classification by the use of the Calladine-Dickerson analysis. In the recognition of restriction enzymes, the methyl group in a certain sequence is considered to play an important role by changing the local conformation of DNA

Soybean hull and enzyme inclusion effects on diet digestibility and growth performance in beef steers consuming corn-based diets.

Science.gov (United States)

Russell, J R; Sexten, W J; Kerley, M S

2016-06-01

A beef feedlot study was conducted to determine the effects of increasing soybean hull (SH) inclusion and enzyme addition on diet digestibility and animal performance. The hypothesis was SH inclusion and enzyme addition would increase fiber digestibility with no negative effect on animal performance. Eight treatments (TRT) were arranged in a 4 × 2 factorial using four diets and two enzyme (ENZ) inclusion rates. The diets were composed primarily of whole shell corn (WSC) with 0%, 7%, 14%, or 28% SH replacing corn. The ENZ was a commercial proprietary mix of , and (Cattlemace, R&D Life Sciences, Menomonie, WI) included in the diets at 0% (S0, S7, S14, S28) or 0.045% DM basis (S0e, S7e, S14e, S28e). Eighty steers (287 ± 31 kg, SD) were stratified by weight and blocked into pens with 1 heavy and 1 light pen per TRT (2 pen/TRT, 5 steers/pen). Steers were fed for 70 d with titanium dioxide included in the diets for the final 15 d. Fecal samples were collected on d 70 to determine diet digestibility. Diets were balanced for AA and RDP requirement based on available ME. Individual DMI was measured using a GrowSafe system. Diet, ENZ, and diet × ENZ effects were analyzed using the MIXED procedure of SAS. Initial BW was applied as a covariate for final BW (FBW), and DMI was included as a covariate for all digestibility measures. The diet × ENZ interaction had no effect on FBW, ADG, DMI, or any digestibility measure ( ≥ 0.11). Steers fed ENZ tended to have greater FBW ( = 0.09) and had numerically greater ADG than steers not fed ENZ. Diet influenced DMI ( digestibility ( ≥ 0.2). Diet had an effect on NDF and ADF digestibility ( ≤ 0.04) which decreased as SH inclusion increased. The addition of ENZ tended to decrease NDF digestibility ( = 0.08) but had no effect on ADF digestibility ( = 0.8). Fiber digestibility in WSC diets did not improve with SH inclusion or ENZ addition but steers fed diets with 14% to 28% of WSC replaced by SH and the addition of 0.045% ENZ

Enhanced resolution of DNA restriction fragments: A procedure by two-dimensional electrophoresis and double-labeling

International Nuclear Information System (INIS)

Yi, M.; Au, L.C.; Ichikawa, N.; Ts'o, P.O.

1990-01-01

A probe-free method was developed to detect DNA rearrangement in bacteria based on the electrophoretic separation of twice-digested restriction fragments of genomic DNA into a two-dimensional (2-D) pattern. The first restriction enzyme digestion was done in solution, followed by electrophoresis of the restriction fragments in one dimension. A second restriction enzyme digestion was carried out in situ in the gel, followed by electrophoresis in a second dimension perpendicular to the first electrophoresis. The 2-D pattern provides for the resolution of 300-400 spots, which are defined and indexed by an x,y coordinate system with size markers. This approach has greatly increased the resolution power over conventional one-dimensional (1-D) electrophoresis. To study DNA rearrangement, a 2-D pattern from a test strain was compared with the 2-D pattern from a reference strain. After the first digestion, genomic DNA fragments from the test strain were labeled with 35S, while those from the reference strain were labeled with 32P. This was done to utilize the difference in the energy emission of 35S and 32P isotopes for autoradiography when two x-ray films were exposed simultaneously on top of the gel after the 2-D electrophoresis. The irradiation from the decay of 35S exposed only the lower film, whereas the irradiation from the decay of 32P exposed both the lower and upper films. Different DNA fragments existed in the test DNA compared with the reference DNA can be identified unambiguously by the differential two 2-D patterns produced on two films upon exposure to the 35S and 32P fragments in the same gel. An appropriate photographic procedure further simplified the process, allowing only the difference in DNA fragments between these two patterns to be shown in the map

Assessment of digestive enzymes activity during the fry development of the endangered Caspian brown trout Salmo caspius.

Science.gov (United States)

Zamani, A; Hajimoradloo, A; Madani, R; Farhangi, M

2009-09-01

The study of digestive enzymes activity at Salmo caspius fry showed that enzymes were available at the moment of mouth opening on the first day post hatching (dph) and the activity of enzymes showed no significant difference from the hatching day 28 dph. An increased activity was seen between 32 and 43 dph and this activity was significantly higher than the activity during the first 28 days. In the primary stages after yolk sac resorption (43-58 dph), enzymes activity showed an increased profile, however none of them showed a significant difference between 43 and 58 dph.

Extraction and identification of α-amylase inhibitor peptides from Nephelium lappacheum and Nephelium mutabile seed protein using gastro-digestive enzymes.

Science.gov (United States)

Evaristus, Natashya Anak; Wan Abdullah, Wan Nadiah; Gan, Chee-Yuen

2018-04-01

The potential of N. lappacheum and N. mutabile seed as a source of α-amylase inhibitor peptides was explored based on the local traditional practice of using the seed. Different gastro-digestive enzymes (i.e. pepsin or chymotrypsin) or a sequential digestion were used to extract the peptides. The effects of digestion time and enzyme to substrate (E:S) ratio on the α-amylase inhibitory activity were investigated. Results showed that chymotrypsin was effective in producing the inhibitor peptides from rambutan seed protein at E:S ratio 1:20 for 1 h, whereas pepsin was more effective for pulasan seed protein under the same condition. A total of 20 and 31 novel inhibitor peptides were identified, respectively. These peptides could bind with the subsites of α-amylase (i.e. Trp58, Trp59, Tyr62, Asp96, Arg195, Asp197, Glu233, His299, Asp300, and His305) and formed a sliding barrier that preventing the formation of enzyme/substrate intermediate leading to lower α-amylase activity. Copyright © 2018 Elsevier Inc. All rights reserved.

Relationship between physiological measurements (SFG -scope for growth-) and the functionality of the digestive gland in Mytilus galloprovincialis.

Science.gov (United States)

Albentosa, Marina; Sánchez-Hernández, Miriam; Campillo, Juan Antonio; Moyano, Francisco Javier

2012-11-01

The present study was aimed to establish the relationship between the functionality of the digestive gland and physiological rates including SFG (scope for growth) in wild mussels, Mytilus galloprovincilis. The experimental set-up consisted in the evaluation of changes in the morphology of the gland, as well as in the activity of some key digestive enzymes (amylase, laminarinase, cellulase and protease) within a broad range of SFG obtained through manipulation of food ration. The higher SFG values were correlated to an increase in both the size of the digestive gland and the activities of enzymes when expressed in relation to individual. In contrast, no clear relations were observed when the activity of enzymes was expressed in relation to soluble protein, with the exception to amylase. The higher protease activities measured in mussels showing lower SFG may reflect an initial stage of catabolic processes intended to compensate the energy deficit produced by food restriction. The potential use of parameters measured in digestive glands in studies of marine pollution was discussed. Copyright © 2012 Elsevier Inc. All rights reserved.

In vitro digestibility of individual amino acids in rumen-undegraded protein: the modified three-step procedure and the immobilized digestive enzyme assay.

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Boucher, S E; Calsamiglia, S; Parsons, C M; Stern, M D; Moreno, M Ruiz; Vázquez-Añón, M; Schwab, C G

2009-08-01

Three soybean meal, 3 SoyPlus (West Central Cooperative, Ralston, IA), 5 distillers dried grains with solubles, and 5 fish meal samples were used to evaluate the modified 3-step in vitro procedure (TSP) and the in vitro immobilized digestive enzyme assay (IDEA; Novus International Inc., St. Louis, MO) for estimating digestibility of AA in rumen-undegraded protein (RUP-AA). In a previous experiment, each sample was ruminally incubated in situ for 16 h, and in vivo digestibility of AA in the intact samples and in the rumen-undegraded residues (RUR) was obtained for all samples using the precision-fed cecectomized rooster assay. For the modified TSP, 5 g of RUR was weighed into polyester bags, which were then heat-sealed and placed into Daisy(II) incubator bottles. Samples were incubated in a pepsin/HCl solution followed by incubation in a pancreatin solution. After this incubation, residues remaining in the bags were analyzed for AA, and digestibility of RUP-AA was calculated based on disappearance from the bags. In vitro RUP-AA digestibility estimates obtained with this procedure were highly correlated to in vivo estimates. Corresponding intact feeds were also analyzed via the pepsin/pancreatin steps of the modified TSP. In vitro estimates of AA digestibility of the feeds were highly correlated to in vivo RUP-AA digestibility, which suggests that the feeds may not need to be ruminally incubated before determining RUP-AA digestibility in vitro. The RUR were also analyzed via the IDEA kits. The IDEA values of the RUR were good predictors of RUP-AA digestibility in soybean meal, SoyPlus, and distillers dried grains with solubles, but the IDEA values were not as good predictors of RUP-AA digestibility in fish meal. However, the IDEA values of intact feed samples were also determined and were highly correlated to in vivo RUP-AA digestibility for all feed types, suggesting that the IDEA value of intact feeds may be a better predictor of RUP-AA digestibility than the IDEA

Effects of water restriction following feeding on nutrient digestibilities, milk yield and composition and blood hormones in lactating Holstein cows under heat stress conditions

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Jalil Ghassemi Nejad

2015-08-01

Full Text Available The effects of water restriction following feeding under heat stress conditions on nutrient digestibilities, milk yield and composition and some blood hormones in lactating Holstein cows were evaluated. The design was completely randomized with 30 high producing lactating Holstein cows (80.8±40.5 DIM which were assigned to two treatment groups (15 cows per treatment. Treatments were free access to water (FAW and 2 h water restriction (2hWR following feeding. Average temperature-humidity index (THI in the farm was over 80 throughout the experiment which defines heat stress conditions. Neutral detergent fibre, organic matter and ether extract digestibilities increased by water restriction (P0.05. Water intake was recorded daily during the digestibility period and was not different between FAW and 2hWR group (P>0.05. Fat corrected milk was higher in 2hWR group than FAW group (P0.05. Somatic cell counts were greater in 2hWR than FAW group (P0.05. Blood prolactin and growth hormone were higher in 2hWR group than the FAW group (P<0.05. It is concluded that water restriction for 2 hours following feeding improved nutrient digestibility of some dietary components and increased milk fat percentage in lactating Holstein cows under heat stress conditions.

Effects of Enzyme Complex Supplementation to a Paddy-based Diet on Performance and Nutrient Digestibility of Meat-type Ducks

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P. Kang

2013-02-01

Full Text Available Paddy rice is rarely used as a feed because of its high fiber content. In this study, two experiments were conducted to study the effects of supplementing an enzyme complex consisting of xylanase, beta-glucanase and cellulase, to paddy-based diets on the performance and nutrient digestibility in meat-type ducks. In the both experiments, meat-type ducks (Cherry Valley were randomly assigned to four treatments. Treatment 1 was a basal diet of corn-soybean; treatment 2 was a basal diet of corn-paddy-soybean; treatment 3, had enzyme complex added to the corn-paddy-soybean basal diet at levels of 0.5 g/kg diet; and treatment 4, had enzyme complex added to the corn-paddy-soybean diet at levels of 1.0 g/kg diet. The results showed that the enzyme complex increased the ADG, and decreased the ADFI and F/G significantly (p0.05. The outcome of this research indicates that the application of enzyme complex made up of xylanase, beta-glucanase, and cellulase, in the corn-paddy-soybean diet, can improve performance and nutrition digestibility in meat-type ducks.

Characterizations of substrate and enzyme specificity of glucoamylase assays of mucosal starch digestion with determinations of group and single biopsy reference values

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Carbohydrate digesting enzyme activities are measured in duodenal biopsies to detect deficiencies of lactase and sucrase activities, however glucoamylase (GA) assays for starch digestion are not included. Because food starch represents half of energy intake in the human diet, assays for starch diges...

Effects of supplemental enzymes on apparent nutrient digestibility in rainbow trout (Oncorhynchus mykiss) fed plant-based diets

DEFF Research Database (Denmark)

Dalsgaard, Anne Johanne Tang; Hjermitslev, Niels Harthøj; Ekmann, Kim Schøn

2010-01-01

in fish feed due to growing demands for and high price variations in fish meal, but high inclusion levels in diets for carnivorous fish are hampered by a great variety of anti-nutritional factors (ANFs), which reduce nutrient utilisation. Exogenous dietary enzymes may potentially help to alleviate...... on the effects of enzymes in fish feed apart from phytase. Phytase works by hydrolyzing phytic acid, and numerous studies have documented that phytase supplementation increases phosphorus availability in fish fed diets with high inclusion levels of plant proteins. Plant derived proteins are increasingly used...... these effects, and the objective of the present study was to evaluate the effects of supplementing protease and pectinase to a diet containing approximately 30% soybean meal, rapeseed meal or sunflower meal on nutrient digestibility in juvenile rainbow trout (Oncorhynchus mykiss). Digestibility trials were...

Restriction fragment length polymorphism of the major histocompatibility complex of the dog.

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Sarmiento, U M; Storb, R F

1988-01-01

Human major histocompatibility complex (HLA) cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the DLA-D region in dogs. Genomic DNA from peripheral blood leucocytes of 23 unrelated DLA-D-homozygous dogs representing nine DLA-D types (defined by mixed leucocyte reaction) was digested with restriction enzymes (Bam HI, Eco RI, Hind III, Pvu II, Taq I, Rsa I, Msp I, Pst I, and Bgl II), separated by agarose gel electrophoresis, and transferred onto Biotrace membrane. The Southern blots were successively hybridized with radiolabeled HLA cDNA probes corresponding to DR, DQ, DP, and DO beta genes. The autoradiograms for all nine enzyme digests displayed multiple bands with the DRb, DQb, and DPb probes while the DOb probe hybridized with one to two bands. The RFLP patterns were highly polymorphic but consistent within each DLA-D type. Standard RFLP patterns were established for nine DLA-D types which could be discriminated from each other by using two enzymes (Rsa I and Pst I) and the HLA-DPb probe. Cluster analysis of the polymorphic restriction fragments detected by the DRb probe revealed four closely related supertypic groups or DLA-DR families: Dw3 + Dw4 + D1, Dw8 + D10, D7 + D16 + D9, and Dw1. This study provides the basis for DLA-D genotyping at a population level by RFLP analysis. These results also suggest that the genetic organization of the DLA-D region may closely resemble that of the HLA complex.

Restriction fragment length polymorphism (RFLP) analysis of PCR products amplified from 18S ribosomal RNA gene of Trypanosoma congolense

International Nuclear Information System (INIS)

Osanyo, A.; Majiwa, P.W.

2006-01-01

Oligonucleotide primers were designed from the conserved nucleotide sequences of 18S ribosomal RNA (18S rRNA) gene of protozoans: Trypanosoma brucei, Leishmania donovani, Triponema aequale and Lagenidium gigantum. The primers were used in polymerace chain reaction (PCR) to generate PCR products of approximately 1 Kb using genomic DNA from T. brucei and the four genotypic groups of T. congolense as template. The five PCR products so produced were digested with several restriction enzymes and hybridized to a DNA probe made from T. brucei PCR product of the same 18S rRNA gene region. Most restriction enzyme digests revealed polymorphism with respect to the location of their recognition sites on the five PCR products. The restriction fragment length polymorphism (RFLP) pattern observed indicate that the 18S rRNA gene sequences of trypanosomes: T. brucei and the four genotypes of T.congolence group are heterogeneous. The results further demonstrate that the region that was amplified can be used in specific identification of trypanosomes species and subspecies.(author)

Digestive peptidase evolution in holometabolous insects led to a divergent group of enzymes in Lepidoptera

KAUST Repository

Dias, Renata O.; Via, Allegra; Brandã o, Marcelo M.; Tramontano, Anna; Silva-Filho, Marcio C.

2015-01-01

© 2015 Elsevier Ltd. Trypsins and chymotrypsins are well-studied serine peptidases that cleave peptide bonds at the carboxyl side of basic and hydrophobic l-amino acids, respectively. These enzymes are largely responsible for the digestion of proteins. Three primary processes regulate the activity of these peptidases: secretion, precursor (zymogen) activation and substrate-binding site recognition. Here, we present a detailed phylogenetic analysis of trypsins and chymotrypsins in three orders of holometabolous insects and reveal divergent characteristics of Lepidoptera enzymes in comparison with those of Coleoptera and Diptera. In particular, trypsin subsite S1 was more hydrophilic in Lepidoptera than in Coleoptera and Diptera, whereas subsites S2-S4 were more hydrophobic, suggesting different substrate preferences. Furthermore, Lepidoptera displayed a lineage-specific trypsin group belonging only to the Noctuidae family. Evidence for facilitated trypsin auto-activation events were also observed in all the insect orders studied, with the characteristic zymogen activation motif complementary to the trypsin active site. In contrast, insect chymotrypsins did not seem to have a peculiar evolutionary history with respect to their mammal counterparts. Overall, our findings suggest that the need for fast digestion allowed holometabolous insects to evolve divergent groups of peptidases with high auto-activation rates, and highlight that the evolution of trypsins led to a most diverse group of enzymes in Lepidoptera.

Digestive peptidase evolution in holometabolous insects led to a divergent group of enzymes in Lepidoptera

KAUST Repository

Dias, Renata O.

2015-03-01

© 2015 Elsevier Ltd. Trypsins and chymotrypsins are well-studied serine peptidases that cleave peptide bonds at the carboxyl side of basic and hydrophobic l-amino acids, respectively. These enzymes are largely responsible for the digestion of proteins. Three primary processes regulate the activity of these peptidases: secretion, precursor (zymogen) activation and substrate-binding site recognition. Here, we present a detailed phylogenetic analysis of trypsins and chymotrypsins in three orders of holometabolous insects and reveal divergent characteristics of Lepidoptera enzymes in comparison with those of Coleoptera and Diptera. In particular, trypsin subsite S1 was more hydrophilic in Lepidoptera than in Coleoptera and Diptera, whereas subsites S2-S4 were more hydrophobic, suggesting different substrate preferences. Furthermore, Lepidoptera displayed a lineage-specific trypsin group belonging only to the Noctuidae family. Evidence for facilitated trypsin auto-activation events were also observed in all the insect orders studied, with the characteristic zymogen activation motif complementary to the trypsin active site. In contrast, insect chymotrypsins did not seem to have a peculiar evolutionary history with respect to their mammal counterparts. Overall, our findings suggest that the need for fast digestion allowed holometabolous insects to evolve divergent groups of peptidases with high auto-activation rates, and highlight that the evolution of trypsins led to a most diverse group of enzymes in Lepidoptera.

Digestive enzyme activity and trophic behavior in two predator aquatic insects (Plecoptera, Perlidae): a comparative study.

Science.gov (United States)

López-Rodríguez, M J; Trenzado, C E; Tierno de Figueroa, J M; Sanz, A

2012-05-01

Plecoptera (Perlidae) are among the major macroinvertebrate predators in stream ecosystems and one of the insect families with lower tolerance to environmental alterations, being usually employed as bioindicators of high water ecological quality. The differences in the trophic roles of the coexisting species have been exclusively studied from their gut contents, while no data are available on the comparative digestive capacity. In the present paper, we make a comparative study of the activity of several digestive enzymes, namely proteases (at different pH), amylase, lipase, trypsin and chymotrypsin, in two species of stoneflies, Perla bipunctata and Dinocras cephalotes, which cohabit in the same stream. The study of digestive enzyme activity together with the analysis of gut contents can contribute to a better understanding of the ecology of these aquatic insects and their role in freshwater food webs. Thus, our results show that the two studied predator species inhabiting in the same stream present specializations on their feeding behaviors, facilitating their coexistence, and also differences in their capacity of use the resources. One of the main findings of this study is that D. cephalotes is able to assimilate a wider trophic resource spectrum and this could be one of the reasons why this species has a wider global distribution in all its geographical range. Copyright © 2012 Elsevier Inc. All rights reserved.

Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis

International Nuclear Information System (INIS)

Watanabe, Kousuke; Emoto, Noriko; Sunohara, Mitsuhiro; Kawakami, Masanori; Kage, Hidenori; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

2010-01-01

Research highlights: → Incubating PCR products at a high temperature causes smears in gel electrophoresis. → Smears interfere with the interpretation of methylation analysis using COBRA. → Treatment with exonuclease I and heat-labile alkaline phosphatase eliminates smears. → The elimination of smears improves the visibility of COBRA. -- Abstract: DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonuclease I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 o C or 65 o C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.

Bioaccessibility and inhibitory effects on digestive enzymes of carnosic acid in sage and rosemary.

Science.gov (United States)

Ercan, Pınar; El, Sedef Nehir

2018-04-28

In this study, the aim was to determine the bioaccessibilities of carnosic acid in sage and rosemary and in vitro inhibitory effects of these samples on lipid and starch digestive enzymes by evaluating the lipase, α-amylase and α-glucosidase enzyme inhibition activities. The content of carnosic acid in rosemary (18.72 ± 0.33 mg/g) was found to be higher than that content of that in sage (3.76 ± 0.13 mg/g) (p sage and rosemary, respectively. The tested sage and rosemary showed inhibitory activity against α-glucosidase (Concentration of inhibitor required to produce a 50% inhibition of the initial rate of reaction - IC 50 88.49 ± 2.35, 76.80 ± 1.68 μg/mL, respectively), α-amylase (IC 50 107.65 ± 12.64, 95.65 ± 2.73 μg/mL, respectively) and lipase (IC 50 6.20 ± 0.63, 4.31 ± 0.62 μg/mL, respectively). Furthermore, to the best of our knowledge, this is the first work that carnosic acid standard equivalent inhibition capacities (CAEIC 50 ) for these food samples were determined and these values were in agreement with the IC 50 values. These results show that sage and rosemary are potent inhibitors of lipase, α-amylase and α-glucosidase digestive enzymes. Copyright © 2018 Elsevier B.V. All rights reserved.

Stock discrimination in Great Lakes Walleye using mitochondrial DNA restriction analysis

International Nuclear Information System (INIS)

Billington, N.; Hebert, P.D.N.

1986-01-01

Over the past two years it has become evident that because of its strict maternal inheritance and rapid rate of evolutionary differentiation, mitochondrial (mt) DNA diversity offers exceptional promise in the discrimination of fish stocks. The current project aims to determine the extent of mt DNA variation among stocks of walleye (Stizostedion vitreum) from the Great Lakes. At this point, mt DNA has been isolated from 68 walleye representing the Thames River stock and a reef breeding stock from western Lake Erie, as well as from individuals of S. canadense, a species which hybridizes with S. vitreum. Mitochondrial DNA was extracted from livers of these fish, purified by CsCl density gradient centrifugation and digested using 20 endonucleases. Polymorphisms were detected with 8 of the enzymes. There was a great deal of variation among fish from both spawning populations, so much so that individual fish could be identified by this technique. No single enzyme allowed discrimination of the two stocks, but restriction pattern variation following Dde I digestion permitted separation of 50% of Lake Erie fish from Thames River stock. Comparison of mt DNA restriction patterns of walleye and sauger showed that two species are easily separable, setting the stage for a more detailed study of hybridization between the taxa

Effect of energy restriction and re-alimentation in Belgian Blue double-muscled beef cows on digestibility and metabolites.

Science.gov (United States)

Fiems, L O; Vanacker, J M; De Boever, J L; van Caelenbergh, W; Aerts, J M; De Brabander, D L

2007-02-01

Four groups of five non-lactating and non-pregnant Belgian Blue double-muscled (BBDM) cows were used to investigate the effect of energy level (E) on digestion, and blood and urine metabolites. The energy levels of the groups, applied indoors during a 140-day restriction period, were 100%, 90%, 80% or 70% of their energy requirements (E100, E90, E80, E70) respectively. Afterwards, animals grazed on the same swards for 203 days (re-alimentation period). Balance trials were conducted at the end of the restriction period (BT1) and at the end of the re-alimentation period (BT2). Blood was sampled at the end of these trials. Diets consisted of maize silage and straw (80/20 on a dry matter basis) and a mineral-vitamin premix, fed at the appropriate E during BT1, or maize silage and a mineral-vitamin premix, fed at 125% of the maintenance requirements, during BT2. Significant increases of the digestibility coefficients were found during BT1 when E decreased, resulting in a better net energy capture of 7% for E70 compared with E100 (p < 0.05). Slightly, but non-significantly higher digestibility coefficients were observed for decreasing E during BT2. Plasma concentrations of glucose and creatinine did not differ between treatments during BT1, while differences were found for triacylglycerols and alpha-amino nitrogen. A tendency for a linear increase was observed for non-esterified fatty acids with decreasing E. Differences in blood metabolite concentrations disappeared in BT2. Urinary creatinine excretion was not affected by E, while body nitrogen loss increased linearly with energy restriction in BT1. No differences were found during BT2, suggesting that non-lactating and non-pregnant BBDM cows are able to adapt to a cyclic change of body weight and body reserves. These data show that restricted cows mobilized body fat as well as body protein. It is concluded that the qualitative aspects of metabolism during energy restriction are comparable in double-muscled cows with

Dietary Immunogen® modulated digestive enzyme activity and immune gene expression in Litopenaeus vannamei post larvae.

Science.gov (United States)

Miandare, Hamed Kolangi; Mirghaed, Ali Taheri; Hosseini, Marjan; Mazloumi, Nastaran; Zargar, Ashkan; Nazari, Sajad

2017-11-01

Pacific white shrimp Litopenaeus vannamei (Boone, 1931) is an important economical shrimp species worldwide, especially in the Middle East region, and farming activities of this species have been largely affected by diseases, mostly viral and bacterial diseases. Scientists have started to use prebiotics for bolstering the immune status of the animal. This study aimed to investigate the influence of Immunogen ® on growth, digestive enzyme activity and immune related gene expression of Litopenaeus vannamei post-larvae. All post-larvae were acclimated to the laboratory condition for 14 days. Upon acclimation, shrimps were fed on different levels of Immunogen ® (0, 0.5, 1 and 1.5 g kg -1 ) for 60 days. No significant differences were detected in weight gain, specific growth rate (SGR) and food conversion ratio (FCR) in shrimp post-larvae in which fed with different levels of Immunogen ® and control diet. The results showed that digestive enzymes activity including protease and lipase increased with different amounts of Immunogen ® in the shrimp diet. Protease activity increased with 1.5 g kg -1 Immunogen ® after 60 days and lipase activity increased with 1 and 1.5 g kg -1 Immunogen ® after 30 and 60 days of the trial respectively (P  0.05). The expression of immune related genes including, prophenoloxidase, crustin and g-type lysozyme increased with diet 1.5 g kg -1 Immunogen ® (P < 0.05) while expression of penaeidin gene increased only with experimental diet 1 g kg -1 of Immunogen ® . These results indicated that increase in digestive enzymes activity and expression of immune related genes could modulate the Immunogen ® in the innate immune system in L. vannamei in this study. Copyright © 2017. Published by Elsevier Ltd.

Simple and sensitive fluorescence assay of restriction endonuclease on graphene oxide

International Nuclear Information System (INIS)

Gang, Jong Back

2015-01-01

Restriction endonucleases hydrolyze internal phosphodiester bonds at specific sites in a DNA sequence. These enzymes are essential in a variety of fields, such as biotechnology and clinical diagnostics. It is of great importance and necessity for the scientific and biomedical use of enzymes to measure endonuclease activity. In this study, graphene oxide (GO) has been used as a platform to measure enzyme activity with high sensitivity. To increase the detection sensitivity of Hinf I, the endonuclease-digested reaction was treated with exonuclease III (Exo III) and a fluorescence assay was conducted to measure the emission. Results showed that Exo III treatment enhanced 2.7-fold signal-to-background ratio for the detection of Hinf I compared with that done without Exo III in the presence of GO

The effect of aerobic exercise training on growth performance, digestive enzyme activities and postprandial metabolic response in juvenile qingbo (Spinibarbus sinensis).

Science.gov (United States)

Li, Xiu-Ming; Yu, Li-Juan; Wang, Chuan; Zeng, Ling-Qing; Cao, Zhen-Dong; Fu, Shi-Jian; Zhang, Yao-Guang

2013-09-01

Continual swimming exercise usually promotes growth in fish at a moderate water velocity. We hypothesized that the improvement in growth in exercise-trained fish may be accompanied by increases in digestive enzyme activity, respiratory capacity and, hence, postprandial metabolism. Juvenile qingbo fish (Spinibarbus sinensis) were subjected to aerobic training for 8weeks at a water velocity of control (3cms(-1)), 1, 2 and 4 body length (bl)s(-1) at a constant temperature of 25°C. The feed intake (FI), food conversion rate (FCR), specific growth rate (SGR), whole-body composition, trypsin and lipase activities, maximal oxygen consumption (M˙O2max) and postprandial M˙O2 response were measured at the end of the training period. Aerobic exercise training induced a significant increase in FI compared with the control group, while the FCR of the 4bls(-1) group was significantly lower than for the other three groups (PFI after long-term training; (3) and aerobic exercise training boosted the activity of digestive enzymes and maximum digestive metabolism, which could favor fast digestion and growth in juvenile S. sinensis. Copyright © 2013 Elsevier Inc. All rights reserved.

A simple enzyme assay for dry matter digestibility and its value in studying food selection by generalist herbivores.

Science.gov (United States)

Choo, Gillian M; Waterman, Peter G; McKey, Doyle B; Gartlan, J Stephen

1981-05-01

The dry matter digestibility of 94 species of leaf was assayed by a simple method involving sequential treatment with pepsin and fungal cellulase enzymes. It was demonstrated that for foliage from rainforest trees of a wide range of dicotyledonous plant families the assay showed high positive correlation with estimates of dry matter digestibility obtained using rumenliquor from a fistulated steer. Both assays were found to reflect negative correlates of digestibility, notably fibre and condensed tannin, rather than the nutritional value of an item. The higher dry matter digestibility of immature leaves relative to mature leaves appeared to be accounted for by their lower fibre content. It is suggested that the pepsin/cellulase assay offers a cheap, quick, routine method of gaining information on the effects of some types of plant secondary compounds (digestibility reducers) on the 'food potential' of different kinds of foliage to herbivores. Its use in studies of herbivory in rainforest areas in relation to analyses for plant secondary compounds and food selection by herbivores is discussed.

The effect of chaya (Cnidoscolus aconitifolius) leaf meal and of exogenous enzymes on amino acid digestibility in broilers.

Science.gov (United States)

Sarmiento-Franco, L; McNab, J M; Pearson, A; Belmar-Casso, R

2003-07-01

1. The apparent ileal nitrogen (N) and amino acid digestibilities in chaya leaf meal (CLM) (Cnidoscolus aconitifolius) with added enzymes, and the same variables in diets containing different amounts of CLM were studied in chickens. 2. In the first experiment pectinase, beta-glucanase, and pectinase + beta-glucanase were added to CLM. In the second experiment, there were three diets based on maize and soybean: 0, 150 and 250 g/kg CLM. 3. Pectinase significantly increased both lysine and overall amino acid digestibilities in CLM. 4. In experiment 2, the amino acid digestibility in birds fed on CLM250 was lower than that from birds fed on either control or CLM150. Only the digestibilities of alanine, arginine and proline were lower in birds fed on CLM150 than in those fed on the control diet. Nitrogen digestibility was lower in birds fed on the CLM250 diet than on either control or CLM150 diets. These findings were attributed to the increasing concentration of fibre with increasing dietary CLM.

Effects of dietary fish oil replacement by vegetable oil on the digestive enzymes activity and intestinal morphology in Meagre, Argyrosomus regius (Asso, 1801

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Fernando Antunes Magalhães

2014-07-01

The results were analyzed by three way factorial. Amylase activity was bigger in FO when compared with VO (Table 1. The same result was observed in chymotrypsin activity. On the other hand, lipase activity was higher in VO. Regarding the levels of lipids, diets with 17% had higher amylase activity than diets with 12%. The inverse was observed in chymotrypsin activity. In relation to lipase activity, no differences were observed on the two levels of lipids studied. No differences in digestive enzymes activities were observed when diets were supplemented with selenium. Epithelium architecture of the posterior intestine was slightly affected by dietary treatments. Higher levels of lipids seem to induce enterocyte vacuolization, and vacuoles seem to be larger when a blend of vegetable oils was used instead of fish oil. No clear role can be attributed to selenium regarding intestinal morphology. In conclusion, our study showed that the source and levels of lipid in diets for meagre have influence in activity of digestible enzymes like amylase, lipase and chymotrypsin. Furthermore, levels of selenium do not cause an alteration in studied digestible enzymes.

One-year monitoring of core biomarker and digestive enzyme responses in transplanted zebra mussels (Dreissena polymorpha).

Science.gov (United States)

Palais, F; Dedourge-Geffard, O; Beaudon, A; Pain-Devin, S; Trapp, J; Geffard, O; Noury, P; Gourlay-Francé, C; Uher, E; Mouneyrac, C; Biagianti-Risbourg, S; Geffard, A

2012-04-01

A 12-month active biomonitoring study was performed in 2008-2009 on the Vesle river basin (Champagne-Ardenne, France) using the freshwater mussel Dreissena polymorpha as a sentinel species; allochthonous mussels originating from a reference site (Commercy) were exposed at four sites (Bouy, Sept-Saulx, Fismes, Ardre) within the Vesle river basin. Selected core biomarkers (acetylcholinesterase (AChE) activity, glutathione-S transferase (GST) activity, metallothionein concentration), along with digestive enzyme activities (amylase, endocellulase) and energy reserve concentrations (glycogen, lipids), were monitored throughout the study in exposed mussels. At the Fismes and Ardre sites (downstream basin), metallic and organic contamination levels were low but still high enough to elicit AChE and GST activity induction in exposed mussels (chemical stress); besides, chemical pollutants had no apparent deleterious effects on mussel condition. At the Bouy and Sept-Saulx sites (upstream basin), mussels obviously suffered from adverse food conditions which seriously impaired individual physiological state and survival (nutritional stress); food scarcity had however no apparent effects on core biomarker responses. Digestive enzyme activities responded to both chemical and nutritional stresses, the increase in energy outputs (general adaptation syndrome-downstream sites) or the decrease in energy inputs (food scarcity-upstream sites) leading to mid- or long-term induction of digestive carbohydrase activities in exposed mussels (energy optimizing strategy). Complex regulation patterns of these activities require nevertheless the use of a multi-marker approach to allow data interpretation. Besides, their sensitivity to natural confounding environmental factors remains to be precised.

Effect of dietary genistein on growth performance, digestive enzyme activity, and body composition of Nile tilapia Oreochromis niloticus

Science.gov (United States)

Chen, Dong; Wang, Wei; Ru, Shaoguo

2015-01-01

An 8-week feeding experiment was performed to evaluate the effect of dietary genistein on growth performance, body composition, and digestive enzymes activity of juvenile Nile tilapia ( Oreochromis niloticus). Four isonitrogenous and isoenergetic diets were formulated containing four graded supplements of genistein: 0, 30, 300, and 3 000 μg/g. Each diet was randomly assigned in triplicate to tanks stocked with 15 juvenile tilapia (10.47±1.24 g). The results show that 30 and 300 μg/g dietary genistein had no significant effect on growth performance of Nile tilapia, but the higher level of genistein (3 000 μg/g) significantly depressed the final body weight and specific growth rate. There was no significant difference in survival rate, feed intake, feed efficiency ratio or whole body composition among all dietary treatments. An assay of digestive enzymes showed that the diet containing 3 000 μg/ggenistein decreased stomach and hepatopancreas protease activity, and amylase activity in the liver and intestine, while a dietary level of 300 μg/g genistein depressed stomach protease and intestine amylase activities. However, no significant difference in stomach amylase activity was found among dietary treatments. Overall, the results of the present study indicate that a high level of dietary genistein (3 000 μg/g, or above) would significantly reduce the growth of Nile tilapia, partly because of its inhibitory effect on the activity of major digestive enzymes. Accordingly, the detrimental effects of genistein, as found in soybean products, should not be ignored when applied as an alternative ingredient source in aquaculture.

Expression of an antimicrobial peptide, digestive enzymes and nutrient transporters in the intestine of E. praecox-infected chickens

Science.gov (United States)

Coccidiosis is a major intestinal disease of poultry, caused by several species of the protozoan Eimeria. The objective of this study was to examine changes in expression of digestive enzymes, nutrient transporters and an antimicrobial peptide following an Eimeria praecox challenge of chickens at d...

Comprehensive evaluation of SNP identification with the Restriction Enzyme-based Reduced Representation Library (RRL method

Directory of Open Access Journals (Sweden)

Du Ye

2012-02-01

Full Text Available Abstract Background Restriction Enzyme-based Reduced Representation Library (RRL method represents a relatively feasible and flexible strategy used for Single Nucleotide Polymorphism (SNP identification in different species. It has remarkable advantage of reducing the complexity of the genome by orders of magnitude. However, comprehensive evaluation for actual efficacy of SNP identification by this method is still unavailable. Results In order to evaluate the efficacy of Restriction Enzyme-based RRL method, we selected Tsp 45I enzyme which covers 266 Mb flanking region of the enzyme recognition site according to in silico simulation on human reference genome, then we sequenced YH RRL after Tsp 45I treatment and obtained reads of which 80.8% were mapped to target region with an 20-fold average coverage, about 96.8% of target region was covered by at least one read and 257 K SNPs were identified in the region using SOAPsnp software. Compared with whole genome resequencing data, we observed false discovery rate (FDR of 13.95% and false negative rate (FNR of 25.90%. The concordance rate of homozygote loci was over 99.8%, but that of heterozygote were only 92.56%. Repeat sequences and bases quality were proved to have a great effect on the accuracy of SNP calling, SNPs in recognition sites contributed evidently to the high FNR and the low concordance rate of heterozygote. Our results indicated that repeat masking and high stringent filter criteria could significantly decrease both FDR and FNR. Conclusions This study demonstrates that Restriction Enzyme-based RRL method was effective for SNP identification. The results highlight the important role of bias and the method-derived defects represented in this method and emphasize the special attentions noteworthy.

Effects of traditional Chinese medicine formula on ruminal fermentation, enzyme activities and nutrient digestibility of beef cattle.

Science.gov (United States)

Zhu, Zhi; Song, Zhen-Hui; Cao, Li-Ting; Wang, Yong; Zhou, Wen-Zhang; Zhou, Pei; Zuo, Fu-Yuan

2018-04-01

This study was conducted to evaluate effects of traditional Chinese medicine formula (TCMF) combined with several herbs on ruminal fermentation, enzyme activities and nutrient digestibility. Twenty finishing bulls were assigned to control or different TCMFs (Yufeisan-1, -2, -3; 2.5% dry matter (DM) in concentrate). Results showed that DM intake was higher (P < 0.05) in the Yufeisan-3 group than others. Compared to control, apparent digestibility of crude protein and neutral detergent fiber were increased (P < 0.05) by Yufeisan-3. No changes were observed in ruminal pH, concentrations of ammonia-N, microbial crude protein and total volatile fatty acid, whereas ratio of acetate to propionate was lower (P < 0.05) and propionate proportion tended to be higher (P < 0.1) in three TCMFs than control. Ruminal xylanase (P = 0.061) and carboxymethylcellulase (P < 0.05) activities were higher in Yufeisan-3 than control. No changes were observed in abundance of total bacteria, fungi and protozoa, whereas Fibrobacter succinogenes (P = 0.062) and Ruminococcus flavefaciens (P < 0.05) were increased and total methanogens was reduced (P = 0.069) by Yufeisan-3 compared to control. Yufeisan-3 improved nutrient digestibility and ruminal enzyme activity, and modified fermentation and microbial community, maybe due to the presence of Herba agastaches, Cortex phellodendri and Gypsum fibrosum. © 2018 Japanese Society of Animal Science.

Digestive enzyme activities are higher in the shortfin mako shark, Isurus oxyrinchus, than in ectothermic sharks as a result of visceral endothermy.

Science.gov (United States)

Newton, Kyle C; Wraith, James; Dickson, Kathryn A

2015-08-01

Lamnid sharks are regionally endothermic fishes that maintain visceral temperatures elevated above the ambient water temperature. Visceral endothermy is thought to increase rates of digestion and food processing and allow thermal niche expansion. We tested the hypothesis that, at in vivo temperatures, the endothermic shortfin mako shark, Isurus oxyrinchus, has higher specific activities of three digestive enzymes-gastric pepsin and pancreatic trypsin and lipase-than the thresher shark, Alopias vulpinus, and the blue shark, Prionace glauca, neither of which can maintain elevated visceral temperatures. Homogenized stomach or pancreas tissue obtained from sharks collected by pelagic longline was incubated at both 15 and 25 °C, at saturating substrate concentrations, to quantify tissue enzymatic activity. The mako had significantly higher enzyme activities at 25 °C than did the thresher and blue sharks at 15 °C. This difference was not a simple temperature effect, because at 25 °C the mako had higher trypsin activity than the blue shark and higher activities for all enzymes than the thresher shark. We also hypothesized that the thermal coefficient, or Q 10 value, would be higher for the mako shark than for the thresher and blue sharks because of its more stable visceral temperature. However, the mako and thresher sharks had similar Q 10 values for all enzymes, perhaps because of their closer phylogenetic relationship. The higher in vivo digestive enzyme activities in the mako shark should result in higher rates of food processing and may represent a selective advantage of regional visceral endothermy.

DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway

NARCIS (Netherlands)

van den Broek, B.; Noom, M.C.; Wuite, G.J.L.

2005-01-01

Type II restriction endonucleases protect bacteria against phage infections by cleaving recognition sites on foreign double-stranded DNA (dsDNA) with extraordinary specificity. This capability arises primarily from large conformational changes in enzyme and/or DNA upon target sequence recognition.

Effect of endogenous proteins and lipids on starch digestibility in rice flour.

Science.gov (United States)

Ye, Jiangping; Hu, Xiuting; Luo, Shunjing; McClements, David Julian; Liang, Lu; Liu, Chengmei

2018-04-01

The composition and structure of the food matrix can have a major impact on the digestion. The aim of this work was to investigate the effects of endogenous proteins and lipids on starch digestibility in rice flour, with an emphasis on establishing the underlying physicochemical mechanisms involved. Native long-grain indica rice flour and rice flour with the lipids and/or proteins removed were subjected to a simulated digestion in vitro. A significant increase in starch digestibility was observed after removal of proteins, lipids, or both. The starch digestibility of the rice flour without lipids was slightly lower than that without proteins, even though the proteins content was about 10-fold higher than the lipids content. Microstructural analysis suggested that the proteins and lipids were normally attached to the surfaces of the starch granules in the native rice flour, thus inhibiting their contact with digestive enzymes. Moreover, the proteins and lipids restricted the swelling of the starch granules, which may have decreased their digestion by reducing their surface areas. In addition, amylose-lipid complex was detected in the rice flour, which is also known to slow down starch digestion. These results have important implications for the design of foods with improved nutritional profiles. Copyright © 2018 Elsevier Ltd. All rights reserved.

Increasing Genome Sampling and Improving SNP Genotyping for Genotyping-by-Sequencing with New Combinations of Restriction Enzymes.

Science.gov (United States)

Fu, Yong-Bi; Peterson, Gregory W; Dong, Yibo

2016-04-07

Genotyping-by-sequencing (GBS) has emerged as a useful genomic approach for exploring genome-wide genetic variation. However, GBS commonly samples a genome unevenly and can generate a substantial amount of missing data. These technical features would limit the power of various GBS-based genetic and genomic analyses. Here we present software called IgCoverage for in silico evaluation of genomic coverage through GBS with an individual or pair of restriction enzymes on one sequenced genome, and report a new set of 21 restriction enzyme combinations that can be applied to enhance GBS applications. These enzyme combinations were developed through an application of IgCoverage on 22 plant, animal, and fungus species with sequenced genomes, and some of them were empirically evaluated with different runs of Illumina MiSeq sequencing in 12 plant species. The in silico analysis of 22 organisms revealed up to eight times more genome coverage for the new combinations consisted of pairing four- or five-cutter restriction enzymes than the commonly used enzyme combination PstI + MspI. The empirical evaluation of the new enzyme combination (HinfI + HpyCH4IV) in 12 plant species showed 1.7-6 times more genome coverage than PstI + MspI, and 2.3 times more genome coverage in dicots than monocots. Also, the SNP genotyping in 12 Arabidopsis and 12 rice plants revealed that HinfI + HpyCH4IV generated 7 and 1.3 times more SNPs (with 0-16.7% missing observations) than PstI + MspI, respectively. These findings demonstrate that these novel enzyme combinations can be utilized to increase genome sampling and improve SNP genotyping in various GBS applications. Copyright © 2016 Fu et al.

Reference genome-independent assessment of mutation density using restriction enzyme-phased sequencing

Directory of Open Access Journals (Sweden)

Monson-Miller Jennifer

2012-02-01

Full Text Available Abstract Background The availability of low cost sequencing has spurred its application to discovery and typing of variation, including variation induced by mutagenesis. Mutation discovery is challenging as it requires a substantial amount of sequencing and analysis to detect very rare changes and distinguish them from noise. Also challenging are the cases when the organism of interest has not been sequenced or is highly divergent from the reference. Results We describe the development of a simple method for reduced representation sequencing. Input DNA was digested with a single restriction enzyme and ligated to Y adapters modified to contain a sequence barcode and to provide a compatible overhang for ligation. We demonstrated the efficiency of this method at SNP discovery using rice and arabidopsis. To test its suitability for the discovery of very rare SNP, one control and three mutagenized rice individuals (1, 5 and 10 mM sodium azide were used to prepare genomic libraries for Illumina sequencers by ligating barcoded adapters to NlaIII restriction sites. For genome-dependent discovery 15-30 million of 80 base reads per individual were aligned to the reference sequence achieving individual sequencing coverage from 7 to 15×. We identified high-confidence base changes by comparing sequences across individuals and identified instances consistent with mutations, i.e. changes that were found in a single treated individual and were solely GC to AT transitions. For genome-independent discovery 70-mers were extracted from the sequence of the control individual and single-copy sequence was identified by comparing the 70-mers across samples to evaluate copy number and variation. This de novo "genome" was used to align the reads and identify mutations as above. Covering approximately 1/5 of the 380 Mb genome of rice we detected mutation densities ranging from 0.6 to 4 per Mb of diploid DNA depending on the mutagenic treatment. Conclusions The

The effect of rolled barley, sodium hydroxide-treated wheat or maize cob silage on digestive enzymes activity in the alimentary tract of dairy cows

DEFF Research Database (Denmark)

Moharrey, A.; Hymøller, Lone; Weisbjerg, Martin Riis

2017-01-01

In the present study digestive enzyme activities were studied in the rumen, intestine and faeces of dairy cows fed rations differing in starch source. Three total mixed rations were prepared for dairy cows with maize cob silage (MCS), sodium hydroxide-treated wheat (SHW) or rolled barley as starch...... DM (2.61 vs 2.91 and 3.15%) and a higher ash content (30.99 vs 29.24 and 24.31%) in the ruminal fluid without affecting enzyme activities. Positive correlation between lipolytic and amylolitic activities in ruminal fluid was stated, which supported the hypothesis that amylolytic bacteria provide...... energy for lipolytic bacteria. So, the enzymes activities in the different parts of the digestive tract were not affected by the different starch sources....

Effects of Hanseniaspora opuntiae C21 on the growth and digestive enzyme activity of juvenile sea cucumber Apostichopus japonicas

Science.gov (United States)

Ma, Yuexin; Liu, Zhiming; Yang, Zhiping; Bao, Pengyun; Zhang, Congyao; Ding, Jianfeng

2014-07-01

The effects of a diet containing Hanseniaspora opuntiae C21 on growth and digestive enzyme activity were estimated in juvenile Apostichopus japonicus. Groups of sea cucumbers were fed diets containing H. opuntiae C21 at 0 (control), 104, 105, and 106 CFU (colony-forming units)/g feed. Results showed that after 45 d the specific growth rate (SGR) of sea cucumbers fed a C21-supplemented diet at 10 4 CFU/g feed was significantly higher than that of the control ( P sea cucumbers. In addition, after feeding the C21-supplemented diets for 15 d, the sea cucumbers were switched to an unsupplemented diet and C21 was confirmed to be capable of colonizing the intestine for at least 31 d after cessation of feeding. In conclusion, C21 was shown to successfully colonize the intestine of juvenile A. japonicus via dietary supplementation, and improve growth and digestive enzyme activity.

Starvation stress affects the interplay among shrimp gut microbiota, digestion and immune activities.

Science.gov (United States)

Dai, Wen-Fang; Zhang, Jin-Jie; Qiu, Qiong-Fen; Chen, Jiong; Yang, Wen; Ni, Sui; Xiong, Jin-Bo

2018-05-24

Aquatic animals are frequently suffered from starvation due to restricted food availability or deprivation. It is currently known that gut microbiota assists host in nutrient acquisition. Thus, exploring the gut microbiota responses would improve our understanding on physiological adaptation to starvation. To achieve this, we investigated how the gut microbiota and shrimp digestion and immune activities were affected under starvation stress. The results showed that the measured digestion activities in starved shrimp were significantly lower than in normal cohorts; while the measured immune activities exhibited an opposite trend. A structural equation modeling (SEM) revealed that changes in the gut bacterial community were directly related to digestive and immune enzyme activities, which in turn markedly affected shrimp growth traits. Notably, several gut bacterial indicators that characterized the shrimp nutrient status were identified, with more abundant opportunistic pathogens in starved shrimp, although there were no statistical differences in the overall diversity and the structures of gut bacterial communities between starved and normal shrimp. Starved shrimp exhibited less connected and cooperative interspecies interaction as compared with normal cohorts. Additionally, the functional pathways involved in carbohydrate and protein digestion, glycan biosynthesis, lipid and enzyme metabolism remarkably decreased in starved shrimp. These attenuations could increase the susceptibility of starved shrimp to pathogens infection. In summary, this study provides novel insights into the interplay among shrimp digestion, immune activities and gut microbiota in response to starvation stress. Copyright © 2018. Published by Elsevier Ltd.

Effects of dietary supplementation with green tea waste on growth, digestive enzyme and lipid metabolism of juvenile hybrid tilapia, Oreochromis niloticus × O. aureus.

Science.gov (United States)

Zheng, Qingmei; Han, Chunyan; Zhong, Yanmei; Wen, Rushu; Zhong, Ming

2017-04-01

An 8-week feeding trial was conducted to evaluate the effects of dietary supplementation with green tea waste (GTW) on growth, digestive enzyme and lipid metabolism of juvenile hybrid tilapia, Oreochromis niloticus × O. aureus. The fish (initial mean body weight, 12.63 ± 0.75 g) were fed five experimental diets that included 0 (control), 0.8, 1.6, 3.2 or 6.4 % of GTW in triplicate aquaria, twice daily. Growth performance, plasma metabolites content and liver and intestine digestive enzyme activities were determined. Fish accepted well all experimental diets during the trial, and no mortality was observed. The weight gain increased (P tilapia to improve growth performance, digestion efficacy and fat metabolism.

Influence of Guava by-Product, Enzyme Supplementation and Gamma Irradiation on Performance and Digestive Utilization of Fattening Rabbits

International Nuclear Information System (INIS)

Mekkawy, S.H.; El-Faramawy, A.A.; Zakaria, S. M.

2000-01-01

A total number of 32 New Zealand white rabbits weighing about 850 g were used to study the influence of guava by-product (GBP)on growth, feed consumption, feed efficiency, carcass dressing percentage, blood parameters and digestive efficiency. Four diets were formulated to provide about 17% crude fiber (CF). Control diet, diet with 16% (GBP), diet with the same percent of (GBP) supplemented with enzyme supplement and the last one also the same with the third diet in addition to treatment by gamma irradiation (3 kGy). The results indicated that there were no significant differences (P<0.05) between the experimental groups and control in growth, feed consumption, feed efficiency and carcass dressing percentage. Blood parameters (total protein, albumin, globulin, total lipids, and alkaline phosphatase) were within normal range through out the groups. Apparent digestibility coefficient of nutrients ( OM, CP, CF, EE and NFE) were significantly higher in rabbits fed the diet of (GBP) and enzyme supplementation. Our data indicate that (GBP) can replace 16% of alfalfa hay without decreasing growth performance of rabbits

[Anaerobic digestion of lignocellulosic biomass with animal digestion mechanisms].

Science.gov (United States)

Wu, Hao; Zhang, Pan-Yue; Guo, Jian-Bin; Wu, Yong-Jie

2013-02-01

Lignocellulosic material is the most abundant renewable resource in the earth. Herbivores and wood-eating insects are highly effective in the digestion of plant cellulose, while anaerobic digestion process simulating animal alimentary tract still remains inefficient. The digestion mechanisms of herbivores and wood-eating insects and the development of anaerobic digestion processes of lignocellulose were reviewed for better understanding of animal digestion mechanisms and their application in design and operation of the anaerobic digestion reactor. Highly effective digestion of lignocellulosic materials in animal digestive system results from the synergistic effect of various digestive enzymes and a series of physical and biochemical reactions. Microbial fermentation system is strongly supported by powerful pretreatment, such as rumination of ruminants, cellulase catalysis and alkali treatment in digestive tract of wood-eating insects. Oxygen concentration gradient along the digestive tract may stimulate the hydrolytic activity of some microorganisms. In addition, the excellent arrangement of solid retention time, digesta flow and end product discharge enhance the animal digestion of wood cellulose. Although anaerobic digestion processes inoculated with rumen microorganisms based rumen digestion mechanisms were developed to treat lignocellulose, the fermentation was more greatly limited by the environmental conditions in the anaerobic digestion reactors than that in rumen or hindgut. Therefore, the anaerobic digestion processes simulating animal digestion mechanisms can effectively enhance the degradation of wood cellulose and other organic solid wastes.

Effect of orally administered betel leaf (Piper betle Linn.) on digestive enzymes of pancreas and intestinal mucosa and on bile production in rats.

Science.gov (United States)

Prabhu, M S; Platel, K; Saraswathi, G; Srinivasan, K

1995-10-01

The influence of two varieties of betel leaf (Piper betle Linn.) namely, the pungent Mysore and non-pungent Ambadi, was examined on digestive enzymes of pancreas and intestinal mucosa and on bile secretion in experimental rats. The betel leaves were administered orally at two doses which were either comparable to human consumption level or 5 times this. The results indicated that while these betel leaves do not influence bile secretion and composition, they have a significant stimulatory influence on pancreatic lipase activity. Besides, the Ambadi variety of betel leaf has a positive stimulatory influence on intestinal digestive enzymes, especially lipase, amylase and disaccharidases. A slight lowering in the activity of these intestinal enzymes was seen when Mysore variety of betel leaf was administered, and this variety also had a negative effect on pancreatic amylase. Further, both the betel leaf varieties have shown decreasing influence on pancreatic trypsin and chymotrypsin activities.

Cloning and analysis of a bifunctional methyltransferase/restriction endonuclease TspGWI, the prototype of a Thermus sp. enzyme family

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Zylicz-Stachula Agnieszka

2009-05-01

Full Text Available Abstract Background Restriction-modification systems are a diverse class of enzymes. They are classified into four major types: I, II, III and IV. We have previously proposed the existence of a Thermus sp. enzyme family, which belongs to type II restriction endonucleases (REases, however, it features also some characteristics of types I and III. Members include related thermophilic endonucleases: TspGWI, TaqII, TspDTI, and Tth111II. Results Here we describe cloning, mutagenesis and analysis of the prototype TspGWI enzyme that recognises the 5'-ACGGA-3' site and cleaves 11/9 nt downstream. We cloned, expressed, and mutagenised the tspgwi gene and investigated the properties of its product, the bifunctional TspGWI restriction/modification enzyme. Since TspGWI does not cleave DNA completely, a cloning method was devised, based on amino acid sequencing of internal proteolytic fragments. The deduced amino acid sequence of the enzyme shares significant sequence similarity with another representative of the Thermus sp. family – TaqII. Interestingly, these enzymes recognise similar, yet different sequences in the DNA. Both enzymes cleave DNA at the same distance, but differ in their ability to cleave single sites and in the requirement of S-adenosylmethionine as an allosteric activator for cleavage. Both the restriction endonuclease (REase and methyltransferase (MTase activities of wild type (wt TspGWI (either recombinant or isolated from Thermus sp. are dependent on the presence of divalent cations. Conclusion TspGWI is a bifunctional protein comprising a tandem arrangement of Type I-like domains; particularly noticeable is the central HsdM-like module comprising a helical domain and a highly conserved S-adenosylmethionine-binding/catalytic MTase domain, containing DPAVGTG and NPPY motifs. TspGWI also possesses an N-terminal PD-(D/EXK nuclease domain related to the corresponding domains in HsdR subunits, but lacks the ATP-dependent translocase module

Experimental assessment of factors influencing dewatering properties of thermophilically digested biosolids

Energy Technology Data Exchange (ETDEWEB)

Zhou, Jianpeng; Mavinic, Donald S.; Kelly, Harlan G.; Ramey, William D.

2003-07-01

Beneficial land application of processed wastewater sludges (biosolids) is a cost-effective, and environmentally sustainable option for the final disposal of sludges, because nutrients and organic matters in the sludge are recovered and reused as a resource. Thermophilic sludge digestion produces Class A biosolids, which can be reused without restrictions. Recent experience from full-scale thermophilic sludge digestion facilities in North America revealed that, dewatering thermophilically digested biosolids required more polymers to condition than mesophilically digested biosolids. This paper reports a laboratory study that investigated factors having significant impacts on dewatering properties of digested biosolids, and assessed the relationship among digestion, dewatering properties, and characteristics of thermophilically digested biosolids. The experimental work used batch-operated, bench-scale aerobic sludge digesters. Dewaterability was measured as Capillary Suction Time (CST). The study found that feed sludge composition significantly affected dewaterability of digested sludge. Higher percentage of the secondary sludge in the feed sludge corresponded to more significant deterioration in dewaterability. The effect of thermophilic digestion temperatures on dewaterabilty was rapid, occurred within 3-hour of digestion, indicting a heat shock effect, rather than a microbiological effect. When a high shear was applied to the sludge in digesters, it resulted In a significant deterioration in dewaterability in the digested sludge. It appears there was a strong correlation between dewaterability and extracellular biopolymers. Enzymes (protease) treatment confirmed that role of extracellular proteins in affecting the dewatering properties of thermophilic biosolids, also revealed the complex nature of biopolymers' effect on dewaterability. Such effects might be due to protein-polysaccharides interactions, hydrogen bonding, or hydrophilic and hydrophobic

Short communication: Effect of casein haplotype on angiotensin-converting enzyme inhibitory and antioxidant capacities of milk casein from Italian Holstein cows before and following in vitro digestion with gastrointestinal enzymes.

Science.gov (United States)

Perna, Annamaria; Simonetti, Amalia; Gambacorta, Emilio

2016-09-01

The aim of this work was to investigate the effect of casein haplotype (αS1, β, and κ) on antioxidative and angiotensin-converting enzyme (ACE) inhibitory capacities of milk casein from Italian Holstein cows before and following in vitro digestion with gastrointestinal enzymes. The antioxidant capacity was measured using 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid and ferric-reducing antioxidant power assays, whereas ACE inhibition was determined by ACE-inhibitory assay. The ACE-inhibitory and antioxidant capacities of milk casein increased during in vitro gastrointestinal digestion. Casein haplotype significantly influenced the antioxidative and ACE-inhibitory capacities of digested casein. In particular, BB-A(2)A(1)-AA casein and BB-A(1)A(1)-AA casein showed the highest ACE-inhibitory capacity, BB-A(2)A(2)-AA casein showed the highest antioxidant capacity, whereas BB-A(2)A(2)-BB casein showed the lowest biological capacity. To date, few studies have been done on the effect of casein haplotype on biological capacity of milk casein, thus the present study sets the basis for a new knowledge that could lead to the production of milk with better nutraceutical properties. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Ruminant Nutrition Symposium: Improving cell wall digestion and animal performance with fibrolytic enzymes.

Science.gov (United States)

Adesogan, A T; Ma, Z X; Romero, J J; Arriola, K G

2014-04-01

This paper aimed to summarize published responses to treatment of cattle diets with exogenous fibrolytic enzymes (EFE), to discuss reasons for variable EFE efficacy in animal trials, to recommend strategies for improving enzyme testing and EFE efficacy in ruminant diets, and to identify proteomic differences between effective and ineffective EFE. A meta-analysis of 20 dairy cow studies with 30 experiments revealed that only a few increased lactational performance and the response was inconsistent. This variability is attributable to several enzyme, feed, animal, and management factors that were discussed in this paper. The variability reflects our limited understanding of the synergistic and sequential interactions between exogenous glycosyl hydrolases, autochthonous ruminal microbes, and endogenous fibrolytic enzymes that are necessary to optimize ruminal fiber digestion. An added complication is that many of the standard methods of assaying EFE activities may over- or underestimate their potential effects because they are based on pure substrate saccharification and do not simulate ruminal conditions. Our recent evaluation of 18 commercial EFE showed that 78 and 83% of them exhibited optimal endoglucanase and xylanase activities, respectively, at 50 °C, and 77 and 61% had optimal activities at pH 4 to 5, respectively, indicating that most would likely act suboptimally in the rumen. Of the many fibrolytic activities that act synergistically to degrade forage fiber, the few usually assayed, typically endoglucanase and xylanase, cannot hydrolyze the recalcitrant phenolic acid-lignin linkages that are the main constraints to ruminal fiber degradation. These factors highlight the futility of random addition of EFE to diets. This paper discusses reasons for the variable animal responses to dietary addition of fibrolytic enzymes, advances explanations for the inconsistency, suggests a strategy to improve enzyme efficacy in ruminant diets, and describes differences

Correlation between the activity of digestive enzymes and nonself recognition in the gut of Eisenia andrei earthworms

Czech Academy of Sciences Publication Activity Database

Procházková, Petra; Šustr, Vladimír; Dvořák, Jiří; Roubalová, Radka; Škanta, František; Pižl, Václav; Bilej, Martin

2013-01-01

Roč. 114, č. 3 (2013), s. 217-221 ISSN 0022-2011 R&D Projects: GA MŠk(CZ) EE2.3.20.0055; GA MŠk(CZ) EE2.3.30.0003; GA ČR GA206/07/0378 Institutional support: RVO:61388971 ; RVO:60077344 Keywords : Digestive enzyme * Earthworm * Eisenia andrei Subject RIV: EC - Immunology; EE - Microbiology, Virology (BC-A) Impact factor: 2.601, year: 2013

Interdomain communication in the endonuclease/motor subunit of type I restriction-modification enzyme EcoR124I

Czech Academy of Sciences Publication Activity Database

Sinha, Dhiraj; Shamayeva, Katerina; Ramasubramani, V.; Řeha, David; Bialevich, V.; Khabiri, Morteza; Guzanová, Alena; Milbar, N.; Weiserová, Marie; Cséfalvay, Eva; Carey, J.; Ettrich, Rüdiger

2014-01-01

Roč. 20, č. 7 (2014), s. 2334 ISSN 1610-2940 R&D Projects: GA ČR GAP207/12/2323 Institutional support: RVO:67179843 ; RVO:61388971 Keywords : DNA restriction enzymes * Molecular modeling * QM/MM calculations * principal components analysis * E. coli * Multisubunit enzyme complex * Correlated loop motions Subject RIV: EH - Ecology, Behaviour; EE - Microbiology, Virology (MBU-M) Impact factor: 1.736, year: 2014

Effects of diets containing vegetable protein concentrates on performance and activity of digestive enzymes in silver catfish (Rhamdia quelen

Directory of Open Access Journals (Sweden)

Naglezi de Menezes Lovatto

2014-02-01

Full Text Available The purpose of study was to evaluate the effect of using protein concentrates crambe and sunflower meal in the diet of silver catfish juveniles, as substitute for animal protein source. A total of 300 silver catfish had been separate in 15 experimental units of 280 L, totaling five treatments with three replications. We evaluated two levels (25% and 50% replacement of the meat and bone meal by protein concentrates of crambe and sunflower meals. Evaluated growth parameters, biological index and digestive enzymes in fish. There was no statistical difference for mass (g and standard length (cm, but the fish diet CPFCr-25% had greater total length (cm. No difference in dry matter, crude protein and total protein deposited (calculated. However, there was a higher concentration of ash in the carcass of the animals fed the control diet and CPFCr-50% in relation to diet CPFG- 50%, in addition, higher levels of lipids in fish fed diet CPFG-50%. No significant differences for hepatosomatic index, digestive somatic index and intestinal quotient of animals subjected to different treatments. The activity of digestive enzymes trypsin and chymotrypsin did not change. There was increased activity of acid protease. The quantitative and qualitative increase in protein concentration from this fraction allows the use of bran protein concentrates crambe and sunflower as substitutes for animal protein source.

Digestive enzymes of some earthworms.

Science.gov (United States)

Mishra, P C; Dash, M C

1980-10-15

4 species of tropical earthworms differed with regard to enzyme activity. The maximum activity of protease and of cellulase occurred in the posterior region of the gut of the earthworms. On the average Octochaetona surensis shows maximum activity and Drawida calebi shows minimum activity for all the enzymes studied.

Improvement in Saccharification Yield of Mixed Rumen Enzymes by Identification of Recalcitrant Cell Wall Constituents Using Enzyme Fingerprinting.

Science.gov (United States)

Badhan, Ajay; Wang, Yu-Xi; Gruninger, Robert; Patton, Donald; Powlowski, Justin; Tsang, Adrian; McAllister, Tim A

2015-01-01

Identification of recalcitrant factors that limit digestion of forages and the development of enzymatic approaches that improve hydrolysis could play a key role in improving the efficiency of meat and milk production in ruminants. Enzyme fingerprinting of barley silage fed to heifers and total tract indigestible fibre residue (TIFR) collected from feces was used to identify cell wall components resistant to total tract digestion. Enzyme fingerprinting results identified acetyl xylan esterases as key to the enhanced ruminal digestion. FTIR analysis also suggested cross-link cell wall polymers as principal components of indigested fiber residues in feces. Based on structural information from enzymatic fingerprinting and FTIR, enzyme pretreatment to enhance glucose yield from barley straw and alfalfa hay upon exposure to mixed rumen-enzymes was developed. Prehydrolysis effects of recombinant fungal fibrolytic hydrolases were analyzed using microassay in combination with statistical experimental design. Recombinant hemicellulases and auxiliary enzymes initiated degradation of plant structural polysaccharides upon application and improved the in vitro saccharification of alfalfa and barley straw by mixed rumen enzymes. The validation results showed that microassay in combination with statistical experimental design can be successfully used to predict effective enzyme pretreatments that can enhance plant cell wall digestion by mixed rumen enzymes.

Evaluation of simultaneous binding of Chromomycin A3 to the multiple sites of DNA by the new restriction enzyme assay.

Science.gov (United States)

Murase, Hirotaka; Noguchi, Tomoharu; Sasaki, Shigeki

2018-06-01

Chromomycin A3 (CMA3) is an aureolic acid-type antitumor antibiotic. CMA3 forms dimeric complexes with divalent cations, such as Mg 2+ , which strongly binds to the GC rich sequence of DNA to inhibit DNA replication and transcription. In this study, the binding property of CMA3 to the DNA sequence containing multiple GC-rich binding sites was investigated by measuring the protection from hydrolysis by the restriction enzymes, AccII and Fnu4HI, for the center of the CGCG site and the 5'-GC↓GGC site, respectively. In contrast to the standard DNase I footprinting method, the DNA substrates are fully hydrolyzed by the restriction enzymes, therefore, the full protection of DNA at all the cleavable sites indicates that CMA3 simultaneously binds to all the binding sites. The restriction enzyme assay has suggested that CMA3 has a high tendency to bind the successive CGCG sites and the CGG repeat. Copyright © 2018 Elsevier Ltd. All rights reserved.

Restriction analysis of genetic variability of Polish isolates of Tomato black ring virus.

Science.gov (United States)

Jończyk, Magdalena; Borodynko, Natasza; Pospieszny, Henryk

2004-01-01

Several different isolates of Tomato black ring virus (TBRV) have been collected in Poland from cucumber, tomato, potato and black locust plants. Biological tests showed some differences in the range of infected plants and the type of symptoms, which was the basis for selection of seven the most biologically different TBRV isolates. According to the sequence of TBRV-MJ, several primer pairs were designed and almost the entire sequence of both genomic RNAs was amplified. The RT-PCR products derived from all tested TBRV isolates were digested by restriction enzymes. On the basis of the restriction patterns, the variable and the conserved regions of the TBRV genome were defined and the relationships between the Polish TBRV isolates established.

THE EFFECTIVITY TEST OF SHEEP RUMEN LIQUOR ENZYME ADDED TO PALM KERNEL MEAL ON ITS DECREASE OF CRUDE FIBER AND APPARENT DIGESTIBILITY COEFFICIENT FOR CATFISH Pangasius hypophthalmus DIET

Directory of Open Access Journals (Sweden)

Wahyu Pamungkas

2011-12-01

Full Text Available Two experiments were conducted to evaluate the hydrolysis of fiber content in palm kernel meal (PKM by sheep rumen liquor enzyme and to know the apparent digestibility coefficient of hydrolyzed PKM for catfish Pangasius hypophthalmus. The first trial examined effectivity of sheep rumen liquor enzyme to decrease crude fiber content of PKM. The added volume of sheep rumen liquor enzyme was 0, 20, 40, 60, 80, and 100 mL/kg PKM and then it was incubated for 0, 12, and 24 hours. A factorial completely randomized experimental design consisted of 2 variables and triplicates were selected. The second trial was conducted to evaluate the apparent digestibility coefficients of hydrolized PKM for catfish. Apparent digestibility coefficients were determined using chromic oxide indicator added to both reference and test diets. The feed ingredients used in the trial were hydrolyzed PKM (PKMe and unhydrolyzed PKM (PKM. Ten fishes with weighing around 20 g were used in the trial and held in 80 l tanks. Feces were collected from three replicate groups of fish using a fecal collection column attached to fish rearing tank. PKM hydrolyzed with 100 mL/kg and incubated for 24 hour showed the lowest crude fiber content (6.99% among the treatments (P<0.05. Apparent digestibility coefficient of hydrolyzed PKM was 57.57% compared with unhydrolyzed PKM 15.31%. Based on the evaluation in those parameters it was concluded that sheep rumen liquor enzyme added to PKM was effective to decrease crude fiber content of PKM and improve apparent digestibility coefficient of PKM for catfish.

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

Science.gov (United States)

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.

Digestive morphophysiology of Gryllodes sigillatus (Orthoptera: Gryllidae).

Science.gov (United States)

Biagio, Fernanda P; Tamaki, Fabio K; Terra, Walter R; Ribeiro, Alberto F

2009-12-01

The evolution of the digestive system in the Order Orthoptera is disclosed from the study of the morphophysiology of the digestive process in its major taxa. This paper deals with a cricket representing the less known suborder Ensifera. Most amylase and trypsin activities occur in crop and caeca, respectively. Maltase and aminopeptidase are found in soluble and membrane-bound forms in caeca, with aminopeptidase also occurring in ventriculus. Amaranth was orally fed to Gryllodes sigillatus adults or injected into their haemolymph. The experiments were performed with starving and feeding insects with identical results. Following feeding of the dye the luminal side of the most anterior ventriculus (and in lesser amounts the midgut caeca) became heavily stained. In injected insects, the haemal side of the most posterior ventriculus was stained. This suggested that the anterior ventriculus is the main site of water absorption (the caeca is a secondary one), whereas the posterior ventriculus secretes water into the gut. Thus, a putative counter-current flux of fluid from posterior to anterior ventriculus may propel digestive enzyme recycling. This was confirmed by the finding that digestive enzymes are excreted at a low rate. The fine structure of midgut caeca and ventriculus cells revealed that they have morphological features that may be related to their involvement in secretion (movement from cell to lumen) and absorption (movement from lumen to cell) of fluids. Furthermore, morphological data showed that both merocrine and apocrine secretory mechanisms occur in midgut cells. The results showed that cricket digestion differs from that in grasshopper in having: (1) more membrane-bound digestive enzymes; (2) protein digestion slightly displaced toward the ventriculus; (3) midgut fluxes, and hence digestive enzyme recycling, in both starved and fed insects.

Effects of chronic exposure to cadmium and temperature, alone or combined, on the threespine stickleback (Gasterosteus aculeatus): Interest of digestive enzymes as biomarkers.

Science.gov (United States)

Hani, Younes Mohamed Ismail; Turies, Cyril; Palluel, Olivier; Delahaut, Laurence; Gaillet, Véronique; Bado-Nilles, Anne; Porcher, Jean-Marc; Geffard, Alain; Dedourge-Geffard, Odile

2018-06-01

The development of predictive, sensitive and reliable biomarkers is of crucial importance for aquatic biomonitoring to assess the effects of chemical substances on aquatic organisms, especially when it comes to combined effects with other stressors (e.g. temperature). The first purpose of the present study was to evaluate the single and combined effects of 90 days of exposure to an environmental cadmium concentration (0.5 μg L -1 ) and two water temperatures (16 and 21 °C) on different parameters. These parameters are involved in (i) the antioxidant system (superoxide dismutase activity -SOD- and total glutathione levels -GSH-), (ii) the energy metabolism, i.e. energy reserves (glycogen, lipids, proteins) and digestive enzymes (trypsin, amylase, intestinal alkaline phosphatase -IAP-), and (iii) biometric parameters (weight, length, Fulton's condition factor, and the gonadosomatic index -GSI-) of threespine stickleback (Gasterosteus aculeatus). The second purpose was to determine the interest of the three digestive enzymes as biomarkers in comparison with the other parameters. The higher temperature (21 °C) impacted the anti-oxidant and energy reserve parameters. In liver, GSH levels increased on day 60, while SOD decreased on days 15 and 90, with a significant decrease of protein and lipid energy reserves on day 90. In muscle, the higher temperature decreased SOD activity only on day 90. G. aculeatus biometric parameters were also impacted by the higher temperature, which limited stickleback growth after 90 days of exposure. In female sticklebacks, the GSI peaked on day 60 and decreased sharply on day 90, while the highest values were reached at day 90 in the control groups, suggesting impaired reproduction in sticklebacks raised at 21 °C. These results suggest that 21 °C is an upper-limit temperature for long-term physiological processes in sticklebacks. In contrast, very low-concentration cadmium exposure had no effect on classical biomarkers

Homology of yeast photoreactivating gene fragment with human genomic digests

International Nuclear Information System (INIS)

Meechan, P.J.; Milam, K.M.; Cleaver, J.E.

1984-01-01

Enzymatic photoreactivation of UV-induced DNA lesions has been demonstrated for a variety of prokaryotic and eukaryotic organisms. Its presence in placental mammals, however, has not been clearly established. The authors attempted to resolve this question by assaying for the presence (or absence) of sequences in human DNA complimentary to a fragment of the photoreactivating gene from S. cerevisiae that has recently been cloned. In another study, DNA from human, chick E. coli and yeast cells was digested with either HindIII of BglII, electrophoresed on a 0.5% agarose gel, transferred (Southern blot) to a nylon membrane and probed for homology against a Sau3A restriction fragment from S. cerevisiae that compliments phr/sup -/ cells. Hybridization to human DNA digests was observed only under relatively non-stringent conditions indicating the gene is not conserved in placental mammals. These results are correlated with current literature data concerning photoreactivating enzymes

DNA synapsis through transient tetramerization triggers cleavage by Ecl18kI restriction enzyme

NARCIS (Netherlands)

Zaremba, M.; Lyubchenko, Y.L.; Laurens, N.; van den Broek, B.; Wuite, G.J.L.; Siksnys, V.

2010-01-01

To cut DNA at their target sites, restriction enzymes assemble into different oligomeric structures. The Ecl18kI endonuclease in the crystal is arranged as a tetramer made of two dimers each bound to a DNA copy. However, free in solution Ecl18kI is a dimer. To find out whether the Ecl18kI dimer or

Molecular identification of similar species of the genus Biomphalaria (Mollusca: Planorbidae determined by a polymerase chain reaction-restriction fragment length polymorphism

Directory of Open Access Journals (Sweden)

Caldeira Roberta Lima

1998-01-01

Full Text Available The freshwater snails Biomphalaria straminea, B. intermedia, B. kuhniana and B. peregrina, are morphologically similar; based on this similarity the first three species were therefore grouped in the complex B. straminea. The morphological identification of these species is based on characters such as vaginal wrinkling, relation between prepuce: penial sheath:deferens vas and number of muscle layers in the penis wall. In this study the polymerase chain reaction restriction fragment length polymorphism technique was used for molecular identification of these molluscs. This technique is based on the amplification of the internal transcribed spacer regions ITS1 e ITS2 of the ribosomal RNA gene and subsequent digestion of these fragments by restriction enzymes. Six enzymes were tested: Dde I, Mnl I, Hae III, Rsa I, Hpa II e Alu I. The restriction patterns obtained with DdeI presented the best profile for separation of the four species of Biomphalaria. The profiles obtained with all the enzymes were used to estimate the genetic distances among the species through analysis of common banding patterns.

Analysis of DNA restriction fragments greater than 5.7 Mb in size from the centromeric region of human chromosomes.

Science.gov (United States)

Arn, P H; Li, X; Smith, C; Hsu, M; Schwartz, D C; Jabs, E W

1991-01-01

Pulsed electrophoresis was used to study the organization of the human centromeric region. Genomic DNA was digested with rare-cutting enzymes. DNA fragments from 0.2 to greater than 5.7 Mb were separated by electrophoresis and hybridized with alphoid and simple DNA repeats. Rare-cutting enzymes (Mlu I, Nar I, Not I, Nru I, Sal I, Sfi I, Sst II) demonstrated fewer restriction sites at centromeric regions than elsewhere in the genome. The enzyme Not I had the fewest restriction sites at centromeric regions. As much as 70% of these sequences from the centromeric region are present in Not I DNA fragments greater than 5.7 and estimated to be as large as 10 Mb in size. Other repetitive sequences such as short interspersed repeated segments (SINEs), long interspersed repeated segments (LINEs), ribosomal DNA, and mini-satellite DNA that are not enriched at the centromeric region, are not enriched in Not I fragments of greater than 5.7 Mb in size.

Correspondence between radioactive and functional methods in the quality control of DNA restriction and modifying enzymes

DEFF Research Database (Denmark)

Trujillo, L E; Pupo, E; Miranda, F

1996-01-01

We evaluated the use of two radiolabeled lambda DNA/Hpa II substrates to detect 5'-->3', 3'-->5' single and double stranded DNA dependent exonuclease and phosphatase activities found as contaminants in restriction and modifying enzyme preparations. Looking for the meaning of the radioactive assay...

Cloning and molecular ontogeny of digestive enzymes in fed and food-deprived developing gilthead seabream (Sparus aurata) larvae.

Science.gov (United States)

Mata-Sotres, José Antonio; Martos-Sitcha, Juan Antonio; Astola, Antonio; Yúfera, Manuel; Martínez-Rodríguez, Gonzalo

2016-01-01

We have determined the expression pattern of key pancreatic enzymes precursors (trypsinogen, try; chymotrypsinogen, ctrb; phospholipase A2, pla2; bile salt-activated lipase, cel; and α-amylase, amy2a) during the larval stage of gilthead seabream (Sparus aurata) up to 60days after hatching (dph). Previously, complete sequences of try, cel, and amy2a were cloned and phylogenetically analyzed. One new isoform was found for cel transcript (cel1b). Expression of all enzyme precursors was detected before the mouth opening. Expression of try and ctrb increased during the first days of development and then maintained high values with some fluctuations during the whole larval stage. The prolipases pla2 and cel1b increased from first-feeding with irregular fluctuation until the end of the experiment. Contrarily, cel1a maintained low expression values during most of the larval stage increasing at the end of the period. Nevertheless, cel1a expression was negligible as compared with cel1b. The expression of amy2a sharply increased during the first week followed by a gradual decrease. In addition, a food-deprivation experiment was performed to find the differences in relation to presence/absence of gut content after the opening of the mouth. The food-deprived larvae died at 10dph. The expression levels of all digestive enzymes increased up to 7dph, declining sharply afterwards. This expression pattern up to 7dph was the same observed in fed larvae, confirming the genetic programming during the early development. Main digestive enzymes in gilthead seabream larvae exhibited the same expression profiles than other marine fish with carnivorous preferences in their juvenile stages. Copyright © 2015 Elsevier Inc. All rights reserved.

Enzyme treatment to decrease solids and improve digestion of ...

African Journals Online (AJOL)

The aim of anaerobic digestion of primary sewage sludge is to convert the carbonaceous material contained in the solids into methane and carbon dioxide. The products of digestion are therefore gases, stabilised sludge solids which are subsequently dewatered and disposed of, and sludge liquor which is generally further ...

Strategies in protein sequencing and characterization: Multi-enzyme digestion coupled with alternate CID/ETD tandem mass spectrometry

Energy Technology Data Exchange (ETDEWEB)

Nardiello, Donatella; Palermo, Carmen, E-mail: carmen.palermo@unifg.it; Natale, Anna; Quinto, Maurizio; Centonze, Diego

2015-01-07

Highlights: • Multi-enzyme digestion for protein sequencing and characterization by CID/ETD. • Simultaneous use of trypsin/chymotrypsin for the maximization of sequence. • Identification of PTMs, sequence variants and species-specific residues. • Increase of accuracy in sequence assignments by orthogonal fragmentation techniques. - Abstract: A strategy based on a simultaneous multi-enzyme digestion coupled with electron transfer dissociation (ETD) and collision-induced dissociation (CID) was developed for protein sequencing and characterization, as a valid alternative platform in ion-trap based proteomics. The effect of different proteolytic procedures using chymotrypsin, trypsin, a combination of both, and Lys-C, was carefully evaluated in terms of number of identified peptides, protein coverage, and score distribution. A systematic comparison between CID and ETD is shown for the analysis of peptides originating from the in-solution digestion of standard caseins. The best results were achieved with a trypsin/chymotrypsin mix combined with CID and ETD operating in alternating mode. A post-database search validation of MS/MS dataset was performed, then, the matched peptides were cross checked by the evaluation of ion scores, rank, number of experimental product ions, and their relative abundances in the MS/MS spectrum. By integrated CID/ETD experiments, high quality-spectra have been obtained, thus allowing a confirmation of spectral information and an increase of accuracy in peptide sequence assignments. Overlapping peptides, produced throughout the proteins, reduce the ambiguity in mapping modifications between natural variants and animal species, and allow the characterization of post translational modifications. The advantages of using the enzymatic mix trypsin/chymotrypsin were confirmed by the nanoLC and CID/ETD tandem mass spectrometry of goat milk proteins, previously separated by two-dimensional gel electrophoresis.

Effects of Pseudoalteromonas sp. BC228 on digestive enzyme activity and immune response of juvenile sea cucumber ( Apostichopus japonicus)

Science.gov (United States)

Ma, Yuexin; Sun, Feixue; Zhang, Congyao; Bao, Pengyun; Cao, Shuqing; Zhang, Meiyan

2014-12-01

A marine bacterium, Pseudoalteromonas sp. BC228 was supplemented to feed in a feeding experiment aiming to determine its ability of enhancing the digestive enzyme activity and immune response of juvenile Apostichopus japonicus. Sea cucumber individuals were fed with the diets containing 0 (control), 105, 107 and 109 CFU g-1 diet of BC228 for 45 days. Results showed that intestinal trypsin and lipase activities were significantly enhanced by 107 and 109 CFU g-1 diet of BC228 in comparison with control ( P sea cucumber fed the diet supplemented with 107 CFU g-1 diet of BC228 was significantly higher than that of those fed control diet ( P sea cucumber, respectively, in comparison with other diets ( P Sea cucumbers, 10 each diet, were challenged with Vibrio splendidus NB13 after 45 days of feeding. It was found that the cumulative incidence and mortality of sea cucumber fed with BC228 containing diets were lower than those of animals fed control diet. Our findings evidenced that BC228 supplemented in diets improved the digestive enzyme activity of juvenile sea cucumber, stimulated its immune response and enhanced its resistance to the infection of V. splendidus.

Effects of pancreatic digestive enzymes, sodium bicarbonate, and a proton pump inhibitor on steatorrhoea caused by pancreatic diseases.

Science.gov (United States)

Nakamura, T; Takebe, K; Kudoh, K; Ishii, M; Imamura, K; Kikuchi, H; Kasai, F; Tandoh, Y; Yamada, N; Arai, Y

1995-01-01

Forty-five patients with pancreatic steatorrhoea (27 with calcified pancreatitis, 13 with non-calcified pancreatitis, two with pancreaticoduodenectomy, one with total pancreatectomy, and two with pancreatic cancer) were divided into four groups and given the following medication for 2 to 4 weeks: 4 to 6 g/day of sodium bicarbonate (group I); 9 g/day of high-lipase pancreatin (lipase, 56,600 U/g, Fédération Internationale Pharmaceutique (FIP); group II); 12 to 24 tablets or 9.0 g of commercial pancreatic enzyme preparations (group III); or 50 mg of omeprazole (group IV). Faecal fat excretion was evaluated before and after drug administration. Faecal fat excretion was reduced by 2.9 g (range, 1.7 to 5.0 g) in group I; 8.8 g (range, 2.9 to 39.9 g) in group II; 10.8 g (range, 2.3 to 21.8 g) in group III; and 4.3 g (range, 3.6 to 5.6 g) in group IV. The pancreatic digestive enzyme preparation was more effective than sodium bicarbonate and agents that raise the pH of the upper small intestine (such as proton-pump inhibitors) in reducing faecal fat excretion. The results indicate that all of the preparations used are effective against mild pancreatic steatorrhoea. If the condition is more advanced, however, a massive dosage of pancreatic digestive enzyme and possibly the combined use of an agent to raise the pH of the upper small intestine are likely to be effective.

INVITRO DIGESTIBILITY OF PROTEIN FROM BARLEY AND OTHER CEREALS

DEFF Research Database (Denmark)

Buchmann, N. B.

1979-01-01

An in vitro method for measuring barley protein digestibility is presented. Samples were first incubated with pepsin in HCl; pancreatin was then added concomitantly with a bacteriostatic borate buffer. After TCA-precipitation, soluble nitrogen was measured. The digestion was unaffected...... by accumulated free amino acids. There were no free amino acids following pepsin treatment, but the essential ones were well liberated by pancreatin. Results for barley grown in the field or in pots, and for decortified barley fractions agreed with true digestibility values determined with rats. Of these samples...... digestibility depended on the type of enzyme and on the enzyme-to-substrate ratio....

Rapid preparation of functional polysaccharides from Pyropia yezoensis by microwave-assistant rapid enzyme digest system.

Science.gov (United States)

Lee, Ji-Hyeok; Kim, Hyung-Ho; Ko, Ju-Young; Jang, Jun-Ho; Kim, Gwang-Hoon; Lee, Jung-Suck; Nah, Jae-Woon; Jeon, You-Jin

2016-11-20

This study describes a simple preparation of functional polysaccharides from Pyropia yezoensis using a microwave-assistant rapid enzyme digest system (MAREDS) with various carbohydrases, and evaluates their antioxidative effects. Polysaccharide hydrolysates were prepared using MAREDS under different hydrolytic conditions of the carbohydrases and microwave powers. Polysaccharides less than 10kDa (Low molecular weight polysaccharides, LMWP, ≤10kDa) were efficiently obtained using an ultrafiltration (molecular weight cut-off of 10kDa). MAREDS increases AMG activation via an increased degree of hydrolysis; the best AMG hydrolysate was prepared using a 10:1 ratio of substrate to enzyme for 2h in MAREDS with 400W. LMWP consisted of galactose (27.3%), glucose (64.5%), and mannose (8.3%) from the AMG hydrolysate had stronger antioxidant effects than the high molecular weight polysaccharides (>10kDa). We rapidly prepared functional LMWPs by using MAREDS with carbohydrases, and suggest that LMWP might be potentially a valuable algal polysaccharide antioxidant. Copyright © 2016 Elsevier Ltd. All rights reserved.

Digestibility of gluten proteins is reduced by baking and enhanced by starch digestion.

Science.gov (United States)

Smith, Frances; Pan, Xiaoyan; Bellido, Vincent; Toole, Geraldine A; Gates, Fred K; Wickham, Martin S J; Shewry, Peter R; Bakalis, Serafim; Padfield, Philip; Mills, E N Clare

2015-10-01

Resistance of proteins to gastrointestinal digestion may play a role in determining immune-mediated adverse reactions to foods. However, digestion studies have largely been restricted to purified proteins and the impact of food processing and food matrices on protein digestibility is poorly understood. Digestibility of a total gliadin fraction (TGF), flour (cv Hereward), and bread was assessed using in vitro batch digestion with simulated oral, gastric, and duodenal phases. Protein digestion was monitored by SDS-PAGE and immunoblotting using monoclonal antibodies specific for celiac-toxic sequences (QQSF, QPFP) and starch digestion by measuring undigested starch. Whereas the TGF was rapidly digested during the gastric phase the gluten proteins in bread were virtually undigested and digested rapidly during the duodenal phase only if amylase was included. Duodenal starch digestion was also slower in the absence of duodenal proteases. The baking process reduces the digestibility of wheat gluten proteins, including those containing sequences active in celiac disease. Starch digestion affects the extent of protein digestion, probably because of gluten-starch complex formation during baking. Digestion studies using purified protein fractions alone are therefore not predictive of digestion in complex food matrices. © 2015 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

ICRPfinder: a fast pattern design algorithm for coding sequences and its application in finding potential restriction enzyme recognition sites

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Stafford Phillip

2009-09-01

Full Text Available Abstract Background Restriction enzymes can produce easily definable segments from DNA sequences by using a variety of cut patterns. There are, however, no software tools that can aid in gene building -- that is, modifying wild-type DNA sequences to express the same wild-type amino acid sequences but with enhanced codons, specific cut sites, unique post-translational modifications, and other engineered-in components for recombinant applications. A fast DNA pattern design algorithm, ICRPfinder, is provided in this paper and applied to find or create potential recognition sites in target coding sequences. Results ICRPfinder is applied to find or create restriction enzyme recognition sites by introducing silent mutations. The algorithm is shown capable of mapping existing cut-sites but importantly it also can generate specified new unique cut-sites within a specified region that are guaranteed not to be present elsewhere in the DNA sequence. Conclusion ICRPfinder is a powerful tool for finding or creating specific DNA patterns in a given target coding sequence. ICRPfinder finds or creates patterns, which can include restriction enzyme recognition sites, without changing the translated protein sequence. ICRPfinder is a browser-based JavaScript application and it can run on any platform, in on-line or off-line mode.

Effect of Probiotic Bacillus megaterium PTB 1.4 on the Population of Intestinal Microflora, Digestive Enzyme Activity and the Growth of Catfish (Clarias sp.

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Wahyu Afrilasari

2016-10-01

Full Text Available This study aimed to analyze the effect of Bacillus megaterium PTB 1.4 on the population of intestinal microflora, digestive enzyme activity, and the growth of catfish. Gnotobiotic and normal fish were used. Treatment using gnotobiotic was divided into gnoto (with feed and 100 μg/mL rifampicin and gnotoplus (with feed, 100 μg/mL rifampicin, and 1% probiotic; whereas treatment using normal fish was divided into normalplus (with feed and 1% probiotic and normal (only feed. The amount of bacteria on gastrointestinal tract was measured 30 days after treatments using the total plate count method. The results indicated no significant difference in bacterial growth between gnotobiotic and normal fish. The total amount of probiotic bacteria with normalplus treatment was significantly different with gnotoplus. The activity of protease and amylase enzymes, and specific growth rate in normalplus treatment were significantly higher (p < 0.05 than other treatments. Bacillus megaterium PTB 1.4 increased the activity of digestive enzymes and the growth of catfish.

Identification of Pork Contamination in Meatballs of Indonesia Local Market Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP Analysis

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Yuny Erwanto

2014-10-01

Full Text Available This research applied and evaluated a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP using cytochrome b gene to detect pork contamination in meatballs from local markets in Surabaya and Yogyakarta regions, Indonesia. To confirm the effectiveness and specificity of this fragment, thirty nine DNA samples from different meatball shops were isolated and amplified, and then the PCR amplicon was digested by BseDI restriction enzyme to detect the presence of pork in meatballs. BseDI restriction enzyme was able to cleave porcine cytochrome b gene into two fragments (131 bp and 228 bp. Testing the meatballs from the local market showed that nine of twenty meatball shops in Yogyakarta region were detected to have pork contamination, but there was no pork contamination in meatball shops in Surabaya region. In conclusion, specific PCR amplification of cytochrome b gen and cleaved by BseDI restriction enzymes seems to be a powerful technique for the identification of pork presence in meatball because of its simplicity, specificity and sensitivity. Furthermore, pork contamination intended for commercial products of sausage, nugget, steak and meat burger can be checked. The procedure is also much cheaper than other methods based on PCR, immunodiffusion and other techniques that need expensive equipment.

Identification of Pork Contamination in Meatballs of Indonesia Local Market Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis.

Science.gov (United States)

Erwanto, Yuny; Abidin, Mohammad Zainal; Sugiyono, Eko Yasin Prasetyo Muslim; Rohman, Abdul

2014-10-01

This research applied and evaluated a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using cytochrome b gene to detect pork contamination in meatballs from local markets in Surabaya and Yogyakarta regions, Indonesia. To confirm the effectiveness and specificity of this fragment, thirty nine DNA samples from different meatball shops were isolated and amplified, and then the PCR amplicon was digested by BseDI restriction enzyme to detect the presence of pork in meatballs. BseDI restriction enzyme was able to cleave porcine cytochrome b gene into two fragments (131 bp and 228 bp). Testing the meatballs from the local market showed that nine of twenty meatball shops in Yogyakarta region were detected to have pork contamination, but there was no pork contamination in meatball shops in Surabaya region. In conclusion, specific PCR amplification of cytochrome b gen and cleaved by BseDI restriction enzymes seems to be a powerful technique for the identification of pork presence in meatball because of its simplicity, specificity and sensitivity. Furthermore, pork contamination intended for commercial products of sausage, nugget, steak and meat burger can be checked. The procedure is also much cheaper than other methods based on PCR, immunodiffusion and other techniques that need expensive equipment.

FEATURES DIGESTION OF STURGEON SPECIES (ACIPENSERIDAE (REVIEW

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M. Simon

2016-09-01

Full Text Available Purpose. To review scientific sources are about the anatomical, physiological and biochemical characteristics of the digestive system and proper digestion process in the sturgeon species (Acipenseridae. Outline the common anatomical and morphological characteristics of the gastrointestinal tract. Consider the activity of digestive enzymes and the influence of various factors. Findings. Review of scientific papers reveals that although the digestion of sturgeon are broadly similar to those of the cartilaginous and bony fish, there are a number of species specificity. In particular, sturgeon enzymes have a wider temperature and hydrogen ranges. It is confirmed that temperature adaptations of digestive system poikilothermic organ-isms are realised mainly thanks to reorganisations of fermental systems. It is shown that enzymes in sturgeons are adjustable, as their activity level significantly changes under the influence of divalent metal ions (Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+. The assumption that evolutionary adaptation of hydrolytic function of intestines of fishes to temperature conditions of an inhabitancy takes place, apparently, is made. The paper describes the effect of sex and age factors on the level of activity of enzymes of sturgeons. Set out the regularities of circadian rhythms of the fish of this family. Showed specific features of the liver and its involvement in lipid metabolism and antioxidant defense system. Practical value. The knowledge of hydrolysis characteristics of a diet of sturgeon species is important for the efficiency estimation of feeding and understanding of evolutionary and ecological aspects of digestion physiology. Systematized data on the digestive system of fish sturgeon species are of interest a wide range of research in two main areas. Firstly, although the sturgeon are relict species, but the adaptation of their digestive system is still going on, allowing you to analyze the evolutionary development of the

Microbial diversity and digestive enzyme activities in the gut of earthworms found in sawmill industries in Abeokuta, Nigeria

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Bamidele Julius A.

2014-09-01

Full Text Available The growing demand for wood has resulted in large volumes of wood wastes that are daily released to the soil from the activities of sawmills in South-Western Nigeria. In an attempt to setup a bioremediation model for sawdust, this study therefore aimed at evaluating microbial diversity, and the level of digestive enzymes in the gut of earthworms (Eudrilus eugeniae, Libyodrilus violaceous and Hyperiodrilus africanus of sawmill origin. Four major sawmills located in Abeokuta (7o9’12” N - 3o19’35” E, namely Lafenwa, Sapon, Isale-Ake and Kotopo sawmills were used for this study. The arboretum of the Federal University of Agriculture, Abeokuta was used as control. Gut microbial analysis was carried out using the pour-plate method while digestive enzyme activities in the earthworm guts were done by the spectrophotometric method. Higher microbial counts (28.5±0.1x10³-97.0±0.1x10³cfu for bacteria and 7.0±0.1x10³-96.0±0.1x10³cfu for fungi and microbial diversity were recorded in the gut of earthworms of the sawmill locations than those of the control site (17.5±0.1x10³cfu for bacteria and 4.5±0.1x10³cfu for fungi. Streptococcus mutans and Proteus spp.were common in the gut of E. eugeniae, and L. violaceous from the study sawmills, while Streptococcus mutans were also identified in H. africanus, but absent in the gut of E. eugeniae from the control site. Cellulase (48.67±0.02mg/g and lipase (1.81±0.01mg/g activities were significantly higher (p<0.05 in the gut of earthworms from the control site than those of the study sawmills. Furthermore, amylase (α and β activity was highest in the gut of earthworms from the sawmills. Variations observed in the gut microbial and digestive enzyme activities of earthworms from the study sawmills as compared to the control site suggests that earthworms, especially E. eugeniae, could be a better organism for use as bioremediator of wood wastes. Rev. Biol. Trop. 62 (3: 1241-1249. Epub 2014 September

Microbial diversity and digestive enzyme activities in the gut of earthworms found in sawmill industries in Abeokuta, Nigeria.

Science.gov (United States)

Bamidele, Julius A; Idowu, Adewunmi B; Ademolu, Kehinde O; Atayese, Adijat O

2014-09-01

The growing demand for wood has resulted in large volumes of wood wastes that are daily released to the soil from the activities of sawmills in South-Western Nigeria. In an attempt to setup a bioremediation model for sawdust, this study therefore aimed at evaluating microbial diversity, and the level of digestive enzymes in the gut of earthworms (Eudrilus eugeniae, Libyodrilus violaceous and Hyperiodrilus africanus) of sawmill origin. Four major sawmills located in Abeokuta (7°9'12" N- 3°19'35" E), namely Lafenwa, Sapon, Isale-Ake and Kotopo sawmills were used for this study. The arboretum of the Federal University of Agriculture, Abeokuta was used as control. Gut microbial analysis was carried out using the pour-plate method while digestive enzyme activities in the earthworm guts were done by the spectrophotometric method. Higher microbial counts (28.5 ± 0.1 x 10(3)-97.0 ± 0.1 x 10(3) cfu for bacteria and 7.0 ± 0.1x 10(3)-96.0 ± 0.1 x 10(3) cfu for fungi) and microbial diversity were recorded in the gut of earthworms of the sawmill locations than those of the control site (17.5 ± 0.1 x10(3) cfu for bacteria and 4.5 ± 0.1 x 10(3) cfu for fungi). Streptococcus mutans and Proteus spp. were common in the gut of E. eugeniae, and L. violaceous from the study sawmills, while Streptococcus mutans were also identified in H. africanus, but absent in the gut of E. eugeniae from the control site. Cellulase (48.67 ± 0.02 mg/g) and lipase (1.81 ± 0.01 mg/g) activities were significantly higher (p earthworms from the control site than those of the study sawmills. Furthermore, amylase (α and β) activity was highest in the gut of earthworms from the sawmills. Variations observed in the gut microbial and digestive enzyme activities of earthworms from the study sawmills as compared to the control site suggests that earthworms, especially E. eugeniae, could be a better organism for use as bioremediator of wood wastes.

Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation.

Science.gov (United States)

Yegnasubramanian, Srinivasan; Lin, Xiaohui; Haffner, Michael C; DeMarzo, Angelo M; Nelson, William G

2006-02-09

Hypermethylation of CpG island (CGI) sequences is a nearly universal somatic genome alteration in cancer. Rapid and sensitive detection of DNA hypermethylation would aid in cancer diagnosis and risk stratification. We present a novel technique, called COMPARE-MS, that can rapidly and quantitatively detect CGI hypermethylation with high sensitivity and specificity in hundreds of samples simultaneously. To quantitate CGI hypermethylation, COMPARE-MS uses real-time PCR of DNA that was first digested by methylation-sensitive restriction enzymes and then precipitated by methyl-binding domain polypeptides immobilized on a magnetic solid matrix. We show that COMPARE-MS could detect five genome equivalents of methylated CGIs in a 1000- to 10,000-fold excess of unmethylated DNA. COMPARE-MS was used to rapidly quantitate hypermethylation at multiple CGIs in >155 prostate tissues, including benign and malignant prostate specimens, and prostate cell lines. This analysis showed that GSTP1, MDR1 and PTGS2 CGI hypermethylation as determined by COMPARE-MS could differentiate between malignant and benign prostate with sensitivities >95% and specificities approaching 100%. This novel technology could significantly improve our ability to detect CGI hypermethylation.

An easily regenerable enzyme reactor prepared from polymerized high internal phase emulsions

International Nuclear Information System (INIS)

Ruan, Guihua; Wu, Zhenwei; Huang, Yipeng; Wei, Meiping; Su, Rihui; Du, Fuyou

2016-01-01

A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith (polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal polymerization of a high internal phase emulsion containing styrene, divinylbenzene and polyglutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The performance of the resultant enzyme reactor was assessed according to the conversion ability of N_α-benzoyl-L-arginine ethyl ester to N_α-benzoyl-L-arginine, and the protein digestibility of bovine serum albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited high enzyme immobilization efficiency and fast and easy-control protein digestibility. BSA and Cyt-C could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with 4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor was a good selector potentially applied in proteomics and catalysis areas. - Graphical abstract: Schematic illustration of preparation of hypercrosslinking polyHIPE immobilized enzyme reactor for on-column protein digestion. - Highlights: • A reactor was prepared and used for enzyme immobilization and continuous on-column protein digestion. • The new polyHIPE IMER was quite suit for protein digestion with good properties. • On-column digestion revealed that the IMER was easy regenerated by HCl without any structure destruction.

An easily regenerable enzyme reactor prepared from polymerized high internal phase emulsions

Energy Technology Data Exchange (ETDEWEB)

Ruan, Guihua, E-mail: guihuaruan@hotmail.com [Guangxi Key Laboratory of Electrochemical and Magnetochemical Functional Materials, College of Chemistry and Bioengineering, Guilin University of Technology, Guangxi 541004 (China); Guangxi Collaborative Innovation Center for Water Pollution Control and Water Safety in Karst Area, Guilin University of Technology, Guilin 541004 (China); Wu, Zhenwei; Huang, Yipeng; Wei, Meiping; Su, Rihui [Guangxi Key Laboratory of Electrochemical and Magnetochemical Functional Materials, College of Chemistry and Bioengineering, Guilin University of Technology, Guangxi 541004 (China); Du, Fuyou, E-mail: dufu2005@126.com [Guangxi Key Laboratory of Electrochemical and Magnetochemical Functional Materials, College of Chemistry and Bioengineering, Guilin University of Technology, Guangxi 541004 (China); Guangxi Collaborative Innovation Center for Water Pollution Control and Water Safety in Karst Area, Guilin University of Technology, Guilin 541004 (China)

2016-04-22

A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith (polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal polymerization of a high internal phase emulsion containing styrene, divinylbenzene and polyglutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The performance of the resultant enzyme reactor was assessed according to the conversion ability of N{sub α}-benzoyl-L-arginine ethyl ester to N{sub α}-benzoyl-L-arginine, and the protein digestibility of bovine serum albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited high enzyme immobilization efficiency and fast and easy-control protein digestibility. BSA and Cyt-C could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with 4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor was a good selector potentially applied in proteomics and catalysis areas. - Graphical abstract: Schematic illustration of preparation of hypercrosslinking polyHIPE immobilized enzyme reactor for on-column protein digestion. - Highlights: • A reactor was prepared and used for enzyme immobilization and continuous on-column protein digestion. • The new polyHIPE IMER was quite suit for protein digestion with good properties. • On-column digestion revealed that the IMER was easy regenerated by HCl without any structure destruction.

EVOLUTIONARY TRANSITIONS IN ENZYME ACTIVITY OF ANT FUNGUS GARDENS

DEFF Research Database (Denmark)

De Fine Licht, Henrik H; Schiøtt, Morten; Mueller, Ulrich G

2010-01-01

an association with a monophyletic clade of specialized symbionts. In conjunction with the transition to specialized symbionts, the ants advanced in colony size and social complexity. Here we provide a comparative study of the functional specialization in extracellular enzyme activities in fungus gardens across...... the attine phylogeny. We show that, relative to sister clades, gardens of higher-attine ants have enhanced activity of protein-digesting enzymes, whereas gardens of leaf-cutting ants also have increased activity of starch-digesting enzymes. However, the enzyme activities of lower-attine fungus gardens...... are targeted primarily towards partial degradation of plant cell walls, reflecting a plesiomorphic state of non-domesticated fungi. The enzyme profiles of the higher-attine and leaf-cutting gardens appear particularly suited to digest fresh plant materials and to access nutrients from live cells without major...

Substantial differences in bias between single-digest and double-digest RAD-seq libraries: A case study.

Science.gov (United States)

Flanagan, Sarah P; Jones, Adam G

2018-03-01

The trade-offs of using single-digest vs. double-digest restriction site-associated DNA sequencing (RAD-seq) protocols have been widely discussed. However, no direct empirical comparisons of the two methods have been conducted. Here, we sampled a single population of Gulf pipefish (Syngnathus scovelli) and genotyped 444 individuals using RAD-seq. Sixty individuals were subjected to single-digest RAD-seq (sdRAD-seq), and the remaining 384 individuals were genotyped using a double-digest RAD-seq (ddRAD-seq) protocol. We analysed the resulting Illumina sequencing data and compared the two genotyping methods when reads were analysed either together or separately. Coverage statistics, observed heterozygosity, and allele frequencies differed significantly between the two protocols, as did the results of selection components analysis. We also performed an in silico digestion of the Gulf pipefish genome and modelled five major sources of bias: PCR duplicates, polymorphic restriction sites, shearing bias, asymmetric sampling (i.e., genotyping fewer individuals with sdRAD-seq than with ddRAD-seq) and higher major allele frequencies. This combination of approaches allowed us to determine that polymorphic restriction sites, an asymmetric sampling scheme, mean allele frequencies and to some extent PCR duplicates all contribute to different estimates of allele frequencies between samples genotyped using sdRAD-seq versus ddRAD-seq. Our finding that sdRAD-seq and ddRAD-seq can result in different allele frequencies has implications for comparisons across studies and techniques that endeavour to identify genomewide signatures of evolutionary processes in natural populations. © 2017 John Wiley & Sons Ltd.

Assessment of enzyme supplementation on growth performance and apparent nutrient digestibility in diets containing undecorticated sunflower seed meal in layer chicks.

Science.gov (United States)

Fafiolu, A O; Oduguwa, O O; Jegede, A V; Tukura, C C; Olarotimi, I D; Teniola, A A; Alabi, J O

2015-08-01

Six hundred and forty one-day-old layer chicks were used to investigate the effect of replacing soybean meal with undecorticated sunflower seed meal protein for protein at 0, 25, 50, and 75% levels. Diets were without enzyme supplementation or with enzyme supplementation with four replications of twenty birds. Growth performance and nutrient utilization were determined. Proximate composition of the undecorticated sunflower seed meal used revealed that undecorticated sunflower seed meal contained 925.9, 204.5, 336.2, 215.1, 52.0 and 192.2g/kg dry matter, crude protein, ether extract, crude fibre, ash and soluble carbohydrates, respectively. Results showed that the final weight of 484.4 g/bird was obtained for birds on 75% undecorticated sunflower seed meal diet, while the lowest value of 472.2g/bird was obtained for birds on 25% undecorticated sunflower seed meal diet. Weight gain per bird per day was not significantly (P > 0.05) affected as the level of undecorticated sunflower seed meal increased in the diets. Feed intake per bird per day increased (P meal inclusion in the diet. However, enzyme supplementation of the diets showed marked (P meal inclusion in the diet while crude protein digestibility progressively reduced (P meal increased in the diet. Ash digestibility values were, however, increased (P meal increased in the diets. Birds on enzyme-supplemented diets consistently showed superior (P meal in the diets of layer chicks showed a similar body weight gain/bird/day with the control. Undecorticated sunflower seed meal used in this study is a good source of crude protein, ether extract, and amino acids and had the potential to serve as feeding stuffs as replacement for soybeans. The nutritive value of undecorticated sunflower seed meal was improved for layer chicks with exogenous enzyme supplementation. © 2015 Poultry Science Association Inc.

Evolutionary transitions in enzyme activity of ant fungus gardens.

Science.gov (United States)

De Fine Licht, Henrik H; Schiøtt, Morten; Mueller, Ulrich G; Boomsma, Jacobus J

2010-07-01

Fungus-growing (attine) ants and their fungal symbionts passed through several evolutionary transitions during their 50 million year old evolutionary history. The basal attine lineages often shifted between two main cultivar clades, whereas the derived higher-attine lineages maintained an association with a monophyletic clade of specialized symbionts. In conjunction with the transition to specialized symbionts, the ants advanced in colony size and social complexity. Here we provide a comparative study of the functional specialization in extracellular enzyme activities in fungus gardens across the attine phylogeny. We show that, relative to sister clades, gardens of higher-attine ants have enhanced activity of protein-digesting enzymes, whereas gardens of leaf-cutting ants also have increased activity of starch-digesting enzymes. However, the enzyme activities of lower-attine fungus gardens are targeted primarily toward partial degradation of plant cell walls, reflecting a plesiomorphic state of nondomesticated fungi. The enzyme profiles of the higher-attine and leaf-cutting gardens appear particularly suited to digest fresh plant materials and to access nutrients from live cells without major breakdown of cell walls. The adaptive significance of the lower-attine symbiont shifts remains unclear. One of these shifts was obligate, but digestive advantages remained ambiguous, whereas the other remained facultative despite providing greater digestive efficiency.

A Mimicking-of-DNA-Methylation-Patterns Pipeline for Overcoming the Restriction Barrier of Bacteria

Science.gov (United States)

Zhang, Guoqiang; Wang, Wenzhao; Deng, Aihua; Sun, Zhaopeng; Zhang, Yun; Liang, Yong; Che, Yongsheng; Wen, Tingyi

2012-01-01

Genetic transformation of bacteria harboring multiple Restriction-Modification (R-M) systems is often difficult using conventional methods. Here, we describe a mimicking-of-DNA-methylation-patterns (MoDMP) pipeline to address this problem in three difficult-to-transform bacterial strains. Twenty-four putative DNA methyltransferases (MTases) from these difficult-to-transform strains were cloned and expressed in an Escherichia coli strain lacking all of the known R-M systems and orphan MTases. Thirteen of these MTases exhibited DNA modification activity in Southwestern dot blot or Liquid Chromatography–Mass Spectrometry (LC–MS) assays. The active MTase genes were assembled into three operons using the Saccharomyces cerevisiae DNA assembler and were co-expressed in the E. coli strain lacking known R-M systems and orphan MTases. Thereafter, results from the dot blot and restriction enzyme digestion assays indicated that the DNA methylation patterns of the difficult-to-transform strains are mimicked in these E. coli hosts. The transformation of the Gram-positive Bacillus amyloliquefaciens TA208 and B. cereus ATCC 10987 strains with the shuttle plasmids prepared from MoDMP hosts showed increased efficiencies (up to four orders of magnitude) compared to those using the plasmids prepared from the E. coli strain lacking known R-M systems and orphan MTases or its parental strain. Additionally, the gene coding for uracil phosphoribosyltransferase (upp) was directly inactivated using non-replicative plasmids prepared from the MoDMP host in B. amyloliquefaciens TA208. Moreover, the Gram-negative chemoautotrophic Nitrobacter hamburgensis strain X14 was transformed and expressed Green Fluorescent Protein (GFP). Finally, the sequence specificities of active MTases were identified by restriction enzyme digestion, making the MoDMP system potentially useful for other strains. The effectiveness of the MoDMP pipeline in different bacterial groups suggests a universal potential

Improvement of Raw-Starch Digestibility of Amylase from Aspergillus awamori by Gamma Radiation Mutation for Alcohol Production

International Nuclear Information System (INIS)

Kanlayakrit, Werasit; Piadang, Nattayana; Maweang, Metinee

2006-09-01

Aspergillus awamori was induced to mutation by gamma ray to improve raw-starch digestibility of amylase enzyme. Twenty fungal colonies were selected base on and amylase and glucoamylase activities including raw starch digestibility. The result showed that isolate A a(i)-2(16) was the best isolate for raw-starch digestion (65.64 %). It produced extracellular amylase enzyme which showed highest raw-starch digestibility more than wild type about 2 folds. Based on enzymes from solid culture showed activities higher from liquid medium. Therefore solid culture is suitable for fungal enzyme production.

Effects of dietary fructooligosaccharide on digestive enzyme activities, intestinal microflora and morphology of male broilers.

Science.gov (United States)

Xu, Z R; Hu, C H; Xia, M S; Zhan, X A; Wang, M Q

2003-06-01

Two hundred forty male Avian Farms broiler chicks, 1 d of age, were randomly allocated to four treatments, each of which had five pens of 12 chicks per pen. The chicks were used to investigate the effects of fructooligosaccharide (FOS) on digestive enzyme activities and intestinal microflora and morphology. The chicks received the same basal diet based on corn-soybean meal, and FOS was added to the basal diet at 0, 2.0, 4.0, and 8.0 g/kg diet at the expense of corn. Addition of 4.0 g/kg FOS to the basal diet significantly increased average daily gain of broilers. The feed-to-gain ratios were significantly decreased for the birds fed diets with 2.0 and 4.0 g/kg FOS versus the control. Addition of 4.0 g/kg FOS enhanced the growth of Bifidobacterium and Lactobacillus, but inhibited Escherichia coli in the small intestinal and cecal digesta. Supplementation of 2.0 or 4.0 g/kg FOS to chicks significantly improved the activities of amylase compared to the control (12.80 or 14.75 vs. 8.42 Somogyi units). A significant increase in the activities of total protease was observed in 4.0 g/kg FOS-treated birds versus controls (83.91 vs. 65.97 units). Morphology data for the duodenum, jejunum, and ileum showed no significant differences for villus height, crypt depth, or microvillus height at the duodenum. By contrast, addition of 4.0 g/kg FOS significantly increased ileal villus height, jejunal and ileal microvillus height, and villus-height-to-crypt-depth ratios at the jejunum and ileum and decreased crypt depth at the jejunum and ileum. However, addition of 8.0 g/kg FOS had no significant effect on growth performance, digestive enzyme activities, intestinal microflora, or morphology.

Trypsin immobilization in ordered porous polymer membranes for effective protein digestion

International Nuclear Information System (INIS)

Qiao, Juan; Kim, Jin Yong; Wang, Yuan Yuan; Qi, Li; Wang, Fu Yi; Moon, Myeong Hee

2016-01-01

Fast and effective protein digestion is a vital process for mass spectrometry (MS) based protein analysis. This study introduces a porous polymer membrane enzyme reactor (PPMER) coupled to nanoflow liquid chromatography-tandem MS (nLC-ESI-MS/MS) for on-line digestion and analysis of proteins. Poly (styrene-co-maleic anhydride) (PS-co-MAn) was fabricated by the breath figure method to make a porous polymer membrane in which the MAn group was covalently bound to enzyme. Based on this strategy, microscale PPMER (μPPMER) was constructed for on-line connection with the nLC-ESI-MS/MS system. Its capability for enzymatic digestion with bovine serum albumin (BSA) was evaluated with varied digestion periods. The on-line proteolysis of BSA and subsequent analysis with μPPMER-nLC-ESI-MS/MS revealed that peptide sequence coverage increased from 10.3% (digestion time 10 min) to 89.1% (digestion time 30 min). μPPMER can efficiently digest proteins due to the microscopic confinement effect, showing its potential application in fast protein identification and protease immobilization. Applications of on-line digestion using μPPMER with human plasma and urinary proteome samples showed that the developed on-line method yielded equivalent or better performance in protein coverage and identified more membrane proteins than the in-solution method. This may be due to easy accommodation of hydrophobic membrane proteins within membrane pores. - Highlights: • A porous polymer membrane enzyme reactor was developed. • Breath figure method was used for the fabrication of porous polymer membrane. • The enzyme reactor was coupled to nLC-ESI-MS/MS for proteins on-line digestion.

Trypsin immobilization in ordered porous polymer membranes for effective protein digestion

Energy Technology Data Exchange (ETDEWEB)

Qiao, Juan [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing 100190 (China); Kim, Jin Yong [Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seoul 120-749 (Korea, Republic of); Wang, Yuan Yuan [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing 100190 (China); Qi, Li, E-mail: qili@iccas.ac.cn [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing 100190 (China); Wang, Fu Yi [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing 100190 (China); Moon, Myeong Hee, E-mail: mhmoon@yonsei.ac.kr [Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seoul 120-749 (Korea, Republic of)

2016-02-04

Fast and effective protein digestion is a vital process for mass spectrometry (MS) based protein analysis. This study introduces a porous polymer membrane enzyme reactor (PPMER) coupled to nanoflow liquid chromatography-tandem MS (nLC-ESI-MS/MS) for on-line digestion and analysis of proteins. Poly (styrene-co-maleic anhydride) (PS-co-MAn) was fabricated by the breath figure method to make a porous polymer membrane in which the MAn group was covalently bound to enzyme. Based on this strategy, microscale PPMER (μPPMER) was constructed for on-line connection with the nLC-ESI-MS/MS system. Its capability for enzymatic digestion with bovine serum albumin (BSA) was evaluated with varied digestion periods. The on-line proteolysis of BSA and subsequent analysis with μPPMER-nLC-ESI-MS/MS revealed that peptide sequence coverage increased from 10.3% (digestion time 10 min) to 89.1% (digestion time 30 min). μPPMER can efficiently digest proteins due to the microscopic confinement effect, showing its potential application in fast protein identification and protease immobilization. Applications of on-line digestion using μPPMER with human plasma and urinary proteome samples showed that the developed on-line method yielded equivalent or better performance in protein coverage and identified more membrane proteins than the in-solution method. This may be due to easy accommodation of hydrophobic membrane proteins within membrane pores. - Highlights: • A porous polymer membrane enzyme reactor was developed. • Breath figure method was used for the fabrication of porous polymer membrane. • The enzyme reactor was coupled to nLC-ESI-MS/MS for proteins on-line digestion.

Digestive physiology comparisons of aquatic invertebrates in the Upper Mississippi River Basin

Science.gov (United States)

Sauey, Blake W.; Amberg, Jon J.; Cooper, Scott T.; Grunwald, Sandra K.; Haro, Roger J.; Gaikowski, Mark

2016-01-01

Limited information is available on the composition of digestive enzymes present in unionid mussels and the zebra mussel, Dreissena polymorpha. Available information is nearly exclusive to species used for culture purposes. A commercially available enzyme assay kit was used to examine the effect of habitat within an ecosystem, season, and species on the activities of several digestive enzymes. We used Amblema plicata to represent native unionids, D. polymorpha, and also Hydropsyche orris as an outgroup to compare differences between mussels and other macroinvertebrates. The data indicated that neither location nor time affect the activities of the digestive enzymes tested; species was the only factor to affect the activity. Differences were found mostly between four enzymes: naphthol-AS-BI-phosphohydrolase, acid phosphatase, alkaline phosphatase, and β-galactosidase.

Application of Pineapple Juice in the Fish Digestion Process for Carcinogenic Liver Fluke Metacercaria Collection

Science.gov (United States)

Sripan, Panupan; Aukkanimart, Ratchadawan; Boonmars, Thidarut; Pranee, Sriraj; Songsri, Jiraporn; Boueroy, Parichart; Khueangchaingkhwang, Sukhonthip; Pumhirunroj, Benjamabhorn; Artchayasawat, Atchara

2017-01-01

Pepsin is common digestive enzyme used for fish digestion in the laboratory to collect trematode metacercariae. In a field study, to survey the infected fish is needed a huge yield of pepsin and it is very expensive. Therefore, our purpose of this study was to investigate the candidate enzyme from pineapple juice which has a digestive enzyme called bromelain, a mixture of proteolytic enzymes, to digest fish in order to harvest metacercariae. Fish were divided into 2 groups: one group in which metacercariae were harvested using acid pepsin as a control and other groups in which the fish was digested using fresh pineapple juices. The results showed that pineapple juice is able to digest fish similarly to pepsin. The Pattavia pineapple juice had the highest number of metacercariae similar to the control. For Trat Si Thong pineapple juice, we found the number of metacercariae was less than control. This result suggests that the Pattavia pineapple juice was optimal juice for fish digestion to metacercaria collection and can be used instread of pepsin acid. PMID:28441786

Random Tagging Genotyping by Sequencing (rtGBS, an Unbiased Approach to Locate Restriction Enzyme Sites across the Target Genome.

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Elena Hilario

Full Text Available Genotyping by sequencing (GBS is a restriction enzyme based targeted approach developed to reduce the genome complexity and discover genetic markers when a priori sequence information is unavailable. Sufficient coverage at each locus is essential to distinguish heterozygous from homozygous sites accurately. The number of GBS samples able to be pooled in one sequencing lane is limited by the number of restriction sites present in the genome and the read depth required at each site per sample for accurate calling of single-nucleotide polymorphisms. Loci bias was observed using a slight modification of the Elshire et al.some restriction enzyme sites were represented in higher proportions while others were poorly represented or absent. This bias could be due to the quality of genomic DNA, the endonuclease and ligase reaction efficiency, the distance between restriction sites, the preferential amplification of small library restriction fragments, or bias towards cluster formation of small amplicons during the sequencing process. To overcome these issues, we have developed a GBS method based on randomly tagging genomic DNA (rtGBS. By randomly landing on the genome, we can, with less bias, find restriction sites that are far apart, and undetected by the standard GBS (stdGBS method. The study comprises two types of biological replicates: six different kiwifruit plants and two independent DNA extractions per plant; and three types of technical replicates: four samples of each DNA extraction, stdGBS vs. rtGBS methods, and two independent library amplifications, each sequenced in separate lanes. A statistically significant unbiased distribution of restriction fragment size by rtGBS showed that this method targeted 49% (39,145 of BamH I sites shared with the reference genome, compared to only 14% (11,513 by stdGBS.

Carbohydrate digestion in Lutzomyia longipalpis' larvae (Diptera - Psychodidae).

Science.gov (United States)

Vale, Vladimir F; Moreira, Bruno H; Moraes, Caroline S; Pereira, Marcos H; Genta, Fernando A; Gontijo, Nelder F

2012-10-01

Lutzomyia longipalpis is the principal species of phlebotomine incriminated as vector of Leishmania infantum, the etiological agent of visceral leishmaniasis in the Americas. Despite its importance as vector, almost nothing related to the larval biology, especially about its digestive system has been published. The objective of the present study was to obtain an overview of carbohydrate digestion by the larvae. Taking in account that phlebotomine larvae live in the soil rich in decaying materials and microorganisms we searched principally for enzymes capable to hydrolyze carbohydrates present in this kind of substrate. The principal carbohydrases encountered in the midgut were partially characterized. One of them is a α-amylase present in the anterior midgut. It is probably involved with the digestion of glycogen, the reserve carbohydrate of fungi. Two other especially active enzymes were present in the posterior midgut, a membrane bound α-glucosidase and a membrane bound trehalase. The first, complete the digestion of glycogen and the other probably acts in the digestion of trehalose, a carbohydrate usually encountered in microorganisms undergoing hydric stress. In a screening done with the use of p-nitrophenyl-derived substrates other less active enzymes were also observed in the midgut. A general view of carbohydrate digestion in L. longipalpis was presented. Our results indicate that soil microorganisms appear to be the main source of nutrients for the larvae. Copyright © 2012 Elsevier Ltd. All rights reserved.

Protein digestion in ruminants

African Journals Online (AJOL)

a balance between synthesis and hydrolysis. Aside from .... be used to follow the synthesis of this protein fraction. (Clarke, 1977a) .... form of digestive enzymes, urea and ammonia (Egan, ..... decreasing urine-nitrogen excretion (Thornton, Bird,.

Digestibility, growth, blood chemistry, and enzyme activity of juvenile Oreochromis niloticus fed isocaloric diets containing animal and plant byproducts

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Magnolia Montoya-Mejía

Full Text Available ABSTRACT In this work, we studied the digestibility, growth, blood chemistry, and enzyme activity of Nile tilapia (Oreochromis niloticus juveniles (0.95±0.18 g using different animal (fish silage meal, whey meal, bovine blood meal, and red crab meal and plant (extruded bean, extruded chickpea meal, coconut paste, Jatropha curcas meal, and chickpea meal dietary byproducts. Nine isocaloric diets (321.92±9.10 kcal g−1 were evaluated for 60 days. The highest digestibility of crude protein values for animal and plant sources were obtained for the whey (93.6 and extruded bean meal (90.5 diets, respectively. The final body weight was higher for the red crab and extruded chickpea meal diets, meanwhile the fish silage and red crab byproducts obtained the highest protein efficiency ratio. Hematocrit was similar among the diets of each byproduct source and presented correlation with growth parameters. The highest glucose, cholesterol, and triglyceride values were obtained for fish silage (138.0, 260.5, and 389.0 mg dL−1, respectively and whey meal (174.5, 242.3, and 284.0 mg dL−1, respectively groups. A positive correlation was found between the digestibility of crude protein of ingredients and chymotrypsin activity. Oreochromis niloticus is able to better utilize fish silage, whey, extruded bean, and extruded chickpea byproducts, adjusting its digestive physiology. Such ingredients can be used for formulating cheaper and efficient tilapia diets.

[Effect of light intensity on the growth and digestive enzyme activity of juvenile sea cucumber Apostichopus japonicas under two kinds of culture methods].

Science.gov (United States)

Wei, Zi-Zhong; Zhao, Wen

2014-01-01

The effects of light intensity (0, 1000, 2000 and 3000 1x) on the growth and digestive enzyme activity of juvenile sea cucumber Apostichopus japonicus under two kinds of culture methods (compound Chinese medicine preparation and microbial preparation) were studied. Results showed that the relative mass gain rate (WGR) and the specific growth rate (SGR) of juvenile sea cucumber were significantly affected by light intensity (P sea cucumber under different light intensities were 2000 1x > 1000 1x > 3000 1x > 0 1x. Under the same light intensity, the growth of juvenile sea cucumber under the two kinds of culture methods were significantly different (P sea cucumber. The order of amylase and lipase activity was 2000 1x > 1000 1x > 3000 1x > 0 1x, while that of protease activity was 1000 1x > 2000 1x > 0 1x > 3000 1x. Under the same light intensity, the digestive enzyme activities of the Chinese medicine treatment were generally higher than those of the microbial treatment.

Effect of potential probiotic Rhodotorula benthica D30 on the growth performance, digestive enzyme activity and immunity in juvenile sea cucumber Apostichopus japonicus.

Science.gov (United States)

Wang, Ji-hui; Zhao, Liu-qun; Liu, Jin-feng; Wang, Han; Xiao, Shan

2015-04-01

The effects of dietary addition of yeast Rhodotorula benthica (R. benthica) D30 which isolated from local sea mud at levels of 0 (control), 10(5), 10(6) and 10(7) CFU/g feed on the growth performance, digestive enzyme activity, immunity and disease resistance of juvenile sea cucumber Apostichopus japonicus were investigated. It was shown that dietary addition of R. benthica D30 significantly increased the growth rates of sea cucumbers (p  0.05). It was observed that adding R. benthica D30 could significantly decrease the cumulative mortality of sea cucumbers. The present study demonstrated that dietary addition of R. benthica D30 could increase growth performance and some digestive enzyme activities, improve immunity and disease resistance of A. japonicus. And the medium (10(6) CFU) and high (10(7) CFU) additional levels showed better effects. It suggests that yeast R. benthica D30 could be a good probiotic for aquaculture. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification of planorbids from Venezuela by polymerase chain reaction amplification and restriction fragment length polymorphism of internal transcriber spacer of the RNA ribosomal gene

Directory of Open Access Journals (Sweden)

Caldeira Roberta L

2000-01-01

Full Text Available Snails of the genus Biomphalaria from Venezuela were subjected to morphological assessment as well as polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP analysis. Morphological identification was carried out by comparison of characters of the shell and the male and female reproductive apparatus. The PCR-RFLP involved amplification of the internal spacer region ITS1 and ITS2 of the RNA ribosomal gene and subsequent digestion of this fragment by the restriction enzymes DdeI, MnlI, HaeIII and MspI. The planorbids were compared with snails of the same species and others reported from Venezuela and present in Brazil, Cuba and Mexico. All the enzymes showed a specific profile for each species, that of DdeI being the clearest. The snails were identified as B. glabrata, B. prona and B. kuhniana.

Detection of the Single Nucleotide Polymorphism at Position rs2735940 in the Human Telomerase Reverse Transcriptase Gene by the Introduction of a New Restriction Enzyme Site for the PCR-RFLP Assay.

Science.gov (United States)

Wang, Sihua; Ding, Mingcui; Duan, Xiaoran; Wang, Tuanwei; Feng, Xiaolei; Wang, Pengpeng; Yao, Wu; Wu, Yongjun; Yan, Zhen; Feng, Feifei; Yu, Songcheng; Wang, Wei

2017-09-01

It has been shown that the single nucleotide polymorphism (SNP) of the rs2735940 site in the human telomerase reverse transcriptase ( hTERT ) gene is associated with increased cancer risk. The traditional method to detect SNP genotypes is polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). However, there is a limitation to utilizing PCR-RFLP due to a lack of proper restriction enzyme sites at many polymorphic loci. This study used an improved PCR-RFLP method with a mismatched base for detection of the SNP rs2735940. A new restriction enzyme cutting site was created by created restriction site PCR (CRS-PCR), and in addition, the restriction enzyme Msp I for CRS-PCR was cheaper than other enzymes. We used this novel assay to determine the allele frequencies in 552 healthy Chinese Han individuals, and found the allele frequencies to be 63% for allele C and 37% for allele T In summary, the modified PCR-RFLP can be used to detect the SNP of rs2735940 with low cost and high efficiency. © 2017 by the Association of Clinical Scientists, Inc.

Cloning and restriction enzyme mapping of ribosomal DNA of Giardia duodenalis, Giardia ardeae and Giardia muris.

Science.gov (United States)

van Keulen, H; Campbell, S R; Erlandsen, S L; Jarroll, E L

1991-06-01

In an attempt to study Giardia at the DNA sequence level, the rRNA genes of three species, Giardia duodenalis, Giardia ardeae and Giardia muris were cloned and restriction enzyme maps were constructed. The rDNA repeats of these Giardia show completely different restriction enzyme recognition patterns. The size of the rDNA repeat ranges from approximately 5.6 kb in G. duodenalis to 7.6 kb in both G. muris and G. ardeae. These size differences are mainly attributable to the variation in length of the spacer. Minor differences exist among these Giardia in the sizes of their small subunit rRNA and the internal transcribed spacer between small and large subunit rRNA. The genetic maps were constructed by sequence analysis of the DNA around the 5' and 3' ends of the mature rRNA genes and between the rRNA covering the 5.8S rRNA gene and internal transcribed spacer. Comparison of the 5.8S rDNA and 3' end of large subunit rDNA from these three Giardia species showed considerable sequence variation, but the rDNA sequences of G. duodenalis and G. ardeae appear more closely related to each other than to G. muris.

Telomere Restriction Fragment (TRF) Analysis.

Science.gov (United States)

Mender, Ilgen; Shay, Jerry W

2015-11-20

restriction enzyme recognition sites within TTAGGG tandem telomeric repeats, therefore digestion of genomic DNA, not telomeric DNA, with a combination of 6 base restriction endonucleases reduces genomic DNA size to less than 800 bp.

Milk glucosidase activity enables suckled pup starch digestion

Science.gov (United States)

Starch requires six enzymes for digestion to free glucose: two amylases (salivary and pancreatic) and four mucosal maltase activities; sucrase-isomaltase and maltase-glucoamylase. All are deficient in suckling rodents. The objective of this study is to test (13)C-starch digestion before weaning by m...

Angiotensin I-Converting Enzyme (ACE Inhibitory Activity and ACE Inhibitory Peptides of Salmon (Salmo salar Protein Hydrolysates Obtained by Human and Porcine Gastrointestinal Enzymes

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Małgorzata Darewicz

2014-08-01

Full Text Available The objectives of the present study were two-fold: first, to detect whether salmon protein fractions possess angiotensin I-converting enzyme (ACE inhibitory properties and whether salmon proteins can release ACE inhibitory peptides during a sequential in vitro hydrolysis (with commercial porcine enzymes and ex vivo digestion (with human gastrointestinal enzymes. Secondly, to evaluate the ACE inhibitory activity of generated hydrolysates. A two-step ex vivo and in vitro model digestion was performed to simulate the human digestion process. Salmon proteins were degraded more efficiently by porcine enzymes than by human gastrointestinal juices and sarcoplasmic proteins were digested/hydrolyzed more easily than myofibrillar proteins. The ex vivo digested myofibrillar and sarcoplasmic duodenal samples showed IC50 values (concentration required to decrease the ACE activity by 50% of 1.06 and 2.16 mg/mL, respectively. The in vitro hydrolyzed myofibrillar and sarcoplasmic samples showed IC50 values of 0.91 and 1.04 mg/mL, respectively. Based on the results of in silico studies, it was possible to identify 9 peptides of the ex vivo hydrolysates and 7 peptides of the in vitro hydrolysates of salmon proteins of 11 selected peptides. In both types of salmon hydrolysates, ACE-inhibitory peptides IW, IY, TVY and VW were identified. In the in vitro salmon protein hydrolysates an ACE-inhibitory peptides VPW and VY were also detected, while ACE-inhibitory peptides ALPHA, IVY and IWHHT were identified in the hydrolysates generated with ex vivo digestion. In our studies, we documented ACE inhibitory in vitro effects of salmon protein hydrolysates obtained by human and as well as porcine gastrointestinal enzymes.

The Secretion and Action of Brush Border Enzymes in the Mammalian Small Intestine.

Science.gov (United States)

Hooton, Diane; Lentle, Roger; Monro, John; Wickham, Martin; Simpson, Robert

2015-01-01

Microvilli are conventionally regarded as an extension of the small intestinal absorptive surface, but they are also, as latterly discovered, a launching pad for brush border digestive enzymes. Recent work has demonstrated that motor elements of the microvillus cytoskeleton operate to displace the apical membrane toward the apex of the microvillus, where it vesiculates and is shed into the periapical space. Catalytically active brush border digestive enzymes remain incorporated within the membranes of these vesicles, which shifts the site of BB digestion from the surface of the enterocyte to the periapical space. This process enables nutrient hydrolysis to occur adjacent to the membrane in a pre-absorptive step. The characterization of BB digestive enzymes is influenced by the way in which these enzymes are anchored to the apical membranes of microvilli, their subsequent shedding in membrane vesicles, and their differing susceptibilities to cleavage from the component membranes. In addition, the presence of active intracellular components of these enzymes complicates their quantitative assay and the elucidation of their dynamics. This review summarizes the ontogeny and regulation of BB digestive enzymes and what is known of their kinetics and their action in the peripheral and axial regions of the small intestinal lumen.

A new restriction endonuclease-based method for highly-specific detection of DNA targets from methicillin-resistant Staphylococcus aureus.

Directory of Open Access Journals (Sweden)

Maria W Smith

Full Text Available PCR multiplexing has proven to be challenging, and thus has provided limited means for pathogen genotyping. We developed a new approach for analysis of PCR amplicons based on restriction endonuclease digestion. The first stage of the restriction enzyme assay is hybridization of a target DNA to immobilized complementary oligonucleotide probes that carry a molecular marker, horseradish peroxidase (HRP. At the second stage, a target-specific restriction enzyme is added, cleaving the target-probe duplex at the corresponding restriction site and releasing the HRP marker into solution, where it is quantified colorimetrically. The assay was tested for detection of the methicillin-resistant Staphylococcus aureus (MRSA pathogen, using the mecA gene as a target. Calibration curves indicated that the limit of detection for both target oligonucleotide and PCR amplicon was approximately 1 nM. Sequences of target oligonucleotides were altered to demonstrate that (i any mutation of the restriction site reduced the signal to zero; (ii double and triple point mutations of sequences flanking the restriction site reduced restriction to 50-80% of the positive control; and (iii a minimum of a 16-bp target-probe dsDNA hybrid was required for significant cleavage. Further experiments showed that the assay could detect the mecA amplicon from an unpurified PCR mixture with detection limits similar to those with standard fluorescence-based qPCR. Furthermore, addition of a large excess of heterologous genomic DNA did not affect amplicon detection. Specificity of the assay is very high because it involves two biorecognition steps. The proposed assay is low-cost and can be completed in less than 1 hour. Thus, we have demonstrated an efficient new approach for pathogen detection and amplicon genotyping in conjunction with various end-point and qPCR applications. The restriction enzyme assay may also be used for parallel analysis of multiple different amplicons from the same

Digestive System (For Teens)

Science.gov (United States)

... in the stomach. Sometimes, though, a bacterium called Helicobacter pylori or the chronic use of certain medications weakens ... can affect the whole gastrointestinal tract from the mouth to the anus as well ... organs to produce enzymes and other substances that aid in digestion. ...

The effect of an angiotensin-converting enzyme inhibitor on water and electrolyte balance in water-restricted sheep

Directory of Open Access Journals (Sweden)

R.A. Meintjies

1999-07-01

Full Text Available The importance of angiotensin II in the regulation of water and electrolyte balance in sheep is questionable. In this trial the effects of an angiotensin-converting enzyme (ACE inhibitor were quantified in sheep on restricted water intake. Comparing the phase of water restriction only with that of water restriction plus ACE inhibition, significant increases were observed during the latter phase in urine volume, sodium and potassium excretion via the urine, sodium concentration in the plasma and osmolar clearance. Urine osmolarity decreased with inhibition of angiotensin II formation while variables such as water, sodium and potassium loss via the faeces were unaffected. Most of the renal effects of ACE inhibition, except the increase in urinary potassium excretion, were explicable in terms of the established functions of angiotensin II. Furthermore, results of this trial indicate that angiotensin II has no significant effect on the intestine in regulating water and electrolyte excretion via the faeces.

Simultaneous and rapid differential diagnosis of Mycoplasma genitalium and Ureaplasma urealyticum based on a polymerase chain reaction-restriction fragment length polymorphism

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R Mirnejad

2011-01-01

Full Text Available Objectives: The aim of this investigation was to simultaneously detect and differentiate Mycoplasma genitalium and Ureaplasma urealyticum in female patients suffering from genital complications by polymerase chain reaction (PCR-restriction fragment length polymorphism (RFLP. Materials and Methods : Genital swabs were taken from 210 patients. They were transported to the laboratory in phosphate-buffered saline. For PCR, samples were analysed with genus-specific MyUu-R and MyUu-F primers. This primer set, which was originally designed in our laboratory, amplified a 465 bp fragment (M. genitalium and a 559 bp fragment (U. urealyticum. Samples containing a band of the expected sizes for the Mycoplasma strains were subjected to digestion with a restriction endonuclease enzyme of TaqI and Cac8I. Results: Of the 210 samples, a total of 100 (47.6% samples were found to be positive for Mycoplasmas (seven M. genitalium isolates, 3.3%; and 89 U. urealyticum isolates, 42.4%, and coinfections with both species were detected in four samples (1.9%. The PCR-RFLP results showed that M. genitalium and U. urealyticum are different by enzyme patterns. Conclusion: PCR-RFLP offers a rapid and easily applicable protocol to simultaneous detection and differentiation of M. genitalium and U. urealyticum from clinical samples when specific primers and restriction enzymes are used.

Formation of reactive aldehydes (MDA, HHE, HNE) during the digestion of cod liver oil: comparison of human and porcine in vitro digestion models.

Science.gov (United States)

Tullberg, Cecilia; Larsson, Karin; Carlsson, Nils-Gunnar; Comi, Irene; Scheers, Nathalie; Vegarud, Gerd; Undeland, Ingrid

2016-03-01

In this work, we investigated lipid oxidation of cod liver oil during gastrointestinal (GI) digestion using two types of in vitro digestion models. In the first type of model, we used human GI juices, while we used digestive enzymes and bile from porcine origin in the second type of model. Human and porcine models were matched with respect to factors important for lipolysis, using a standardized digestion protocol. The digests were analysed for reactive oxidation products: malondialdehyde (MDA), 4-hydroxy-trans-2-nonenal (HNE), and 4-hydroxy-trans-2-hexenal (HHE) by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS), and for free fatty acids (FFA) obtained during the digestion by gas chromatography-mass spectrometry (GC-MS). The formation of the oxidation products MDA, HHE, and HNE was low during the gastric digestion, however, it increased during the duodenal digestion. The formation of the oxidation products reached higher levels when digestive juices of human origin were used (60 μM of MDA, 0.96 μM of HHE, and 1.6 μM of HNE) compared to when using enzymes and bile of porcine origin (9.8, and 0.36 μM of MDA; 0.16, and 0.026 μM of HHE; 0.23, and 0.005 μM of HNE, respectively, in porcine models I and II). In all models, FFA release was only detected during the intestinal step, and reached up to 31% of total fatty acids (FA). The findings in this work may be of importance when designing oxidation oriented lipid digestion studies.

Development of a universal double-digest RAD sequencing approach for a group of nonmodel, ecologically and economically important insect and fish taxa.

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Burford Reiskind, M O; Coyle, K; Daniels, H V; Labadie, P; Reiskind, M H; Roberts, N B; Roberts, R B; Schaff, J; Vargo, E L

2016-11-01

The generation of genome-scale data is critical for a wide range of questions in basic biology using model organisms, but also in questions of applied biology in nonmodel organisms (agriculture, natural resources, conservation and public health biology). Using a genome-scale approach on a diverse group of nonmodel organisms and with the goal of lowering costs of the method, we modified a multiplexed, high-throughput genomic scan technique utilizing two restriction enzymes. We analysed several pairs of restriction enzymes and completed double-digestion RAD sequencing libraries for nine different species and five genera of insects and fish. We found one particular enzyme pair produced consistently higher number of sequence-able fragments across all nine species. Building libraries off this enzyme pair, we found a range of usable SNPs between 4000 and 37 000 SNPS per species and we found a greater number of usable SNPs using reference genomes than de novo pipelines in STACKS. We also found fewer reads in the Read 2 fragments from the paired-end Illumina Hiseq run. Overall, the results of this study provide empirical evidence of the utility of this method for producing consistent data for diverse nonmodel species and suggest specific considerations for sequencing analysis strategies. © 2016 John Wiley & Sons Ltd.

Maternal protein restriction affects gene expression and enzyme activity of intestinal disaccharidases in adult rat offspring

International Nuclear Information System (INIS)

Pinheiro, D.F.; Pacheco, P.D.G.; Alvarenga, P.V.; Buratini, J. Jr; Castilho, A.C.S.; Lima, P.F.; Sartori, D.R.S.; Vicentini-Paulino, M.L.M.

2013-01-01

This study investigated the consequences of intrauterine protein restriction on the gastrointestinal tract and particularly on the gene expression and activity of intestinal disaccharidases in the adult offspring. Wistar rat dams were fed isocaloric diets containing 6% protein (restricted, n = 8) or 17% protein (control, n = 8) throughout gestation. Male offspring (n = 5-8 in each group) were evaluated at 3 or 16 weeks of age. Maternal protein restriction during pregnancy produced offspring with growth restriction from birth (5.7 ± 0.1 vs 6.3 ± 0.1 g; mean ± SE) to weaning (42.4 ± 1.3 vs 49.1 ± 1.6 g), although at 16 weeks of age their body weight was similar to control (421.7 ± 8.9 and 428.5 ± 8.5 g). Maternal protein restriction also increased lactase activity in the proximal (0.23 ± 0.02 vs 0.15 ± 0.02), medial (0.30 ± 0.06 vs 0.14 ± 0.01) and distal (0.43 ± 0.07 vs 0.07 ± 0.02 U·g -1 ·min -1 ) small intestine, and mRNA lactase abundance in the proximal intestine (7.96 ± 1.11 vs 2.38 ± 0.47 relative units) of 3-week-old offspring rats. In addition, maternal protein restriction increased sucrase activity (1.20 ± 0.02 vs 0.91 ± 0.02 U·g -1 ·min -1 ) and sucrase mRNA abundance (4.48 ± 0.51 vs 1.95 ± 0.17 relative units) in the duodenum of 16-week-old rats. In conclusion, the present study shows for the first time that intrauterine protein restriction affects gene expression of intestinal enzymes in offspring

Maternal protein restriction affects gene expression and enzyme activity of intestinal disaccharidases in adult rat offspring

Energy Technology Data Exchange (ETDEWEB)

Pinheiro, D.F.; Pacheco, P.D.G.; Alvarenga, P.V.; Buratini, J. Jr; Castilho, A.C.S.; Lima, P.F.; Sartori, D.R.S.; Vicentini-Paulino, M.L.M. [Departamento de Fisiologia, Instituto de Biociências, Universidade Estadual Paulista, Botucatu, SP (Brazil)

2013-03-15

This study investigated the consequences of intrauterine protein restriction on the gastrointestinal tract and particularly on the gene expression and activity of intestinal disaccharidases in the adult offspring. Wistar rat dams were fed isocaloric diets containing 6% protein (restricted, n = 8) or 17% protein (control, n = 8) throughout gestation. Male offspring (n = 5-8 in each group) were evaluated at 3 or 16 weeks of age. Maternal protein restriction during pregnancy produced offspring with growth restriction from birth (5.7 ± 0.1 vs 6.3 ± 0.1 g; mean ± SE) to weaning (42.4 ± 1.3 vs 49.1 ± 1.6 g), although at 16 weeks of age their body weight was similar to control (421.7 ± 8.9 and 428.5 ± 8.5 g). Maternal protein restriction also increased lactase activity in the proximal (0.23 ± 0.02 vs 0.15 ± 0.02), medial (0.30 ± 0.06 vs 0.14 ± 0.01) and distal (0.43 ± 0.07 vs 0.07 ± 0.02 U·g{sup -1}·min{sup -1}) small intestine, and mRNA lactase abundance in the proximal intestine (7.96 ± 1.11 vs 2.38 ± 0.47 relative units) of 3-week-old offspring rats. In addition, maternal protein restriction increased sucrase activity (1.20 ± 0.02 vs 0.91 ± 0.02 U·g{sup -1}·min{sup -1}) and sucrase mRNA abundance (4.48 ± 0.51 vs 1.95 ± 0.17 relative units) in the duodenum of 16-week-old rats. In conclusion, the present study shows for the first time that intrauterine protein restriction affects gene expression of intestinal enzymes in offspring.

Re-evaluating the kinetics of ATP hydrolysis during initiation of DNA sliding by Type III restriction enzymes.

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Tóth, Júlia; Bollins, Jack; Szczelkun, Mark D

2015-12-15

DNA cleavage by the Type III restriction enzymes requires long-range protein communication between recognition sites facilitated by thermally-driven 1D diffusion. This 'DNA sliding' is initiated by hydrolysis of multiple ATPs catalysed by a helicase-like domain. Two distinct ATPase phases were observed using short oligoduplex substrates; the rapid consumption of ∼10 ATPs coupled to a protein conformation switch followed by a slower phase, the duration of which was dictated by the rate of dissociation from the recognition site. Here, we show that the second ATPase phase is both variable and only observable when DNA ends are proximal to the recognition site. On DNA with sites more distant from the ends, a single ATPase phase coupled to the conformation switch was observed and subsequent site dissociation required little or no further ATP hydrolysis. The overall DNA dissociation kinetics (encompassing site release, DNA sliding and escape via a DNA end) were not influenced by the second phase. Although the data simplifies the ATP hydrolysis scheme for Type III restriction enzymes, questions remain as to why multiple ATPs are hydrolysed to prepare for DNA sliding. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Digestion of starch in a dynamic small intestinal model.

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Jaime-Fonseca, M R; Gouseti, O; Fryer, P J; Wickham, M S J; Bakalis, S

2016-12-01

The rate and extent of starch digestion have been linked with important health aspects, such as control of obesity and type-2 diabetes. In vitro techniques are often used to study digestion and simulated nutrient absorption; however, the effect of gut motility is often disregarded. The present work aims at studying fundamentals of starch digestion, e.g. the effect of viscosity on digestibility, taking into account both biochemical and engineering (gut motility) parameters. New small intestinal model (SIM) that realistically mimics gut motility (segmentation) was used to study digestibility and simulated oligosaccharide bio accessibility of (a) model starch solutions; (b) bread formulations. First, the model was compared with the rigorously mixed stirred tank reactor (STR). Then the effects of enzyme concentration/flow rate, starch concentration, and digesta viscosity (addition of guar gum) were evaluated. Compared to the STR, the SIM showed presence of lag phase when no digestive processes could be detected. The effects of enzyme concentration and flow rate appeared to be marginal in the region of mass transfer limited reactions. Addition of guar gum reduced simulated glucose absorption by up to 45 % in model starch solutions and by 35 % in bread formulations, indicating the importance of chyme rheology on nutrient bioaccessibility. Overall, the work highlights the significance of gut motility in digestive processes and offers a powerful tool in nutritional studies that, additionally to biochemical, considers engineering aspects of digestion. The potential to modulate food digestibility and nutrient bioaccessibility by altering food formulation is indicated.

Molecular detection and PCR-RFLP analysis using Pst1 and Alu1 of multidrug resistant Klebsiella pneumoniae causing urinary tract infection in women in the eastern part of Bangladesh

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Golam Mahmudunnabi

2018-06-01

Full Text Available Klebsiella pneumoniae is the second leading causative agent of UTI. In this study, a rapid combined polymerase chain reaction and restriction fragment length polymorphism analysis was developed to identify K. pneumoniae in women, infected with urinary tract infection in the Sylhet city of Bangladesh. Analysis of 11 isolates from women at the age range of 20–55 from three different hospitals were done firstly by amplification with K. pneumoniae specific ITS primers. All of the 11 collected isolates were amplified in PCR and showed the expected 136 bp products. Then, restriction fragment length polymorphism analysis of 11 isolates were conducted after PCR amplification by 16s rRNA universal primers, followed by subsequent digestion and incubation with two restriction enzymes, Pst1 and Alu1. Seven out of 11 isolates were digested by Pst1 restriction enzymes, six isolates digested by Alu1, and while others were negative for both enzymes. Data results reveal that, women at age between 25 and 50 were digested by both enzymes. A woman aged over than 50 was negative while bellow 20 was digested by only Pst1. The results could pave the tactic for further research in the detection of K. pneumoniae from UTI infected women. Keywords: Klebsiella pneumoniae, ITS-primer, MDR isolates, PCR-RFLP analysis

Digestion-ligation-only Hi-C is an efficient and cost-effective method for chromosome conformation capture.

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Lin, Da; Hong, Ping; Zhang, Siheng; Xu, Weize; Jamal, Muhammad; Yan, Keji; Lei, Yingying; Li, Liang; Ruan, Yijun; Fu, Zhen F; Li, Guoliang; Cao, Gang

2018-05-01

Chromosome conformation capture (3C) technologies can be used to investigate 3D genomic structures. However, high background noise, high costs, and a lack of straightforward noise evaluation in current methods impede the advancement of 3D genomic research. Here we developed a simple digestion-ligation-only Hi-C (DLO Hi-C) technology to explore the 3D landscape of the genome. This method requires only two rounds of digestion and ligation, without the need for biotin labeling and pulldown. Non-ligated DNA was efficiently removed in a cost-effective step by purifying specific linker-ligated DNA fragments. Notably, random ligation could be quickly evaluated in an early quality-control step before sequencing. Moreover, an in situ version of DLO Hi-C using a four-cutter restriction enzyme has been developed. We applied DLO Hi-C to delineate the genomic architecture of THP-1 and K562 cells and uncovered chromosomal translocations. This technology may facilitate investigation of genomic organization, gene regulation, and (meta)genome assembly.

Dry anaerobic digestion of lignocellulosic and protein residues

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Maryam M Kabir

2015-12-01

Full Text Available Utilisation of wheat straw and wool textile waste in dry anaerobic digestion (AD process was investigated. Dry-AD of the individual substrates as well as co-digestion of those were evaluated using different total solid (TS contents ranging between 6 to 30%. Additionally, the effects of the addition of nutrients and cellulose- or protein-degrading enzymes on the performance of the AD process were also investigated. Dry-AD of the wheat straw resulted in methane yields of 0.081 – 0.200 Nm3CH4/kgVS with the lowest and highest values obtained at 30 and 21% TS, respectively. The addition of the cellulolytic enzymes could significantly increase the yield in the reactor containing 13% TS (0.231 Nm3CH4/kg VS. Likewise, degradation of wool textile waste was enhanced significantly at TS of 13% with the addition of the protein-degrading enzyme (0.131 Nm3CH4/kg VS. Furthermore, the co-digestion of these two substrates showed higher methane yields compared with the methane potentials calculated for the individual fractions at all the investigated TS contents due to synergetic effects and better nutritional balance.

Cellulase digestibility of pretreated biomass is limited by cellulose accessibility.

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Jeoh, Tina; Ishizawa, Claudia I; Davis, Mark F; Himmel, Michael E; Adney, William S; Johnson, David K

2007-09-01

Attempts to correlate the physical and chemical properties of biomass to its susceptibility to enzyme digestion are often inconclusive or contradictory depending on variables such as the type of substrate, the pretreatment conditions and measurement techniques. In this study, we present a direct method for measuring the key factors governing cellulose digestibility in a biomass sample by directly probing cellulase binding and activity using a purified cellobiohydrolase (Cel7A) from Trichoderma reesei. Fluorescence-labeled T. reesei Cel7A was used to assay pretreated corn stover samples and pure cellulosic substrates to identify barriers to accessibility by this important component of cellulase preparations. The results showed cellulose conversion improved when T. reesei Cel7A bound in higher concentrations, indicating that the enzyme had greater access to the substrate. Factors such as the pretreatment severity, drying after pretreatment, and cellulose crystallinity were found to directly impact enzyme accessibility. This study provides direct evidence to support the notion that the best pretreatment schemes for rendering biomass more digestible to cellobiohydrolase enzymes are those that improve access to the cellulose in biomass cell walls, as well as those able to reduce the crystallinity of cell wall cellulose.

Biogas performance from co-digestion of Taihu algae and kitchen wastes

International Nuclear Information System (INIS)

Zhao, Ming-Xing; Ruan, Wen-Quan

2013-01-01

Highlights: • Co-digestion mode improves the biogas yield of Taihu algae and kitchen wastes. • Neutral protease enzyme reached maximum in algae only group. • The activity of dehydrogenase enzyme in mixed substrate groups was higher than that of algae and kitchen wastes only group. - Abstract: Co-digestion of Taihu algae with high carbon content substrate can balance the nutrients in the fermentation process. In this study, optimal mixing ratio for co-digestion of Taihu algae and kitchen wastes were investigated in order to improve biogas production potential. The results indicated that the biogas yield reached 388.6 mL/gTS at C/N15:1 group, which was 1.29 and 1.18 times of algae and kitchen wastes only. The maximum concentration of VFA reached 4239 mg/L on 8th day in kitchen wastes group, which was 1.21 times of algae group. Neutral protease enzyme activity in algae group reached maximum of 904.2 μg/(gTS h), while dehydrogenase enzyme at C/N 15:1 group reached maximum of 3402.2 μgTF/(gTS h). The feasibility of adjusting the C/N with co-digestion of Taihu algae and kitchen wastes to increase biogas production was demonstrated. Remarkably, the C/N of 15:1 was found to be the most appropriate ratio

Synergistic function of four novel thermostable glycoside hydrolases from a long-term enriched thermophilic methanogenic digester

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Meng eWang

2015-05-01

Full Text Available In biofuel production from lignocellulose, low thermostability and product inhibition strongly restrict the enzyme activities and production process. Application of multiple thermostable glycoside hydrolases, forming an enzyme cocktail, can result in a synergistic action and therefore improve production efficiency and reduce operational costs. Therefore, increasing enzyme thermostabilities and compatibility are important for the biofuel industry. In this study, we reported the screening, cloning and biochemical characterization of four novel thermostable lignocellulose hydrolases from a metagenomic library of a long-term dry thermophilic methanogenic digester community, which were highly compatible with optimal conditions and specific activities. The optimal temperatures of the four enzymes, β-xylosidase, xylanase, β-glucosidase, and cellulase ranged from 60°C to 75°C, and over 80% residual activities were observed after 2 h incubation at 50°C. Mixtures of these hydrolases retained high residual synergistic activities after incubation with cellulose, xylan, and steam-exploded corncob at 50°C for 72 h. In addition, about 55% dry weight of steam-exploded corncob was hydrolyzed to glucose and xylose by the synergistic action of the four enzymes at 50°C for 48 h. This work suggested that since different enzymes from a same ecosystem could be more compatible, screening enzymes from a long-term enriching community could be a favorable strategy.

Effects of xylitol on carbohydrate digesting enzymes activity, intestinal glucose absorption and muscle glucose uptake: a multi-mode study.

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Chukwuma, Chika Ifeanyi; Islam, Md Shahidul

2015-03-01

The present study investigated the possible mechanism(s) behind the effects of xylitol on carbohydrate digesting enzymes activity, muscle glucose uptake and intestinal glucose absorption using in vitro, ex vivo and in vivo experimental models. The effects of increasing concentrations of xylitol (2.5%-40% or 164.31 mM-2628.99 mM) on alpha amylase and alpha glucosidase activity in vitro and intestinal glucose absorption and muscle glucose uptake were investigated under ex vivo conditions. Additionally, the effects of an oral bolus dose of xylitol (1 g per kg BW) on gastric emptying and intestinal glucose absorption and digesta transit in the different segments of the intestinal tract were investigated in normal and type 2 diabetic rats at 1 hour after dose administration, when phenol red was used as a recovery marker. Xylitol exhibited concentration-dependent inhibition of alpha amylase (IC₅₀ = 1364.04 mM) and alpha glucosidase (IC₅₀ = 1127.52 mM) activity in vitro and small intestinal glucose absorption under ex vivo condition. Xylitol also increased dose dependent muscle glucose uptake with and without insulin, although the uptake was not significantly affected by the addition of insulin. Oral single bolus dose of xylitol significantly delayed gastric emptying, inhibited intestinal glucose absorption but increased the intestinal digesta transit rate in both normal and diabetic rats compared to their respective controls. The data of this study suggest that xylitol reduces intestinal glucose absorption via inhibiting major carbohydrate digesting enzymes, slowing gastric emptying and fastening the intestinal transit rate, but increases muscle glucose uptake in normal and type 2 diabetic rats.

Electricity purchase agreements and distributed energy policies for anaerobic digesters

International Nuclear Information System (INIS)

Binkley, David; Harsh, Stephen; Wolf, Christopher A.; Safferman, Steven; Kirk, Dana

2013-01-01

Anaerobic digestion is increasingly recognized for its ability to produce renewable energy and reduce greenhouse gas emissions from livestock operations. In 2010, there were 2645 U.S. dairy farms with herd sizes large enough to support anaerobic digesters, yet only 156 systems were in operation (U.S. Environmental Protection Agency (U.S. EPA), 2010a. Market Opportunities for Biogas Recovery Systems at U.S. Livestock Facilities. AgSTAR Program; U.S. Environmental Protection Agency (U.S. EPA), 2011. Operational Anaerobic Digesters, Sorted by State (Dairy). AgSTAR Program.). This study analyzes the net present value of digester systems under alternative electricity purchase agreements and how returns are affected by standby charges, net metering policies and the use of feed-in-tariffs. In order for digester potential to be fully realized on a state or national level, changes to distributed energy policy are required. Results indicated that standby charges can reduce revenues from offsetting electricity by an average of nearly 20%. Net metering rules limit participation among larger farms and negatively affect profitability by restricting engine–generator size. Lastly, the effectiveness of a fixed price feed-in-tariff policy for digesters is significantly affected by project size differentiation. Digester energy policies are similar nationwide, making this study useful for government regulatory agencies and digester owners throughout the U.S. - Highlights: ► Anaerobic digester net present value was examined over a range of herd sizes. ► Standby charges reduce electricity sales revenues by an average of nearly 20%. ► Net metering rules reduce profitability by restricting engine–generator size. ► Feed-in-tariffs for digesters are significantly affected by project size.

Extraction of intracellular protein from Chlorella pyrenoidosa using a combination of ethanol soaking, enzyme digest, ultrasonication and homogenization techniques.

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Zhang, Ruilin; Chen, Jian; Zhang, Xuewu

2018-01-01

Due to the rigid cell wall of Chlorella species, it is still challenging to effectively extract significant amounts of protein. Mass methods were used for the extraction of intracellular protein from microalgae with biological, mechanical and chemical approaches. In this study, based on comparison of different extraction methods, a new protocol was established to maximize extract amounts of protein, which was involved in ethanol soaking, enzyme digest, ultrasonication and homogenization techniques. Under the optimized conditions, 72.4% of protein was extracted from the microalgae Chlorella pyrenoidosa, which should contribute to the research and development of Chlorella protein in functional food and medicine. Copyright © 2017 Elsevier Ltd. All rights reserved.

Screening exogenous fibrolytic enzyme preparations for improved in vitro digestibility of bermudagrass haylage.

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Romero, J J; Zarate, M A; Arriola, K G; Gonzalez, C F; Silva-Sanchez, C; Staples, C R; Adesogan, A T

2015-04-01

Our objectives were to evaluate the effects of 12 exogenous fibrolytic enzyme products (EFE) on ruminal in vitro neutral detergent fiber digestibility (NDFD) and preingestive hydrolysis of a 4-wk regrowth of bermudagrass haylage (BH), to examine the accuracy of predicting NDFD with EFE activity measures, and to examine the protein composition of the most and least effective EFE at increasing NDFD. In experiment 1, effects of 12 EFE on NDFD of BH were tested. Enzymes were applied in quadruplicate to culture tubes containing ground BH. The suspension was incubated for 24 h at 25 °C before addition of rumen fluid media and further incubation for 24 h at 39 °C. The experiment was repeated twice. In addition, regression relationships between EFE activity measures and NDFD were examined. Compared with the values for the control, 9 EFE-treated substrates had greater NDFD (37.8 to 40.4 vs. 35.6%), 6 had greater total VFA concentration (59.1 to 61.2 vs. 55.4 mM), and 4 had lower acetate-to-propionate ratios (3.03 to 3.16 vs. 3.24). In experiment 2, EFE effects on preingestive fiber hydrolysis were evaluated by incubating enzyme-treated and untreated bermudagrass suspensions in quadruplicate for 24 h at 25 °C and examining fiber hydrolysis measures. Compared with values for the control, 3 EFE reduced neutral detergent fiber concentration (62.8 to 63.7 vs. 67.3%), 10 increased release of water-soluble carbohydrates (26.8 to 58.5 vs. 22.8 mg/g), and 8 increased release of ferulic acid (210 to 391 vs. 198 μg/g). Regression analyses revealed that enzyme activities accurately [coefficient of determination (R(2)) = 0.98] predicted preingestive hydrolysis measures (water-soluble carbohydrates, ferulic acid), moderately (R(2) = 0.47) predicted neutral detergent fiber hydrolysis, but poorly (R(2) ≤ 0.1) predicted dry matter and NDFD. In experiment 3, proteomic tools were used to examine the protein composition of the most and least effective EFE at improving NDFD. Relative to

Digestion of isolated legume cells in a stomach-duodenum model: three mechanisms limit starch and protein hydrolysis.

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Bhattarai, Rewati R; Dhital, Sushil; Wu, Peng; Chen, Xiao Dong; Gidley, Michael J

2017-07-19

Retention of intact plant cells to the end of the small intestine leads to transport of entrapped macronutrients such as starch and protein for colonic microbial fermentation, and is a promising mechanism to increase the content of resistant starch in diets. However, the effect of gastro-intestinal bio-mechanical processing on the intactness of plant cells and the subsequent resistance to enzymatic digestion of intracellular starch and protein are not well understood. In this study, intact cells isolated from legume cotyledons are digested in a laboratory model which mimics the mechanical and biochemical conditions of the rat stomach and duodenum. The resulting digesta are characterised in terms of cell (wall) integrity as well as intracellular starch and protein hydrolysis. The cells remained essentially intact in the model with negligible (ca. 2-3%) starch or protein digestion; however when the cells were mechanically broken and digested in the model, the hydrolysis was increased to 45-50% suggesting that intact cellular structures could survive the mixing regimes in the model stomach and duodenum sufficiently to prevent digestive enzyme access. Apart from intact cell walls providing effective barrier properties, they also limit digestibility by restricting starch gelatinisation during cooking, and significant non-specific binding of α-amylase is observed to both intact and broken cell wall components, providing a third mechanism hindering starch hydrolysis. The study suggests that the preservation of intactness of plant cells, such as from legumes, could be a viable approach to achieve the targeted delivery of resistant starch to the colon.

Validation of an in vitro model for predicting rumen and total-tract fiber digestibility in dairy cows fed corn silages with different in vitro neutral detergent fiber digestibilities at 2 levels of dry matter intake.

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Lopes, F; Cook, D E; Combs, D K

2015-01-01

An in vivo study was performed to validate an in vitro procedure that predicts rate of fiber digestion and total-tract neutral detergent fiber digestibility (TTNDFD). Two corn silages that differed in fiber digestibility were used in this trial. The corn silage with lower fiber digestibility (LFDCS) had the TTNDFD prediction of 36.0% of total NDF, whereas TTNDFD for the corn silage with higher fiber digestibility (HFDCS) was 44.9% of total neutral detergent fiber (NDF). Two diets (1 with LFDCS and 1 with HFDCS) were formulated and analyzed using the in vitro assay to predict the TTNDFD and rumen potentially digestible NDF (pdNDF) digestion rate. Similar diets were fed to 8 ruminally cannulated, multiparous, high-producing dairy cows in 2 replicated 4×4 Latin squares with 21-d periods. A 2×2 factorial arrangement of treatments was used with main effects of intake (restricted to approximately 90% of ad libitum intake vs. ad libitum) and corn silage of different fiber digestibility. Treatments were restricted and ad libitum LFDCS as well as restricted and ad libitum HFDCS. The input and output values predicted from the in vitro model were compared with in vivo measurements. The pdNDF intake predicted by the in vitro model was similar to pdNDF intake observed in vivo. Also, the pdNDF digestion rate predicted in vitro was similar to what was observed in vivo. The in vitro method predicted TTNDFD of 50.2% for HFDCS and 42.9% for LFDCS as a percentage of total NDF in the diets, whereas the in vivo measurements of TTNDFD averaged 50.3 and 48.6% of total NDF for the HFDCS and LFDCS diets, respectively. The in vitro TTNDFD assay predicted total-tract NDF digestibility of HFDCS diets similar to the digestibility observed in vivo, but for LFDCS diets the assay underestimated the digestibility compared with in vivo. When the in vitro and in vivo measurements were compared without intake effect (ad libitum and restricted) considering only diet effect of silage fiber

Bacillus Probiotic Enzymes: External Auxiliary Apparatus to Avoid Digestive Deficiencies, Water Pollution, Diseases, and Economic Problems in Marine Cultivated Animals.

Science.gov (United States)

Olmos Soto, Jorge

Exploitation of marine fishes is the main source of several life-supporting feed compounds such as proteins, lipids, and carbohydrates that maintain the production of most trading marine organisms by aquaculture. However, at this rate the marine inventory will go to the end soon, since fishery resources are finite. In this sense, the availability of the principal ingredients obtained from marine fishes is going to decrease considerably, increasing the diet prices and affecting the economy of this activity. Therefore, aquaculture industry needs to find nonexpensive land unconventional resources of protein, carbohydrates, and lipids and use bacterial probiotics to improve digestion-assimilation of these unfamiliar compounds. Bacillus subtilis is a cosmopolitan probiotic bacterium with a great enzymatic profile that could improve nutrient digestion-assimilation, induce healthy growth, and avoid water pollution, decreasing economic problems and increasing yields in the aquaculture industry. In this chapter, we present how Bacillus enzymes can help marine animals to assimilate nutrients from unconventional and economic plant resources. © 2017 Elsevier Inc. All rights reserved.

Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis.

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Turk, Nenad; Milas, Zoran; Mojcec, Vesna; Ruzic-Sabljic, Eva; Staresina, Vilim; Stritof, Zrinka; Habus, Josipa; Postic, Daniele

2009-11-01

A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analysed. For serovar identification, the microscopic agglutination test (MAT) and pulsed-field gel electrophoresis (PFGE) were used. To identify isolates on genomic species level, PCR-based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the following serogroup affinities: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331-bp-long fragment of rrs (16S rRNA gene) digested with endonucleases MnlI and DdeI and real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to Leptospira kirschneri, Icterohaemorrhagiae isolates to Leptospira interrogans and Javanica isolates to Leptospira borgpetersenii. Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analysed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate the diversity of leptospires circulating in Croatia. We point out the usefulness of a combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires.

Maltase-glucoamylase modulates gluconeogenesis and sucrase-isomaltase dominates starch digestion glucogenesis

Science.gov (United States)

Six enzyme activities are needed to digest starch to absorbable free glucose; 2 luminal alpha-amylases (AMY) and 4 mucosal maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) subunit activities are involved in the digestion. The AMY activities break down starch to soluble oligomeric dextrins; mu...

Biochemical, transcriptomic and proteomic analyses of digestion in the scorpion Tityus serrulatus: insights into function and evolution of digestion in an ancient arthropod.

Science.gov (United States)

Fuzita, Felipe J; Pinkse, Martijn W H; Patane, José S L; Juliano, Maria A; Verhaert, Peter D E M; Lopes, Adriana R

2015-01-01

Scorpions are among the oldest terrestrial arthropods and they have passed through small morphological changes during their evolutionary history on land. They are efficient predators capable of capturing and consuming large preys and due to envenomation these animals can become a human health challenge. Understanding the physiology of scorpions can not only lead to evolutionary insights but also is a crucial step in the development of control strategies. However, the digestive process in scorpions has been scarcely studied. In this work, we describe the combinatory use of next generation sequencing, proteomic analysis and biochemical assays in order to investigate the digestive process in the yellow scorpion Tityus serrulatus, mainly focusing in the initial protein digestion. The transcriptome generated database allowed the quantitative identification by mass spectrometry of different enzymes and proteins involved in digestion. All the results suggested that cysteine cathepsins play an important role in protein digestion. Two digestive cysteine cathepsins were isolated and characterized presenting acidic characteristics (pH optima and stability), zymogen conversion to the mature form after acidic activation and a cross-class inhibition by pepstatin. A more elucidative picture of the molecular mechanism of digestion in a scorpion was proposed based on our results from Tityus serrulatus. The midgut and midgut glands (MMG) are composed by secretory and digestive cells. In fasting animals, the secretory granules are ready for the next predation event, containing enzymes needed for alkaline extra-oral digestion which will compose the digestive fluid, such as trypsins, astacins and chitinase. The digestive vacuoles are filled with an acidic proteolytic cocktail to the intracellular digestion composed by cathepsins L, B, F, D and legumain. Other proteins as lipases, carbohydrases, ctenitoxins and a chitolectin with a perithrophin domain were also detected. Evolutionarily

A new assay based on terminal restriction fragment length polymorphism of homocitrate synthase gene fragments for Candida species identification.

Science.gov (United States)

Szemiako, Kasjan; Śledzińska, Anna; Krawczyk, Beata

2017-08-01

Candida sp. have been responsible for an increasing number of infections, especially in patients with immunodeficiency. Species-specific differentiation of Candida sp. is difficult in routine diagnosis. This identification can have a highly significant association in therapy and prophylaxis. This work has shown a new application of the terminal restriction fragment length polymorphism (t-RFLP) method in the molecular identification of six species of Candida, which are the most common causes of fungal infections. Specific for fungi homocitrate synthase gene was chosen as a molecular target for amplification. The use of three restriction enzymes, DraI, RsaI, and BglII, for amplicon digestion can generate species-specific fluorescence labeled DNA fragment profiles, which can be used to determine the diagnostic algorithm. The designed method can be a cost-efficient high-throughput molecular technique for the identification of six clinically important Candida species.

Digestion and microbial protein synthesis in sheep as affected by ...

African Journals Online (AJOL)

Useni , Alain

enzyme (EFE) on the in vitro gas production (GP) and ANKOM digestion systems on the mixture of milled ... determine the EFE effect on the DM, CP and NDF digestion of a mixture of lucerne hay and wheat straw .... and the microbial protein synthesis (MPS) measured as purine derivates (RNA equivalent in µg/DM g) on.

Effects of Bacillus subtilis on the growth performance, digestive enzymes, immune gene expression and disease resistance of white shrimp, Litopenaeus vannamei.

Science.gov (United States)

Zokaeifar, Hadi; Balcázar, José Luis; Saad, Che Roos; Kamarudin, Mohd Salleh; Sijam, Kamaruzaman; Arshad, Aziz; Nejat, Naghmeh

2012-10-01

We studied the effect of two probiotic Bacillus subtilis strains on the growth performance, digestive enzyme activity, immune gene expression and disease resistance of juvenile white shrimp (Litopenaeus vannamei). A mixture of two probiotic strains, L10 and G1 in equal proportions, was administered at two different doses 10(5) (BM5) and 10(8) (BM8) CFU g(-1) feed to shrimp for eight weeks. In comparison to untreated control group, final weight, weight gain and digestive enzyme activity were significantly greater in shrimp fed BM5 and BM8 diets. Significant differences for specific growth rate (SGR) and survival were recorded in shrimp fed BM8 diet as compared with the control; however, no significant differences were recorded for food conversion ratio (FCR) among all the experimental groups. Eight weeks after the start of the feeding period, shrimp were challenged with Vibrio harveyi. Statistical analysis revealed significant differences in shrimp survival between probiotic and control groups. Cumulative mortality of the control group was 63.3%, whereas cumulative mortality of the shrimp that had been given probiotics was 20.0% with BM8 and 33.3% with BM5. Subsequently, real-time PCR was employed to determine the mRNA levels of prophenoloxidase (proPO), peroxinectin (PE), lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) and serine protein (SP). The expression of all immune-related genes studied was significantly up-regulated (P growth performance and disease resistance through an enhanced immune response in shrimp. Copyright © 2012 Elsevier Ltd. All rights reserved.

Adenylyl cyclases in the digestive system.

Science.gov (United States)

Sabbatini, Maria Eugenia; Gorelick, Fred; Glaser, Shannon

2014-06-01

Adenylyl cyclases (ACs) are a group of widely distributed enzymes whose functions are very diverse. There are nine known transmembrane AC isoforms activated by Gαs. Each has its own pattern of expression in the digestive system and differential regulation of function by Ca(2+) and other intracellular signals. In addition to the transmembrane isoforms, one AC is soluble and exhibits distinct regulation. In this review, the basic structure, regulation and physiological roles of ACs in the digestive system are discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

Research Applications of Proteolytic Enzymes in Molecular Biology

Directory of Open Access Journals (Sweden)

József Tőzsér

2013-11-01

Full Text Available Proteolytic enzymes (also termed peptidases, proteases and proteinases are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. Proteases are extensively applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications require their use, including production of Klenow fragments, peptide synthesis, digestion of unwanted proteins during nucleic acid purification, cell culturing and tissue dissociation, preparation of recombinant antibody fragments for research, diagnostics and therapy, exploration of the structure-function relationships by structural studies, removal of affinity tags from fusion proteins in recombinant protein techniques, peptide sequencing and proteolytic digestion of proteins in proteomics. The aim of this paper is to review the molecular biological aspects of proteolytic enzymes and summarize their applications in the life sciences.

Robust Trypsin Coating on Electrospun Polymer Nanofibers in Rigorous Conditions and Its Uses for Protein Digestion

Energy Technology Data Exchange (ETDEWEB)

Ahn, Hye-Kyung; Kim, Byoung Chan; Jun, Seung-Hyun; Chang, Mun Seock; Lopez-Ferrer, Daniel; Smith, Richard D.; Gu, Man Bock; Lee, Sang-Won; Kim, Beom S.; Kim, Jungbae

2010-12-15

An efficient protein digestion in proteomic analysis requires the stabilization of proteases such as trypsin. In the present work, trypsin was stabilized in the form of enzyme coating on electrospun polymer nanofibers (EC-TR), which crosslinks additional trypsin molecules onto covalently-attached trypsin (CA-TR). EC-TR showed better stability than CA-TR in rigorous conditions, such as at high temperatures of 40 °C and 50 °C, in the presence of organic co-solvents, and at various pH's. For example, the half-lives of CA-TR and EC-TR were 0.24 and 163.20 hours at 40 ºC, respectively. The improved stability of EC-TR can be explained by covalent-linkages on the surface of trypsin molecules, which effectively inhibits the denaturation, autolysis, and leaching of trypsin. The protein digestion was performed at 40 °C by using both CA-TR and EC-TR in digesting a model protein, enolase. EC-TR showed better performance and stability than CA-TR by maintaining good performance of enolase digestion under recycled uses for a period of one week. In the same condition, CA-TR showed poor performance from the beginning, and could not be used for digestion at all after a few usages. The enzyme coating approach is anticipated to be successfully employed not only for protein digestion in proteomic analysis, but also for various other fields where the poor enzyme stability presently hampers the practical applications of enzymes.

Production of saccharifying enzyme using the wastewater of a shochu distillery

Energy Technology Data Exchange (ETDEWEB)

Morimura, S.; Kida, K.; Yakita, Y.; Sonoda, Y. (Kumamoto University, Kumamoto (Japan). Faculty of Engineering); Myoga, H. (Organo Co. LTd., Tokyo (Japan))

1991-05-25

A saccharifying enzyme was produced using wastewater from a shochu distillery. Since the wastewater contained highly concentrated volatile fatty acids and those severely inhibited cell growth at low pH as converted to their free forms, the initial pH ranging from 4.5 to 6.0 was optimum. It was suggested that cell autolysis facilitated the release of the saccharifying enzyme, however, a released protease digested the enzyme with a subsequent decrease in activity. The enzyme was purified easily, and the purified enzyme was homogeneous as analyzed by disc electrophoresis. The enzyme was characterized by a molecular weight of 54,000 Da, an isoelectric point of pH 3.6, and the optimum reaction temperature and pH of 50-55{degree}C and 4.5-5.5, respectively. The enzyme could digest no raw starch, and the hydrolyzate of soluble starch by the enzyme was composed of two to four oligosaccharides. Based on above results and the amino acid sequence in a N-terminal, the enzyme produced was concluded to be {alpha}-amylase. 11 refs., 8 figs., 6 tabs.

Xylanase Increased the Ileal Digestibility of Non-Starch Polysaccharides and Concentration of Low Molecular Weight Non-Digestible Carbohydrates in Pigs Fed High Levels of wheat DDGS

DEFF Research Database (Denmark)

Pedersen, Mads Brøgger; Yu, Shukun; Arent, Susan

2015-01-01

The objective was to study the effect of a commercially available xylanase (CAX), an experimental xylanase (EX), and EX in combination with protease (EXP) on the degradation of nondigestible carbohydrates (NDC) and apparent ileal digestibility (AID) of nutrients in wheat distillers dried grains...... with solubles (wDDGS). The control and 3 enzyme diets contained 96% wDDGS supplemented with vitamins, minerals, l-lysine, and chromic oxide as a digestibility marker in addition to enzyme premix. Eight ileal cannulated pigs were fed 4 experimental diets containing 96% wDDGS—a control diet or 1 of 3 diets...

Kinetics of Cellulose Digestion by Fibrobacter succinogenes S85

OpenAIRE

Maglione, G.; Russell, J. B.; Wilson, D. B.

1997-01-01

Growing cultures of Fibrobacter succinogenes S85 digested cellulose at a rapid rate, but nongrowing cells and cell extracts did not have detectable crystalline cellulase activity. Cells that had been growing exponentially on cellobiose initiated cellulose digestion and succinate production immediately, and cellulose-dependent succinate production could be used as an index of enzyme activity against crystalline cellulose. Cells incubated with cellulose never produced detectable cellobiose, and...

Modulation of digestive physiology and biochemistry in Mytilus californianus in response to feeding level acclimation and microhabitat

Directory of Open Access Journals (Sweden)

Kwasi M. Connor

2016-09-01

Full Text Available The intertidal mussel Mytilus californianus is a critical foundation species that is exposed to fluctuations in the environment along tidal- and wave-exposure gradients. We investigated feeding and digestion in mussels under laboratory conditions and across environmental gradients in the field. We assessed whether mussels adopt a rate-maximization (higher ingestion and lower assimilation or a yield-maximization acquisition (lower ingestion and higher assimilation strategy under laboratory conditions by measuring feeding physiology and digestive enzyme activities. We used digestive enzyme activity to define resource acquisition strategies in laboratory studies, then measured digestive enzyme activities in three microhabitats at the extreme ends of the tidal- and wave-exposure gradients within a stretch of shore (<20 m projected sea-ward. Our laboratory results indicated that mussels benefit from a high assimilation efficiency when food concentration is low and have a low assimilation efficiency when food concentration is high. Additionally, enzyme activities of carbohydrases amylase, laminarinase and cellulase were elevated when food concentration was high. The protease trypsin, however, did not increase with increasing food concentration. In field conditions, low-shore mussels surprisingly did not have high enzyme activities. Rather, high-shore mussels exhibited higher cellulase activities than low-shore mussels. Similarly, trypsin activity in the high-shore-wave-sheltered microhabitat was higher than that in high-shore-wave-exposed. As expected, mussels experienced increasing thermal stress as a function of reduced submergence from low to high shore and shelter from wave-splash. Our findings suggest that mussels compensate for limited feeding opportunities and thermal stress by modulating digestive enzyme activities.

Phytase, a new life for an “old” enzyme

Science.gov (United States)

Phytase represents a group of phosphohydrolytic enzymes that initiate stepwise removals of phosphate from phytate. Simple-stomached species such as swine, poultry, and fish require extrinsic phytase to digest phytate: the major form of phosphorus in plant feeds. Consequently, this enzyme is suppleme...

Relevant pH and lipase for in vitro models of gastric digestion.

Science.gov (United States)

Sams, Laura; Paume, Julie; Giallo, Jacqueline; Carrière, Frédéric

2016-01-01

The development of in vitro digestion models relies on the availability of in vivo data such as digestive enzyme levels and pH values recorded in the course of meal digestion. The variations of these parameters along the GI tract are important for designing dynamic digestion models but also static models for which the choice of representative conditions of the gastric and intestinal conditions is critical. Simulating gastric digestion with a static model and a single set of parameters is particularly challenging because the variations in pH and enzyme concentration occurring in the stomach are much broader than those occurring in the small intestine. A review of the literature on this topic reveals that most models of gastric digestion use very low pH values that are not representative of the fed conditions. This is illustrated here by showing the variations in gastric pH as a function of meal gastric emptying instead of time. This representation highlights those pH values that are the most relevant for testing meal digestion in the stomach. Gastric lipolysis is still largely ignored or is performed with microbial lipases. In vivo data on gastric lipase and lipolysis have however been collected in humans and dogs during test meals. The biochemical characterization of gastric lipase has shown that this enzyme is rather unique among lipases: (i) stability and activity in the pH range 2 to 7 with an optimum at pH 4-5.4; (ii) high tensioactivity that allows resistance to bile salts and penetration into phospholipid layers covering TAG droplets; (iii) sn-3 stereospecificity for TAG hydrolysis; and (iv) resistance to pepsin. Most of these properties have been known for more than two decades and should provide a rational basis for the replacement of gastric lipase by other lipases when gastric lipase is not available.

Double digest revisited : Complexity and Approximability in the Presence of Noisy Data

NARCIS (Netherlands)

Cieliebak, Mark; Eidenbenz, Stephan; Woeginger, Gerhard; Warnow, Tandy; Zhu, Binhai

2003-01-01

We revisit the Double Digest problem, which occurs in sequencing of large DNA strings and consists of reconstructing the relative positions of cut sites from two different enzymes: we first show that Double Digest is strongly NP-complete, improving upon previous results that only showed weak

Caloric restriction counteracts age-related changes in the activities of sorbitol metabolizing enzymes from mouse liver

Science.gov (United States)

Hagopian, Kevork; Ramsey, Jon J.; Weindruch, Richard

2009-01-01

The influence of caloric restriction (CR) on hepatic sorbitol-metabolizing enzyme activities was investigated in young and old mice. Aldose reductase and sorbitol dehydrogenase activities were significantly lower in old CR mice than in old controls. Young CR mice showed decreased aldose reductase activity and a trend towards decreased sorbitol dehydrogenase when compared to controls. Metabolites of the pathway, namely sorbitol, glucose and fructose were decreased by CR in young and old mice. Pyruvate levels were decreased by CR in both young and old mice, while lactate decreased only in old CR. Malate levels increased in old CR but remained unchanged in young CR, when compared with controls. Accordingly, the lactae/pyruvate and malate/pyruvate ratios in young and old CR mice were increased, indicating increased NADH/NAD and NADPH/NADP redox couples, respectively. The results indicate that decreased glucose levels under CR conditions lead to decreased sorbitol pathway enzyme activities and metabolite levels, and could contribute to the beneficial effects of long-term CR through decreased sorbitol levels and NADPH sparing. PMID:18953666

Application of residual polysaccharide-degrading enzymes in dried shiitake mushrooms as an enzyme preparation in food processing.

Science.gov (United States)

Tatsumi, E; Konishi, Y; Tsujiyama, S

2016-11-01

To examine the activities of residual enzymes in dried shiitake mushrooms, which are a traditional foodstuff in Japanese cuisine, for possible applications in food processing. Polysaccharide-degrading enzymes remained intact in dried shiitake mushrooms and the activities of amylase, β-glucosidase and pectinase were high. A potato digestion was tested using dried shiitake powder. The enzymes reacted with potato tuber specimens to solubilize sugars even under a heterogeneous solid-state condition and that their reaction modes were different at 38 and 50 °C. Dried shiitake mushrooms have a potential use in food processing as an enzyme preparation.

Evaluation of Asteraceae herbal extracts in the management of diabetes and obesity. Contribution of caffeoylquinic acids on the inhibition of digestive enzymes activity and formation of advanced glycation end-products (in vitro).

Science.gov (United States)

Spínola, Vítor; Castilho, Paula C

2017-11-01

The study was performed to assess, for the first time, the in vitro anti-diabetic potential of ten Asteraceae plant extracts to inhibit the activity of digestive enzymes (α-amylase, α-, β-glucosidases and lipase) responsible for hydrolysis/digestion of sugar and lipids. Prevention of advanced glycation end-products (AGEs) formation was evaluated in bovine serum albumin/ribose glycation reaction model. The phytochemical profiles and caffeoylquinic acids (CQAs) contents were determined for the methanolic extract of each plant. Analyzed plant extracts exhibited significant inhibitory activity against key digestive enzymes linked to type II diabetes and obesity. A strong inhibition was observed for glucosidases and mild activity towards amylase and lipase (compared to reference compounds). Moreover, some extracts exhibited potent ability to prevent formation of AGEs, implicated in some diabetic complications. Caffeoylquinic acids were dominant in all plant extracts and findings demonstrate that these compounds are the most relevant hypoglycemic and anti-glycation agents. From the obtained results, Argyranthemum pinnatifidum, Helichrysum melaleucum, and Phagnalon lowei are good candidates for further development of phyto-pharmaceutical preparations as complementary therapy for diabetes and obesity control. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structural Orders of Wheat Starch Do Not Determine the In Vitro Enzymatic Digestibility.

Science.gov (United States)

Wang, Shujun; Wang, Shaokang; Liu, Lu; Wang, Shuo; Copeland, Les

2017-03-01

In this study, we elucidated the underlying mechanisms that are responsible for the rate-limiting step for wheat starch digestion. Wheat starch samples with a degree of gelatinization (DG) ranging from 0 to 100% were prepared. As DG increased, the ordered structures of the starch were disrupted increasingly. In contrast, almost all of the increase in the rate and extent of in vitro enzymatic digestion coincided with a DG of only 6% and a minor loss of structural order. As DG increased beyond 6%, digestibility of the starch increased only slightly. We propose that the access and binding of enzymes to starch is greatly increased with only a small DG, which is followed by the simultaneous hydrolysis of crystalline and amorphous areas in gelatinized starch. In vitro enzymatic digestibility of starch was determined predominantly by enzyme binding to starch rather than the ordered structures of starch.

The role of lipid and carbohydrate digestive enzyme inhibitors in the management of obesity: a review of current and emerging therapeutic agents

Directory of Open Access Journals (Sweden)

Tucci S

2010-05-01

Full Text Available Sonia A Tucci, Emma J Boyland, Jason CG HalfordKissileff Laboratory for the Study of Human Ingestive Behaviour, School of Psychology, University of Liverpool, Liverpool, UKAbstract: Obesity is a global epidemic associated with significant morbidity and mortality in adults and ill health in children. A proven successful approach in weight management has been the disruption of nutrient digestion, with orlistat having been used to treat obesity for the last 10 years. Although orlistat-induced weight loss remains modest, it produces meaningful reductions in risk factors for obesity-related conditions such as diabetes and cardiovascular disease. Moreover, this lipase inhibitor is free of the serious side effects that have dogged appetite-suppressing drugs. This success had driven investigation into new generation nutraceuticals, supplements and pharmaceutical agents that inhibit the breakdown of complex carbohydrates and fats within the gut. This review focuses on agents purported to inhibit intestinal enzymes responsible for macronutrient digestion. Except for some synthetic products, the majority of agents reviewed are either botanical extracts or bacterial products. Currently, carbohydrate digestion inhibitors are under development to improve glycemic control and these may also induce some weight loss. However, colonic fermentation induced side effects, such as excess gas production, remain an issue for these compounds. The α-glucosidase inhibitor acarbose, and the α-amylase inhibitor phaseolamine, have been used in humans with some promising results relating to weight loss. Nonetheless, few of these agents have made it into clinical studies and without any clinical proof of concept or proven efficacy it is unlikely any will enter the market soon.Keywords: lipase, amylase, saccharidases, overweight, orlistat, Alli®, digestion, body weight

Decreasing the amount of trypsin in in-gel digestion leads to diminished chemical noise and improved protein identifications.

Science.gov (United States)

Hu, Mo; Liu, Yanhua; Yu, Kaiwen; Liu, Xiaoyun

2014-09-23

Pre-fractionation by gel electrophoresis is often combined with liquid chromatography-mass spectrometry (LC-MS) for large-scale profiling of complex protein samples. An essential component of this widely applied proteomic platform is in-gel protein digestion. In nearly two decades of practicing this approach, an extremely high level of trypsin has been utilized due to the consideration of slow enzyme diffusion into the gel matrix. Here we report that trypsin autolysis products contribute to the bulk of chemical noise in in-gel digestion and remarkably we found evidence that the amount of trypsin can be slashed by an order of magnitude with comparable digestion performance. By revising perhaps the most critical element of this decade-old digestion protocol, the proteomics community relying on gel separation prior to LC-MS analysis will benefit instantly from much lowered cost due to enzyme expenditure. More importantly, substantially reduced chemical noise (i.e., trypsin self-cleavage products) as a result of less enzyme usage translates into more protein identifications when limited amounts of samples are the interest of interrogation. In-gel digestion is one of the most widely used methods in proteomics. An exceedingly high level of trypsin has been utilized due to the consideration of slow enzyme diffusion into the gel matrix. This requirement has been faithfully kept in nearly two decades of practicing this approach. Here we report that trypsin concentration can be slashed by at least an order of magnitude while still providing comparable digestion performance. Thus the proteomics community relying on gel separation prior to LC-MS analysis will benefit instantly from much lowered enzyme cost. More importantly, substantially reduced chemical noise (i.e., trypsin autolysis products) due to less enzyme usage translates into ~30% more protein identifications when limited amounts of protein samples are analyzed. Copyright © 2014 Elsevier B.V. All rights reserved.

Complexo enzimático para suínos: digestão, metabolismo, desempenho e impacto ambiental Enzyme complex for swine: nutrient digestion, metabolism, performance and environmental impact

Directory of Open Access Journals (Sweden)

Urbano dos Santos Ruiz

2008-03-01

Full Text Available Foram conduzidos dois experimentos para avaliar a suplementação enzimática (amilase, pentosanase, celulase, protease e a-galactosidase em rações à base de milho e farelo de soja para suínos. No Exp 1, foram determinadas as digestibilidades aparentes da energia, matéria seca, proteína e das fibras das rações, com ou sem o complexo enzimático, para suínos machos castrados, com 19,96 ± 2,90 kg de peso vivo. Foi utilizado o método da coleta total de fezes e as rações foram formuladas com níveis reduzidos de energia, proteína e aminoácidos. No Exp 2, foram mensurados o desempenho, a excreção de sólidos totais e voláteis, matéria mineral, nitrogênio, macro e microminerais nas fezes, em relação ao desempenho, de suínos machos castrados dos 50 aos 151 dias de idade, com peso médio inicial de 18,34 ± 1,35 kg. Foram utilizadas três dietas, sendo uma ração formulada para atender ou exceder as exigências nutricionais dos animais, de acordo com o NRC (1998, e outras duas com níveis reduzidos de energia, proteína e aminoácidos, suplementadas ou não com o complexo enzimático. Foi utilizado o delineamento em blocos casualizados. A suplementação enzimática em rações contendo milho e farelo de soja não promoveu incrementos na digestibilidade de nutrientes, não melhorou o desempenho dos animais e também não reduziu a excreção de resíduos pelas fezes. Mais estudos são necessários para testar novas matrizes e diferentes níveis do complexo enzimático.Two experiments were conducted to evaluate dietary enzyme supplementation (amylase, pentosanase, celulase and a-galactosidase in corn-soy diets for swine. In the Exp 1, the apparent digestibilities of energy, dry matter, protein and fibers of the feeds were determined, using a nutritional matrix with or without enzyme supplementation for barrows with 19.96 ± 2.90 kg body weight. The total feces collection method was used and the diets were formulated with reduced

Carbohydrases in the digestive system of the spined soldier bug, Podisus maculiventris (Say) (Hemiptera: Pentatomidae).

Science.gov (United States)

Ghamari, Mahboob; Hosseininaveh, Vahid; Darvishzadeh, Ali; Chougule, Nanasaheb P

2014-04-01

The spined soldier bug, Podisus maculiventris, is a generalist predator of insects and has been used in biological control. However, information on the digestion of food in this insect is lacking. Therefore, we have studied the digestive system in P. maculiventris, and further characterized carbohydrases in the digestive tract. The midgut of all developmental stages was composed of anterior, median, and posterior regions. The volumes of the anterior midgut decreased and the median midgut increased in older instars and adults, suggesting a more important role of the median midgut in food digestion. However, carbohydrase activities were predominant in the anterior midgut. In comparing the specific activity of carbohydrases, α-amylase activity was more in the salivary glands (with two distinct activity bands in zymograms), and glucosidase and galactosidase activities were more in the midgut. Salivary α-amylases were detected in the prey hemolymph, demonstrating the role of these enzymes in extra-oral digestion. However, the catalytic efficiency of midgut α-amylase activity was approximately twofold more than that of the salivary gland enzymes, and was more efficient in digesting soluble starch than glycogen. Midgut α-amylases were developmentally regulated, as one isoform was found in first instar compared to three isoforms in fifth instar nymphs. Starvation significantly affected carbohydrase activities in the midgut, and acarbose inhibited α-amylases from both the salivary glands and midgut in vitro and in vivo. The structural diversity and developmental regulation of carbohydrases in the digestive system of P. maculiventris demonstrate the importance of these enzymes in extra-oral and intra-tract digestion, and may explain the capability of the hemipteran to utilize diverse food sources. © 2014 Wiley Periodicals, Inc.

Combined techniques for characterising pasta structure reveals how the gluten network slows enzymic digestion rate.

Science.gov (United States)

Zou, Wei; Sissons, Mike; Gidley, Michael J; Gilbert, Robert G; Warren, Frederick J

2015-12-01

The aim of the present study is to characterise the influence of gluten structure on the kinetics of starch hydrolysis in pasta. Spaghetti and powdered pasta were prepared from three different cultivars of durum semolina, and starch was also purified from each cultivar. Digestion kinetic parameters were obtained through logarithm-of-slope analysis, allowing identification of sequential digestion steps. Purified starch and semolina were digested following a single first-order rate constant, while pasta and powdered pasta followed two sequential first-order rate constants. Rate coefficients were altered by pepsin hydrolysis. Confocal microscopy revealed that, following cooking, starch granules were completely swollen for starch, semolina and pasta powder samples. In pasta, they were completely swollen in the external regions, partially swollen in the intermediate region and almost intact in the pasta strand centre. Gluten entrapment accounts for sequential kinetic steps in starch digestion of pasta; the compact microstructure of pasta also reduces digestion rates. Copyright © 2015 Elsevier Ltd. All rights reserved.

Optimizing Restriction Site Placement for Synthetic Genomes

Science.gov (United States)

Montes, Pablo; Memelli, Heraldo; Ward, Charles; Kim, Joondong; Mitchell, Joseph S. B.; Skiena, Steven

Restriction enzymes are the workhorses of molecular biology. We introduce a new problem that arises in the course of our project to design virus variants to serve as potential vaccines: we wish to modify virus-length genomes to introduce large numbers of unique restriction enzyme recognition sites while preserving wild-type function by substitution of synonymous codons. We show that the resulting problem is NP-Complete, give an exponential-time algorithm, and propose effective heuristics, which we show give excellent results for five sample viral genomes. Our resulting modified genomes have several times more unique restriction sites and reduce the maximum gap between adjacent sites by three to nine-fold.

A digestive prolyl carboxypeptidase in Tenebrio molitor larvae.

Science.gov (United States)

Goptar, Irina A; Shagin, Dmitry A; Shagina, Irina A; Mudrik, Elena S; Smirnova, Yulia A; Zhuzhikov, Dmitry P; Belozersky, Mikhail A; Dunaevsky, Yakov E; Oppert, Brenda; Filippova, Irina Yu; Elpidina, Elena N

2013-06-01

Prolyl carboxypeptidase (PRCP) is a lysosomal proline specific serine peptidase that also plays a vital role in the regulation of physiological processes in mammals. In this report, we isolate and characterize the first PRCP in an insect. PRCP was purified from the anterior midgut of larvae of a stored product pest, Tenebrio molitor, using a three-step chromatography strategy, and it was determined that the purified enzyme was a dimer. The cDNA of PRCP was cloned and sequenced, and the predicted protein was identical to the proteomic sequences of the purified enzyme. The substrate specificity and kinetic parameters of the enzyme were determined. The T. molitor PRCP participates in the hydrolysis of the insect's major dietary proteins, gliadins, and is the first PRCP to be ascribed a digestive function. Our collective data suggest that the evolutionary enrichment of the digestive peptidase complex in insects with an area of acidic to neutral pH in the midgut is a result of the incorporation of lysosomal peptidases, including PRCP. Published by Elsevier Ltd.

Application of solid waste from anaerobic digestion of poultry litter in Agrocybe aegerita cultivation: mushroom production, lignocellulolytic enzymes activity and substrate utilization.

Science.gov (United States)

Isikhuemhen, Omoanghe S; Mikiashvili, Nona A; Kelkar, Vinaya

2009-06-01

The degradation and utilization of solid waste (SW) from anaerobic digestion of poultry litter by Agrocybe aegerita was evaluated through mushroom production, loss of organic matter (LOM), lignocellulolytic enzymes activity, lignocellulose degradation and mushroom nutrients content. Among the substrate combinations (SCs) tested, substrates composed of 10-20% SW, 70-80% wheat straw and 10% millet was found to produce the highest mushroom yield (770.5 and 642.9 g per 1.5 kg of substrate). LOM in all SCs tested varied between 8.8 and 48.2%. A. aegerita appears to degrade macromolecule components (0.6-21.8% lignin, 33.1-55.2% cellulose and 14-53.9% hemicellulose) during cultivation on the different SCs. Among the seven extracellular enzymes monitored, laccase, peroxidase and CMCase activities were higher before fruiting; while xylanase showed higher activities after fruiting. A source of carbohydrates (e.g., millet) in the substrate is needed in order to obtain yield and biological efficiency comparable to other commercially cultivated exotic mushrooms.

Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

OpenAIRE

van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D

2015-01-01

The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1-2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand brea...

Effects of mercury on lysosomal protein digestion in the kidney proximal tubule

International Nuclear Information System (INIS)

Madsen, K.M.; Christensen, E.I.

1978-01-01

The effect of mercury on renal lysosomal protein digestion was studied after administration of mercury in vitro and in vivo. Mercuric chloride or methylmercury chloride was added in vitro to lysosomal enzymes isolated from normal rats, and subsequently, digestion experiments were carried out using 125 I-labeled lysozyme or cytochrome c as substrate proteins. Both mercury compounds produced a concentration-dependent inhibition of the degradation of the proteins, mercuric chloride being the strongest inhibitor. Mercuric chloride was also administered to rats in vivo for 5 to 8 months. Renal lysosomal enzymes from these animals also had a decreased ability to digest the two substrate proteins. Furthermore, the digestion of lysozyme intravenously injected into mercury-intoxicated rats was decreased in renal cortical slices incubated in vitro. Electron microscope autoradiography showed that intravenously injected labeled lysozyme was located primarily over lysosomes in proximal tubule cells 1 hour after injection in both control animals and mercury-intoxicated rats. These results suggest a decreased catabolism of low molecular weight proteins in the kidney during chronic mercury intoxication

Effects of small peptides, probiotics, prebiotics, and synbiotics on growth performance, digestive enzymes, and oxidative stress in orange-spotted grouper, Epinephelus coioides, juveniles reared in artificial seawater

Science.gov (United States)

Wang, Tao; Cheng, Yongzhou; Chen, Xiaoyan; Liu, Zhaopu; Long, Xiaohua

2017-01-01

Aquaculture production efficiency may increase by using feed additives. This study investigated the effects of different dietary additives [w/w: 2% small peptides, 0.01% probiotics ( Bacillus licheniformis) and 0.2% prebiotics (inulin)] on growth performance, digestive enzyme activities, and oxidative stress in juvenile Epinephelus coioides reared in artificial seawater of two salt concentrations (13.5 vs. 28.5). Weight gain rate was significantly higher in fish fed the diet supplemented with small peptides, B. licheniformis, inulin, or synbiotics than that in fish fed the basal diet; the greatest weight gain rate was found in fish fed the small peptide treatment [56.0% higher than basal diet]. Higher feed efficiency was detected in fish fed the diet supplemented with small peptides than that of fish in the other dietary treatments. Total protease activity in the stomach and intestines was highest in fish fed the small peptide-treated diet, whereas lipase activity was highest in those fed synbiotics (combination of Bacillus licheniformis and inulin) than that in fish fed the other treatments. Antioxidant enzyme (total superoxide dismutase and catalase) activities and hepatic malondialdehyde content were higher in fish receiving the dietary supplements and maintained in artificial seawater containing 13.5 salinity compared with those in the control (28.5). Hepatic catalase activity in grouper fed the diets with small peptides or synbiotics decreased significantly compared with that in control fish. Overall, the three types of additives improved growth rate of juvenile grouper and digestive enzymes activities to varying degrees but did not effectively improve antioxidant capacity under low-salinity stress conditions.

The Protein Composition of the Digestive Fluid from the Venus Flytrap Sheds Light on Prey Digestion Mechanisms*

Science.gov (United States)

Schulze, Waltraud X.; Sanggaard, Kristian W.; Kreuzer, Ines; Knudsen, Anders D.; Bemm, Felix; Thøgersen, Ida B.; Bräutigam, Andrea; Thomsen, Line R.; Schliesky, Simon; Dyrlund, Thomas F.; Escalante-Perez, Maria; Becker, Dirk; Schultz, Jörg; Karring, Henrik; Weber, Andreas; Højrup, Peter; Hedrich, Rainer; Enghild, Jan J.

2012-01-01

The Venus flytrap (Dionaea muscipula) is one of the most well-known carnivorous plants because of its unique ability to capture small animals, usually insects or spiders, through a unique snap-trapping mechanism. The animals are subsequently killed and digested so that the plants can assimilate nutrients, as they grow in mineral-deficient soils. We deep sequenced the cDNA from Dionaea traps to obtain transcript libraries, which were used in the mass spectrometry-based identification of the proteins secreted during digestion. The identified proteins consisted of peroxidases, nucleases, phosphatases, phospholipases, a glucanase, chitinases, and proteolytic enzymes, including four cysteine proteases, two aspartic proteases, and a serine carboxypeptidase. The majority of the most abundant proteins were categorized as pathogenesis-related proteins, suggesting that the plant's digestive system evolved from defense-related processes. This in-depth characterization of a highly specialized secreted fluid from a carnivorous plant provides new information about the plant's prey digestion mechanism and the evolutionary processes driving its defense pathways and nutrient acquisition. PMID:22891002

A MIDGUT DIGESTIVE PHOSPHOLIPASE A2 IN LARVAL MOSQUITOES, AEDES ALBOPICTUS AND CULEX QUINQUEFASCIATUS

Science.gov (United States)

Phospholipase A2 (PLA2) is a secretory digestive enzyme that hydrolyzes ester bond at sn-2 position of dietary phospholipids, creating free fatty acid and lysophopholipid. The free fatty acids (arachidonic acid) are absorbed into midgut cells. Aedes albopictus and Culex quinquefasciatus digestive PL...

Azadirachtin induced larval avoidance and antifeeding by disruption of food intake and digestive enzymes in Drosophila melanogaster (Diptera: Drosophilidae).

Science.gov (United States)

Bezzar-Bendjazia, Radia; Kilani-Morakchi, Samira; Maroua, Ferdenache; Aribi, Nadia

2017-11-01

Botanical insecticides are a promising alternative to reduce the harmful effects of synthetic chemicals. Among the botanical biopesticides, azadirachtin obtained from the Indian neem tree Azadirachta indica A. Juss. (Meliaceae) is probably the biorational insecticide with greatest agriculture use nowadays due to its broad insecticide activity. The current study, evaluated the lethal and sublethal effects of azadirachtin on larval avoidance, food intake and digestive enzymes of Drosophila melanogaster larvae as biological model. Azadirachtin was applied topically at two doses LD 25 (0.28μg) and LD 50 (0.67μg) on early third instars larvae. Results evaluated 24h after treatment showed that larvae exhibited significant repellence to azadirachtin and prefer keeping in untreated arenas rather than moving to treated one. In addition, azadirachtin avoidance was more marked in larvae previously treated with this compound as compared with naïf larvae (controls). Moreover, azadirachtin treatment decreased significantly the amount of larval food intake. Finally, azadirachtin reduced significantly the activity of larval α-amylase, chitinase and protease and increased the activity of lipase. This finding showed that azadirachtin induced behavioral and physiological disruption affecting the ability of the insect to digest food. This rapid installation of avoidance and long term antifeedancy might reinforce the action of azadirachtin and provide a new behavioral strategy for integrated pest management programs. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterisation of protein families in spider digestive fluids and their role in extra-oral digestion.

Science.gov (United States)

Walter, André; Bechsgaard, Jesper; Scavenius, Carsten; Dyrlund, Thomas S; Sanggaard, Kristian W; Enghild, Jan J; Bilde, Trine

2017-08-10

Spiders are predaceous arthropods that are capable of subduing and consuming relatively large prey items compared to their own body size. For this purpose, spiders have evolved potent venoms to immobilise prey and digestive fluids that break down nutrients inside the prey's body by means of extra-oral digestion (EOD). Both secretions contain an array of active proteins, and an overlap of some components has been anecdotally reported, but not quantified. We systematically investigated the extent of such protein overlap. As venom injection and EOD succeed each other, we further infer functional explanations, and, by comparing two spider species belonging to different clades, assess its adaptive significance for spider EOD in general. We describe the protein composition of the digestive fluids of the mygalomorph Acanthoscurria geniculata and the araneomorph Stegodyphus mimosarum, in comparison with previously published data on a third spider species. We found a number of similar hydrolases being highly abundant in all three species. Among them, members of the family of astacin-like metalloproteases were particularly abundant. While the importance of these proteases in spider venom and digestive fluid was previously noted, we now highlight their widespread use across different spider taxa. Finally, we found species specific differences in the protein overlap between venom and digestive fluid, with the difference being significantly greater in S. mimosarum compared to A. geniculata. The injection of venom precedes the injection with digestive fluid, and the overlap of proteins between venom and digestive fluid suggests an early involvement in EOD. Species specific differences in the overlap may reflect differences in ecology between our two study species. The protein composition of the digestive fluid of all the three species we compared is highly similar, suggesting that the cocktail of enzymes is highly conserved and adapted to spider EOD.

Testing the effect of different enzyme blends on increasing the biogas yield of straw and digested manure fibers

DEFF Research Database (Denmark)

Njoku, Stephen Ikechukwu; Jurado, Esperanza; Malmgren-Hansen, Bjørn

In this study, enzymatic treatment was tested to increase the biogas yield of wheat straw (WS) and digested manure fibers (DMF) in the Re-Injection Loop Concept, which combines anaerobic digestion with solid separation to enhance the biogas yield per ton of manure by: 1. Digestion of the easily d...... degradable fraction of manure in the biogas process. 2. Separation of the residual recalcitrant digested fiber fraction project. 3. Ultrasound and/or enzymatic treatment of the digested fiber fraction. 4. Recirculation of the treated fiber fraction into the reactor.......In this study, enzymatic treatment was tested to increase the biogas yield of wheat straw (WS) and digested manure fibers (DMF) in the Re-Injection Loop Concept, which combines anaerobic digestion with solid separation to enhance the biogas yield per ton of manure by: 1. Digestion of the easily...

Mammalian Mucosal ?-Glucosidases Coordinate with ?-Amylase in the Initial Starch Hydrolysis Stage to Have a Role in Starch Digestion beyond Glucogenesis

OpenAIRE

Dhital, Sushil; Lin, Amy Hui-Mei; Hamaker, Bruce R.; Gidley, Michael J.; Muniandy, Anbuhkani

2013-01-01

Starch digestion in the human body is typically viewed in a sequential manner beginning with α-amylase and followed by α-glucosidase to produce glucose. This report indicates that the two enzyme types can act synergistically to digest granular starch structure. The aim of this study was to investigate how the mucosal α-glucosidases act with α-amylase to digest granular starch. Two types of enzyme extracts, pancreatic and intestinal extracts, were applied. The pancreatic extract containing pre...

A titration approach to identify the capacity for starch digestion in milk-fed calves

NARCIS (Netherlands)

Gilbert, M. S.; van den Borne, J. J. G. C.; Berends, H.; Pantophlet, A. J.; Schols, H. A.; Gerrits, W. J. J.

Calf milk replacers (MR) commonly contain 40% to 50% lactose. For economic reasons, starch is of interest as a lactose replacer. Compared with lactose, starch digestion is generally low in calves. It is, however, unknown which enzyme limits the rate of starch digestion. The objectives were to

Pattern analysis approach reveals restriction enzyme cutting abnormalities and other cDNA library construction artifacts using raw EST data

Directory of Open Access Journals (Sweden)

Zhou Sun

2012-05-01

Full Text Available Abstract Background Expressed Sequence Tag (EST sequences are widely used in applications such as genome annotation, gene discovery and gene expression studies. However, some of GenBank dbEST sequences have proven to be “unclean”. Identification of cDNA termini/ends and their structures in raw ESTs not only facilitates data quality control and accurate delineation of transcription ends, but also furthers our understanding of the potential sources of data abnormalities/errors present in the wet-lab procedures for cDNA library construction. Results After analyzing a total of 309,976 raw Pinus taeda ESTs, we uncovered many distinct variations of cDNA termini, some of which prove to be good indicators of wet-lab artifacts, and characterized each raw EST by its cDNA terminus structure patterns. In contrast to the expected patterns, many ESTs displayed complex and/or abnormal patterns that represent potential wet-lab errors such as: a failure of one or both of the restriction enzymes to cut the plasmid vector; a failure of the restriction enzymes to cut the vector at the correct positions; the insertion of two cDNA inserts into a single vector; the insertion of multiple and/or concatenated adapters/linkers; the presence of 3′-end terminal structures in designated 5′-end sequences or vice versa; and so on. With a close examination of these artifacts, many problematic ESTs that have been deposited into public databases by conventional bioinformatics pipelines or tools could be cleaned or filtered by our methodology. We developed a software tool for Abnormality Filtering and Sequence Trimming for ESTs (AFST, http://code.google.com/p/afst/ using a pattern analysis approach. To compare AFST with other pipelines that submitted ESTs into dbEST, we reprocessed 230,783 Pinus taeda and 38,709 Arachis hypogaea GenBank ESTs. We found 7.4% of Pinus taeda and 29.2% of Arachis hypogaea GenBank ESTs are “unclean” or abnormal, all of which could be cleaned

Effects of dietary amylose/amylopectin ratio on growth performance, feed utilization, digestive enzymes, and postprandial metabolic responses in juvenile obscure puffer Takifugu obscurus.

Science.gov (United States)

Liu, Xiang-he; Ye, Chao-xia; Ye, Ji-dan; Shen, Bi-duan; Wang, Chun-yan; Wang, An-li

2014-10-01

The effect of dietary amylose/amylopectin (AM/AP) ratio on growth, feed utilization, digestive enzyme activities, plasma parameters, and postprandial blood glucose responses was evaluated in juvenile obscure puffer, Takifugu obscurus. Five isonitrogenous (430 g kg(-1) crude protein) and isolipidic (90 g kg(-1) crude lipid) diets containing an equal starch level (250 g kg(-1) starch) with different AM/AP ratio diets of 0/25, 3/22, 6/19, 9/16 and 12/13 were formulated. Each experimental diet was fed to triplicate groups (25 fish per tank), twice daily during a period of 60 days. After the growth trial, a postprandial blood response test was carried out. Fish fed diet 6/19 showed best growth, feed efficiency and protein efficiency ratio. Hepatosomatic index, plasma total cholesterol concentration, liver glycogen and lipid content, and gluconokinase, pyruvate kinase and fructose-1,6-bisphosphatase activities were lower in fish fed highest AM/AP diet (12/13) than in fish fed the low-amylose diets. Activities of liver and intestinal trypsin in fish fed diet 3/22 and diet 6/19 were higher than in fish fed diet 9/16 and diet 12/13. Activities of liver and intestinal amylase and intestinal lipase, and starch digestibility were negatively correlated with dietary AM/AP ratio. Fish fed diet 3/22 and diet 6/19 showed higher plasma total amino acid concentration than fish fed the other diets, while plasma urea nitrogen concentration and activities of alanine aminotransferase and aspartate aminotransferase showed the opposite trend. Equal values were found for viscerosomatic index and condition factor, whole body and muscle composition, plasma high-density and low-density lipoprotein cholesterol concentrations, and activities of lipase and hexokinase and glucose-6-phosphatase in liver. Postprandial plasma glucose and triglyceride peak value of fish fed diet 12/13 were lower than in fish fed the low-amylose diets, and the peak time of plasma glucose was later than in fish fed the

Digestive challenges for vertebrate animals: Microbial diversity, cardiorespiratory coupling, and dietary specialization

DEFF Research Database (Denmark)

Barboza, P.S.; Bennett, A.; Lignot, H.-H.

2010-01-01

and digestive functions, and (3) the evolution of dietary specialization. Herbivores consume, digest, and detoxify complex diets by using a wide variety of enzymes expressed by bacteria, predominantly in the phyla Firmicutes and Bacteroidetes. Carnivores, such as snakes that feed intermittently, sometimes...... characteristics of the diet and the level of food intake. In this article, we discuss three themes that affect the ability of an animal to alter digestive function in relation to novel substrates and changing food supply: (1) the fermentative digestion in herbivores, (2) the integration of cardiopulmonary...... process very large meals that require compensatory adjustments in blood flow, acid secretion, and regulation of acid‐base homeostasis. Snakes and birds that specialize in simple diets of prey or nectar retain their ability to digest a wider selection of prey. The digestive system continues...

Distribution and characteristics of endogenous digestive enzymes in the red-eared slider turtle, Trachemys scripta elegans.

Science.gov (United States)

Sun, Jian-Yi; Du, Jie; Qian, Li-Chun; Jing, Ming-Yan; Weng, Xiao-Yan

2007-08-01

Distribution and properties of the main digestive enzymes including protease and amylase, from stomach, pancreas and the anterior, middle and posterior intestine of the adult red-eared slider turtle Trachemys scripta elegans were studied at various pHs and temperatures. The optimum temperature and pH for protease in stomach, pancreas and the anterior, middle and posterior intestine were 40 degrees C, 2.5; 50 degrees C, 8.0; 50 degrees C, 7.0; 50 degrees C, 8.0; and 50 degrees C, 8.5; respectively. The optimum temperature and pH for amylase in stomach, pancreas and anterior, middle and posterior intestine were 40 degrees C, 8.0; 30 degrees C, 7.5; 40 degrees C, 7.0; 50 degrees C, 8.0; and 50 degrees C, 8.0; respectively. Under the optimum conditions, the order of protease activity from high to low was of pancreas, stomach and the anterior, posterior and middle intestine; the activity of amylase in descending order was of anterior intestine, pancreas, posterior intestine, middle intestine and stomach.

Hemicellulose conversion by anaerobic digestion

Energy Technology Data Exchange (ETDEWEB)

Ghosh, S; Honry, M P; Christopher, R W

1985-01-01

This research was undertaken to study the digestibility of the hemicellulose fractions of an aquatic biomass, a land-based biomass and a biomass-waste blend under various fermentation conditions. The conversion of hemicellulose was higher than those of cellulose and protein under the mesophilic condition. Hemicellulose was converted at a much lower efficency than cellulose during thermophilic digestion. In contrast, cellulose conversion was about the same under mesophilic and thermophilic conditions. Cellulose was utilized in preference to hemicellulose during mesophilic fermentation of nitrogen-supplemented Bermuda grass. It was speculated that Bermuda grass cellulose was converted at a higher efficiency than hemicellulose in the pressure of external nitrogen because the metabolism of the breakdown product (glucose) of cellulose required the least investment of enzymes and energy. 4 references.

PCR Amplification of Ribosomal DNA for Species Identification in the Plant Pathogen Genus Phytophthora

Science.gov (United States)

Ristaino, Jean B.; Madritch, Michael; Trout, Carol L.; Parra, Gregory

1998-01-01

We have developed a PCR procedure to amplify DNA for quick identification of the economically important species from each of the six taxonomic groups in the plant pathogen genus Phytophthora. This procedure involves amplification of the 5.8S ribosomal DNA gene and internal transcribed spacers (ITS) with the ITS primers ITS 5 and ITS 4. Restriction digests of the amplified DNA products were conducted with the restriction enzymes RsaI, MspI, and HaeIII. Restriction fragment patterns were similar after digestions with RsaI for the following species: P. capsici and P. citricola; P. infestans, P. cactorum, and P. mirabilis; P. fragariae, P. cinnamomi, and P. megasperma from peach; P. palmivora, P. citrophthora, P. erythroseptica, and P. cryptogea; and P. megasperma from raspberry and P. sojae. Restriction digests with MspI separated P. capsici from P. citricola and separated P. cactorum from P. infestans and P. mirabilis. Restriction digests with HaeIII separated P. citrophthora from P. cryptogea, P. cinnamomi from P. fragariae and P. megasperma on peach, P. palmivora from P. citrophthora, and P. megasperma on raspberry from P. sojae. P. infestans and P. mirabilis digests were identical and P. cryptogea and P. erythroseptica digests were identical with all restriction enzymes tested. A unique DNA sequence from the ITS region I in P. capsici was used to develop a primer called PCAP. The PCAP primer was used in PCRs with ITS 1 and amplified only isolates of P. capsici, P. citricola, and P. citrophthora and not 13 other species in the genus. Restriction digests with MspI separated P. capsici from the other two species. PCR was superior to traditional isolation methods for detection of P. capsici in infected bell pepper tissue in field samples. The techniques described will provide a powerful tool for identification of the major species in the genus Phytophthora. PMID:9501434

Digestive enzyme as benchmark for insecticide resistance development in Culex pipiens larvae to chemical and bacteriologic insecticides.

Science.gov (United States)

Kamel, Nashwa H; Bahgat, Iman M; El Kady, Gamal A

2013-04-01

This work monitored changes in some digestive enzymes (trypsin and aminopeptidase) associated with the building up of resistance in Cx. pipiens larvae to two chemical insecticides (methomyl and/or malathion) and one biological insecticide (Bacillus thuringiensis-H14 or B.t H 14). The LC50 value of methomyl for both field- and the 12th generation (F12) of the selected strain was 1.789 ppm and 8.925 ppm respectively. The LC50 value of malathion for both field and the F12 of the selected strain was 0.082 ppm and 0.156 ppm respectively, and those of B.t H14 of field strain and the F12 was 2.550ppm & 2.395ppm respectively. The specific activity of trypsin enzyme in control susceptible colony was 20.806 +/- 0.452micromol/min/mg protein; but at F4 and F8 for malathion and methomyl treated larvae were 10.810 +/- 0.860 & 15.616+/-0.408 micromol/min/mg protein, respectively. Trypsin activity of F12 in treated larvae with B.t.H14 was 2.097 +/- 0.587 microiol/min/mg protein. Aminopeptidase specific activity for susceptible control larvae was 173.05 +/- 1.3111 micromol/min/mg protein. This activity decreased to 145.15 +/- 4.12, 152.497 +/- 6.775 & 102.04 +/- 3.58a micromol/min/mg protein after larval (F 12) treatment with methomyl, malathion and B.t H 14 respectively.

Inside the guts of wood-eating catfishes: can they digest wood?

Science.gov (United States)

German, Donovan P

2009-11-01

To better understand the structure and function of the gastrointestinal (GI) tracts of wood-eating catfishes, the gross morphology, length, and microvilli surface area (MVSA) of the intestines of wild-caught Panaque nocturnus, P. cf. nigrolineatus "Marañon", and Hypostomus pyrineusi were measured, and contrasted against these same metrics of a closely related detritivore, Pterygoplichthys disjunctivus. All four species had anatomically unspecialized intestines with no kinks, valves, or ceca of any kind. The wood-eating catfishes had body size-corrected intestinal lengths that were 35% shorter than the detritivore. The MVSA of all four species decreased distally in the intestine, indicating that nutrient absorption preferentially takes place in the proximal and mid-intestine, consistent with digestive enzyme activity and luminal carbohydrate profiles for these same species. Wild-caught Pt. disjunctivus, and P. nigrolineatus obtained via the aquarium trade, poorly digested wood cellulose (<33% digestibility) in laboratory feeding trials, lost weight when consuming wood, and passed stained wood through their digestive tracts in less than 4 h. Furthermore, no selective retention of small particles was observed in either species in any region of the gut. Collectively, these results corroborate digestive enzyme activity profiles and gastrointestinal fermentation levels in the fishes' GI tracts, suggesting that the wood-eating catfishes are not true xylivores such as beavers and termites, but rather, are detritivores like so many other fishes from the family Loricariidae.

Fast conversion of scFv to Fab antibodies using type IIs restriction enzymes.

Science.gov (United States)

Sanmark, Hanna; Huovinen, Tuomas; Matikka, Tero; Pettersson, Tiina; Lahti, Maria; Lamminmäki, Urpo

2015-11-01

Single chain variable fragment (scFv) antibody libraries are widely used for developing novel bioaffinity reagents, although Fab or IgG molecules are the preferred antibody formats in many final applications. Therefore, rapid conversion methods for combining multiple DNA fragments are needed to attach constant domains to the scFv derived variable domains. In this study we describe a fast and easy cloning method for the conversion of single framework scFv fragments to Fab fragments using type IIS restriction enzymes. All cloning steps excluding plating of the Fab transformants can be done in 96 well plates and the procedure can be completed in one working day. The concept was tested by converting 69 scFv clones into Fab format on 96 well plates, which resulted in 93% success rate. The method is particularly useful as a high-throughput tool for the conversion of the chosen scFv clones into Fab molecules in order to analyze them as early as possible, as the conversion can significantly affect the binding properties of the chosen clones. Copyright © 2015 Elsevier B.V. All rights reserved.

CNCM I-745 Improves Intestinal Enzyme Function: A Trophic Effects Review

Directory of Open Access Journals (Sweden)

Margret I Moré

2018-02-01

Full Text Available Several properties of the probiotic medicinal yeast Saccharomyces boulardii CNCM I-745 contribute to its efficacy to prevent or treat diarrhoea. Besides immunologic effects, pathogen-binding and anti-toxin effects, as well as positive effects on the microbiota, S boulardii CNCM I-745 also has pronounced effects on digestive enzymes of the brush border membrane, known as trophic effects. The latter are the focus of this review. Literature has been reviewed after searching Medline and PMC databases. All relevant non-clinical and clinical studies are summarized. S. boulardii CNCM I-745 synthesizes and secretes polyamines, which have a role in cell proliferation and differentiation. The administration of polyamines or S. boulardii CNCM I-745 enhances the expression of intestinal digestive enzymes as well as nutrient uptake transporters. The signalling mechanisms leading to enzyme activation are not fully understood. However, polyamines have direct nucleic acid–binding capacity with regulatory impact. S. boulardii CNCM I-745 induces signalling via the mitogen-activated protein kinase pathway. In addition, effects on the phosphatidylinositol-3 kinase (PI3K pathway have been reported. As an additional direct effect, S. boulardii CNCM I-745 secretes certain enzymes, which enhance nutrient acquisition for the yeast and the host. The increased availability of digestive enzymes seems to be one of the mechanisms by which S. boulardii CNCM I-745 counteracts diarrhoea; however, also people with certain enzyme deficiencies may profit from its administration. More studies are needed to fully understand the mechanisms of trophic activation by the probiotic yeast.

An Investigation of Cellulose Digesting Bacteria in the Panda Gut Microbiome

Science.gov (United States)

Lu, M.; Leung, F. C.

2014-12-01

The Giant Panda (Ailuropoda melanoleuca) diet consists primarily of bamboo leaves, stems and shoots. However, the Giant Panda lacks genes for the enzymes needed to digest cellulose, the core component of bamboo. Thus, it is hypothesized that the cellulolytic digestion necessary for maintaining the Giant Panda diet is carried out by microbial symbionts in the panda gut microbiota. Fecal microbiota is used as surrogate index for gut microbiota since the Giant Panda is listed by the World Conservation Union as a Threatened Species. Two bacterial isolates with potential cellulolytic activity were isolated from Giant Panda fecal samples and cultured on selective media CMC (carboxymethyl cellulose) agar and CMC-Congo Red agar using various methods of inoculation. After incubation, clearance zones around colonies were observed and used as qualitative assays for cellulose digestion. Polymerase chain reaction amplification of the 16S rRNA gene was completed and species identification was done based on the BLAST result of 16S rRNA sequence obtained using Sanger sequencing. Once the cellulase activity is confirmed, genomic DNA of the bacteria will be extracted and used for whole genome shotgun sequencing. Illumina next generation sequencing platform will be adopted as it yields high-throughput information, providing a better understanding of cellulose digestion and the molecular genetic pathways to renewable sources of biofuels. Researchers have identified multiple cellulose-digesting microbes in the Giant Panda gut, but few have applied such bacteria in converting cellulose into glucose to create biofuel. Cellulosic ethanol, a biofuel, is produced through the fermentation of lignocellulosic biomasses. This anaerobic process is aided by cellulose-digesting enzymes. Certain microbes, such as those present in the Giant Panda gut, can produce enzymes that cleave the glycosidic bonds of cellulose (C6H10O5) into glucose molecules (C6H12O6), which can then be fermented into ethanol

Investigation of digestion Kinetics in commercial starches using in-situ small-angle neutron scattering

International Nuclear Information System (INIS)

Blazek, Jaroslav; Gilbert, Elliot Paul

2009-01-01

Full text: The digestion of starch has been the subject of many investigations, mostly involving in vitro measurement of the susceptibility of starches to attack by different enzymes, rather than measuring actual digestibility in vivo. The rate and extent of amylolytic hydrolysis of granular starches is known to vary according to botanical origin. Granule characteristics considered to influence susceptibility to attack by alpha-amylase include crystallinity, granule size and available specific surface, amylose content, porosity, structural inhomogeneities and degree of integrity. Most in-vitro studies of granular starch digestion have been limited to samples for which aliquots have been removed from the reaction mixture at various time intervals and freeze-dried to be subsequently characterized using a range of techniques. It remains unclear whether sample preparation creates artefacts in the samples. In this study, we have studied the kinetics of starch digestion of several commercial granular starches by time-resolved small-angle neutron scattering using an in-situ digestion chamber allowing, for the first time, to follow structural changes of starch in the course of digestion directly in the digestion mixture. Additionally, samples before and after digestion were studied by x-ray diffraction, small-angle x-ray scattering, differential scanning calorimetry and microscopy. Microscopy revealed that studied starches, which varied in their amylose content and digestion kinetics, followed different modes of attack The multidisciplinary approach allowed the nanostructural changes detected by small-angle scattering in the course of enzymic breakdown to be correlated with changes in crystallinity and functional properties.

Synergism between ultrasonic pretreatment and white rot fungal enzymes on biodegradation of wheat chaff.

Science.gov (United States)

Sabarez, Henry; Oliver, Christine Maree; Mawson, Raymond; Dumsday, Geoff; Singh, Tanoj; Bitto, Natalie; McSweeney, Chris; Augustin, Mary Ann

2014-11-01

Lignocellulosic biomass samples (wheat chaff) were pretreated by ultrasound (US) (40kHz/0.5Wcm(-2)/10min and 400kHz/0.5Wcm(-2)/10min applied sequentially) prior to digestion by enzyme extracts obtained from fermentation of the biomass with white rot fungi (Phanerochaete chrysosporium or Trametes sp.). The accessibility of the cellulosic components in wheat chaff was increased, as demonstrated by the increased concentration of sugars produced by exposure to the ultrasound treatment prior to enzyme addition. Pretreatment with ultrasound increased the concentration of lignin degradation products (guaiacol and syringol) obtained from wheat chaff after enzyme addition. In vitro digestibility of wheat chaff was also enhanced by the ultrasonics pretreatment in combination with treatment with enzyme extracts. Degradation was enhanced with the use of a mixture of the enzyme extracts compared to that for a single enzyme extract. Copyright © 2014. Published by Elsevier B.V.

Mutagenesis of the redox-active disulfide in mercuric ion reductase: Catalysis by mutant enzymes restricted to flavin redox chemistry

International Nuclear Information System (INIS)

Distefano, M.D.; Au, K.G.; Walsh, C.T.

1989-01-01

Mercuric reductase, a flavoenzyme that possesses a redox-active cystine, Cys 135 Cys 140 , catalyzes the reduction of Hg(II) to Hg(0) by NADPH. As a probe of mechanism, the authors have constructed mutants lacking a redox-active disulfide by eliminating Cys 135 (Ala 135 Cys 140 ), Cys 14 (Cys 135 Ala 140 ), or both (Ala 135 Ala 140 ). Additionally, they have made double mutants that lack Cys 135 (Ala 135 Cys 139 Cys 140 ) or Cys 140 (Cys 135 Cys 139 Ala 140 ) but introduce a new Cys in place of Gly 139 with the aim of constructing dithiol pairs in the active site that do not form a redox-active disulfide. The resulting mutant enzymes all lack redox-active disulfides and are hence restricted to FAD/FADH 2 redox chemistry. Each mutant enzyme possesses unique physical and spectroscopic properties that reflect subtle differences in the FAD microenvironment. Preliminary evidence for the Ala 135 Cys 139 Cys 14 mutant enzyme suggests that this protein forms a disulfide between the two adjacent Cys residues. Hg(II) titration experiments that correlate the extent of charge-transfer quenching with Hg(II) binding indicate that the Ala 135 Cys 140 protein binds Hg(II) with substantially less avidity than does the wild-type enzyme. All mutant mercuric reductases catalyze transhydrogenation and oxygen reduction reactions through obligatory reduced flavin intermediates at rates comparable to or greater than that of the wild-type enzyme. In multiple-turnover assays which monitored the production of Hg(0), two of the mutant enzymes were observed to proceed through at least 30 turnovers at rates ca. 1000-fold slower than that of wild-type mercuric reductase. They conclude that the Cys 135 and Cys 140 thiols serve as Hg(II) ligands that orient the Hg(II) for subsequent reduction by a reduced flavin intermediate

ENZYME RESISTANCE OF GENETICALLY MODIFIED STARCH POTATOES

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A. Sh. Mannapova

2015-01-01

Full Text Available Here in this article the justification of expediency of enzyme resistant starch use in therapeutic food products is presented . Enzyme resistant starch is capable to resist to enzymatic hydrolysis in a small intestine of a person, has a low glycemic index, leads to decrease of postprandial concentration of glucose, cholesterol, triglycerides in blood and insulin reaction, to improvement of sensitivity of all organism to insulin, to increase in sense of fulness and to reduction of adjournment of fats. Resistant starch makes bifidogenшс impact on microflora of a intestine of the person, leads to increase of a quantity of lactobacillus and bifidobacterium and to increased production of butyric acid in a large intestine. In this regard the enzyme resistant starch is an important component in food for prevention and curing of human diseases such as diabetes, obesity, colitis, a cancer of large and direct intestine. One method is specified by authors for imitation of starch digestion in a human body. This method is based on the definition of an enzyme resistance of starch in vitro by its hydrolysis to glucose with application of a glucoamylase and digestive enzyme preparation Pancreatin. This method is used in researches of an enzyme resistance of starch, of genetically modified potato, high amylose corn starch Hi-Maize 1043 and HYLON VII (National Starch Food Innovation, USA, amylopectin and amylose. It is shown that the enzyme resistance of the starch emitted from genetically modified potatoes conforms to the enzyme resistance of the high amylose corn starch “Hi-Maize 1043 and HYLON VII starch”, (National Starch Food Innovation, the USA relating to the II type of enzyme resistant starch. It is established that amylopectin doesn't have the enzyme resistant properties. The results of researches are presented. They allow us to make the following conclusion: amylose in comparison with amylopectin possesses higher enzyme resistance and gives to

Significant effect of NSP-ase enzyme supplementation in sunflower meal-based diet on the growth and nutrient digestibility in broilers.

Science.gov (United States)

Bilal, M; Mirza, M A; Kaleem, M; Saeed, M; Reyad-Ul-Ferdous, Md; Abd El-Hack, M E

2017-04-01

The response of broiler chickens to 3 levels of sunflower meal and 2 levels of NSP-ase enzyme combination (with and without) was investigated in 3 × 2 factorial arrangement under complete randomized design (CRD). A total of 240 Hubbard broiler chicks were fed on practical mash diets having 2950 kcal of ME and 21% CP from 1 to 42 days of age. The BW gain was not significantly reduced when 25% SFM was added in the diets during 1 to 42 days of age. Supplementation of NSP-ase in broiler diets (day 1-42 overall) demonstrated non-significant differences (p Replacement of SBM with SFM or inclusion of SFM at higher level (25%) increased/deteriorated FCR. The addition of exogenous NSP-ase showed a significant improvement (p < 0.01) in feed:gain. The improvement was clearly demonstrated when SFM was added to the experimental diet at 15% or even 20%. Supplementation of NSP-ase at the 25% inclusion level could not, however, sustain the beneficial effect, which was possibly due to excessively high dietary CF. No difference was noted across the treatments regarding carcass response. Relative gizzard weight and intestinal weight were observed to be improved in birds consuming higher levels of SFM (p = 0.00). The digestibility of CF was observed to improve when SFM was used at 20% and 25% in the diets. No improvement in the digestibility of CF was observed with NSP-ase supplementation, which meant other factors were clearly involved. Supplementation of NSP-ase improved FCR up to 20% SFM. At 25% SFM, no improvement in the digestibility of CF was observed with NSP-ase supplementation. Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.

Optimisation of substrate blends in anaerobic co-digestion using adaptive linear programming.

Science.gov (United States)

García-Gen, Santiago; Rodríguez, Jorge; Lema, Juan M

2014-12-01

Anaerobic co-digestion of multiple substrates has the potential to enhance biogas productivity by making use of the complementary characteristics of different substrates. A blending strategy based on a linear programming optimisation method is proposed aiming at maximising COD conversion into methane, but simultaneously maintaining a digestate and biogas quality. The method incorporates experimental and heuristic information to define the objective function and the linear restrictions. The active constraints are continuously adapted (by relaxing the restriction boundaries) such that further optimisations in terms of methane productivity can be achieved. The feasibility of the blends calculated with this methodology was previously tested and accurately predicted with an ADM1-based co-digestion model. This was validated in a continuously operated pilot plant, treating for several months different mixtures of glycerine, gelatine and pig manure at organic loading rates from 1.50 to 4.93 gCOD/Ld and hydraulic retention times between 32 and 40 days at mesophilic conditions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Qualitative feed restriction for heavy swines: effect on digestibility and weight of organs of digestive tract, and environmental impact of feces Restrição alimentar qualitativa para suínos pesados: efeito sobre digestibilidade e peso dos órgãos do trato digestório e impacto ambiental das fezes

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A.L. Fraga

2009-12-01

Full Text Available A total of 50 barrows were used to evaluate qualitative feed restriction on digestibility of dietary components, weight of organs of digestive tract, and composition and production of feces. Five experimental diets, with increasing levels of qualitative feed restriction of 0, 5, 10, 15, and 20% were used. There was linear reduction (PForam utilizados 50 suínos machos castrados para avaliar o efeito da restrição alimentar qualitativa sobre a digestibilidade dos componentes dietéticos, os órgãos do trato digestório, a composição e a produção fecal. Foram utilizadas cinco dietas experimentais, com níveis de restrição qualitativa de 0, 5, 10, 15 e 20%. Houve redução linear (P<0,001 para todos os coeficientes de digestibilidade, com exceção da fibra em detergente ácido, que apresentou resposta quadrática (P<0,05. Os teores de sólidos totais (P<0,01 e voláteis (P<0,05, e minerais totais (P<0,001 nas fezes aumentaram com os níveis de restrição alimentar, enquanto os níveis de K (P<0,05, Cu (P<0,01 e de N, P, Na, Ca, Mg, Fe e Zn (P<0,001, apresentaram resposta quadrática. A excreção diária de fezes, sólidos totais e voláteis, minerais totais, N, P, K, Mn e Cu (P<0,001, Ca, Na, Mg e Fe (P<0,05 apresentaram aumento em função do nível da restrição alimentar qualitativa. A restrição qualitativa pode ser alternativa para destinação de resíduos da agroindústria, conferindo boas propriedades às fezes suínas, no que diz respeito à utilização para adubação de culturas.

Comparison of lipases for in vitro models of gastric digestion

DEFF Research Database (Denmark)

Sassene, P J; Fanø, M; Mu, H

2016-01-01

Lipase (ROL), Rabbit Gastric Lipase (RGL) and recombinant HGL (rHGL), were used to catalyze the in vitro digestion of two infant formulas (a medium-chain triacylglyceride enriched formula (MC-IF) and a predominantly long-chain triacylglyceride formula (LC-IF)). Digesta were withdrawn after 0, 5, 15, 30......, 60 min of gastric digestion and after 90 or 180 min of intestinal digestion with or without the presence of pancreatic enzymes, respectively. The digesta were analyzed by scanning electron microscopy and gas chromatography to quantify the release of fatty acids (FAs). Digestions of both formulas......, catalyzed by ROL, showed that the extent of gastric digestion was higher than expected from previously published in vivo data. ROL was furthermore insensitive to FA chain length and all FAs were released at the same pace. RGL and rHGL favoured the release of MC-FAs in both formulas, but rHGL did also...

EFFECTS OF DIFFERENT TYPES OF FEED ON GROWTH, SURVIVAL AND DIGESTIVE ENZYME ACTIVITY OF EARLY JUVENILE-STAGE MARBLE GOBY, Oxyeleotris marmoratus

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Muhammad Darwis

2008-06-01

Full Text Available The stable mass seed production technique of marble goby, Oxyeleotris marmoratus has not yet been established due to high mortality occurring on the early juvenile stage and/or after 40 days after hatching (d AH. In order to establish the mass seed production technique, O. marmoratus juveniles of 40 d AH were reared for 60 days to determine growth, survival and digestive enzyme activity at different types of feed (trash fish, artificial feed and Artemia sp. nauplii. Specific growth rate (5.32 and survival rate (91.3% in juveniles fed Artemia sp. nauplii showed significantly higher (P<0.05 than those of juveniles fed trash fish and artificial feed. At the end of the experiment, the mean specific trypsin, amylase and lipase activities of juveniles fed Artemia sp. nauplii were recorded as 0.04 mU mg protein-1m-1, 0.114 mg maltose mg protein-1h-1 and 9.58 m Units, respectively, and they were significantly higher than those of juveniles fed trash fish and artificial feed. In the present study it was shown that O. marmoratus early juveniles had more efficient digestibility and absorption the nutrient when fed with Artemia.

Advanced Applications of Robotics in Digestive Surgery

Science.gov (United States)

Patriti, Alberto; Addeo, Pietro; Buchs, Nicolas; Casciola, Luciano; Morel, Philippe

2011-01-01

Laparoscopy is widely recognized as feasible and safe approach to many oncologic and benign digestive conditions and is associated with an improved early outcome. Robotic surgery promises to overcome intrinsic limitations of laparoscopic surgery by a three-dimensional view and wristed instruments widening indications for a minimally invasive approach. To date, the more interesting applications of robotic surgery are those operations restricted to one abdominal quadrant and requiring a fine dissection and digestive reconstruction. While robot-assisted rectal and gastric surgery are becoming well-accepted options among the surgical community, applications of robotics in hepato-biliary and pancreatic surgery are still debated. PMID:23905029

The BsaHI restriction-modification system: Cloning, sequencing and analysis of conserved motifs

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Roberts Richard J

2008-05-01

Full Text Available Abstract Background Restriction and modification enzymes typically recognise short DNA sequences of between two and eight bases in length. Understanding the mechanism of this recognition represents a significant challenge that we begin to address for the BsaHI restriction-modification system, which recognises the six base sequence GRCGYC. Results The DNA sequences of the genes for the BsaHI methyltransferase, bsaHIM, and restriction endonuclease, bsaHIR, have been determined (GenBank accession #EU386360, cloned and expressed in E. coli. Both the restriction endonuclease and methyltransferase enzymes share significant similarity with a group of 6 other enzymes comprising the restriction-modification systems HgiDI and HgiGI and the putative HindVP, NlaCORFDP, NpuORFC228P and SplZORFNP restriction-modification systems. A sequence alignment of these homologues shows that their amino acid sequences are largely conserved and highlights several motifs of interest. We target one such conserved motif, reading SPERRFD, at the C-terminal end of the bsaHIR gene. A mutational analysis of these amino acids indicates that the motif is crucial for enzymatic activity. Sequence alignment of the methyltransferase gene reveals a short motif within the target recognition domain that is conserved among enzymes recognising the same sequences. Thus, this motif may be used as a diagnostic tool to define the recognition sequences of the cytosine C5 methyltransferases. Conclusion We have cloned and sequenced the BsaHI restriction and modification enzymes. We have identified a region of the R. BsaHI enzyme that is crucial for its activity. Analysis of the amino acid sequence of the BsaHI methyltransferase enzyme led us to propose two new motifs that can be used in the diagnosis of the recognition sequence of the cytosine C5-methyltransferases.

Melatonin Promotes Cheliped Regeneration, Digestive Enzyme Function, and Immunity Following Autotomy in the Chinese Mitten Crab, Eriocheir sinensis

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Cong Zhang

2018-03-01

Full Text Available In the pond culture of juvenile Eriocheir sinensis, a high limb-impairment rate seriously affects the culture success. Therefore, it is particularly important to artificially promote limb regeneration. This study evaluated the effects of melatonin on cheliped regeneration, digestive ability, and immunity, as well as its relationship with the eyestalk. It was found that the injection of melatonin significantly increased the limb regeneration rate compared with the saline group (P < 0.05. The qRT-PCR results of growth-related genes showed that the level of EcR-mRNA (ecdysteroid receptor and Chi-mRNA (chitinase expression was significantly increased following the melatonin injection, while the expression of MIH-mRNA (molt-inhibiting hormone was significantly decreased (P < 0.05. Melatonin significantly increased lipase activity (P < 0.05. We observed that the survival rates of limb-impaired and unilateral eyestalk-ablated crabs were substantially improved following melatonin treatment, whereas the survival of the unilateral eyestalk-ablated crabs was significantly decreased compared with the control group (P < 0.05. Furthermore, the results of serum immune and antioxidant capacity revealed that melatonin significantly increased the total hemocyte counts (THC, hemocyanin content, total antioxidant capacity (T-AOC, acid phosphatase (ACP, and glutathione peroxidase activity (GSH-Px, whereas the immune-related parameters were significantly decreased in eyestalk-ablated crabs (P < 0.05. Therefore, these findings indicate that melatonin exerts a protective effect on organism injury, which could promote limb regeneration by up-regulating the expression of growth-related genes, improve digestive enzyme activity, and strengthen the immune response, particularly antioxidant capacity.

Physiogenomic analysis of weight loss induced by dietary carbohydrate restriction

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Wood Richard J

2006-05-01

Full Text Available Abstract Background Diets that restrict carbohydrate (CHO have proven to be a successful dietary treatment of obesity for many people, but the degree of weight loss varies across individuals. The extent to which genetic factors associate with the magnitude of weight loss induced by CHO restriction is unknown. We examined associations among polymorphisms in candidate genes and weight loss in order to understand the physiological factors influencing body weight responses to CHO restriction. Methods We screened for genetic associations with weight loss in 86 healthy adults who were instructed to restrict CHO to a level that induced a small level of ketosis (CHO ~10% of total energy. A total of 27 single nucleotide polymorphisms (SNPs were selected from 15 candidate genes involved in fat digestion/metabolism, intracellular glucose metabolism, lipoprotein remodeling, and appetite regulation. Multiple linear regression was used to rank the SNPs according to probability of association, and the most significant associations were analyzed in greater detail. Results Mean weight loss was 6.4 kg. SNPs in the gastric lipase (LIPF, hepatic glycogen synthase (GYS2, cholesteryl ester transfer protein (CETP and galanin (GAL genes were significantly associated with weight loss. Conclusion A strong association between weight loss induced by dietary CHO restriction and variability in genes regulating fat digestion, hepatic glucose metabolism, intravascular lipoprotein remodeling, and appetite were detected. These discoveries could provide clues to important physiologic adaptations underlying the body mass response to CHO restriction.

Enzymatic digestibility of peptides cross-linked by ionizing radiation

International Nuclear Information System (INIS)

Dizdaroglu, M.; Gajewski, E.; Simic, M.G.

1984-01-01

Digestibility by proteolytic enzymes of peptides cross-linked by ionizing radiation was investigated. Small peptides of alanine and phenylalanine were chosen as model compounds and aminopeptidases and carboxypeptidases were used as proteolytic enzymes. Peptides exposed to γ-radiation in aqueous solution were analysed by high-performance liquid chromatography before and after hydrolysis by aminopeptidase M, leucine aminopeptidase carboxypeptidase A and carboxypeptidase Y. The results obtained clearly demonstrate the different actions of these enzymes on cross-linked aliphatic and aromatic peptides. Peptide bonds of cross-linked dipeptides of alanine were completely resistant to enzymatic hydrolysis whereas the enzymes, except for carboxypeptidase Y, cleaved all peptide bonds of cross-linked peptides of phenylalanine. The actions of the enzymes on these particular compounds are discussed in detail. (author)

Levels of digestible protein to surubim (Pseudoplatystoma sp. reared in net cages

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Claucia Aparecida Honorato

2014-10-01

Full Text Available The Pseudoplatystoma sp. is species of carnivorous fish that require special attention in the diet offered. This work had the objective to determine the digestible protein requirement of juvenile the Pseudoplatystoma sp. reared in net cages. The test consisted of four isoenergetic diets (2606.69 ± 39.16 kcal kg-1 of digestible energy containing increasing levels of digestible protein (23, 24, 26 and 28%PD provided to juveniles of surubim (157.35±11.23g for five months. The parameters of growth, fillet composition, metabolic liver enzymes and morphometry of the intestine and liver were analyzed in completely randomized design with four treatments and four replicates. An increase of protein in the diet provided better weight gain. The metabolic liver enzymes increased in fish fed 24PD. The histopathological changes were not observed in the liver of the fish. The bowel histology showed adaptation to increased protein in the diet until the 26 level PD. juveniles of Pseudoplatystoma sp. Were demanding in digestible protein, showing the best results of production performance and nutrient use efficiency with the diet containing 28%PD.

Fat digestion and absorption in spice-pretreated rats.

Science.gov (United States)

Prakash, Usha N S; Srinivasan, Krishnapura

2012-02-01

A few common spices are known to stimulate secretion of bile with higher amount of bile acids which play a major role in digestion and absorption of dietary lipids. It would be appropriate to verify if these spices enable efficient digestion and absorption during high-fat intake. In this context, dietary ginger (0.05%), piperine (0.02%), capsaicin (0.015%), and curcumin (0.5%) were examined for their influence on bile secretion, digestive enzymes of pancreas and absorption of dietary fat in high-fat (30%) fed Wistar rats for 8 weeks. These spices enhanced the activity of pancreatic lipase, amylase, trypsin and chymotrypsin by 22-57%, 32-51%, 63-81% and 12-38%, respectively. Dietary intake of spices along with high-fat enhanced fat absorption. These dietary spices increased bile secretion with higher bile acid content. Stimulation of lipid mobilisation from adipose tissue was suggested by the decrease in perirenal adipose tissue weight by dietary capsaicin and piperine. This was also accompanied by prevention of the accumulation of triglyceride in liver and serum in high-fat fed rats. Activities of key lipogenic enzymes in liver were reduced which was accompanied by an increased activity of hormone-sensitive lipase. Thus, dietary ginger and other spice compounds enhance fat digestion and absorption in high-fat fed situation through enhanced secretion of bile salts and a stimulation of the activity pancreatic lipase. At the same time, the energy expenditure is facilitated by these spices to prevent the accumulation of absorbed fat. Copyright © 2011 Society of Chemical Industry.

On-site hydrolytic enzymes production from fungal co-cultivation of Bermuda grass and corn cob.

Science.gov (United States)

Amaro-Reyes, Aldo; Gracida, Jorge; Huizache-Peña, Nelson; Elizondo-García, Norberto; Salazar-Martínez, José; García Almendárez, Blanca E; Regalado, Carlos

2016-07-01

Solid state fermentation (SSF) is used to produce industrial enzymes. The objective of this study was to use a co-culture of Aspergillus niger GS1 and Trichoderma reesei, grown on a mixture of Bermuda grass and corn cob to obtain fermented forage (FF) rich in hydrolytic enzymes, as a value added ingredient for animal feed. FPase, amylase and xylanase productivities (dry matter, DM) were 8.8, 181.4, and 42.1Ug(-1)h(-1), respectively (1U=reducing sugars released min(-1)), after 12-16h of SSF with C/N=60. Cellulose, hemicellulose and lignin decreased 1.6-, 2.7- and 1.9-fold (DM), respectively. In vitro ruminal and true digestibility of DM was improved 2.4- and 1.4-fold. Ruminal digestion of FF reduced 1.32-fold the acetate:propionate ratio, which may reduce the environmental impact of ruminants feeding. On-site hydrolytic enzymes productivity using SSF without enzymes extraction could be of economic potential for digestibility improvement in animal feed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

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Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

2016-01-01

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology. PMID:26861509

Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

Science.gov (United States)

Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

2016-02-01

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

Feeding behavior and digestive physiology in sea cucumber Apostichopus japonicus.

Science.gov (United States)

Sun, Jiamin; Zhang, Libin; Pan, Yang; Lin, Chenggang; Wang, Fang; Kan, Rentao; Yang, Hongsheng

2015-02-01

The feeding behavior and digestive physiology of the sea cucumber, Apostichopus japonicus are not well understood. A better understanding may provide useful information for the development of the aquaculture of this species. In this article the tentacle locomotion, feeding rhythms, ingestion rate (IR), feces production rate (FPR) and digestive enzyme activities were studied in three size groups (small, medium and large) of sea cucumber under a 12h light/12h dark cycle. Frame-by-frame video analysis revealed that all size groups had similar feeding strategies using a grasping motion to pick up sediment particles. The tentacle insertion rates of the large size group were significantly faster than those of the small and medium-sized groups (Psea cucumber were nocturnal and their feeding peaks occurred at 02:00-04:00. The medium and large-sized groups also had a second feeding peak during the day. Both IR and FPR in all groups were significantly higher at night than those during the daytime (P<0.05). Additionally, the peak activities of digestive enzymes were 2-4h earlier than the peak of feeding. Taken together, these results demonstrated that the light/dark cycle was a powerful environment factor that influenced biological rhythms of A. japonicus, which had the ability to optimize the digestive processes for a forthcoming ingestion. Copyright © 2014 Elsevier Inc. All rights reserved.

Development Of Electronic Digestive System Module For Effective Teaching And Learning

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Liman Aminu Doko

2017-07-01

Full Text Available The digestive system hence digestion of food is usually one of the topics taught at the secondary and tertiary levels of education. Often this topic is taught using teaching aid in the form of diagrams or charts drawn on plane papers. The inanimate nature of these teaching aid employed makes learning less interesting and comprehension difficult. This paper presents the design and construction of a semi animated digestive module with remote control that visualizes the movement and process of food digestion in the body. Basically the system consists of carved wooden digestive organs with light emitting diodes LEDs carefully fixed on the path of digestion. A remote control is also built to aid remote access to the module. These LEDs start to blink indicating swallowing from the mouth down to the anus illustrating the process of digestion which also involves the production of enzymes. A comparison of with the improved teaching aid will make conventional types showed that it aroused student interest during teaching and learning process. It also reduced too much abstract explanation. Thus making teaching more efficient.

Effect of adding cofactors to exogenous fibrolytic enzymes on preingestive hydrolysis, in vitro digestibility, and fermentation of bermudagrass haylage.

Science.gov (United States)

Romero, J J; Ma, Z X; Gonzalez, C F; Adesogan, A T

2015-07-01

Our objectives were to examine if adding metal ion cofactors (COF) to exogenous fibrolytic enzymes (EFE) would increase the beneficial effects of the EFE on the preingestive hydrolysis and in vitro digestibility and fermentation of bermudagrass haylage. In experiment 1, 5 COF (Mn(2+), Co(2+), Fe(2+), Ca(2+), and Mg(2+)) were screened to select the best candidates for synergistically enhancing release of water-soluble carbohydrates (WSC) from bermudagrass haylage by 5 EFE. The 5 EFE (1A, 2A, 11C, 13D, and 15D) were sourced from Trichoderma reesei and Aspergillus oryzae and they were the most effective of 12 EFE at increasing the neutral detergent fiber digestibility of bermudagrass haylage in a previous trial. Adding 1mM of each of the COF to EFE 2A or 11C synergistically increased release of WSC from bermudagrass haylage, as did adding (1mM) Fe(2+) to 1A, Mn(2+), Co(2+), or Fe(2+) to 13D, or Co(2+)or Fe(2+) to 15D. The greatest release of WSC responses were obtained by adding Mn(2+) to 11C (38%) or by adding Fe(2+) to 2A or 13D (10 and 21.9%, respectively). In experiment 2, the effect of increasing the COF dose on in vitro digestibility and fermentation of bermudagrass haylage was examined using the best EFE-COF combinations from experiment 1. Effects of adding increasing doses of these COF on EFE-mediated changes in vitro digestibility depended on the COF-EFE combination. Adding 10mM Mn(2+) alone to bermudagrass haylage increased DMD and NDFD by 2.7 and 6.3% and adding 11C alone increased these measures by 6.6 and 15.5%, respectively. However, adding 10mM Mn(2+) with 11C resulted in 3.5 and 8.1% increases in DMD and NDFD, respectively, beyond the increases caused by adding 11C alone. Adding Fe(2+) to 2A had no effects on EFE-mediated digestibility responses, but 2A prevented adverse effects of adding Fe(2+) alone on DMD and NDFD. In contrast, adding Fe(2+) to 13D reduced the increases in DMD and NDFD caused by adding the EFE alone. This study shows that adding COF

Physicochemical properties and starch digestibility of Chinese noodles in relation to optimal cooking time.

Science.gov (United States)

Ye, Xiaoting; Sui, Zhongquan

2016-03-01

Changes in the physicochemical properties and starch digestibility of white salted noodles (WSN) at different cooking stage were investigated. The noodles were dried in fresh air and then cooked for 2-12 min by boiling in distilled water to determine the properties of cooking quality, textural properties and optical characteristic. For starch digestibility, dry noodles were milled and sieved into various particle size classes ranging from 0.5 mm to 5.0 mm, and hydrolyzed by porcine pancreatic α-amylase. The optimal cooking time of WSN determined by squeezing between glasses was 6 min. The results showed that the kinetics of solvation of starch and protein molecules were responsible for changes of the physicochemical properties of WSN during cooking. The susceptibility of starch to α-amylase was influenced by the cooking time, particle size and enzyme treatment. The greater value of rapidly digestible starch (RDS) and lower value of slowly digestible starch (SDS) and resistant starch (RS) were reached at the optimal cooking stage ranging between 63.14-71.97%, 2.47-10.74% and 23.94-26.88%, respectively, indicating the susceptibility on hydrolysis by enzyme was important in defining the cooked stage. The study suggested that cooking quality and digestibility were not correlated but the texture greatly controls the digestibility of the noodles. Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of The Phytase Enzyme Addition in The Artificial Feed on Digestibility of Feed, Feed Conversion Ratio and Growth of Gift Tilapia Saline Fish (Oreochromis niloticus) Nursery Stadia I

Science.gov (United States)

Rachmawati, Diana; Samidjan, Istiyanto; Elfitasari, Tita

2018-02-01

The purpose of this study was to determine the effect of adding the phytase enzyme in the artificial feed on digestibility of feed, feed conversion ratio and growth of gift tilapia saline fish (Oreochromis niloticus) nursery stadia I. The fish samples in this study used gift tilapia saline fish (O. niloticus) with an average weight of 0,62 ± 0,008 g/fish and the stocking density of 1 fish1 L. Experimental method used in this study was completely randomized design with 4 treatments and 3 repetitions. The treatments were by adding phytase enzyme in artificial feed with the different level of doses those were A (0 FTU kg1 feed), B (500 FTU kg1 feed), C (1000 FTU kg1 feed) and D (1500 FTU kg1 feed). The results show that the addition of phytase enzyme was significantly (P0.05) affected on Survival Rate (SR) of gift tilapia saline fish. The optimum doses of phytase enzyme on RGR, FCR, PER, ADCP and ADCF of gift tilapia saline fish ranged from 1060 to 1100 FTU kg-1 feed.

Crosslinked Enzyme Aggregates in Hierarchically-Ordered Mesoporous Silica: A Simple and Effective Method for Enzyme Stabilization

Energy Technology Data Exchange (ETDEWEB)

Kim, Moon Il; Kim, Jungbae; Lee, Jinwoo; Jia, Hongfei; Na, Hyon Bin; Youn, Jongkyu; Kwak, Ja Hun; Dohnalkova, Alice; Grate, Jay W.; Wang, Ping; Hyeon, Taeghwan; Park, Hyun-Gyu; Chang, Ho Nam

2007-02-01

alpha-chymotrypsin (CT) and lipase (LP) were immobilized in hierarchically-ordered mesocellular mesoporous silica (HMMS) in a simple but effective way for the enzyme stabilization, which was achieved by the enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of nanometer scale crosslinked enzyme aggregates (CLEAs) entrapped in the mesocellular pores of HMMS (37 nm), which did not leach out of HMMS through narrow mesoporous channels (13 nm). CLEA of alpha-chymotrypsin (CLEA-CT) in HMMS showed a high enzyme loading capacity and significantly increased enzyme stability. No activity decrease of CLEA-CT was observed for two weeks under even rigorously shaking condition, while adsorbed CT in HMMS and free CT showed a rapid inactivation due to the enzyme leaching and presumably autolysis, respectively. With the CLEA-CT in HMMS, however, there was no tryptic digestion observed suggesting that the CLEA-CT is not susceptible to autolysis. Moreover, CLEA of lipase (CLEA-LP) in HMMS retained 30% specific activity of free lipase with greatly enhanced stability. This work demonstrates that HMMS can be efficiently employed as host materials for enzyme immobilization leading to highly enhanced stability of the immobilized enzymes with high enzyme loading and activity.

Host Specificity of Salmonella typhimurium Deoxyribonucleic Acid Restriction and Modification

Science.gov (United States)

Slocum, Harvey; Boyer, Herbert W.

1973-01-01

The restriction and modification genes of Salmonella typhimurium which lie near the thr locus were transferred to a restrictionless mutant of Escherichia coli. These genes were found to be allelic to the E. coli K, B, and A restriction and modification genes. E. coli recombinants with the restriction and modification host specificity of S. typhimurium restricted phage λ that had been modified by each of the seven known host specificities of E. coli at efficiency of plating levels of about 10−2. Phage λ modified with the S. typhimurium host specificity was restricted by six of the seven E. coli host specificities but not by the RII (fi− R-factor controlled) host specificity. It is proposed that the restriction and modification enzymes of this S. typhimurium host specificity have two substrates, one of which is a substrate for the RII host specificity enzymes. PMID:4570605

Feed intake limitation strategies for the growing rabbit: effect on feeding behaviour, welfare, performance, digestive physiology and health: a review.

Science.gov (United States)

Gidenne, T; Combes, S; Fortun-Lamothe, L

2012-09-01

This review aims to present the different effects produced by a post-weaning intake limitation strategy on the growing rabbit, now largely used by French professional rabbit breeders. Although a quantitative feed restriction leads to slower growth, feed conversion (FC) is improved, particularly when the rabbits are again fed freely, as compensatory growth occurs. This better FC or the healthy rabbit is because of better digestion resulting from slower passage through the intestine, whereas the digestive physiology is slightly modified (morphometry of the intestinal mucosa, fermentation pattern, microbiota). Meat quality and carcass characteristics are not greatly affected by feed restriction, except for a lower dressing-out percentage. One of the main advantages of limiting post-weaning intake of the rabbit is to reduce the mortality and morbidity rate due to digestive disorders (particularly epizootic rabbit enteropathy syndrome). The consequences for animal welfare are debatable, as feed restriction probably leads to hunger, but it reduces the incidence of digestive troubles after weaning. However, the growing rabbit adapts very well to an intake limitation strategy, without any aggressive behaviour for congener. In conclusion, restriction strategies could improve profitability of rabbit breeding, but they should be adapted to any specific breeding situation, according to the national market, feed prices, etc.

PENGARUH DEGRADASI ENZIM PROTEOLITIK TERHADAP AKTIVITAS ANGIOTENSIN CONVERTING ENZYME INHIBITOR BEKASAM DENGAN Lactobacillus plantarum B1765 (The Effect of Degradation of Proteolitic Enzyme on Angiotensin Converting Enzyme Inhibitor Activity of Bekasam with Lactobacillus plantarum B1765

Directory of Open Access Journals (Sweden)

Prima Retno Wikandari

2016-10-01

Full Text Available This research studied the effect of digestive enzyme degradation on the Angiotensin Converting Enzyme Inhibitor (ACEI activity and the stability of bekasam peptide and ACEI activity. Water extract of bekasam was subjected to pepsin and trypsin. The stability of peptide was measured from the changes of peptide concentration before and after treatment by those enzymes. The stability of ACEI activity was measured by hypuric acid liberated from Hip-His-Leu as ACE substrate and determined by spectrophotometer. The results showed that proteolytic enzyme degradation did not affect the concentration of peptide (p>0,05 and the mean concentration 36.72. It was closely related with the ACEI activity that did not change significantly before and after digestion by pepsin and trypsin (p>0,05 and the mean ACEI activity was 70.73. It showed that ACEI activity of bekasam did not change by the degradation of digestive enzyme. Keywords: bekasam, fermented fish, peptides, ACEI activity ABSTRAK Penelitian ini bertujuan untuk mengkaji pengaruh degradasi enzim pencernaan proteolitik terhadap stabilitas peptida dan aktivitas Angiotensin Converting Enzyme Inhibitor (ACEI bekasam yang difermentasi dengan kultur starter Lactobacillus plantarum B1765. Terhadap ekstrak bekasam diberi perlakuan enzim proteolitik pepsin dan tripsin. Pengujian stabilitas peptida diukur dengan ada tidaknya perubahan jumlah peptida setelah perlakuan enzim menggunakan metode formol, sedangkan aktivitas ACEI dilakukan dengan mengetahui jumlah asam hipurat dari substrat Hip-His-Leu yang dibebaskan oleh ACE diukur dengan spektrofotometer. Hasil pengujian menunjukkan perlakuan enzim proteolitik tidak berpengaruh pada konsentrasi peptida dengan p>0,05 dengan nilai rata-rata konsentrasi peptida sebesar 36,72. Hal ini berkorelasi dengan aktivitas ACEI yang juga menunjukkan tidak ada pengaruh antara perlakuan sebelum dan setelah degradasi enzim (p>0,05 dengan rata-rata aktivitas ACEI sebesar 70,73. Hasil

Rheostatic control of tryptic digestion in a microscale fluidic system

International Nuclear Information System (INIS)

Percy, Andrew J.; Schriemer, David C.

2010-01-01

Integrated fluidic systems that unite bottom-up and top-down proteomic approaches have the potential to deliver complete protein characterization. To circumvent fraction collection, as is conducted in current blended approaches, a technique to regulate digestion efficiency in a flow-through system is required. The present study examined the concept of regulating tryptic digestion in an immobilized enzyme reactor (IMER), incorporating mixed solvent systems for digestion acceleration. Using ovalbumin, cytochrome c, and myoglobin as protein standards, we demonstrate that tryptic digestion can be efficiently regulated between complete digestion and no digestion extremes by oscillating between 45 and 0% acetonitrile in the fluid stream. Solvent composition was tuned using programmable solvent waveforms in a closed system consisting of the IMER, a sample delivery stream, a dual gradient pumping system and a mass spectrometer. Operation in this rheostatic digestion mode provides access to novel peptide mass maps (due to substrate unfolding hysteresis) as well as the intact protein, in a reproducible and stable fashion. Although cycle times were on the order of 90 s for testing purposes, we show that regulated digestion is sufficiently rapid to be limited by solvent switching efficiency and kinetics of substrate unfolding/folding. Thus, regulated digestion should be useful in blending bottom-up and top-down proteomics in a single closed fluidic system.

Accelerated protein digestion and amino acid absorption after Roux-en-Y gastric bypass

DEFF Research Database (Denmark)

Bojsen-Møller, Anna Kirstine; Jacobsen, Siv H; Dirksen, Carsten

2015-01-01

BACKGROUND: Roux-en-Y gastric bypass (RYGB) involves exclusion of major parts of the stomach and changes in admixture of gastro-pancreatic enzymes, which could have a major impact on protein digestion and amino acid absorption. OBJECTIVE: We investigated the effect of RYGB on amino acid appearance......: RYGB accelerates caseinate digestion and amino acid absorption, resulting in faster and higher but more transient postprandial elevation of plasma amino acids. Changes are likely mediated by accelerated intestinal nutrient entry and clearly demonstrate that protein digestion is not impaired after RYGB...

Effect of exogenous xylanase, amylase, and protease as single or combined activities on nutrient digestibility and growth performance of broilers fed corn/soy diets.

Science.gov (United States)

Amerah, A M; Romero, L F; Awati, A; Ravindran, V

2017-04-01

Two trials (a 42-d performance and a 21-d cohort digestibility) were conducted to evaluate the performance and nutrient digestibility of broilers fed corn diets supplemented with exogenous xylanase, amylase, and protease as single or combined activities. A nutritionally adequate, positive control (PC) diet was formulated. The negative control (NC) diet was formulated to be lower in metabolizable energy (∼86 kcal/kg diet) and digestible amino acids (1 to 2%) compared to PC. The other 4 treatments were based on the NC and they were either supplemented with xylanase (X), amylase (A), protease (P), or a combination of X, A, and P (XAP; to provide 2,000 U of X, 200 U of A, and 4,000 U of P/kg diet). All diets were marginal in AvP and Ca and contained a background of phytase (1,000 FTU/kg). In each trial, male broiler (Ross 308) chicks were allocated to the 5 treatments (10 replicates of 20 birds/pen and 9 replicates of 8 birds/cage for the performance and digestibility trials, respectively). In the digestibility trial, ileal digesta was collected on d21 for the determination of nutrient utilization. Data were subjected to one-way ANOVA and means were separated by Tukey's HSD test. Only the XAP improved (P digestibility and apparent ileal digestible energy (AIDE). Both P and XAP improved N retention. The relative improvement in energy digestibility due to enzyme supplementation was greater at the ileal level than that measured in the excreta. The measured changes on AIDE due to supplemental enzymes were much higher than the sum of calculated contributions from starch, fat, and protein. Supplementation of all enzymes reduced (P  0.05) by dietary treatments. Both X and XAP had lower (P  0.05) FCR compared to PC. In conclusion, these results suggest a synergistic effect between X, A and P on broiler performance and nutrient digestibility. In the current study, AIDE measurements appeared to overestimate the enzyme response. Calculation of the energy contribution by

Immunohistochemical localization of hepatopancreatic phospholipase A2 in Hexaplex Trunculus digestive cells

Science.gov (United States)

2011-01-01

Background Mammalian sPLA2-IB localization cell are well characterized. In contrast, much less is known about aquatic primitive ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes and the mode of digestion of lipid food. Results The marine snail digestive phospholipase A2 (mSDPLA2) has been previously purified from snail hepatopancreas. The specific polyclonal antibodies were prepared and used for immunohistochimical and immunofluorescence analysis in order to determine the cellular location of mSDPLA2. Our results showed essentially that mSDPLA2 was detected inside in specific vesicles tentatively named (mSDPLA2+) granules of the digestive cells. No immunolabelling was observed in secretory zymogene-like cells. This immunocytolocalization indicates that lipid digestion in the snail might occur in specific granules inside the digestive cells. Conclusion The cellular location of mSDPLA2 suggests that intracellular phospholipids digestion, like other food components digestion of snail diet, occurs in these digestive cells. The hepatopancreas of H. trunculus has been pointed out as the main region for digestion, absorption and storage of lipids. PMID:21631952

The ontogeny of nutrient transporter and digestive enzyme gene expression in domestic pigeon (Columba livia) intestine and yolk sac membrane during pre- and posthatch development.

Science.gov (United States)

Dong, X Y; Wang, Y M; Yuan, C; Zou, X T

2012-08-01

To better understand the digestive capacity in domestic pigeons (Columba livia), this study was conducted to evaluate nutrient transporters and digestive enzymes gene expression in small intestine and yolk sac membrane (YSM) during pre- and posthatch development. We investigated the oligopeptide transporter Pept1, sodium glucose transporter SGLT1, glucose transporter GLUT2, aminopeptidase-N (APN), and sucrase-isomaltase (SI). Intestine was collected at embryo d 12, 14, and 16, day of hatch, and d 1, 3, 5, 8, and 14 posthatch. The YSM was collected at embryo d 12, 14, 16, and day of hatch. The cDNA fragments for Pept1, SGLT1, GLUT2, APN, and SI were isolated and cloned using reverse-transcription PCR. The sequences data showed that these genes were highly identical to the gene of chicken. The mRNA expression of each gene was assayed using real-time PCR. Expression of intestinal nutrient transporters increased linearly (Ppigeons and establish a foundation for future research on the nutrients requirements for young pigeons.

Impact of levels of total digestible nutrients on microbiome, enzyme profile and degradation of feeds in buffalo rumen.

Science.gov (United States)

Kala, Anju; Kamra, D N; Kumar, Avinash; Agarwal, Neeta; Chaudhary, L C; Joshi, C G

2017-01-01

The present study was aimed at understanding a shift in rumen microbiome of buffaloes fed various levels of total digestible nutrients. To understand the process, the metagenomics of rumen microbes, in vivo and in vitro rumen fermentation studies were carried out. Three rumen fistulated adult male Murrah buffaloes were fed three isonitrogenous diets varying in total digestible nutrients (70, 85 and 100% of TDN requirement) in 3X3 switch over design. On dry matter basis, wheat straw/ roughage content were 81, 63 and 51% and that of maize grain was 8, 16 and 21% in three diets respectively. After 20 d of feeding, rumen liquor and rumen contents were sampled just before (0h) and 4h post feeding. Ruminococcus flavefaciens and R. albus (estimated with real time PCR) were higher in high roughage diets. The predominant phyla in all the three groups were Bacteroidetes, Firmicutes followed by Proteobacteria, Actinobacteria and Fibrobacteres. A core group of more than fifty rumen bacteria was present in all the animals with very little variations due to level of TDN. The most predominant bacterial genera reported in order of decreasing abundance were: Prevotella, Bacteroides, Clostridium, Ruminococcus, Eubacterium, Parabacteroides, Fibrobacter, Butyrivibrio etc. The higher diversity of the enyzmes families GH 23, GH 28, GH 39, GH 97, GH 106, and GH 127 (the enzymes active in fibre and starch degradation) were significantly higher on 100%TDN diet while CE 14 (required for the hydrolysis of bond between carbohydrate and lignin) was higher on low TDN (70%) diet, indicating ester bond cleavage was better in animals fed high roughage (wheat straw) diet.

Altering the selection capabilities of common cloning vectors via restriction enzyme mediated gene disruption

Science.gov (United States)

2013-01-01

Background The cloning of gene sequences forms the basis for many molecular biological studies. One important step in the cloning process is the isolation of bacterial transformants carrying vector DNA. This involves a vector-encoded selectable marker gene, which in most cases, confers resistance to an antibiotic. However, there are a number of circumstances in which a different selectable marker is required or may be preferable. Such situations can include restrictions to host strain choice, two phase cloning experiments and mutagenesis experiments, issues that result in additional unnecessary cloning steps, in which the DNA needs to be subcloned into a vector with a suitable selectable marker. Results We have used restriction enzyme mediated gene disruption to modify the selectable marker gene of a given vector by cloning a different selectable marker gene into the original marker present in that vector. Cloning a new selectable marker into a pre-existing marker was found to change the selection phenotype conferred by that vector, which we were able to demonstrate using multiple commonly used vectors and multiple resistance markers. This methodology was also successfully applied not only to cloning vectors, but also to expression vectors while keeping the expression characteristics of the vector unaltered. Conclusions Changing the selectable marker of a given vector has a number of advantages and applications. This rapid and efficient method could be used for co-expression of recombinant proteins, optimisation of two phase cloning procedures, as well as multiple genetic manipulations within the same host strain without the need to remove a pre-existing selectable marker in a previously genetically modified strain. PMID:23497512

Laboratory Exercise: Study of Digestive and Regulatory Processes through the Exploration of Fasted and Postprandial Blood Glucose

Science.gov (United States)

Hopper, Mari K.; Maurer, Luke W.

2013-01-01

Digestive physiology laboratory exercises often explore the regulation of enzyme action rather than systems physiology. This laboratory exercise provides a systems approach to digestive and regulatory processes through the exploration of postprandial blood glucose levels. In the present exercise, students enrolled in an undergraduate animal…

Cooking temperature is a key determinant of in vitro meat protein digestion rate: investigation of underlying mechanisms.

Science.gov (United States)

Bax, Marie-Laure; Aubry, Laurent; Ferreira, Claude; Daudin, Jean-Dominique; Gatellier, Philippe; Rémond, Didier; Santé-Lhoutellier, Véronique

2012-03-14

The present study aimed to evaluate the digestion rate and nutritional quality of pig muscle proteins in relation to different meat processes (aging, mincing, and cooking). Under our experimental conditions, aging and mincing had little impact on protein digestion. Heat treatments had different temperature-dependent effects on the meat protein digestion rate and degradation potential. At 70 °C, the proteins underwent denaturation that enhanced the speed of pepsin digestion by increasing enzyme accessibility to protein cleavage sites. Above 100 °C, oxidation-related protein aggregation slowed pepsin digestion but improved meat protein overall digestibility. The digestion parameters defined here open new insights on the dynamics governing the in vitro digestion of meat protein. However, the effect of cooking temperature on protein digestion observed in vitro needs to be confirmed in vivo.

Dynamics of digestive proteolytic system during blood feeding of the hard tick Ixodes ricinus

Directory of Open Access Journals (Sweden)

Sojka Daniel

2010-12-01

Full Text Available Abstract Background Ticks are vectors of a wide variety of pathogens causing severe diseases in humans and domestic animals. Intestinal digestion of the host blood is an essential process of tick physiology and also a limiting factor for pathogen transmission since the tick gut represents the primary site for pathogen infection and proliferation. Using the model tick Ixodes ricinus, the European Lyme disease vector, we have previously demonstrated by genetic and biochemical analyses that host blood is degraded in the tick gut by a network of acidic peptidases of the aspartic and cysteine classes. Results This study reveals the digestive machinery of the I. ricinus during the course of blood-feeding on the host. The dynamic profiling of concentrations, activities and mRNA expressions of the major digestive enzymes demonstrates that the de novo synthesis of peptidases triggers the dramatic increase of the hemoglobinolytic activity along the feeding period. Overall hemoglobinolysis, as well as the activity of digestive peptidases are negligible at the early stage of feeding, but increase dramatically towards the end of the slow feeding period, reaching maxima in fully fed ticks. This finding contradicts the established opinion that blood digestion is reduced at the end of engorgement. Furthermore, we show that the digestive proteolysis is localized intracellularly throughout the whole duration of feeding. Conclusions Results suggest that the egressing proteolytic system in the early stage of feeding and digestion is a potential target for efficient impairment, most likely by blocking its components via antibodies present in the host blood. Therefore, digestive enzymes are promising candidates for development of novel 'anti-tick' vaccines capable of tick control and even transmission of tick-borne pathogens.

Saccharomyces boulardii CNCM I-745 Improves Intestinal Enzyme Function: A Trophic Effects Review.

Science.gov (United States)

Moré, Margret I; Vandenplas, Yvan

2018-01-01

Several properties of the probiotic medicinal yeast Saccharomyces boulardii CNCM I-745 contribute to its efficacy to prevent or treat diarrhoea. Besides immunologic effects, pathogen-binding and anti-toxin effects, as well as positive effects on the microbiota, S boulardii CNCM I-745 also has pronounced effects on digestive enzymes of the brush border membrane, known as trophic effects. The latter are the focus of this review. Literature has been reviewed after searching Medline and PMC databases. All relevant non-clinical and clinical studies are summarized. S. boulardii CNCM I-745 synthesizes and secretes polyamines, which have a role in cell proliferation and differentiation. The administration of polyamines or S. boulardii CNCM I-745 enhances the expression of intestinal digestive enzymes as well as nutrient uptake transporters. The signalling mechanisms leading to enzyme activation are not fully understood. However, polyamines have direct nucleic acid-binding capacity with regulatory impact. S. boulardii CNCM I-745 induces signalling via the mitogen-activated protein kinase pathway. In addition, effects on the phosphatidylinositol-3 kinase (PI3K) pathway have been reported. As an additional direct effect, S. boulardii CNCM I-745 secretes certain enzymes, which enhance nutrient acquisition for the yeast and the host. The increased availability of digestive enzymes seems to be one of the mechanisms by which S. boulardii CNCM I-745 counteracts diarrhoea; however, also people with certain enzyme deficiencies may profit from its administration. More studies are needed to fully understand the mechanisms of trophic activation by the probiotic yeast.

Survival of Saccharomyces cerevisiae after treatment with the restriction endonuclease Alu I

International Nuclear Information System (INIS)

Winckler, K.; Bach, B.; Obe, G.

1988-01-01

Treatment of yeast cells proficient in the repair of radiation damage (Saccharomyces cervisiae) with the restriction endonuclease Alu I leads to a positive dose-effect relationship between inactivation level and enzyme concentration. The data suggest an uptake of the active restriction enzyme into the cells and a relationship between induction of DNA double-strand breaks and cell killing. (author)

Rhythmicity and plasticity of digestive physiology in a euryhaline teleost fish, permit (Trachinotus falcatus)

DEFF Research Database (Denmark)

Lazado, Carlo Cabacang; Pedersen, Per Bovbjerg; Nguyen, Huy Quang

2017-01-01

Digestive physiology is considered to be under circadian control, but there is little evidence in teleost fish. The present study explored the rhythmicity and plasticity to feeding schedules of enzymatic digestion in a candidate aquaculture fish, the permit (Trachinotus falcatus). The first...... experiment identified the rhythms of digestive factors throughout the light-dark (LD) cycle. Gastric luminal pH and pepsin activity showed significant daily variation albeit not rhythmic. These dynamic changes were likewise observed in several digestive enzymes, in which the activities of intestinal protease......, chymotrypsin and lipase exhibited significant daily rhythms. In the second experiment, the existence of feed anticipatory activity in the digestive factors was investigated by subjecting the fish to either periodic or random feeding. Anticipatory gastric acidification prior to feeding was identified...

DNA double-strand break measurement in mammalian cells by pulsed-field gel electrophoresis: an approach using restriction enzymes and gene probing

International Nuclear Information System (INIS)

Loebrich, M.; Ikpeme, S.; Kiefer, J.

1994-01-01

DNA samples prepared from human SP 3 cells, which had not been exposed to various doses of X-ray, were treated with NotI restriction endonuclease before being run in a contour-clamped homogeneous electrophoresis system. The restriction enzyme cuts the DNA at defined positions delivering DNA sizes which can be resolved by pulsed-field gel electrophoresis (PFGE). In order to investigate only one of the DNA fragments, a human lactoferrin cDNA, pHL-41, was hybridized to the DNA separated by PFGE. As a result, only the DNA fragment which contains the hybridized gene was detected resulting in a one-band pattern. The decrease of this band was found to be exponential with increasing radiation dose. From the slope, a double-strand break induction rate of (6.3±0.7) x 10 -3 /Mbp/Gy was deduced for 80 kV X-rays. (Author)

Highly Stable Trypsin-Aggregate Coatings on Polymer Nanofibers for Repeated Protein Digestion

Energy Technology Data Exchange (ETDEWEB)

Kim, Byoung Chan; Lopez-Ferrer, Daniel; Lee, Sang-mok; Ahn, Hye-kyung; Nair, Sujith; Kim, Seong H.; Kim, Beom S.; Petritis, Konstantinos; Camp, David G.; Grate, Jay W.; Smith, Richard D.; Koo, Yoon-mo; Gu, Man Bock; Kim, Jungbae

2009-04-01

A stable and robust trypsin-based biocatalytic system was developed and demonstrated for proteomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This new process produced a 300-fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was also resistant to autolysis, enabling repeated digestions of bovine serum albumin over 40 days and successful peptide identification by LC-MS/MS. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e. chymotrypsin), which makes it suitable for use in “real-world” proteomic applications. Overall, the biocatalytic nanofibers with enzyme aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.

A new methodology for the determination of enzyme activity based on carbon nanotubes and glucose oxidase.

Science.gov (United States)

Yeşiller, Gülden; Sezgintürk, Mustafa Kemal

2015-11-10

In this research, a novel enzyme activity analysis methodology is introduced as a new perspective for this area. The activity of elastase enzyme, which is a digestive enzyme mostly of found in the digestive system of vertebrates, was determined by an electrochemical device composed of carbon nanotubes and a second enzyme, glucose oxidase, which was used as a signal generator enzyme. In this novel methodology, a complex bioactive layer was constructed by using carbon nanotubes, glucose oxidase and a supporting protein, gelatin on a solid, conductive substrate. The activity of elastase was determined by monitoring the hydrolysis rate of elastase enzyme in the bioactive layer. As a result of this hydrolysis of elastase, glucose oxidase was dissociated from the bioactive layer, and following this the electrochemical signal due to glucose oxidase was decreased. The progressive elastase-catalyzed digestion of the bioactive layer containing glucose oxidase decreased the layer's enzymatic efficiency, resulting in a decrease of the glucose oxidation current as a function of the enzyme activity. The ratio of the decrease was correlated to elastase activity level. In this study, optimization experiments of bioactive components and characterization of the resulting new electrochemical device were carried out. A linear calibration range from 0.0303U/mL to 0.0729U/mL of elastase was reported. Real sample analyses were also carried out by the new electrochemical device. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparative Digestive Physiology

Science.gov (United States)

Karasov, William H.; Douglas, Angela E.

2015-01-01

In vertebrates and invertebrates, morphological and functional features of gastrointestinal (GI) tracts generally reflect food chemistry, such as content of carbohydrates, proteins, fats, and material(s) refractory to rapid digestion (e.g., cellulose). The expression of digestive enzymes and nutrient transporters approximately matches the dietary load of their respective substrates, with relatively modest excess capacity. Mechanisms explaining differences in hydrolase activity between populations and species include gene copy number variations and single-nucleotide polymorphisms. Transcriptional and posttranscriptional adjustments mediate phenotypic changes in the expression of hydrolases and transporters in response to dietary signals. Many species respond to higher food intake by flexibly increasing digestive compartment size. Fermentative processes by symbiotic microorganisms are important for cellulose degradation but are relatively slow, so animals that rely on those processes typically possess special enlarged compartment(s) to maintain a microbiota and other GI structures that slow digesta flow. The taxon richness of the gut microbiota, usually identified by 16S rRNA gene sequencing, is typically an order of magnitude greater in vertebrates than invertebrates, and the interspecific variation in microbial composition is strongly influenced by diet. Many of the nutrient transporters are orthologous across different animal phyla, though functional details may vary (e.g., glucose and amino acid transport with K+ rather than Na+ as a counter ion). Paracellular absorption is important in many birds. Natural toxins are ubiquitous in foods and may influence key features such as digesta transit, enzymatic breakdown, microbial fermentation, and absorption PMID:23720328

Intactness of cell wall structure controls the in vitro digestion of starch in legumes.

Science.gov (United States)

Dhital, Sushil; Bhattarai, Rewati R; Gorham, John; Gidley, Michael J

2016-03-01

Increasing the level of starch that is not digested by the end of the small intestine and therefore enters the colon ('resistant starch') is a major opportunity for improving the nutritional profile of foods. One mechanism that has been shown to be successful is entrapment of starch within an intact plant tissue structure. However, the level of tissue intactness required for resistance to amylase digestion has not been defined. In this study, intact cells were isolated from a range of legumes after thermal treatment at 60 °C (starch not gelatinised) or 95 °C (starch gelatinised) followed by hydrolysis using pancreatic alpha amylase. It was found that intact cells, isolated at either temperature, were impervious to amylase. However, application of mechanical force damaged the cell wall and made starch accessible to digestive enzymes. This shows that the access of enzymes to the entrapped swollen starch is the rate limiting step controlling hydrolysis of starch in cooked legumes. The results suggest that a single cell wall could be sufficient to provide an effective delivery of starch to the large intestine with consequent nutritional benefits, provided that mechanical damage during digestion is avoided.

Distant homology between yeast photoreactivating gene fragment and human genomic digests

International Nuclear Information System (INIS)

Meechan, P.J.; Milam, K.M.; Cleaver, J.E.

1985-01-01

Hybridization of DNA coding for the yeast DNA photolyase to human genomic DNA appears to allow one to determine whether a conserved enzyme is coded for in human cells. Under stringent conditions (68 0 C), hybridization is not found between the cloned yeast fragment (YEp13-phr1) and human or chick genomic digests. At less stringent conditions (60 0 C), hybridization is observed with chick digests, indicating evolutionary divergence even among organisms capable of photo-reactivation. At 50 0 C, weak hybridization with human digests was observed, indicating further divergence from the cloned gene. Data concerning the precise extent of homology and methods to clone the chick gene for use as another probe are discussed

Environmental impacts of anaerobic digestion and the use of anaerobic residues as soil amendment

Energy Technology Data Exchange (ETDEWEB)

Mosey, F.E. [VFA Services Ltd., Herts (United Kingdom)

1996-01-01

This paper defines the environmental role of anaerobic digestion within the overall objective of recovering energy from renewable biomass resources. Examples and opportunities for incorporating anaerobic digestion into biomass-to-energy schemes are discussed, together with environmental aspects of anaerobic digestion plants. These include visual, public amenity, pathogens and public health, odor control, and gaseous emissions. Digestate disposal and the benefits of restrictions on recycling organic wastes and biomass residues back to the land are discussed, particularly as they relate to American and European codes of practice and environmental legislation. The paper concludes that anaerobic digestion, if performed in purpose-designed reactors that efficiently recover and use biogas, is an environmentally benign process that can enhance energy recovery and aid the beneficial land use of plant residues in many biomass-to-energy schemes.

Improvement of raw starch digestibility by ion-beam mutation of Aspergillus awamori

Energy Technology Data Exchange (ETDEWEB)

Amsal, Aryanti [National Atomic Energy Agency, Jakarta (Indonesia); Takigami, Machiko; Ito, Hitoshi

1998-09-01

Aspergillus awamori possess the ability to express raw starch digestibility. For the effective utilization of starchy crops produced in South-Asian countries, it is important to achieve the digestion of raw starchs for industrial fermentation process. In this study, higher ratio of mutant strains of Aspergillus awamori IFO4033 were isolated by irradiation of C{sup 5+} ion-beam on freeze dried spores with improvement of enzyme production for two-to threefold in the extracellular {alpha}-amylase compared with gamma-irradiation. The digestibility of raw starch from cassava, sago and sukun increased remarkably about two-to threefold by some mutant strains obtained from irradiation of C{sup 5+} ion-beam. (author)

Improvement of raw starch digestibility by ion-beam mutation of Aspergillus awamori

International Nuclear Information System (INIS)

Amsal, Aryanti; Takigami, Machiko; Ito, Hitoshi

1998-01-01

Aspergillus awamori possess the ability to express raw starch digestibility. For the effective utilization of starchy crops produced in South-Asian countries, it is important to achieve the digestion of raw starchs for industrial fermentation process. In this study, higher ratio of mutant strains of Aspergillus awamori IFO4033 were isolated by irradiation of C 5+ ion-beam on freeze dried spores with improvement of enzyme production for two-to threefold in the extracellular α-amylase compared with gamma-irradiation. The digestibility of raw starch from cassava, sago and sukun increased remarkably about two-to threefold by some mutant strains obtained from irradiation of C 5+ ion-beam. (author)

Differentiation between activity of digestive enzymes of Brachionus calyciflorus and extracellular enzymes of its epizooic bacteria

Directory of Open Access Journals (Sweden)

Wilko H. AHLRICHS

2009-08-01

Full Text Available The rotifer Brachionus calyciflorus was examined by scanning electron microscopy (SEM for surface-attached, i.e. epizootic, bacteria to ascertain their specific localization and thus find out if we could discern between rotifer and bacterial enzyme activity. The lorica of B. calyciflorus was colonized by one distinct type of bacteria, which originated from the algal culture used for rotifer feeding. The corona, posterior epidermis and foot of all inspected individuals were always without attached bacteria. The density of the attached bacteria was higher with the increasing age of B. calyciflorus: while young individuals were colonized by ~ tens of bacterial cells, older ones had on average hundreds to thousands of attached bacteria. We hypothesize that epizooic bacteria may produce the ectoenzymes phosphatases and β-N-acetylhexosaminidases on the lorica, but not on the corona of B. calyciflorus. Since enzyme activities of epizooic bacteria may influence the values and interpretation of bulk rotifer enzyme activities, we should take the bacterial contribution into account.

Exogenous DNA internalisation by sperm cells is improved by combining lipofection and restriction enzyme mediated integration.

Science.gov (United States)

Churchil, R R; Gupta, J; Singh, A; Sharma, D

2011-06-01

1. Three types of exogenous DNA inserts, i.e. complete linearised pVIVO2-GFP/LacZ vector (9620 bp), the LacZ gene (5317 bp) and the GFP gene (2152 bp) were used to transfect chicken spermatozoa through simple incubation of sperm cells with insert. 2. PCR assay, Dot Blot hybridisation and Southern hybridisation showed the successful internalisation of exogenous DNA by chicken sperm cells. 3. Lipofection and Restriction Enzyme Mediated Integration (REMI) were used to improve the rate of internalisation of exogenous DNA by sperm cells. 4. Results from dot blot as well as Southern hybridisation were semi-quantified and improved exogenous DNA uptake by sperm cells through lipofection and REMI. Stronger signals were observed from hybridisation of LacZ as well as GFP specific probe with the DNA from lipofected exogenous DNA transfected sperm DNA in comparison with those transfected with nude exogenous DNA.

Enzymatic Digestion of Chronic Wasting Disease Prions Bound to Soil

Science.gov (United States)

SAUNDERS, SAMUEL E.; BARTZ, JASON C.; VERCAUTEREN, KURT C.; BARTELT-HUNT, SHANNON L.

2010-01-01

Chronic wasting disease (CWD) and sheep scrapie can be transmitted via indirect environmental routes, and it is known that soil can serve as a reservoir of prion infectivity. Given the strong interaction between the prion protein (PrP) and soil, we hypothesized that binding to soil enhances prion resistance to enzymatic digestion, thereby facilitating prion longevity in the environment and providing protection from host degradation. We characterized the performance of a commercially available subtilisin enzyme, the Prionzyme, to degrade soil-bound and unbound CWD and HY TME PrP as a function of pH, temperature, and treatment time. The subtilisin enzyme effectively degraded PrP adsorbed to a wide range of soils and soil minerals below the limits of detection. Signal loss occurred rapidly at high pH (12.5) and within 7 d under conditions representative of the natural environment (pH 7.4, 22°C). We observed no apparent difference in enzyme effectiveness between bound and unbound CWD PrP. Our results show that although adsorbed prions do retain relative resistance to enzymatic digestion compared with other brain homogenate proteins, they can be effectively degraded when bound to soil. Our results also suggest a topical application of a subtilisin enzyme solution may be an effective decontamination method to limit disease transmission via environmental ‘hot spots’ of prion infectivity. PMID:20450190

Use of yeast spores for microencapsulation of enzymes.

Science.gov (United States)

Shi, Libing; Li, Zijie; Tachikawa, Hiroyuki; Gao, Xiao-Dong; Nakanishi, Hideki

2014-08-01

Here, we report a novel method to produce microencapsulated enzymes using Saccharomyces cerevisiae spores. In sporulating cells, soluble secreted proteins are transported to the spore wall. Previous work has shown that the spore wall is capable of retaining soluble proteins because its outer layers work as a diffusion barrier. Accordingly, a red fluorescent protein (RFP) fusion of the α-galactosidase, Mel1, expressed in spores was observed in the spore wall even after spores were subjected to a high-salt wash in the presence of detergent. In vegetative cells, however, the cell wall cannot retain the RFP fusion. Although the spore wall prevents diffusion of proteins, it is likely that smaller molecules, such as sugars, pass through it. In fact, spores can contain much higher α-galactosidase activity to digest melibiose than vegetative cells. When present in the spore wall, the enzyme acquires resistance to environmental stresses including enzymatic digestion and high temperatures. The outer layers of the spore wall are required to retain enzymes but also decrease accessibility of the substrates. However, mutants with mild spore wall defects can retain and stabilize the enzyme while still permitting access to the substrate. In addition to Mel1, we also show that spores can retain the invertase. Interestingly the encapsulated invertase has significantly lower activity toward raffinose than toward sucrose.This suggests that substrate selectivity could be altered by the encapsulation.

Nanostructuring Biomaterials with Specific Activities towards Digestive Enzymes for Controlled Gastrointestinal Absorption of Lipophilic Bioactive Molecules.

Science.gov (United States)

Joyce, Paul; Whitby, Catherine P; Prestidge, Clive A

2016-11-01

This review describes the development of novel lipid-based biomaterials that modulate fat digestion for the enhanced uptake of encapsulated lipophilic bioactive compounds (e.g. drugs and vitamins). Specific focus is directed towards analysing how key material characteristics affect the biological function of digestive lipases and manipulate lipolytic digestion. The mechanism of lipase action is a complex, interfacial process, whereby hydrolysis can be controlled by the ability for lipase to access and adsorb to the lipid-in-water interface. However, significant conjecture exists within the literature regarding parameters that influence the activities of digestive lipases. Important findings from recent investigations that strategically examined the interplay between the interfacial composition of the lipid microenvironment and lipolysis kinetics in simulated biophysical environments are presented. The correlation between lipolysis and the rate of solubilisation and absorption of lipophilic compounds in the gastrointestinal tract (GIT) is detailed. Greater insights into the mechanism of lipase action have provided a new approach for designing colloidal carriers that orally deliver poorly soluble compounds, directly impacting the pharmaceutical and food industries. Copyright © 2016 Elsevier B.V. All rights reserved.

Profiling of proteolytic enzymes in the gut of the tick Ixodes ricinus reveals an evolutionarily conserved network of aspartic and cysteine peptidases

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Mareš Michael

2008-03-01

Full Text Available Abstract Background Ticks are vectors for a variety of viral, bacterial and parasitic diseases in human and domestic animals. To survive and reproduce ticks feed on host blood, yet our understanding of the intestinal proteolytic machinery used to derive absorbable nutrients from the blood meal is poor. Intestinal digestive processes are limiting factors for pathogen transmission since the tick gut presents the primary site of infection. Moreover, digestive enzymes may find practical application as anti-tick vaccine targets. Results Using the hard tick, Ixodes ricinus, we performed a functional activity scan of the peptidase complement in gut tissue extracts that demonstrated the presence of five types of peptidases of the cysteine and aspartic classes. We followed up with genetic screens of gut-derived cDNA to identify and clone genes encoding the cysteine peptidases cathepsins B, L and C, an asparaginyl endopeptidase (legumain, and the aspartic peptidase, cathepsin D. By RT-PCR, expression of asparaginyl endopeptidase and cathepsins B and D was restricted to gut tissue and to those developmental stages feeding on blood. Conclusion Overall, our results demonstrate the presence of a network of cysteine and aspartic peptidases that conceivably operates to digest host blood proteins in a concerted manner. Significantly, the peptidase components of this digestive network are orthologous to those described in other parasites, including nematodes and flatworms. Accordingly, the present data and those available for other tick species support the notion of an evolutionary conservation of a cysteine/aspartic peptidase system for digestion that includes ticks, but differs from that of insects relying on serine peptidases.

Effect of Barley and Enzyme on Performance, Carcass, Enzyme Activity and Digestion Parameters of Broilers

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majid kalantar

2016-04-01

Full Text Available Introduction Corn has been recently used for producing ethanol fuel in the major corn-producing countries such as the US and Brazil. Recent diversion of corn for biofuel production along with the increased world's demand for this feedstuff has resulted in unprecedented rise in feed cost for poultry worldwide. Alternative grains such as wheat and barley can be successfully replaced for corn in poultry diets. These cereal grains can locally grow in many parts of the world as they have remarkably lower water requirement than corn. Wheat and barley are generally used as major sources of energy in poultry diets. Though the major components of these grains are starch and proteins, they have considerable content of non-starch polysaccharides (NSPs, derived from the cell walls (Olukosi et al. 2007; Mirzaie et al. 2012. NSPs are generally considered as anti-nutritional factors (Jamroz et al. 2002. The content and structure of NSP polymers vary between different grains, which consequently affect their nutritive value (Olukosi et al. 2007.Wheat and barley are generally used as major sources of energy in poultry diets. The major components of these grains are starch and proteins, they have considerable content of non-starch polysaccharides (NSPs, derived from the cell walls. NSPs are generally considered as anti-nutritional factors. The content and structure of NSP polymers vary between different grains, which consequently affect their nutritive value. The major part of NSPs in barley comprises polymers of (1→3 (1→4-β- glucans which could impede growth factors and consequently carcass quality through lowering the rate and amount of available nutrients in the mucosal surface of the intestinal. Materials and Methods This experiment was conducted to evaluate the effect of corn and barley based diets supplemented with multi-enzyme on growth, carcass, pancreas enzyme activity and physiological characteristics of broilers. A total number of 375 one day old

Enzymological Characterization of Atm, the First Laccase from Agrobacterium sp. S5-1, with the Ability to Enhance In Vitro digestibility of Maize Straw.

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Wei Si

Full Text Available Laccase is an enzyme that catalyzes oxidation of phenolic compounds, diamines and aromatic amines. In this study, a novel laccase-like gene (atm in a ligninolyitic isolate Agrobacterium sp. S5-1 from soil humus was identified and heterologously expressed in Escherichia coli. Atm exhibited its maximal activity at pH 4.5 and at 50°C. This enzyme was tolerant to high temperature, a broad range of pH, heavy metal ions (Co3+, Mn2+, Cu2+ and Ni2+, 20 mM and all tested organic solvents. Furthermore, Atm significantly (p<0.05 increased dry matter digestibility of maize straw from 23.44% to 27.96% and from 29.53% to 37.10% after 8 or 24 h of digestion and improved acid detergent fiber digestibility from 5.81% to 10.33% and from 12.80% to 19.07% after 8 or 24 h of digestion, respectively. The combination of Atm and fibrolytic enzymes significantly (p<0.05 enhanced neutral detergent fiber digestibility from 19.02% to 24.55% after 24 h of digestion respectively. Results showed treatment with Atm effectively improved in vitro digestibility of maize straw, thus suggesting that Atm has an application potential for bioconversion of lignin rich agricultural byproducts into animal feed and cellulosic ethanol.

Enzymes in Human Milk.

Science.gov (United States)

Dallas, David C; German, J Bruce

2017-01-01

Milk proteins are a complex and diverse source of biological activities. Beyond their function, intact milk proteins also act as carriers of encrypted functional sequences that, when released as peptides, exert biological functions, including antimicrobial and immunomodulatory activity, which could contribute to the infant's competitive success. Research has now revealed that the release of these functional peptides begins within the mammary gland itself. A complex array of proteases produced in mother's milk has been shown to be active in the milk, releasing these peptides. Moreover, our recent research demonstrates that these milk proteases continue to digest milk proteins within the infant's stomach, possibly even to a larger extent than the infant's own proteases. As the neonate has relatively low digestive capacity, the activity of milk proteases in the infant may provide important assistance to digesting milk proteins. The coordinated release of these encrypted sequences is accomplished by selective proteolytic action provided by an array of native milk proteases and infant-produced enzymes. The task for scientists is now to discover the selective advantages of this protein-protease-based peptide release system. © 2017 Nestec Ltd., Vevey/S. Karger AG, Basel.

Comparison of intestinal microbiota and activities of digestive and immune-related enzymes of sea cucumber Apostichopus japonicus in two habitats

Science.gov (United States)

Wang, Qi; Zhang, Xiumei; Chen, Muyan; Li, Wentao; Zhang, Peidong

2017-09-01

Sea cucumber Apostichopus japonicus stock enhancement by releasing hatchery-produced seeds is a management tool used to recover its population under natural environmental conditions. To assess the suitability of releasing sites, we examined the microbiota of the gut contents of A. japonicus from two populations (one in sandy-muddy seagrass beds and one in rocky intertidal reefs) and the microbiota in their surrounding sediments. The activities of digestive and immune-related enzymes in the A. japonicus were also examined. The results indicated that higher bacterial richness and Shannon diversity index were observed in all the seagrass-bed samples. There were significant differences in intestinal and sediment microorganisms between the two habitats, with a 2.87 times higher abundance of Firmicutes in the seagrass bed sediments than that in the reefs. Meanwhile, Bacteroidetes and Actinobacteria were significantly higher abundant in the gut content of A. japonicus from seagrass bed than those from the reefs. In addition, the seagrass-bed samples exhibited a relatively higher abundance of potential probiotics. Principal coordinates analysis and heatmap showed the bacterial communities were classified into two groups corresponding to the two habitat types. Moreover, compared to A. japonicus obtained from rocky intertidal habitat, those obtained from the seagrass bed showed higher lysozyme, superoxide dismutase and protease activities. Our results suggest that bacterial communities present in seagrass beds might enhance the digestive function and immunity of A. japonicus. Therefore, compared with the rocky intertidal reef, seagrass bed seems to be more beneficial for the survival of A. japonicus.

Studying furosemide solubilization using an in vitro model simulating gastrointestinal digestion and drug solubilization in neonates and young infants

DEFF Research Database (Denmark)

Klitgaard, Mette; Sassene, Philip Jonas; Selen, Arzu

2017-01-01

-2months). METHODS: The utilized in vitro model was designed to mimic the digestion and drug solubilization processes occurring in the stomach, and the small intestine of the neonate and young infant population, using physiologically relevant media, volumes and digestive enzymes. Overall the experimental...

Short-term calorie restriction feminizes the mRNA profiles of drug metabolizing enzymes and transporters in livers of mice

International Nuclear Information System (INIS)

Fu, Zidong Donna; Klaassen, Curtis D.

2014-01-01

Calorie restriction (CR) is one of the most effective anti-aging interventions in mammals. A modern theory suggests that aging results from a decline in detoxification capabilities and thus accumulation of damaged macromolecules. The present study aimed to determine how short-term CR alters mRNA profiles of genes that encode metabolism and detoxification machinery in the liver. Male C57BL/6 mice were fed CR (0, 15, 30, or 40%) diets for one month, followed by mRNA quantification of 98 xenobiotic processing genes (XPGs) in the liver, including 7 uptake transporters, 39 phase-I enzymes, 37 phase-II enzymes, 10 efflux transporters, and 5 transcription factors. In general, 15% CR did not alter mRNAs of most XPGs, whereas 30 and 40% CR altered over half of the XPGs (32 increased and 29 decreased). CR up-regulated some phase-I enzymes (fold increase), such as Cyp4a14 (12), Por (2.3), Nqo1 (1.4), Fmo2 (5.4), and Fmo3 (346), and numerous number of phase-II enzymes, such as Sult1a1 (1.2), Sult1d1 (2.0), Sult1e1 (33), Sult3a1 (2.2), Gsta4 (1.3), Gstm2 (1.3), Gstm3 (1.7), and Mgst3 (2.2). CR feminized the mRNA profiles of 32 XPGs in livers of male mice. For instance, CR decreased the male-predominantly expressed Oatp1a1 (97%) and increased the female-predominantly expressed Oatp1a4 (11). In conclusion, short-term CR alters the mRNA levels of over half of the 98 XPGs quantified in livers of male mice, and over half of these alterations appear to be due to feminization of the liver. - Highlights: • Utilized a graded CR model in male mice • The mRNA profiles of xenobiotic processing genes (XPGs) in liver were investigated. • CR up-regulates many phase-II enzymes. • CR tends to feminize the mRNA profiles of XPGs

Short-term calorie restriction feminizes the mRNA profiles of drug metabolizing enzymes and transporters in livers of mice

Energy Technology Data Exchange (ETDEWEB)

Fu, Zidong Donna [Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Klaassen, Curtis D., E-mail: cklaasse@kumc.edu [Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS 66160 (United States)

2014-01-01

Calorie restriction (CR) is one of the most effective anti-aging interventions in mammals. A modern theory suggests that aging results from a decline in detoxification capabilities and thus accumulation of damaged macromolecules. The present study aimed to determine how short-term CR alters mRNA profiles of genes that encode metabolism and detoxification machinery in the liver. Male C57BL/6 mice were fed CR (0, 15, 30, or 40%) diets for one month, followed by mRNA quantification of 98 xenobiotic processing genes (XPGs) in the liver, including 7 uptake transporters, 39 phase-I enzymes, 37 phase-II enzymes, 10 efflux transporters, and 5 transcription factors. In general, 15% CR did not alter mRNAs of most XPGs, whereas 30 and 40% CR altered over half of the XPGs (32 increased and 29 decreased). CR up-regulated some phase-I enzymes (fold increase), such as Cyp4a14 (12), Por (2.3), Nqo1 (1.4), Fmo2 (5.4), and Fmo3 (346), and numerous number of phase-II enzymes, such as Sult1a1 (1.2), Sult1d1 (2.0), Sult1e1 (33), Sult3a1 (2.2), Gsta4 (1.3), Gstm2 (1.3), Gstm3 (1.7), and Mgst3 (2.2). CR feminized the mRNA profiles of 32 XPGs in livers of male mice. For instance, CR decreased the male-predominantly expressed Oatp1a1 (97%) and increased the female-predominantly expressed Oatp1a4 (11). In conclusion, short-term CR alters the mRNA levels of over half of the 98 XPGs quantified in livers of male mice, and over half of these alterations appear to be due to feminization of the liver. - Highlights: • Utilized a graded CR model in male mice • The mRNA profiles of xenobiotic processing genes (XPGs) in liver were investigated. • CR up-regulates many phase-II enzymes. • CR tends to feminize the mRNA profiles of XPGs.

Partial characterisation of digestive proteases of the Mayan cichlid Cichlasoma urophthalmus.

Science.gov (United States)

Cuenca-Soria, C A; Álvarez-González, C A; Ortiz-Galindo, J L; Nolasco-Soria, H; Tovar-Ramírez, D; Guerrero-Zárate, R; Castillo-Domínguez, A; Perera-García, M A; Hernández-Gómez, R; Gisbert, E

2014-06-01

The characterisation of digestive proteases in native freshwater fish such as the Mayan cichlid Cichlasoma urophthalmus provides scientific elements that may be used to design balanced feed that matches with the digestive capacity of the fish. The purpose of this study was to characterise the digestive proteases, including the effect of the pH and the temperature on enzyme activity and stability, as well as the effect of inhibitors using multienzymatic extracts of the stomach and intestine of C. urophthalmus juveniles. Results showed that the optimum activities of the acid and alkaline proteases occurred at pH values of 3 and 9, respectively, whereas their optimum temperatures were 55 and 65 °C, respectively. The acid proteases were most stable at pH values of 2–3 and at temperatures of 35–45 °C, whereas the alkaline proteases were most stable at pH values of 6–9 and at 25–55 °C. The inhibition assays recorded a residual activity of 4% with pepstatin A for the acid proteases. The inhibition of the alkaline proteases was greater than 80% with TPCK, TLCK, EDTA and ovalbumin, and of 60 and 43.8% with PMSF and SBT1, respectively. The results obtained in this study make it possible to state that C. urophthalmus has a sufficiently complete digestive enzyme machinery to degrade food items characteristic of an omnivorous fish species, although specimens showed a tendency to carnivory.

Real-time monitoring of peptic and tryptic digestions of bovine {beta}-casein using quartz crystal microbalance

Energy Technology Data Exchange (ETDEWEB)

Huenerbein, Andreas [Institute of Pharmaceutics and Biopharmaceutics, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale) (Germany)]. E-mail: andreas.huenerbein@pharmazie.uni-halle.de; Schmelzer, Christian E.H. [Institute of Pharmaceutics and Biopharmaceutics, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale) (Germany); Neubert, Reinhard H.H. [Institute of Pharmaceutics and Biopharmaceutics, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale) (Germany)

2007-02-12

In this study peptic and tryptic digestions of bovine {beta}-casein were investigated using quartz crystal microbalance (QCM). {beta}-Casein, which was used as a model protein, was immobilized on the surface of the QCM sensor where its degradation caused shifts in the resonant frequency. Atomic force microscopy was applied for the characterization of the protein layer. Different pH-values for peptic or tryptic digestions were chosen to visualize their effect on enzyme activity. Lower frequency shifts were observed at pH-values deviating from those at the maximum enzyme activity. In the case of the peptic digestion the frequency shift at pH 4 was more than 10 times smaller than those at pH 2. The frequency shifts for tryptic digestions at pH 5.4 and pH 6.4 were about two thirds compared to that obtained for the digestion at pH 7.4. The identification of peptides using MALDI-ToF mass spectrometry was used for verification of the proteolyses of the immobilized protein. Furthermore, it was shown that the QCM technique allows close observation of the effect of different pH-values on the immobilized casein layer. All in all, QCM facilitates the monitoring of the progress of enzymatic reactions in real-time.

Real-time monitoring of peptic and tryptic digestions of bovine β-casein using quartz crystal microbalance

International Nuclear Information System (INIS)

Huenerbein, Andreas; Schmelzer, Christian E.H.; Neubert, Reinhard H.H.

2007-01-01

In this study peptic and tryptic digestions of bovine β-casein were investigated using quartz crystal microbalance (QCM). β-Casein, which was used as a model protein, was immobilized on the surface of the QCM sensor where its degradation caused shifts in the resonant frequency. Atomic force microscopy was applied for the characterization of the protein layer. Different pH-values for peptic or tryptic digestions were chosen to visualize their effect on enzyme activity. Lower frequency shifts were observed at pH-values deviating from those at the maximum enzyme activity. In the case of the peptic digestion the frequency shift at pH 4 was more than 10 times smaller than those at pH 2. The frequency shifts for tryptic digestions at pH 5.4 and pH 6.4 were about two thirds compared to that obtained for the digestion at pH 7.4. The identification of peptides using MALDI-ToF mass spectrometry was used for verification of the proteolyses of the immobilized protein. Furthermore, it was shown that the QCM technique allows close observation of the effect of different pH-values on the immobilized casein layer. All in all, QCM facilitates the monitoring of the progress of enzymatic reactions in real-time

Microflora of hydrobionts digestive tract in Kaunas water storage reservoir

International Nuclear Information System (INIS)

Shyvokiene, J.; Mickiene, L.; Mileriene, E.

1996-01-01

Microbiological and ichthiological investigations carried out in 1990 and 1992 showed the variability of bacterial cenoses in the digestive tract of hydrobionts before and after setting in motion Kruonis hydro pumped storage. The studies also showed that microorganisms of the digestive tract of the hydrobionts investigated were involved in the degradation of nutritional substrates and could serve as indicators of an anthropogenic effect. Before setting in motion the hydro pumped storage hydrocarbon-degrading bacteria (HDB) were detected in the digestive tract of the freshwater shrimps, opossum shrimps, sticklebacks, zebra mussels and roaches. The greatest number of HDB was found in the digestive tract of the roach while in perches they were not detected. However after setting in motion the hydro pumped storage , high numbers of HDB were determined in the digestive tracts of all the hydrobionts investigated. It has been shown that the function of bacterial digestion is conditioned not only by the nutrition specificity of the macroorganism, but on its environment as well. With the aid of enzymes secreted by microorganisms organic compounds difficult to assimilate are transformed into valuable nutrients. Besides, the functional activity of microorganisms of the digestive tract of the hydrobionts indicate the intensity of the digestive process and physiological state of their organism. Therefore, when investigating fish stocks in hydrosystems one must evaluate inner resources of their organism, i.e. functional activities and the activity of digestive tract microorganisms, their quantitative and qualitative composition, relationship with the macroorganism, its growth rate and environment. (author). 15 refs., 5 tabs

Short-term calorie restriction feminizes the mRNA profiles of drug metabolizing enzymes and transporters in livers of mice.

Science.gov (United States)

Fu, Zidong Donna; Klaassen, Curtis D

2014-01-01

Calorie restriction (CR) is one of the most effective anti-aging interventions in mammals. A modern theory suggests that aging results from a decline in detoxification capabilities and thus accumulation of damaged macromolecules. The present study aimed to determine how short-term CR alters mRNA profiles of genes that encode metabolism and detoxification machinery in the liver. Male C57BL/6 mice were fed CR (0, 15, 30, or 40%) diets for one month, followed by mRNA quantification of 98 xenobiotic processing genes (XPGs) in the liver, including 7 uptake transporters, 39 phase-I enzymes, 37 phase-II enzymes, 10 efflux transporters, and 5 transcription factors. In general, 15% CR did not alter mRNAs of most XPGs, whereas 30 and 40% CR altered over half of the XPGs (32 increased and 29 decreased). CR up-regulated some phase-I enzymes (fold increase), such as Cyp4a14 (12), Por (2.3), Nqo1 (1.4), Fmo2 (5.4), and Fmo3 (346), and numerous number of phase-II enzymes, such as Sult1a1 (1.2), Sult1d1 (2.0), Sult1e1 (33), Sult3a1 (2.2), Gsta4 (1.3), Gstm2 (1.3), Gstm3 (1.7), and Mgst3 (2.2). CR feminized the mRNA profiles of 32 XPGs in livers of male mice. For instance, CR decreased the male-predominantly expressed Oatp1a1 (97%) and increased the female-predominantly expressed Oatp1a4 (11). In conclusion, short-term CR alters the mRNA levels of over half of the 98 XPGs quantified in livers of male mice, and over half of these alterations appear to be due to feminization of the liver. Copyright © 2013 Elsevier Inc. All rights reserved.

Genotyping of the Holstein-Friesian crossbred cattle for CD18 gene using PCR-RFLP

Directory of Open Access Journals (Sweden)

A. S. Khade

2014-05-01

Full Text Available Aim: The present study was undertaken in Holstein-Friesian (HF crossbred cattle with the objective to find out genotype of HF crossbred cattle for Bovine Leucocyte Adhesion Deficiency (BLAD by using PCR-RFLP. Materials and Methods: 50 blood samples were collected from HF crossbred cattle and subjected to PCR. The amplified PCR products were digested using Taq I restriction enzyme at 65 oC overnight. After restriction digestion, the final PCR products were electrophoresed on 2.5 % agarose gel. Results: All the 50 animals under present investigation were found to be normal as the amplified PCR product upon digestion with Taq I restriction enzyme, revealed two bands of 313 bp and 54 bp for normal animals. Conclusions: In the present investigation D128G carrier frequency was found to be 0 %. However, recent reports suggest that the mutant gene has already been observed in the HF crossbred cattle population of India, which makes it necessary to screen the animals to avoid the risk of spreading BLAD in the breeding cattle population.

Influence of feed provisioning prior to digesta sampling on precaecal amino acid digestibility in broiler chickens.

Science.gov (United States)

Siegert, Wolfgang; Ganzer, Christian; Kluth, Holger; Rodehutscord, Markus

2018-06-01

A regression approach was applied to determine the influence of feed provisioning prior to digesta sampling on precaecal (pc) amino acid (AA) digestibility in broiler chickens. Soybean meal was used as an example test ingredient. Five feed-provisioning protocols were investigated, four with restricted provision and one with ad libitum provision. When provision was restricted, feed was provided for 30 min after a withdrawal period of 12 h. Digesta were sampled 1, 2, 4 and 6 h after feeding commenced. A diet containing 300 g maize starch/kg was prepared. Half or all the maize starch was replaced with soybean meal in two other diets. Average pc digestibility of all determined AA in the soybean meal was 86% for the 4 and 6-h protocols and 66% and 60% for the 2 and 1-h protocols, respectively. Average pc AA digestibility of soybean meal was 76% for ad libitum feed provision. Feed provisioning also influenced the determined variance. Variance in digestibility ranked in magnitude 1 h > ad libitum > 2 h > 6 h > 4 h for all AA. Owing to the considerable influence of feed-provisioning protocols found in this study, comparisons of pc AA digestibility between studies applying different protocols prior to digesta sampling must be treated with caution. Digestibility experiments aimed at providing estimates for practical feed formulation should use feed-provisioning procedures similar to those used in practice.

Maternal protein restriction induces alterations in insulin signaling and ATP sensitive potassium channel protein in hypothalami of intrauterine growth restriction fetal rats.

Science.gov (United States)

Liu, Xiaomei; Qi, Ying; Gao, Hong; Jiao, Yisheng; Gu, Hui; Miao, Jianing; Yuan, Zhengwei

2013-01-01

It is well recognized that intrauterine growth restriction leads to the development of insulin resistance and type 2 diabetes mellitus in adulthood. To investigate the mechanisms behind this "metabolic imprinting" phenomenon, we examined the impact of maternal undernutrition on insulin signaling pathway and the ATP sensitive potassium channel expression in the hypothalamus of intrauterine growth restriction fetus. Intrauterine growth restriction rat model was developed through maternal low protein diet. The expression and activated levels of insulin signaling molecules and K(ATP) protein in the hypothalami which were dissected at 20 days of gestation, were analyzed by western blot and real time PCR. The tyrosine phosphorylation levels of the insulin receptor substrate 2 and phosphatidylinositol 3'-kinase p85α in the hypothalami of intrauterine growth restriction fetus were markedly reduced. There was also a downregulation of the hypothalamic ATP sensitive potassium channel subunit, sulfonylurea receptor 1, which conveys the insulin signaling. Moreover, the abundances of gluconeogenesis enzymes were increased in the intrauterine growth restriction livers, though no correlation was observed between sulfonylurea receptor 1 and gluconeogenesis enzymes. Our data suggested that aberrant intrauterine milieu impaired insulin signaling in the hypothalamus, and these alterations early in life might contribute to the predisposition of the intrauterine growth restriction fetus toward the adult metabolic disorders.

Effect of DHA supplementation on digestible starch utilization by rainbow trout.

Science.gov (United States)

Tapia-Salazar, M; Bureau, W; Panserat, S; Corraze, G; Bureau, D P

2006-01-01

Rainbow trout has a limited ability to utilize digestible carbohydrates efficiently. Trout feeds generally contain high levels of DHA, a fatty acid known to inhibit a number of glycolytic and lipogenic enzymes in animals. A study was conducted to determine whether carbohydrate utilization by rainbow trout might be affected by dietary DHA level. Two low-carbohydrate (digestible carbohydrate) basal diets were formulated to contain 1 (adequate) or 4 (excess) g/100 g DHA diet respectively. The two basal diets were diluted with increasing levels of digestible starch (0 %, 10 %, 20 % and 30 %, respectively) to produce eight diets. These diets were fed to fish for 12 weeks at 15 degrees C according to a pair-fed protocol that consisted of feeding the same amount of basal diet but different amounts of starch. Live weight, N and lipid gains, hepatic glycogen and plasma glucose values significantly increased, whereas feed efficiency (gain:feed) significantly decreased, with increasing starch intake (Pdigestible N intake) improved with starch supplementation but was not affected by DHA level (P>0.05). Starch increased the activity of glucokinase, pyruvate kinase, glucose 6-phosphate dehydrogenase and fatty acid synthase (P<0.05) but did not affect hexokinase and malic enzyme activity. DHA had no effect on growth but increased plasma glucose and reduced carcass lipid and liver glycogen contents (P<0.05). Glycolytic and lipogenic enzymes were not affected by DHA level, except for pyruvate kinase, which was reduced by increasing DHA level. These results suggest only a marginal effect of dietary DHA on the ability of fish to utilize carbohydrate.

Effects of dietary supplementation of multi-enzyme complex on the ...

African Journals Online (AJOL)

Jane

2011-07-25

Jul 25, 2011 ... and protein escape digestion, reach the midgut and undergo fermentation ... soybean broiler starter diet with an enzyme preparation containing a mixture .... due to the rapid food rate and the deficiency of the necessary innate ...

Extrusion induced low-order starch matrices: Enzymic hydrolysis and structure.

Science.gov (United States)

Zhang, Bin; Dhital, Sushil; Flanagan, Bernadine M; Luckman, Paul; Halley, Peter J; Gidley, Michael J

2015-12-10

Waxy, normal and highwaymen maize starches were extruded with water as sole plasticizer to achieve low-order starch matrices. Of the three starches, we found that only high-amylose extrudate showed lower digestion rate/extent than starches cooked in excess water. The ordered structure of high-amylose starches in cooked and extruded forms was similar, as judged by NMR, XRD and DSC techniques, but enzyme resistance was much greater for extruded forms. Size exclusion chromatography suggested that longer chains were involved in enzyme resistance. We propose that the local molecular density of packing of amylose chains can control the digestion kinetics rather than just crystallinity, with the principle being that density sufficient to either prevent/limit binding and/or slow down catalysis can be achieved by dense amorphous packing. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

Science.gov (United States)

van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D.

2015-01-01

The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1–2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand breaks are introduced at random wherever two translocating enzymes form a so-called collision complex following long-range communication between a pair of target sites in inverted (head-to-head) repeat. Paradoxically, structural models for collision suggest that the nuclease domains are too far apart (>30 bp) to dimerise and produce a double-strand DNA break using just two strand-cleavage events. Here, we examined the organisation of different collision complexes and how these lead to nuclease activation. We mapped DNA cleavage when a translocating enzyme collides with a static enzyme bound to its site. By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD. Direct nuclease dimerization is not required. To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA. This study lends further support to a model where DNA breaks are generated by multiple random nicks due to mobility of a collision complex with an overall DNA-binding footprint of ∼30 bp. PMID:26507855

Experimental Strategy to Discover Microbes with Gluten-degrading Enzyme Activities.

Science.gov (United States)

Helmerhorst, Eva J; Wei, Guoxian

2014-05-05

Gluten proteins contained in the cereals barley, rye and wheat cause an inflammatory disorder called celiac disease in genetically predisposed individuals. Certain immunogenic gluten domains are resistant to degradation by mammalian digestive enzymes. Enzymes with the ability to target such domains are potentially of clinical use. Of particular interest are gluten-degrading enzymes that would be naturally present in the human body, e.g. associated with resident microbial species. This manuscript describes a selective gluten agar approach and four enzyme activity assays, including a gliadin zymogram assay, designed for the selection and discovery of novel gluten-degrading microorganisms from human biological samples. Resident and harmless bacteria and/or their derived enzymes could potentially find novel applications in the treatment of celiac disease, in the form of a probiotic agent or as a dietary enzyme supplement.

Engineering Digestion: Multiscale Processes of Food Digestion.

Science.gov (United States)

Bornhorst, Gail M; Gouseti, Ourania; Wickham, Martin S J; Bakalis, Serafim

2016-03-01

Food digestion is a complex, multiscale process that has recently become of interest to the food industry due to the developing links between food and health or disease. Food digestion can be studied by using either in vitro or in vivo models, each having certain advantages or disadvantages. The recent interest in food digestion has resulted in a large number of studies in this area, yet few have provided an in-depth, quantitative description of digestion processes. To provide a framework to develop these quantitative comparisons, a summary is given here between digestion processes and parallel unit operations in the food and chemical industry. Characterization parameters and phenomena are suggested for each step of digestion. In addition to the quantitative characterization of digestion processes, the multiscale aspect of digestion must also be considered. In both food systems and the gastrointestinal tract, multiple length scales are involved in food breakdown, mixing, absorption. These different length scales influence digestion processes independently as well as through interrelated mechanisms. To facilitate optimized development of functional food products, a multiscale, engineering approach may be taken to describe food digestion processes. A framework for this approach is described in this review, as well as examples that demonstrate the importance of process characterization as well as the multiple, interrelated length scales in the digestion process. © 2016 Institute of Food Technologists®

The digestive system of the stony coral Stylophora pistillata.

Science.gov (United States)

Raz-Bahat, M; Douek, J; Moiseeva, E; Peters, E C; Rinkevich, B

2017-05-01

Because hermatypic species use symbiotic algal photosynthesis, most of the literature in this field focuses on this autotrophic mode and very little research has studied the morphology of the coral's digestive system or the digestion process of particulate food. Using histology and histochemestry, our research reveals that Stylophora pistillata's digestive system is concentrated at the corals' peristome, actinopharynx and mesenterial filaments (MF). We used in-situ hybridization (ISH) of the RNA transcript of the gene that codes for the S. pistillata digestive enzyme, chymotrypsinogen, to shed light on the functionality of the digestive system. Both the histochemistry and the ISH pointed to the MF being specialized digestive organs, equipped with large numbers of acidophilic and basophilic granular gland cells, as well as acidophilic non-granular gland cells, some of which produce chymotrypsinogen. We identified two types of MF: short, trilobed MF and unilobed, long and convoluted MF. Each S. pistillata polyp harbors two long convoluted MF and 10 short MF. While the short MF have neither secreting nor stinging cells, each of the convoluted MF display gradual cytological changes along their longitudinal axis, alternating between stinging and secreting cells and three distinctive types of secretory cells. These observations indicate the important digestive role of the long convoluted MF. They also indicate the existence of novel feeding compartments in the gastric cavity of the polyp, primarily in the nutritionally active peristome, in the actinopharynx and in three regions of the MF that differ from each other in their cellular components, general morphology and chymotrypsinogen excretion.

Survival of Lactobacillus rhamnosus strains inoculated in cheese matrix during simulated human digestion.

Science.gov (United States)

Pitino, Iole; Randazzo, Cinzia L; Cross, Kathryn L; Parker, Mary L; Bisignano, Carlo; Wickham, Martin S J; Mandalari, Giuseppina; Caggia, Cinzia

2012-08-01

Survival of probiotic bacteria during transit through the gastrointestinal (GI) tract is influenced by a number of environmental variables including stomach acidity, bile salts, digestive enzymes and food matrix. This study assessed survival of seven selected Lactobacillus rhamnosus strains delivered within a model cheese system to the human upper GI tract using a dynamic gastric model (DGM). Good survival rates for all tested strains were recorded during both simulated gastric and duodenal digestion. Strains H12, H25 and N24 demonstrated higher survival capacities during gastric digestion than L. rhamnosus GG strain used as control, with H12 and N24 continuing to grow during duodenal digestion. Strains L. rhamnosus F17, N24 and R61 showed adhesion properties to both HT-29 and Caco-2 cells. The ability to attach to the cheese matrix during digestion was confirmed by scanning electron microscopy, also indicating production of extracellular polysaccharides as a response to acid stress. Copyright © 2012 Elsevier Ltd. All rights reserved.

Molecular characterisation of Mycovellosiella koepki, the causal ...

African Journals Online (AJOL)

KEN

2007-02-05

Feb 5, 2007 ... then annealing at 52°C for 1 min and finally 72°C for primer extension. The final primer elongation segment of the run was extended to 10 min at 72°C. Amplified products were digested with restriction enzymes according to the manufacturer's instructions. (Boehringer Mannheim). Digested DNA fragments ...

Purification and characterization of angiotensin-1 converting enzyme

African Journals Online (AJOL)

user

2013-04-10

Apr 10, 2013 ... assay, each separated sample (50 μl) was added to the enzyme solution (50 μl) and then .... barriers in the human body might limit or enhance the activity of the .... tyrosine, proline or phenylalanine at the carboxy-terminal .... egg white hydrolysates: Effect of a simulated intestinal digestion. Food Chem.

Effects of supplemental microbial phytase enzyme on performance ...

African Journals Online (AJOL)

This experiment was conducted to investigate the effects of supplemental phytase in a corn-wheatsoybean meal basal diet on phosphorus (P) digestibility and performance of broiler chicks. 378 one-day old broiler chicks (Ross 308) were allocated to 3×3 factorial arrangements with three levels of phytase enzyme (0, 500 ...

Comparisons of blood biochemical parameters, digestive enzyme activities and volatile fatty acid profile between Meishan and Yorkshire piglets

Directory of Open Access Journals (Sweden)

Shouqing Ma

2015-12-01

Full Text Available This study was conducted to compare physiological characteristics between Meishan and Yorkshire piglets in their early lives. Six healthy purebred Meishan sows and Yorkshire sows with close farrowing dates were used in this research. The piglets sucked their respective sow's milk for 14 days, then they were slaughtered to collect samples of blood, pancreas, contents of stomach, jejunum, cecum, colon as well as feces for analysis of blood biochemical parameters, digestive enzymes, and volatile fatty acid (VFA. The results showed that Yorkshire piglets had higher concentrations of high-density lipoprotein cholesterol (HDL-C and total cholesterol (TC (P < 0.05. Gastric lipase activity was higher in Meishan piglets but Yorkshire piglets had higher lactase activity (P < 0.05. The total VFA together with acetate and propionate in cecum and colon were higher in Meishan piglets than in Yorkshire piglets (P < 0.05, but acetate in jejunum and ratio of acetate to propionate in colon were lower in Meishan piglets than in Yorkshire piglets (P < 0.05. In conclusion, in early suckling period, significant differences exist in host metabolism and intestinal microbial metabolism between Meishan and Yorkshire piglets.

Antioxidant and antidiabetic properties of tartary buckwheat rice flavonoids after in vitro digestion*

Science.gov (United States)

Bao, Tao; Wang, Ye; Li, Yu-ting; Gowd, Vemana; Niu, Xin-he; Yang, Hai-ying; Chen, Li-shui; Chen, Wei; Sun, Chong-de

2016-01-01

Oxidative stress and diabetes have a tendency to alter protein, lipid, and DNA moieties. One of the strategic methods used to reduce diabetes-associated oxidative stress is to inhibit the carbohydrate-digesting enzymes, thereby decreasing gastrointestinal glucose production. Plant-derived natural antioxidant molecules are considered a therapeutic tool in the treatment of oxidative stress and diabetes. The objective of this study was to identify tartary buckwheat rice flavonoids and evaluate the effect of in vitro digestion on their antioxidant and antidiabetic properties. High performance liquid chromatography (HPLC) analysis indicated the presence of rutin as a major component and quercitrin as a minor component of both digested and non-digested flavonoids. Both extracts showed a significant antioxidant capacity, but digested flavonoids showed reduced activity compared to non-digested. There were some decreases of the antioxidant activities (2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazy (DPPH) radical, and ferric reducing antioxidant power (FRAP)) of digested tartary buckwheat rice flavonoids compared with non-digested. Flavonoids from both groups significantly inhibited reactive oxygen species (ROS) production and α-glucosidase activity. Both digested and non-digested flavonoids markedly increased glucose consumption and glycogen content in HepG2 cells. Tartary buckwheat rice flavonoids showed appreciable antioxidant and antidiabetic properties, even after digestion. Tartary buckwheat rice appears to be a promising functional food with potent antioxidant and antidiabetic properties. PMID:27921399

Antioxidant and antidiabetic properties of tartary buckwheat rice flavonoids after in vitro digestion.

Science.gov (United States)

Bao, Tao; Wang, Ye; Li, Yu-Ting; Gowd, Vemana; Niu, Xin-He; Yang, Hai-Ying; Chen, Li-Shui; Chen, Wei; Sun, Chong-de

Oxidative stress and diabetes have a tendency to alter protein, lipid, and DNA moieties. One of the strategic methods used to reduce diabetes-associated oxidative stress is to inhibit the carbohydrate-digesting enzymes, thereby decreasing gastrointestinal glucose production. Plant-derived natural antioxidant molecules are considered a therapeutic tool in the treatment of oxidative stress and diabetes. The objective of this study was to identify tartary buckwheat rice flavonoids and evaluate the effect of in vitro digestion on their antioxidant and antidiabetic properties. High performance liquid chromatography (HPLC) analysis indicated the presence of rutin as a major component and quercitrin as a minor component of both digested and non-digested flavonoids. Both extracts showed a significant antioxidant capacity, but digested flavonoids showed reduced activity compared to non-digested. There were some decreases of the antioxidant activities (2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazy (DPPH) radical, and ferric reducing antioxidant power (FRAP)) of digested tartary buckwheat rice flavonoids compared with non-digested. Flavonoids from both groups significantly inhibited reactive oxygen species (ROS) production and α-glucosidase activity. Both digested and non-digested flavonoids markedly increased glucose consumption and glycogen content in HepG2 cells. Tartary buckwheat rice flavonoids showed appreciable antioxidant and antidiabetic properties, even after digestion. Tartary buckwheat rice appears to be a promising functional food with potent antioxidant and antidiabetic properties.

A first step towards a consensus static in vitro model for simulating full-term infant digestion.

Science.gov (United States)

Ménard, O; Bourlieu, C; De Oliveira, S C; Dellarosa, N; Laghi, L; Carrière, F; Capozzi, F; Dupont, D; Deglaire, A

2018-02-01

In vitro alternatives to clinical trials are used for studying human food digestion. For simulating infant digestion, only a few models, lacking physiological relevance, are available. Thanks to an extensive literature review of the in vivo infant digestive conditions, a gastrointestinal static in vitro model was developed for infants born at term and aged 28days. The model was applied to the digestion of a commercial infant formula. Kinetics of digestion, as well as the structural evolution, were compared with those obtained while submitting the same formula to the adult international consensus protocol of in vitro static digestion. The kinetics of proteolysis and lipolysis differed according to the physiological stage resulting mainly from the reduced level of enzymes and bile salts, as well as the higher gastric pH in the infant model. This in vitro static model of infant digestion is of interest for scientists, food or pharmaceutical manufacturers. Copyright © 2017 Elsevier Ltd. All rights reserved.

Formulation of enzyme blends to maximize the hydrolysis of alkaline peroxide pretreated alfalfa hay and barley straw by rumen enzymes and commercial cellulases.

Science.gov (United States)

Badhan, Ajay; Wang, Yuxi; Gruninger, Robert; Patton, Donald; Powlowski, Justin; Tsang, Adrian; McAllister, Tim

2014-04-26

Efficient conversion of lignocellulosic biomass to fermentable sugars requires the synergistic action of multiple enzymes; consequently enzyme mixtures must be properly formulated for effective hydrolysis. The nature of an optimal enzyme blends depends on the type of pretreatment employed as well the characteristics of the substrate. In this study, statistical experimental design was used to develop mixtures of recombinant glycosyl hydrolases from thermophilic and anaerobic fungi that enhanced the digestion of alkaline peroxide treated alfalfa hay and barley straw by mixed rumen enzymes as well as commercial cellulases (Accelerase 1500, A1500; Accelerase XC, AXC). Combinations of feruloyl and acetyl xylan esterases (FAE1a; AXE16A_ASPNG), endoglucanase GH7 (EGL7A_THITE) and polygalacturonase (PGA28A_ASPNG) with rumen enzymes improved straw digestion. Inclusion of pectinase (PGA28A_ASPNG), endoxylanase (XYN11A_THITE), feruloyl esterase (FAE1a) and β-glucosidase (E-BGLUC) with A1500 or endoglucanase GH7 (EGL7A_THITE) and β-xylosidase (E-BXSRB) with AXC increased glucose release from alfalfa hay. Glucose yield from straw was improved when FAE1a and endoglucanase GH7 (EGL7A_THITE) were added to A1500, while FAE1a and AXE16A_ASPNG enhanced the activity of AXC on straw. Xylose release from alfalfa hay was augmented by supplementing A1500 with E-BGLUC, or AXC with EGL7A_THITE and XYN11A_THITE. Adding arabinofuranosidase (ABF54B_ASPNG) and esterases (AXE16A_ASPNG; AXE16B_ASPNG) to A1500, or FAE1a and AXE16A_ASPNG to AXC enhanced xylose release from barley straw, a response confirmed in a scaled up assay. The efficacy of commercial enzyme mixtures as well as mixed enzymes from the rumen was improved through formulation with synergetic recombinant enzymes. This approach reliably identified supplemental enzymes that enhanced sugar release from alkaline pretreated alfalfa hay and barley straw.

Structural and Digestion Properties of Soluble-, Slowly Digestible and Resistant Maltodextrin from Cassava Starch by Enzymatic Modification

DEFF Research Database (Denmark)

Sorndech, Waraporn

The combination of branching enzyme (BE) and amylomaltase (AM) were selected to modify cassava starch. AM were used to elongate the glucan chains in order to enhance BE activity to create branching linkages. Cassava starch were gelatinized and incubated with BE or AMBE or BEAMBE or simultaneous...... AM and BE. The molecular analysis of the products including amylopectin chain length distribution, content of α-1,6 glucosidic linkages, absolute molecular weight distribution and digestibility were examined. Only BE catalysis showed 7.8% of branching linkages. The sequential AMBE-treated starch...... showed 9.9%-10.0% branching linkages, while the sequential BEAMBE-treated starch gained 10.9%-13.1% of branching linkages. Moreover, the sequential AMBE and BEAMBE-treated starch retarded the digestion rate of α-amylase and glucoamylase. Overall, sequential BEAMBE catalysis resulted in more...

Characteristics of chitinase isolated from different part of snakehead fish (Channa striata) digestive tract

Science.gov (United States)

Baehaki, A.; Lestari, S. D.; Wahidman, Y.; Gofar, N.

2018-01-01

Naturally, snakehead fish (Channa striata) is a prodigious carnivore feeding mainly on live animals, including small shrimp. Based on its feeding habits, the digestrive tracts of snakehead is considered as auspicious source of various enzumes including chitinase. The purpose of this study was to partially characterize chitinase enzyme isolated from digestive tract of snakehead fish. Two parts of digestive tract, stomach and intestine were used as enzymes’ source. The results showed that chitinase activity from the stomach was higher than chitinase activity from the intestine. The pH and temperature optimum of chitinase activity from digestive tract (the stomach and the intestine) were 6.0 and 70 °C, respectively.

Effect of the dose of exogenous fibrolytic enzyme preparations on preingestive fiber hydrolysis, ruminal fermentation, and in vitro digestibility of bermudagrass haylage.

Science.gov (United States)

Romero, J J; Zarate, M A; Adesogan, A T

2015-01-01

Our objectives were to evaluate the effects of the dose rates of 5 Trichoderma reesei and Aspergillus oryzae exogenous fibrolytic enzymes (EFE; 1A, 2A, 11C, 13D, and 15D) on in vitro digestibility, fermentation characteristics, and preingestive hydrolysis of bermudagrass haylage and to identify the optimal dose of each EFE for subsequent in vitro and in vivo studies. In experiment 1, EFE were diluted in citrate-phosphate buffer (pH 6) and applied in quadruplicate in each of 2 runs at 0× (control), 0.5×, 1×, 2×, and 3×; where 1× was the respective manufacturer-recommended dose (2.25, 2.25, 10, 15, and 15g of EFE/kg of dry matter). The suspension was incubated for 24h at 25°C and for a further 24h at 39°C after the addition of ruminal fluid. In experiment 2, a similar approach to that in experiment 1 was used to evaluate simulated preingestive effects, except that sodium azide (0.02% wt/vol) was added to the EFE solution. The suspension was incubated for 24h at 25°C and then 15mL of water was added before filtration to extract water-soluble compounds. For both experiments, data for each enzyme were analyzed separately as a completely randomized block design with a model that included effects of EFE dose, run, and their interaction. In experiment 1, increasing the EFE dose rate nonlinearly increased the DM digestibility of 1A, 2A, 11C, and 13D and the neutral detergent fiber digestibility (NDFD) of 1A, 2A, 11C, and 13D. Optimal doses of 1A, 2A, 11C, 13D, and 15D, as indicated by the greatest increases in NDFD at the lowest dose tested, were 2×, 2×, 1×, 0.5×, and 0.5×, respectively. Increasing the dose rate of 2A, 11C, and 13D nonlinearly increased concentrations of total volatile fatty acids and propionate (mM), decreased their acetate-to-propionate ratios and linearly decreased those of samples treated with 1A and 15D. In experiment 2, increasing the dose rate of each EFE nonlinearly decreased concentrations of netural detergent fiber; also, increasing

Nondetectability of restriction fragments and independence of DNA fragment sizes within and between loci in RFLP typing of DNA

Energy Technology Data Exchange (ETDEWEB)

Chakraborty, R.; Zhong, Y.; Jin, L. (Univ. of Texas Health Science Center, Houston, TX (United States)); Budowle, B. (FBI Academy, Quantico, VA (United States))

1994-08-01

The authors provide experimental evidence showing that, during the restriction-enzyme digestion of DNA samples, some of the HaeIII-digested DNA fragments are small enough to prevent their reliable sizing on a Southern gel. As a result of such nondetectability of DNA fragments, individuals who show a single-band DNA profile at a VNTR locus may not necessarily be true homozygotes. In a population database, when the presence of such nondetectable alleles is ignored, they show that a pseudodependence of alleles within as well as across loci may occur. Using a known statistical method, under the hypothesis of independence of alleles within loci, they derive an efficient estimate of null allele frequency, which may be subsequently used for testing allelic independence within and across loci. The estimates of null allele frequencies, thus derived, are shown to agree with direct experimental data on the frequencies of HaeIII-null alleles. Incorporation of null alleles into the analysis of the forensic VNTR database suggests that the assumptions of allelic independence within and between loci are appropriate. In contrast, a failure to incorporate the occurrence of null alleles would provide a wrong inference regarding the independence of alleles within and between loci. 47 refs., 2 figs., 4 tabs.

The adsorption of α-amylase on barley proteins affects the in vitro digestion of starch in barley flour.

Science.gov (United States)

Yu, Wenwen; Zou, Wei; Dhital, Sushil; Wu, Peng; Gidley, Michael J; Fox, Glen P; Gilbert, Robert G

2018-02-15

The conversion of barley starch to sugars is a complex enzymic process. Most previous work concerned the biotechnical aspect of in situ barley enzymes. However, the interactions among the macromolecular substrates and their effects on enzymic catalysis has been little examined. Here, we explore the mechanisms whereby interactions of protein and starch in barley flour affect the kinetics of enzymatic hydrolysis of starch in an in vitro system, using digestion rate data and structural analysis by confocal microscopy. The degradation kinetics of both uncooked barley flour and of purified starches are found to be two-step sequential processes. Barley proteins, especially the water-soluble component, are found to retard the digestion of starch degraded by α-amylase: the enzyme binds with water-insoluble protein and with starch granules, leading to reduced starch hydrolysis. These findings are of potential industrial value in both the brewing and food industries. Copyright © 2017 Elsevier Ltd. All rights reserved.

The foodomics approach for the evaluation of protein bioaccessibility in processed meat upon in vitro digestion.

Science.gov (United States)

Bordoni, Alessandra; Laghi, Luca; Babini, Elena; Di Nunzio, Mattia; Picone, Gianfranco; Ciampa, Alessandra; Valli, Veronica; Danesi, Francesca; Capozzi, Francesco

2014-06-01

The present work describes a foodomics protocol coupling an in vitro static simulation of digestion to a combination of omics techniques, to grant an overview of the protein digestibility of a meat-based food, namely Bresaola. The proteolytic activity mediated by the digestive enzymes is evaluated through Bradford and SDS-PAGE assays, combined to NMR relaxometry and spectroscopy, to obtain information ranging from the microscopic to the molecular level, respectively. The simple proteomics tool adopted here points out that a clear increase of bioaccessible proteins occurs in the gastric phase, rapidly disappearing during the following duodenal digestion. However, SDS-PAGE and the Bradford assay cannot follow the fate of the digested proteins when the products are sized meat matrix. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A holistic view of dietary carbohydrate utilization in lobster: digestion, postprandial nutrient flux, and metabolism.

Science.gov (United States)

Rodríguez-Viera, Leandro; Perera, Erick; Casuso, Antonio; Perdomo-Morales, Rolando; Gutierrez, Odilia; Scull, Idania; Carrillo, Olimpia; Martos-Sitcha, Juan A; García-Galano, Tsai; Mancera, Juan Miguel

2014-01-01

Crustaceans exhibit a remarkable variation in their feeding habits and food type, but most knowledge on carbohydrate digestion and utilization in this group has come from research on few species. The aim of this study was to make an integrative analysis of dietary carbohydrate utilization in the spiny lobster Panulirus argus. We used complementary methodologies such as different assessments of digestibility, activity measurements of digestive and metabolic enzymes, and post-feeding flux of nutrients and metabolites. Several carbohydrates were well digested by the lobster, but maize starch was less digestible than all other starches studied, and its inclusion in diet affected protein digestibility. Most intense hydrolysis of carbohydrates in the gastric chamber of lobster occurred between 2-6 h after ingestion and afterwards free glucose increased in hemolymph. The inclusion of wheat in diet produced a slow clearance of glucose from the gastric fluid and a gradual increase in hemolymph glucose. More intense hydrolysis of protein in the gastric chamber occurred 6-12 h after ingestion and then amino acids tended to increase in hemolymph. Triglyceride concentration in hemolymph rose earlier in wheat-fed lobsters than in lobsters fed other carbohydrates, but it decreased the most 24 h later. Analyses of metabolite levels and activities of different metabolic enzymes revealed that intermolt lobsters had a low capacity to store and use glycogen, although it was slightly higher in wheat-fed lobsters. Lobsters fed maize and rice diets increased amino acid catabolism, while wheat-fed lobsters exhibited higher utilization of fatty acids. Multivariate analysis confirmed that the type of carbohydrate ingested had a profound effect on overall metabolism. Although we found no evidence of a protein-sparing effect of dietary carbohydrate, differences in the kinetics of their digestion and absorption impacted lobster metabolism determining the fate of other nutrients.

A holistic view of dietary carbohydrate utilization in lobster: digestion, postprandial nutrient flux, and metabolism.

Directory of Open Access Journals (Sweden)

Leandro Rodríguez-Viera

Full Text Available Crustaceans exhibit a remarkable variation in their feeding habits and food type, but most knowledge on carbohydrate digestion and utilization in this group has come from research on few species. The aim of this study was to make an integrative analysis of dietary carbohydrate utilization in the spiny lobster Panulirus argus. We used complementary methodologies such as different assessments of digestibility, activity measurements of digestive and metabolic enzymes, and post-feeding flux of nutrients and metabolites. Several carbohydrates were well digested by the lobster, but maize starch was less digestible than all other starches studied, and its inclusion in diet affected protein digestibility. Most intense hydrolysis of carbohydrates in the gastric chamber of lobster occurred between 2-6 h after ingestion and afterwards free glucose increased in hemolymph. The inclusion of wheat in diet produced a slow clearance of glucose from the gastric fluid and a gradual increase in hemolymph glucose. More intense hydrolysis of protein in the gastric chamber occurred 6-12 h after ingestion and then amino acids tended to increase in hemolymph. Triglyceride concentration in hemolymph rose earlier in wheat-fed lobsters than in lobsters fed other carbohydrates, but it decreased the most 24 h later. Analyses of metabolite levels and activities of different metabolic enzymes revealed that intermolt lobsters had a low capacity to store and use glycogen, although it was slightly higher in wheat-fed lobsters. Lobsters fed maize and rice diets increased amino acid catabolism, while wheat-fed lobsters exhibited higher utilization of fatty acids. Multivariate analysis confirmed that the type of carbohydrate ingested had a profound effect on overall metabolism. Although we found no evidence of a protein-sparing effect of dietary carbohydrate, differences in the kinetics of their digestion and absorption impacted lobster metabolism determining the fate of other

Digestion and microbial protein synthesis in sheep as affected by ...

African Journals Online (AJOL)

Additional to the in situ study, the effects of an exogenous fibrolytic enzyme (EFE) on the in vitro gas production (GP) and ANKOM digestion systems on the mixture of milled LH and WS were determined. The substrate was pre-treated with distilled water (control) or EFE (treatment) 12 hours prior to incubation to allow ...

Identification and Relative Quantification of Bioactive Peptides Sequentially Released during Simulated Gastrointestinal Digestion of Commercial Kefir.

Science.gov (United States)

Liu, Yufang; Pischetsrieder, Monika

2017-03-08

Health-promoting effects of kefir may be partially caused by bioactive peptides. To evaluate their formation or degradation during gastrointestinal digestion, we monitored changes of the peptide profile in a model of (1) oral, (2) gastric, and (3) small intestinal digestion of kefir. Matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analyses revealed clearly different profiles between digests 2/3 and kefir/digest 1. Subsequent ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry identified 92 peptides in total (25, 25, 43, and 30, partly overlapping in kefir and digests 1, 2, and 3, respectively), including 16 peptides with ascribed bioactivity. Relative quantification in scheduled multiple reaction monitoring mode showed that many bioactive peptides were released by simulated digestion. Most prominently, the concentration of angiotensin-converting enzyme inhibitor β-casein 203-209 increased approximately 10 000-fold after combined oral, gastric, and intestinal digestion. Thus, physiological digestive processes may promote bioactive peptide formation from proteins and oligopeptides in kefir. Furthermore, bioactive peptides present in certain compartments of the gastrointestinal tract may exert local physiological effects.

In vitro evaluation on neutral detergent fiber and cellulose digestion by post-ruminal microorganisms in goats.

Science.gov (United States)

Jiao, Jinzhen; Wang, Pengpeng; He, Zhixiong; Tang, Shaoxun; Zhou, Chuanshe; Han, Xuefeng; Wang, Min; Wu, Duanqin; Kang, Jinhe; Tan, Zhiliang

2014-07-01

Post-ruminal digestion of fiber has received much less attention than its ruminal digestion. Using in vitro incubation techniques, the present study explored whether variations in fiber digestion occurred in different segments of the post-ruminal tract and whether fiber structure could influence its digestibility. A split plot design was conducted with gut segments (jejunum, ileum, cecum and colon) as main plot and substrates (neutral detergent fiber (NDF) and cellulose (CEL)) as subplot. With the same substrate, the final asymptotic gas volume (V(F)), gas production at t(i) (V(t(i)), digestibility, microbial crude protein (MCP), total bacteria number (TBN), total short-chain fatty acids (TSCFA) and xylanase in incocula from the cecum and colon exceeded (P production, digestibility, enzyme activities and SCFA but lower pH and NH3-N. The current results imply that the intestinal contents from the cecum and colon have greater potential to digest fiber than those from the jejunum and ileum, and CEL is more easily digested in the post-ruminal tract than NDF. © 2013 Society of Chemical Industry.

Mammalian mucosal α-glucosidases coordinate with α-amylase in the initial starch hydrolysis stage to have a role in starch digestion beyond glucogenesis.

Science.gov (United States)

Dhital, Sushil; Lin, Amy Hui-Mei; Hamaker, Bruce R; Gidley, Michael J; Muniandy, Anbuhkani

2013-01-01

Starch digestion in the human body is typically viewed in a sequential manner beginning with α-amylase and followed by α-glucosidase to produce glucose. This report indicates that the two enzyme types can act synergistically to digest granular starch structure. The aim of this study was to investigate how the mucosal α-glucosidases act with α-amylase to digest granular starch. Two types of enzyme extracts, pancreatic and intestinal extracts, were applied. The pancreatic extract containing predominantly α-amylase, and intestinal extract containing a combination of α-amylase and mucosal α-glucosidase activities, were applied to three granular maize starches with different amylose contents in an in vitro system. Relative glucogenesis, released maltooligosaccharide amounts, and structural changes of degraded residues were examined. Pancreatic extract-treated starches showed a hydrolysis limit over the 12 h incubation period with residues having a higher gelatinization temperature than the native starch. α-Amylase combined with the mucosal α-glucosidases in the intestinal extract showed higher glucogenesis as expected, but also higher maltooligosaccharide amounts indicating an overall greater degree of granular starch breakdown. Starch residues after intestinal extract digestion showed more starch fragmentation, higher gelatinization temperature, higher crystallinity (without any change in polymorph), and an increase of intermediate-sized or small-sized fractions of starch molecules, but did not show preferential hydrolysis of either amylose or amylopectin. Direct digestion of granular starch by mammalian recombinant mucosal α-glucosidases was observed which shows that these enzymes may work either independently or together with α-amylase to digest starch. Thus, mucosal α-glucosidases can have a synergistic effect with α-amylase on granular starch digestion, consistent with a role in overall starch digestion beyond their primary glucogenesis function.

Mammalian mucosal α-glucosidases coordinate with α-amylase in the initial starch hydrolysis stage to have a role in starch digestion beyond glucogenesis.

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Sushil Dhital

Full Text Available Starch digestion in the human body is typically viewed in a sequential manner beginning with α-amylase and followed by α-glucosidase to produce glucose. This report indicates that the two enzyme types can act synergistically to digest granular starch structure. The aim of this study was to investigate how the mucosal α-glucosidases act with α-amylase to digest granular starch. Two types of enzyme extracts, pancreatic and intestinal extracts, were applied. The pancreatic extract containing predominantly α-amylase, and intestinal extract containing a combination of α-amylase and mucosal α-glucosidase activities, were applied to three granular maize starches with different amylose contents in an in vitro system. Relative glucogenesis, released maltooligosaccharide amounts, and structural changes of degraded residues were examined. Pancreatic extract-treated starches showed a hydrolysis limit over the 12 h incubation period with residues having a higher gelatinization temperature than the native starch. α-Amylase combined with the mucosal α-glucosidases in the intestinal extract showed higher glucogenesis as expected, but also higher maltooligosaccharide amounts indicating an overall greater degree of granular starch breakdown. Starch residues after intestinal extract digestion showed more starch fragmentation, higher gelatinization temperature, higher crystallinity (without any change in polymorph, and an increase of intermediate-sized or small-sized fractions of starch molecules, but did not show preferential hydrolysis of either amylose or amylopectin. Direct digestion of granular starch by mammalian recombinant mucosal α-glucosidases was observed which shows that these enzymes may work either independently or together with α-amylase to digest starch. Thus, mucosal α-glucosidases can have a synergistic effect with α-amylase on granular starch digestion, consistent with a role in overall starch digestion beyond their primary

Mammalian Mucosal α-Glucosidases Coordinate with α-Amylase in the Initial Starch Hydrolysis Stage to Have a Role in Starch Digestion beyond Glucogenesis

Science.gov (United States)

Dhital, Sushil; Lin, Amy Hui-Mei; Hamaker, Bruce R.; Gidley, Michael J.; Muniandy, Anbuhkani

2013-01-01

Starch digestion in the human body is typically viewed in a sequential manner beginning with α-amylase and followed by α-glucosidase to produce glucose. This report indicates that the two enzyme types can act synergistically to digest granular starch structure. The aim of this study was to investigate how the mucosal α-glucosidases act with α-amylase to digest granular starch. Two types of enzyme extracts, pancreatic and intestinal extracts, were applied. The pancreatic extract containing predominantly α-amylase, and intestinal extract containing a combination of α-amylase and mucosal α-glucosidase activities, were applied to three granular maize starches with different amylose contents in an in vitro system. Relative glucogenesis, released maltooligosaccharide amounts, and structural changes of degraded residues were examined. Pancreatic extract-treated starches showed a hydrolysis limit over the 12 h incubation period with residues having a higher gelatinization temperature than the native starch. α-Amylase combined with the mucosal α-glucosidases in the intestinal extract showed higher glucogenesis as expected, but also higher maltooligosaccharide amounts indicating an overall greater degree of granular starch breakdown. Starch residues after intestinal extract digestion showed more starch fragmentation, higher gelatinization temperature, higher crystallinity (without any change in polymorph), and an increase of intermediate-sized or small-sized fractions of starch molecules, but did not show preferential hydrolysis of either amylose or amylopectin. Direct digestion of granular starch by mammalian recombinant mucosal α-glucosidases was observed which shows that these enzymes may work either independently or together with α-amylase to digest starch. Thus, mucosal α-glucosidases can have a synergistic effect with α-amylase on granular starch digestion, consistent with a role in overall starch digestion beyond their primary glucogenesis function. PMID

Effect of dietary protein restriction on renal ammonia metabolism

Science.gov (United States)

Lee, Hyun-Wook; Osis, Gunars; Handlogten, Mary E.; Guo, Hui; Verlander, Jill W.

2015-01-01

Dietary protein restriction has multiple benefits in kidney disease. Because protein intake is a major determinant of endogenous acid production, it is important that net acid excretion change in parallel during protein restriction. Ammonia is the primary component of net acid excretion, and inappropriate ammonia excretion can lead to negative nitrogen balance. Accordingly, we examined ammonia excretion in response to protein restriction and then we determined the molecular mechanism of the changes observed. Wild-type C57Bl/6 mice fed a 20% protein diet and then changed to 6% protein developed an 85% reduction in ammonia excretion within 2 days, which persisted during a 10-day study. The expression of multiple proteins involved in renal ammonia metabolism was altered, including the ammonia-generating enzymes phosphate-dependent glutaminase (PDG) and phosphoenolpyruvate carboxykinase (PEPCK) and the ammonia-metabolizing enzyme glutamine synthetase. Rhbg, an ammonia transporter, increased in expression in the inner stripe of outer medullary collecting duct intercalated cell (OMCDis-IC). However, collecting duct-specific Rhbg deletion did not alter the response to protein restriction. Rhcg deletion did not alter ammonia excretion in response to dietary protein restriction. These results indicate 1) dietary protein restriction decreases renal ammonia excretion through coordinated regulation of multiple components of ammonia metabolism; 2) increased Rhbg expression in the OMCDis-IC may indicate a biological role in addition to ammonia transport; and 3) Rhcg expression is not necessary to decrease ammonia excretion during dietary protein restriction. PMID:25925252

Improvement of Protein Digestibility in Jatropha curcas Seed Cakes by Gamma Irradiation

International Nuclear Information System (INIS)

Sudprasert, Wanwisa; Pakkong, Pannee; Thiengtham, Jamroen; Chandang, Pipatpong

2011-06-01

Full text: The effect of gamma radiation on protein digestibility of Jatropha curcas press cake was investigated using in vitro digestibility technique. Six varieties of Jatropha curcas seeds were subjected to cobalt-60 gamma radiation at doses of 10-100 kGy. All treated seeds were defatted by screw press. In vitro protein digest abilities in defatted seeds were assayed using trinitrobenzene sulphonic acid (TNBS) method, by which the contents of alpha amino induced from the function of enzymes were determined using L-alanine as a reference standard. It was found that irradiation treatment at 60 kGy significantly increased the protein digestibility by 15-92%. Also, the results showed that moisture, crude protein, fat and ash contents were unchanged by irradiation, whereas fiber was significantly decreased (p < 0.05). Therefore, irradiation could serve as a possible processing method for protein utilization improvement in defatted Jatropha curcas seeds before using as a protein supplement in animal feed

Suppression of APOBEC3-mediated restriction of HIV-1 by Vif

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Yuqing eFeng

2014-08-01

Full Text Available The APOBEC3 restriction factors are a family of deoxycytidine deaminases that are able to suppress replication of viruses with a single-stranded DNA intermediate by inducing mutagenesis and functional inactivation of the virus. Of the seven human APOBEC3 enzymes, only APOBEC3-D, -F, -G, and -H appear relevant to restriction of HIV-1 in CD4+ T cells and will be the focus of this review. The restriction of HIV-1 occurs most potently in the absence of HIV-1 Vif that induces polyubiquitination and degradation of APOBEC3 enzymes through the proteasome pathway. To restrict HIV-1, APOBEC3 enzymes must be encapsidated into budding virions. Upon infection of the target cell during reverse transcription of the HIV-1 RNA into (-DNA APOBEC3 enzymes deaminate cytosines to forms uracils in single-stranded (- DNA regions. Upon replication of the (-DNA to (+DNA, the HIV-1 reverse transcriptase incorporates adenines opposite the uracils thereby inducing C/G to T/A mutations that can functionally inactivate HIV-1. APOBEC3G is the most studied APOBEC3 enzyme and it is known that Vif attempts to thwart APOBEC3 function not only by inducing its proteasomal degradation but by several degradation-independent mechanisms such as inhibiting APOBEC3G virion encapsidation, mRNA translation, and for those APOBEC3G molecules that still become virion encapsidated, Vif can inhibit APOBEC3G mutagenic activity. Although most Vif variants can induce efficient degradation of APOBEC3-D, -F, and -G, there appears to be differential sensitivity to Vif-mediated degradation for APOBEC3H. This review examines APOBEC3-mediated HIV restriction mechanisms, how Vif acts as a substrate receptor for a Cullin5 ubiquitin ligase complex to induce degradation of APOBEC3s, and the determinants and functional consequences of the APOBEC3 and Vif interaction from a biological and biochemical perspective.

ISOLATION AND LIGNOCELLULOLYTIC ACTIVITIES OF FIBER-DIGESTING BACTERIA FROM DIGESTIVE TRACT OF TERMITE (Cryptothermes sp.

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B.I.M. Tampoebolon

2015-09-01

Full Text Available The objectives of this study were to obtain the fiber-digesting bacteria isolates from termitedigestive tract and to determine the optimum conditions of growth and production of cellulase, xylanaseand ligninase enzyme of isolate. The first study was conducted to isolate and select the fiber-digestingbacteria from the digestive tract of termites based on the highest activity of cellulolytic (S, xylanolytic(X and lignolytic (L. The second study was optimation of the growth conditions of bacteria and theenzyme production due to effect of rice straw substrate and nitrogen. The material used were dry woodtermites, rice straw, and culture medium. The design used was a completely randomized factorial design,in which the first factor was rice straw substrate (1, 2, and 3% W/V, while the second factor wasnitrogen (0.1, 0.2 and 0.3% W/V. Variables measured were cellulase, xylanase and ligninase activities.Results of the first sudy showed that the isolates obtained consisted of 3 types, those were cellulolyticbacteria (S1, S2, and S3, 3 types of bacteria xylanolytic (X1, X2, and X3 and 3 types of bacteria lignolytic(L1, L2, and L3. Meanwhile, results of the second study showed that isolates of S2, X3, and L1 had thehighest activity, those were 1.894 U/mL, 1.722 U/mL and 0.314 U/mL, respectively. In conclusion, the addition of 1% level of rice straw substrate and 0.3% of nitrogen showed the highest enzyme activity oncellulase, xylanase and ligninase.

Aged refuse enhances anaerobic digestion of waste activated sludge.

Science.gov (United States)

Zhao, Jianwei; Gui, Lin; Wang, Qilin; Liu, Yiwen; Wang, Dongbo; Ni, Bing-Jie; Li, Xiaoming; Xu, Rui; Zeng, Guangming; Yang, Qi

2017-10-15

In this work, a low-cost alternative approach (i.e., adding aged refuse (AR) into waste activated sludge) to significantly enhance anaerobic digestion of sludge was reported. Experimental results showed that with the addition dosage of AR increasing from 0 to 400 mg/g dry sludge soluble chemical oxygen demand (COD) increased from 1150 to 5240 mg/L at the digestion time of 5 d, while the maximal production of volatile fatty acids (VFA) increased from 82.6 to 183.9 mg COD/g volatile suspended solids. Although further increase of AR addition decreased the concentrations of both soluble COD and VFA, their contents in these systems with AR addition at any concentration investigated were still higher than those in the blank, which resulted in higher methane yields in these systems. Mechanism studies revealed that pertinent addition of AR promoted solubilization, hydrolysis, and acidogenesis processes and did not affect methanogenesis significantly. It was found that varieties of enzymes and anaerobes in AR were primary reason for the enhancement of anaerobic digestion. Humic substances in AR benefited hydrolysis and acidogenesis but inhibited methanogenesis. The effect of heavy metals in AR on sludge anaerobic digestion was dosage dependent. Sludge anaerobic digestion was enhanced by appropriate amounts of heavy metals but inhibited by excessive amounts of heavy metals. The relative abundances of microorganisms responsible for sludge hydrolysis and acidogenesis were also observed to be improved in the system with AR addition, which was consistent with the performance of anaerobic digestion. Copyright © 2017 Elsevier Ltd. All rights reserved.

Carbon balances for in vitro digestion an fermentation of potential roughages for pregnant sows

NARCIS (Netherlands)

Becker, P.M.; Gelder, van A.H.; Wikselaar, van P.G.; Jongbloed, A.W.; Cone, J.W.

2003-01-01

Ad libitum feeding of pregnant sows requires satiating, intake-restricting feed components to prevent sows from getting excessively fat. Because hindgut fermentation starts only after and proceeds much slower than enzymatic digestion in the small intestine, fermentation products might, as nutrients,

Digestive Diseases

Science.gov (United States)

... cells and provide energy. This process is called digestion. Your digestive system is a series of hollow organs joined ... are also involved. They produce juices to help digestion. There are many types of digestive disorders. The ...

Mining anaerobic digester consortia metagenomes for secreted carbohydrate active enzymes

DEFF Research Database (Denmark)

Wilkens, Casper; Busk, Peter Kamp; Pilgaard, Bo

thermophilic and mesophilic ADs a wide variety of carbohydrate active enzyme functions were discovered in the metagenomic sequencing of the microbial consortia. The most dominating type of glycoside hydrolases were β-glucosidases (up to 27%), α-amylases (up to 10%), α-glucosidases (up to 8%), α......, and food wastes (Alvarado et al., 2014). The processes and the roles of the microorganisms that are involved in biomass conversion and methane production in ADs are still not fully understood. We are investigating thermophilic and mesophilic ADs that use wastewater surplus sludge for methane production...... was done with the Peptide Pattern Recognition (PPR) program (Busk and Lange, 2013), which is a novel non-alignment based approach that can predict function of e.g. CAZymes. PPR identifies a set of short conserved sequences, which can be used as a finger print when mining genomes for novel enzymes. In both...

Monitoring digestibility of forages for herbivores: a new application for an old approach

Science.gov (United States)

Vansomeren, Lindsey L.; Barboza, Perry S.; Thompson, Daniel P.; Gustine, David D.

2015-01-01

Ruminant populations are often limited by how well individuals are able to acquire nutrients for growth, maintenance, and reproduction. Nutrient supply to the animal is dictated by the concentration of nutrients in feeds and the efficiency of digesting those nutrients (i.e., digestibility). Many different methods have been used to measure digestibility of forages for wild herbivores, all of which rely on collecting rumen fluid from animals or incubation within animals. Animal-based methods can provide useful estimates, but the approach is limited by the expense of fistulated animals, wide variation in digestibility among animals, and contamination from endogenous and microbial sources that impairs the estimation of nutrient digestibility. We tested an in vitro method using a two-stage procedure using purified enzymes. The first stage, a 6 h acid–pepsin treatment, was followed by a combined 72 h amylase–cellulase or amylase–Viscozyme treatment. We then validated our estimates using in sacco and in vivo methods to digest samples of the same forages. In vitro estimates of dry matter (DM) digestibility were correlated with estimates of in sacco and in vivo DM digestibility (both P content declined below 0.03–0.06 g/g of DM. The in vitro method is well suited to monitoring forage quality over multiple years because it is reproducible, can be used with minimal investment by other laboratories without animal facilities, and can measure digestibility of individual nutrients such as N.

Hemolysis, Elevated Liver Enzymes, and Low Platelets, Severe Fetal Growth Restriction, Postpartum Subarachnoid Hemorrhage, and Craniotomy: A Rare Case Report and Systematic Review

Directory of Open Access Journals (Sweden)

Shadi Rezai

2017-01-01

Full Text Available Introduction. Hemolysis, elevated liver enzymes, and low platelets (HELLP syndrome is a relatively uncommon but traumatic condition occurring in the later stage of pregnancy as a complication of severe preeclampsia or eclampsia. Prompt brain computed tomography (CT or magnetic resonance imaging (MRI and a multidisciplinary management approach are required to improve perinatal outcome. Case. A 37-year-old, Gravida 6, Para 1-0-4-1, Hispanic female with a history of chronic hypertension presented at 26 weeks and 6 days of gestational age. She was noted to have hemolysis, elevated liver enzymes, and low platelets (HELLP syndrome accompanied by fetal growth restriction (FGR, during ultrasound evaluation, warranting premature delivery. The infant was delivered in stable condition suffering no permanent neurological deficit. Conclusion. HELLP syndrome is an uncommon and traumatic obstetric event which can lead to neurological deficits if not managed in a responsive and rapid manner. The central aggravating factor seems to be hypertension induced preeclamptic or eclamptic episode and complications thereof. The syndrome itself is manifested by hemolytic anemia, increased liver enzymes, and decreasing platelet counts with a majority of neurological defects resulting from hemorrhagic stroke or subarachnoid hemorrhage (SAH. To minimize adverse perinatal outcomes, obstetric management of this medical complication must include rapid clinical assessment, diagnostic examination, and neurosurgery consultation.

Changes in physicochemical properties and in vitro starch digestion of native and extruded maize flours subjected to branching enzyme and maltogenic α-amylase treatment.

Science.gov (United States)

Román, Laura; Martínez, Mario M; Rosell, Cristina M; Gómez, Manuel

2017-08-01

Extrusion is an increasingly used type of processing which combined with enzymatic action could open extended possibilities for obtaining clean label modified flours. In this study, native and extruded maize flours were modified using branching enzyme (B) and a combination of branching enzyme and maltogenic α-amylase (BMA) in order to modulate their hydrolysis properties. The microstructure, pasting properties, in vitro starch hydrolysis and resistant starch content of the flours were investigated. Whereas BMA treatment led to greater number of holes on the granule surface in native samples, B and BMA extruded samples showed rougher surfaces with cavities. A reduction in the retrogradation trend was observed for B and BMA native flours, in opposition to the flat pasting profile of their extruded counterparts. The glucose release increased gradually for native flours as the time of reaction did, whereas for extruded flours a fast increase of glucose release was observed during the first minutes of reaction, and kept till the end, indicating a greater accessibility to their porous structure. These results suggested that, in enzymatically treated extruded samples, changes produced at larger hierarchical levels in their starch structure could have masked a slowdown in the starch digestion properties. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecular bass for a malic enzyme null mutation

International Nuclear Information System (INIS)

Brown, M.L.; Wise, L.S.; Rubin, C.S.

1987-01-01

Many tissues from normal (wt) mice have cytosolic malic enzyme (ME) activity and express two mRNAs (2 and 3.1 kb) that code for a single ME polypeptide. Mod-1 null (M-n) mice lack cytosolic ME activity, but express 2.5 and 3.6 kb mRNAs that hybridize with wt ME cDNAs. To investigate the basis for the ME deficiency cDNAs corresponding to M-n ME RNA were cloned. A λgt11 library was prepared using M-n liver mRNA as a template. Wt ME cDNA probes hybridized with several recombinant phages and a 2kb insert with an atypical (non-wt) restriction pattern was subcloned in pGEM 1 and sequenced. The M-n ME cDNA contains an internal directly repeated sequence that corresponds to nts 1109-1617 in the coding region of wt ME cDNA. A restriction fragment from M-n ME cDNA that includes the first 204 bp of repeated sequence and 306 bp of contiguous 5' sequence was subcloned into pGEM 1 and used as a template for synthesizing 32 P-labeled anti-sense RNA. After hybridization with M-n liver RNA the 510 nt transcript was resistant to RNA digestion; after hybridization with wt RNA only fragments corresponding to the normally non-contiguous 204 bp and 306 bp segments of the insert were protected. Thus the partial duplication of coding sequence in M-n ME mRNA is confirmed. Analyses of intron-exon organization in the relevant regions of the wt and M-n ME genes will provide further insights into the mechanism underlying the ME null mutation

Effects of exogenous phytase and xylanase, individually or in combination, and pelleting on nutrient digestibility, available energy content of wheat and performance of growing pigs fed wheat-based diets.

Science.gov (United States)

Yang, Y Y; Fan, Y F; Cao, Y H; Guo, P P; Dong, B; Ma, Y X

2017-01-01

Two experiments were conducted to determine the effects of adding exogenous phytase and xylanase, individually or in combination, as well as pelleting on nutrient digestibility, available energy content of wheat and the performance of growing pigs fed wheat-based diets. In Experiment 1, forty-eight barrows with an initial body weight of 35.9±0.6 kg were randomly assigned to a 2×4 factorial experiment with the main effects being feed form (pellet vs meal) and enzyme supplementation (none, 10,000 U/kg phytase, 4,000 U/kg xylanase or 10,000 U/kg phytase plus 4,000 U/kg xylanase). The basal diet contained 97.8% wheat. Pigs were placed in metabolic cages for a 7-d adaptation period followed by a 5-d total collection of feces and urine. Nutrient digestibility and available energy content were determined. Experiment 2 was conducted to evaluate the effects of pelleting and enzymes on performance of wheat for growing pigs. In this experiment, 180 growing pigs (35.2±9.0 kg BW) were allocated to 1 of 6 treatments according to a 2×3 factorial treatment arrangement with the main effects being feed form (meal vs pellet) and enzyme supplementation (0, 2,500 or 5,000 U/kg xylanase). In Experiment 1, there were no interactions between feed form and enzyme supplementation. Pelleting reduced the digestibility of acid detergent fiber (ADF) by 6.4 percentage units (pdigestibility of energy by 0.6 percentage units (pdigestibility of crude protein by 0.5 percentage units (p = 0.07) compared with diets in mash form. The addition of phytase improved the digestibility of phosphorus (pdigestibility of crude protein by 1.0 percentage units (p = 0.09) and increased the digestibility of neutral detergent fiber (NDF) (pdigestibility of phosphorus (pdigestibility (pdigestibility but decreased ADF digestibility. Adding xylanase increased crude protein digestibility and pig performance. Phytase increased the apparent total tract digestibility of phosphorus and calcium. The combination of

Transcriptome analysis of the digestive system of a wood-feeding termite (Coptotermes formosanus) revealed a unique mechanism for effective biomass degradation.

Science.gov (United States)

Geng, Alei; Cheng, Yanbing; Wang, Yongli; Zhu, Daochen; Le, Yilin; Wu, Jian; Xie, Rongrong; Yuan, Joshua S; Sun, Jianzhong

2018-01-01

Wood-feeding termite, Coptotermes formosanus Shiraki, represents a highly efficient system for biomass deconstruction and utilization. However, the detailed mechanisms of lignin modification and carbohydrate degradation in this system are still largely elusive. In order to reveal the inherent mechanisms for efficient biomass degradation, four different organs (salivary glands, foregut, midgut, and hindgut) within a complete digestive system of a lower termite, C. formosanus , were dissected and collected. Comparative transcriptomics was carried out to analyze these organs using high-throughput RNA sequencing. A total of 71,117 unigenes were successfully assembled, and the comparative transcriptome analyses revealed significant differential distributions of GH (glycosyl hydrolase) genes and auxiliary redox enzyme genes in different digestive organs. Among the GH genes in the salivary glands, the most abundant were GH9, GH22, and GH1 genes. The corresponding enzymes may have secreted into the foregut and midgut to initiate the hydrolysis of biomass and to achieve a lignin-carbohydrate co-deconstruction system. As the most diverse GH families, GH7 and GH5 were primarily identified from the symbiotic protists in the hindgut. These enzymes could play a synergistic role with the endogenous enzymes from the host termite for biomass degradation. Moreover, twelve out of fourteen genes coding auxiliary redox enzymes from the host termite origin were induced by the feeding of lignin-rich diets. This indicated that these genes may be involved in lignin component deconstruction with its redox network during biomass pretreatment. These findings demonstrate that the termite digestive system synergized the hydrolysis and redox reactions in a programmatic process, through different parts of its gut system, to achieve a maximized utilization of carbohydrates. The detailed unique mechanisms identified from the termite digestive system may provide new insights for advanced design of

Effect of heat–moisture treatment on digestibility of different cultivars of sweet potato (Ipomea batatas (L.) Lam) starch

Science.gov (United States)

Senanayake, Suraji; Gunaratne, Anil; Ranaweera, K K D S; Bamunuarachchi, Arthur

2014-01-01

Different heat–moisture levels were applied to native starches from different cultivars of sweet potatoes available in Sri Lanka (Wariyapola red, Wariyapola white, Pallepola variety, Malaysian variety and CARI 273) to study the digestibility level. Samples were treated with 20, 25, and 30% moisture at 85°C and 120°C for 6 h and in vitro starch digestibility was tested with porcine pancreatin enzyme. A range of 19.3–23.5% digestibility was shown by the native starches with no significant difference (P digestibility level of the hydrothermally modified starches and the moisture content showed a positive impact on the digestibility. Heat–moisture treatment at 85°C brought an overall increase in digestibility and temperature beyond 85°C had a negative impact. No significant difference (P digestibility was observed with 20% and 25% moisture at 85°C and increased level were seen at 85°C and 30% moisture. PMID:25473497

Microbial and nutritional regulation of high-solids anaerobic mono-digestion of fruit and vegetable wastes.

Science.gov (United States)

Mu, Hui; Li, Yan; Zhao, Yuxiao; Zhang, Xiaodong; Hua, Dongliang; Xu, Haipeng; Jin, Fuqiang

2018-02-01

The anaerobic digestion of single fruit and vegetable wastes (FVW) can be easily interrupted by rapid acidogenesis and inhibition of methanogen, and the digestion system tends to be particularly unstable at high solid content. In this study, the anaerobic digestion of FVW in batch experiments under mesophilic condition at a high solid concentration of 10% was successfully conducted to overcome the acidogenesis problem through several modifications. Firstly, compared with the conventional anaerobic sludge (CAS), the acclimated anaerobic granular sludge (AGS) was found to be a better inoculum due to its higher Archaea abundance. Secondly, waste activated sludge (WAS) was chosen to co-digest with FVW, because WAS had abundant proteins that could generate intermediate ammonium. The ammonium could neutralize the accumulated volatile fatty acids (VFAs) and prevent the pH value of the digestion system from rapidly decreasing. Co-digestion of FVW and WAS with TS ratio of 60:40 gave the highest biogas yield of 562 mL/g-VS and the highest methane yield of 362 mL/g-VS. Key parameters in the digestion process, including VFAs concentration, pH, enzyme activity, and microbial activity, were also examined.

Nepenthesin protease activity indicates digestive fluid dynamics in carnivorous nepenthes plants.

Directory of Open Access Journals (Sweden)

Franziska Buch

Full Text Available Carnivorous plants use different morphological features to attract, trap and digest prey, mainly insects. Plants from the genus Nepenthes possess specialized leaves called pitchers that function as pitfall-traps. These pitchers are filled with a digestive fluid that is generated by the plants themselves. In order to digest caught prey in their pitchers, Nepenthes plants produce various hydrolytic enzymes including aspartic proteases, nepenthesins (Nep. Knowledge about the generation and induction of these proteases is limited. Here, by employing a FRET (fluorescent resonance energy transfer-based technique that uses a synthetic fluorescent substrate an easy and rapid detection of protease activities in the digestive fluids of various Nepenthes species was feasible. Biochemical studies and the heterologously expressed Nep II from Nepenthes mirabilis proved that the proteolytic activity relied on aspartic proteases, however an acid-mediated auto-activation mechanism was necessary. Employing the FRET-based approach, the induction and dynamics of nepenthesin in the digestive pitcher fluid of various Nepenthes plants could be studied directly with insect (Drosophila melanogaster prey or plant material. Moreover, we observed that proteolytic activity was induced by the phytohormone jasmonic acid but not by salicylic acid suggesting that jasmonate-dependent signaling pathways are involved in plant carnivory.

Authentication of beef, carabeef, chevon, mutton and pork by a PCR-RFLP assay of mitochondrial cytb gene.

Science.gov (United States)

Kumar, Deepak; Singh, S P; Karabasanavar, Nagappa S; Singh, Rashmi; Umapathi, V

2014-11-01

Authentication of meat assumes significance in view of religious, quality assurance, food safety, public health, conservation and legal concerns. Here, we describe a PCR-RFLP (Polymerase Chain Reaction- Restriction Fragment Length Polymorphism) assay targeting mitochondrial cytochrome-b gene for the identification of meats of five most common food animals namely cattle, buffalo, goat, sheep and pig. A pair of forward and reverse primers (VPH-F & VPH-R) amplifying a conserved region (168-776 bp) of mitochondrial cytochrome-b (cytb) gene for targeted species was designed which yielded a 609 bp PCR amplicon. Further, restriction enzyme digestion of the amplicons with Alu1 and Taq1 restriction enzymes resulted in a distinctive digestion pattern that was able to discriminate each species. The repeatability of the PCR-RFLP assay was validated ten times with consistent results observed. The developed assay can be used in routine diagnostic laboratories to differentiate the meats of closely related domestic livestock species namely cattle from buffalo and sheep from goat.

High levels of maize in broiler diets with or without microbial enzyme ...

African Journals Online (AJOL)

mbhuiya3

2013-03-13

Mar 13, 2013 ... High levels of maize in broiler diets with or without microbial enzyme .... to improve carbohydrate digestion and availability of phosphorus from ... grinding machine and stored at –4 ºC in airtight containers for chemical analysis ...

Supplementing enzymes to extruded, soybean based diet improves breakdown of non-starch polysaccharides in rainbow trout (Oncorhynchus mykiss)

DEFF Research Database (Denmark)

Dalsgaard, Anne Johanne Tang; Knudsen, Knud Erik Bach; Verlhac, Viviane

2016-01-01

Plant-based feed ingredients typically contain remnants of dietary fibres [DF; non-starch polysaccharides (NSP) and lignin] that have various antinutritive effects in carnivorous fish. Exogenous enzymes have been shown to improve the apparent digestibility coefficients (ADC) of plant-based diets...... presumably by assisting in the breakdown of NSP. This study examined the effects on NSP degradation when supplementing β-glucanase, xylanase, protease or a mix of the three enzymes to an extruded, juvenile rainbow trout (Oncorhynchus mykiss) diet containing 344 g kg−1 de-hulled, solvent-extracted soybean...... meal (SBM). The NSP content in the non-supplemented control diet and in faecal samples from the dietary treatment groups was analysed to determine the recovery/apparent digestibility of cellulose and total non-cellulosic polysaccharide (T-NCP) sugar monomers. The enzymes had significant, positive...

Rotifer digestive enzymes: direct detection using the ELF technique

Czech Academy of Sciences Publication Activity Database

Štrojsová, M.; Vrba, Jaroslav

2007-01-01

Roč. 593, č. 1 (2007), s. 159-165 ISSN 0018-8158. [International Symposium on Rotifers /11./. Mexiko City, 08.03.2006-13.03.2006] R&D Projects: GA AV ČR(CZ) IAA6017202; GA AV ČR(CZ) IAA600170602; GA AV ČR(CZ) 1QS600170504 Institutional research plan: CEZ:AV0Z60170517 Keywords : rotifers * enzyme localization * phosphatase * lipase * .beta.-N-acetylhexosaminidase Subject RIV: DA - Hydrology ; Limnology Impact factor: 1.201, year: 2007

Human cellular restriction factors that target HIV-1 replication

Directory of Open Access Journals (Sweden)

Jeang Kuan-Teh

2009-09-01

Full Text Available Abstract Recent findings have highlighted roles played by innate cellular factors in restricting intracellular viral replication. In this review, we discuss in brief the activities of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G, bone marrow stromal cell antigen 2 (BST-2, cyclophilin A, tripartite motif protein 5 alpha (Trim5α, and cellular microRNAs as examples of host restriction factors that target HIV-1. We point to countermeasures encoded by HIV-1 for moderating the potency of these cellular restriction functions.

Comparative Analysis of End Point Enzymatic Digests of Arabino-Xylan Isolated from Switchgrass (Panicum virgatum L of Varying Maturities using LC-MSn

Directory of Open Access Journals (Sweden)

Michael J. Bowman

2012-11-01

Full Text Available Switchgrass (Panicum virgatum L., SG is a perennial grass presently used for forage and being developed as a bioenergy crop for conversion of cell wall carbohydrates to biofuels. Up to 50% of the cell wall associated carbohydrates are xylan. SG was analyzed for xylan structural features at variable harvest maturities. Xylan from each of three maturities was isolated using classical alkaline extraction to yield fractions (Xyl A and B with varying compositional ratios. The Xyl B fraction was observed to decrease with plant age. Xylan samples were subsequently prepared for structure analysis by digesting with pure endo-xylanase, which preserved side-groups, or a commercial carbohydrase preparation favored for biomass conversion work. Enzymatic digestion products were successfully permethylated and analyzed by reverse-phase liquid chromatography with mass spectrometric detection (RP-HPLC-MSn. This method is advantageous compared to prior work on plant biomass because it avoids isolation of individual arabinoxylan oligomers. The use of RP-HPLC- MSn differentiated 14 structural oligosaccharides (d.p. 3–9 from the monocomponent enzyme digestion and nine oligosaccharide structures (d.p. 3–9 from hydrolysis with a cellulase enzyme cocktail. The distribution of arabinoxylan oligomers varied depending upon the enzyme(s applied but did not vary with harvest maturity.

Analysis of pyrimidine dimer content of isolated DNA by nuclease digestion

International Nuclear Information System (INIS)

Farland, W.H.; Sutherland, B.M.

1980-01-01

Isolated DNA is highly susceptible to degradation by exogenous nucleases. Complete digestion is possible with a number of well-characterized enzymes from a variety of sources. Treatment of DNA with a battery of enzymes including both phosphodiesterase and phosphatase activities yields a mixture of nucleosides and inorganic phosphate (P/sub i/) as a final product. Unlike native DNA, ultraviolet-irradiated DNA is resistant to complete digestion. Setlow et al. demonstrated that the structural changes in the DNA responsible for the nuclease resistance were the formation of cyclobutyl pyrimidine dimers, the major photoproduct in UV-irradiated DNA. Using venom phosphodiesterase, they demonstrated that UV irradiation of DNA affected both the rate and extent of enzymatic hydrolysis. In addition, it was demonstrated that the major nuclease-resistant product of this hydrolysis was an oligonucleotide containing dimerized pyrimidines. Treatment of the DNA to split the dimers, either photochemically or photoenzymatically, rendered the polymer more susceptible to hydrolysis by the phosphodiesterase. The specificity of photoreactivating enzyme for pyrimidine dimers lends support to the role of these structures in conferring nuclease resistance to UV-irradiated DNA. The nuclease resistance of DNA containing dimers has been the basis of several assays for the measurement of these photoproducts. Sutherland and Chamberlin reported the development of a rapid and sensitive assay for dimers in 32 P-labeled DNA

Novel regenerative large-volume immobilized enzyme reactor: preparation, characterization and application.

Science.gov (United States)

Ruan, Guihua; Wei, Meiping; Chen, Zhengyi; Su, Rihui; Du, Fuyou; Zheng, Yanjie

2014-09-15

A novel large-volume immobilized enzyme reactor (IMER) on small column was prepared with organic-inorganic hybrid silica particles and applied for fast (10 min) and oriented digestion of protein. At first, a thin enzyme support layer was formed in the bottom of the small column by polymerization with α-methacrylic acid and dimethacrylate. After that, amino SiO2 particles was prepared by the sol-gel method with tetraethoxysilane and 3-aminopropyltriethoxysilane. Subsequently, the amino SiO2 particles were activated by glutaraldehyde for covalent immobilization of trypsin. Digestive capability of large-volume IMER for proteins was investigated by using bovine serum albumin (BSA), cytochrome c (Cyt-c) as model proteins. Results showed that although the sequence coverage of the BSA (20%) and Cyt-c (19%) was low, the large-volume IMER could produce peptides with stable specific sequence at 101-105, 156-160, 205-209, 212-218, 229-232, 257-263 and 473-451 of the amino sequence of BSA when digesting 1mg/mL BSA. Eight of common peptides were observed during each of the ten runs of large-volume IMER. Besides, the IMER could be easily regenerated by reactivating with GA and cross-linking with trypsin after breaking the -C=N- bond by 0.01 M HCl. The sequence coverage of BSA from regenerated IMER increased to 25% comparing the non-regenerated IMER (17%). 14 common peptides. accounting for 87.5% of first use of IMER, were produced both with IMER and regenerated IMER. When the IMER was applied for ginkgo albumin digestion, the sequence coverage of two main proteins of ginkgo, ginnacin and legumin, was 56% and 55%, respectively. (Reviewer 2) Above all, the fast and selective digestion property of the large-volume IMER indicated that the regenerative IMER could be tentatively used for the production of potential bioactive peptides and the study of oriented protein digestion. Copyright © 2014 Elsevier B.V. All rights reserved.

A model for cell wall dissolution in mating yeast cells: polarized secretion and restricted diffusion of cell wall remodeling enzymes induces local dissolution.

Science.gov (United States)

Huberman, Lori B; Murray, Andrew W

2014-01-01

Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells.

A Model for Cell Wall Dissolution in Mating Yeast Cells: Polarized Secretion and Restricted Diffusion of Cell Wall Remodeling Enzymes Induces Local Dissolution

Science.gov (United States)

Huberman, Lori B.; Murray, Andrew W.

2014-01-01

Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells. PMID:25329559

Co-Digestion of Napier Grass and Its Silage with Cow Dung for Methane Production

Directory of Open Access Journals (Sweden)

Wipa Prapinagsorn

2017-10-01

Full Text Available Methane production from co-digestion of grass with cow dung and silage with cow dung was conducted by a bioaugmentation technique. For self-fermentation, maximum methane yield (MY of 176.66 and 184.94 mL CH4/g-VSadded were achieved at a ratio of grass to cow dung and silage to cow dung of 1:1, respectively. A higher maximum MY of 179.59 and 208.11 mL CH4/g-VSadded was obtained from co-digestion of grass with cow dung and silage with cow dung bioaugmented with anaerobic sludge at a ratio of 3:1. The solid residue left over after co-digestion at a ratio of 3:1 was pretreated by alkaline plus enzyme before used to produce methane and a maximum MY of 333.63 and 301.38 mL CH4/g-VSadded, respectively, was achieved. Overall power generated from co-digestion of grass with cow dung plus pretreated solid residues and co-digestion of silage with cow dung plus pretreated solid residues were 0.0397 and 0.007 watt, respectively.

Peptide profiling and the bioactivity character of yogurt in the simulated gastrointestinal digestion.

Science.gov (United States)

Jin, Yan; Yu, Yang; Qi, Yanxia; Wang, Fangjun; Yan, Jiaze; Zou, Hanfa

2016-06-01

by the abundance of the parent proteins but by whether the enzymes favored the protein. In summary, peptide profiling and bioactivities of yogurt in simulated gastrointestinal digestion helped to elucidate the health benefits of yogurt peptides. The results further revealed that pre-digestion of milk by lactic acid bacteria are complementary to generate bioactive peptides and to provide particular yogurt functions. Copyright © 2016 Elsevier B.V. All rights reserved.

PHYSICOCHEMICAL PROPERTIES OF THE PROTEOLYTIC ENZYME FROM THE LATEX OF THE MILKWEED, ASCLEPIAS SPECIOSA TORR. SOME COMPARISONS WITH OTHER PROTEASES

Science.gov (United States)

Winnick, Theodore; Davis, Alva R.; Greenberg, David M.

1940-01-01

1. The kinetics of milk clotting by asclepain, the protease of Asclepias speciosa, were investigated. At higher concentrations of enzyme, the clotting time was inversely proportional to the enzyme concentration. 2. The digestion of casein and hemoglobin in 6.6 M urea by asclepain follows the second order reaction rate. The rate was roughly second order for casein in water. 3. Evaluation of the nature of the enzyme-substrate intermediate indicates that one molecule of asclepain combines with one molecule of casein or hemoglobin in urea solution. 4. Inhibition by the reaction products was deduced from the fact that the digestion velocity of hemoglobin in urea solution varied with the asclepain concentration in agreement with the Schütz-Borissov rule. PMID:19873155

Post-translational suppression of expression of intestinal brush border enzymes by fructose

DEFF Research Database (Denmark)

Danielsen, E M

1989-01-01

The two major dietary sugars, fructose and sucrose, were found to suppress effectively the biosynthetic renewal of brush border enzymes in the gut. When studied in cultured explants of pig small intestine mucosa, 10-50 mM concentrations of fructose completely prevented the expression of mature...... cotranslational glycosylation that in turn triggers a rapid proteolytic breakdown. Our findings suggest that renewal of digestive brush border enzymes is transiently suppressed during intake of fructose- or sucrose-rich meals....

A new restriction endonuclease from Citrobacter freundii

OpenAIRE

Janulaitis, A.A.; Stakenas, P.S.; Lebedenko, E.N.; Berlin, Yu.A.

1982-01-01

CfrI, a new restriction endonuclease of unique substrate specificity, has been isolated from a Citrobacter freundii strain. The enzyme recognizes a degenerated sequence PyGGCCPu in double-strand DNA and cleaves it between Py and G residues to yield 5′ -protruding tetranucleotide ends GGCC.

In vitro tissue-digesting properties of krill enzymes compared with fibrinolysin/DNAse, papain and placebo

NARCIS (Netherlands)

Mekkes, J. R.; Le Poole, I. C.; Das, P. K.; Kammeyer, A.; Westerhof, W.

1997-01-01

Wound debridement, the removal of necrotic tissue, can be achieved with proteolytic enzymes. Recently, a new multi-enzyme preparation, krill enzyme, isolated from Antarctic shrimp-like organisms (Euphausia superba), was reported to possess powerful proteolytic activity towards protein substrates. In

Synchronization to light and mealtime of daily rhythms of locomotor activity, plasma glucose and digestive enzymes in the Nile tilapia (Oreochromis niloticus).

Science.gov (United States)

Guerra-Santos, Bartira; López-Olmeda, José Fernando; de Mattos, Bruno Olivetti; Baião, Alice Borba; Pereira, Denise Soledade Peixoto; Sánchez-Vázquez, Francisco Javier; Cerqueira, Robson Bahia; Albinati, Ricardo Castelo Branco; Fortes-Silva, Rodrigo

2017-02-01

The light-dark cycle and feeding can be the most important factors acting as synchronizers of biological rhythms. In this research we aimed to evaluate synchronization to feeding schedule of daily rhythms of locomotor activity and digestive enzymes of tilapia. For that purpose, 120 tilapias (65.0±0.6g) were distributed in 12 tanks (10 fish per tank) and divided into two groups. One group was fed once a day at 11:00h (zeitgeber time, ZT6) (ML group) and the other group was fed at 23:00h (ZT18) (MD group). The fish were anesthetized to collect samples of blood, stomach and midgut at 4-hour intervals over a period of 24h. Fish fed at ML showed a diurnal locomotor activity (74% of the total daily activity occurring during the light phase) and synchronization to the feeding schedule, as this group showed anticipation to the feeding time. Fish fed at MD showed a disruption in the pattern of locomotor activity and became less diurnal (59%). Alkaline protease activity in the midgut showed daily rhythm with the achrophase at the beginning of the dark phase in both ML and MD groups. Acid protease and amylase did not show significant daily rhythms. Plasma glucose showed a daily rhythm with the achrophase shifted by 12h in the ML and MD groups. These results revealed that the feeding time and light cycle synchronize differently the daily rhythms of behavior, digestive physiology and plasma metabolites in the Nile tilapia, which indicate the plasticity of the circadian system and its synchronizers. Copyright © 2016 Elsevier Inc. All rights reserved.