Jensen, Jesper Langholm; Mølgaard, Anne; Poulsen, Jens-Christian Navarro;
Bovine and camel chymosin are aspartic peptidases that are used industrially in cheese production. They cleave the Phe105-Met106 bond of the milk protein κ-casein, releasing its predominantly negatively charged C-terminus, which leads to the separation of the milk into curds and whey. Despite...... having 85% sequence identity, camel chymosin shows a 70% higher milk-clotting activity than bovine chymosin towards bovine milk. The activities, structures, thermal stabilities and glycosylation patterns of bovine and camel chymosin obtained by fermentation in Aspergillus niger have been examined...... differential scanning calorimetry revealed a slightly higher thermal stability of camel chymosin compared with bovine chymosin. The crystal structure of a doubly glycosylated variant of camel chymosin was determined at a resolution of 1.6 Å and the crystal structure of unglycosylated bovine chymosin...
Jensen, Jesper Langholm; Mølgaard, Anne; Poulsen, Jens-Christian Navarro;
Bovine and camel chymosin are aspartic peptidases that are used industrially in cheese production. They cleave the Phe105-Met106 bond of the milk protein κ-casein, releasing its predominantly negatively charged C-terminus, which leads to the separation of the milk into curds and whey. Despite...... chymosin. Both enzymes possess local positively charged patches on their surface that can play a role in interactions with the overall negatively charged C-terminus of κ-casein. Camel chymosin contains two additional positive patches that favour interaction with the substrate. The improved electrostatic...
Luo, Fan; Jiang, Wei Hua; Yang, Yuan Xiao; Li, Jiang; Jiang, Ming Feng
Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector pPICZαA, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production. PMID:27004812
Børsting, Mette Winther; Qvist, Karsten B.; Ardö, Ylva Margareta
Retained coagulant in cheese initiates casein breakdown and influences cheese structure and flavour formation. This study investigated the influence of milk pH on retention of camel chymosin in curd and compared it with bovine chymosin. Milk at five different pH levels was coagulated with same...... coagulation activity. The retention of camel chymosin in curd was rather constant at ∼20% between pH 6.65 and 6.00, while it increased almost linear from 2 to 21% for bovine chymosin. The lower pH dependence for retention of camel chymosin than of bovine chymosin may be explained by a lower negative charge...
Jensen, Jesper Langholm
The central step in cheese making is the separation of milk into curd and whey. This can be done enzymatically by hydrolysis of the Phe105-Met106 bond or nearby bonds in bovine κ-casein, which releases its hydrophilic C-terminal leading to coagulation of the milk. The preferred enzyme...... and characterised, and turned out to have an even higher activity and specificity towards the Phe105-Met106 bond than bovine chymosin. The sequences of bovine and camel chymosin are 85% identical, and yet they have significantly different cheese making properties. The aim of the project was to explain...... this difference through the study of the structures of bovine and camel chymosin, and preparation of catalytically inactive enzymes in complex with substrate. Their milk-clotting activities was determined using the traditional assay on skimmed milk, and a fluorescence resonance energy transfer (FRET) assay...
Langholm Jensen, Jesper [University of Copenhagen, (Denmark); Chr. Hansen A/S, Bøge Allé 10-12, DK-2970 Hørsholm (Denmark); Mølgaard, Anne; Navarro Poulsen, Jens-Christian [University of Copenhagen, (Denmark); Harboe, Marianne Kirsten [Chr. Hansen A/S, Bøge Allé 10-12, DK-2970 Hørsholm (Denmark); Simonsen, Jens Bæk [University of Copenhagen, (Denmark); Lorentzen, Andrea Maria; Hjernø, Karin [University of Southern Denmark, (Denmark); Brink, Johannes M. van den; Qvist, Karsten Bruun [Chr. Hansen A/S, Bøge Allé 10-12, DK-2970 Hørsholm (Denmark); Larsen, Sine, E-mail: firstname.lastname@example.org [University of Copenhagen, (Denmark)
Analysis of the crystal structures of the two milk-clotting enzymes bovine and camel chymosin has revealed that the better milk-clotting activity towards bovine milk of camel chymosin compared with bovine chymosin is related to variations in their surface charges and their substrate-binding clefts. Bovine and camel chymosin are aspartic peptidases that are used industrially in cheese production. They cleave the Phe105-Met106 bond of the milk protein κ-casein, releasing its predominantly negatively charged C-terminus, which leads to the separation of the milk into curds and whey. Despite having 85% sequence identity, camel chymosin shows a 70% higher milk-clotting activity than bovine chymosin towards bovine milk. The activities, structures, thermal stabilities and glycosylation patterns of bovine and camel chymosin obtained by fermentation in Aspergillus niger have been examined. Different variants of the enzymes were isolated by hydrophobic interaction chromatography and showed variations in their glycosylation, N-terminal sequences and activities. Glycosylation at Asn291 and the loss of the first three residues of camel chymosin significantly decreased its activity. Thermal differential scanning calorimetry revealed a slightly higher thermal stability of camel chymosin compared with bovine chymosin. The crystal structure of a doubly glycosylated variant of camel chymosin was determined at a resolution of 1.6 Å and the crystal structure of unglycosylated bovine chymosin was redetermined at a slightly higher resolution (1.8 Å) than previously determined structures. Camel and bovine chymosin share the same overall fold, except for the antiparallel central β-sheet that connects the N-terminal and C-terminal domains. In bovine chymosin the N-terminus forms one of the strands which is lacking in camel chymosin. This difference leads to an increase in the flexibility of the relative orientation of the two domains in the camel enzyme. Variations in the amino acids
Møller, Ulla Kristine; Jensen, Lars Thorbjørn; Mosekilde, Leif;
Calcium intake and absorption is important for bone health. In a randomized double-blind cross-over trial, we investigated effects of adding chymosin to milk on the intestinal calcium absorption as measured by renal calcium excretion and indices of calcium homeostasis. The primary outcome...... of the study was 24-h renal calcium excretion that is considered a proxy measure of the amount of calcium absorbed from the intestine. We studied 125 healthy men and women, aged 34 (25-45) years on two separate days. On each day, a light breakfast was served together with 500 ml of semi-skimmed milk to which...... not depend on plasma 25-hydroxyvitamin D levels. Chymosin added to milk increases renal calcium excretion in the hours following intake without affecting plasma levels of calcium or calciotropic hormones. The effect most likely represents enhanced intestinal calcium absorption shortly after intake. Further...
Palmer, David S.; Christensen, Anders Uhrenholt; Sørensen, Jesper;
Bovine chymosin is an aspartic protease that selectively cleaves the milk protein κ-casein. The enzyme is widely used to promote milk clotting in cheese manufacturing. We have developed models of residues 97-112 of bovine κ-casein complexed with bovine chymosin, using ligand docking, conformation...
... is secreted by cells into fermentation broth and converted to chymosin by acid treatment. All... (fermentation-derived). 184.1685 Section 184.1685 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... (animal-derived) and chymosin preparation (fermentation-derived). (a)(1) Rennet and bovine rennet...
Esteve, C L; Lucey, J A; Pires, E M
Rheological properties of reconstituted skim milk coagulated with plant coagulants Cynara cardunculus L., Cynara humilis L. and chymosin was monitored by dynamic low amplitude oscillation. There are no published reports on the modelling of the gelation behaviour of milk by plant coagulants. Three mathematical models, Scott Blair. Douillard and Carlson, were fitted to the storage modulus (G') as function of time curves. For all coagulants. Scott Blair model was the most efficient in modelling the gelation process, and gave both the smallest residuals and standard error of residuals, Se (P gels with slightly higher G' values than chymosin, but after longer incubation times chymosin gels had higher G' values. It was concluded that the Scott Blair model was the best equation to follow the gelation of milk induced by both plant coagulants as well as chymosin. Modelling is an important and useful method for comparing the gelation process in gels formed by different types of coagulants. PMID:11694051
Full Text Available The present study was conducted to develop and validate a specific radioimmunoassay system for measurement of bovine chymosin B (bChyB concentrations in plasma samples. Bovine ChyB was used for immunization of rabbits and as standard and tracer. Chymosin B concentrations were measured in plasma samples from two groups of calves (Group 1: calves sampled from birth to 24 hours; Group 2: calves sampled from Day 1 to 21 after birth and from one cow during the peri-partum period. Detection limit of the assay was 9.0 ng/ml. Recovery was higher than 89.3%. Repeatability and reproducibility ranged from 1.52% to 5.23% and from 1.52% to 12.57% respectively. No cross-reaction was found with pepsinogen A from bovine, porcine or human origins. In Group 1, bChyB concentrations increased from 47.3 ± 45.1 ng/ml (5 min after birth to 325.5 ± 161.2 ng/ml (12 hours after birth, then no significant change was observed till 24 hours after birth (293.0 ± 161.5 ng/ml. In Group 2, concentrations decreased from Day 1 (455.3 ± 191.1 ng/ml to Day 21 (117.9 ± 85.1 ng/ml. In adult cow, mean concentration was 136.0 ± 32.3 ng/ml. In conclusion, bChyB is able to cross the stomach basal membrane and to reach the blood circulation at detectable levels in both young calves and adult cows.
Rolet-Répécaud, O; Arnould, C; Dupont, D; Gavoye, S; Beuvier, E; Achilleos, C
Chymosin is the major enzyme of natural rennet, traditionally used in cheese making for its high milk-clotting activity. For technical reasons, an accurate characterization of rennet should include its total clotting activity and also its enzymatic composition. Monoclonal antibodies specific to chymosin were obtained from mice immunized with purified bovine chymosin, and an inhibition enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of chymosin in solution. No cross-reactivity was observed with other milk-clotting enzymes commonly used in cheese making. The limit of detection and limit of quantification were 125 and 400 ng/mL, respectively. The values of precision within and among runs were 7.23 and 7.39%, respectively, and satisfying recovery, from 92 to 119%, was found for spiked samples. The inhibition ELISA was successfully applied to commercial rennets, and the results were consistent with those obtained using the standard chromatographic method (IDF 110: A, 1987).
刘佟; 崔艳华; 张兰威; 曲晓军
凝乳酶是一种最早在未断奶的小牛胃中发现的天门冬氨酸蛋白酶,可专一地切割乳中κ-酪蛋白的Phe105-Met106之间的肽键,破坏酪蛋白胶束使牛奶凝结,凝乳酶的凝乳能力及蛋白水解能力使其成为干酪生产中形成质构和特殊风味的关键性酶,被广泛地应用于奶酪和酸奶的制作.本文以牛凝乳酶为例介绍了凝乳酶的结构、理化特性和凝乳机理,综述了凝乳酶主要来源以及不同来源凝乳酶之间酶性质差异,旨在为凝乳酶研究提供些许参考.%Chymosin (rennin; EC 22.214.171.124) belongs to a aspartic proteinase family which was found in unweaned bovine stomachs at first. It can specifically cleave the K-casein at the Phel05-Metl06 peptide bond to broke the casein micelles causing milk-clotting. In cheese produc-tion,chymosin plays an important role as the key enzyme in milk-clotting and processing of texture and flavour. The structure, physicochemi-cal characteristics and milk coagulation mechanism of bovine chymosin were reviewed in this paper. Otherwise, the source of chymosin and the diversity from different sources were discussed.
Chymosin is one of the key enzymes during the production of cheese. With the development of cheese industry, chymosin made by traditional method is not sufficient for its production, so the researchers are looking for the new sources of cbymosin. MJ229 can produce chymosin, that was isolated from glutinous rice wine which was a traditional food in south of China. The enzyme liquor of MJ229 solid - state fermentation was concentrated through hollow fiber ultrafiltration membrane with aperture 60kDa and 6kDa during this experiment. The volume was concentrated 15. 6 times.The enzyme was purified 2. 15 times and the yield radio was 82. 6％. The concentration was purified by Sephadex G 100. The milk -clotting activity was 2200. 0U/mg after purification and it was confirmed by SDS - PAGE pattern that showed the chymosin was nearly purified with a molecular mass of 35.0kDa.%凝乳酶是干酪生产中的关键性酶,随着世界干酪工业的不断发展,传统来源的凝乳酶已远不能满足生产需要,各国研究者不断地寻找新的来源.MJ229是从我国传统食品江米酒酒醪中筛选得到的一株凝乳酶高产菌株.本实验利用截留分子量为60kDa和6kDa的中空纤维超滤膜对MJ229固态发酵物中凝乳酶的浸提液进行浓缩,浸提液体积被浓缩了15.6倍,凝乳酶纯化倍数达2.15倍,活力回收率为82.6%.进一步通过SephadexG-100纯化,得到电泳纯的凝乳酶,活力为2200.0U/mg,同时测得其分子量为35kDa.
Noseda, Diego Gabriel; Recúpero, Matías Nicolás; Blasco, Martín; Ortiz, Gastón Ezequiel; Galvagno, Miguel Angel
The codon sequence optimized bovine prochymosin B gene was cloned under the control of the alcohol oxidase 1 promoter (AOX1) in the vector pPIC9K and integrated into the genome of the methylotrophic yeast Pichia (Komagataella) pastoris (P. pastoris) strain GS115. A transformant clone that showed resistance to over 4 mg G418/ml and displayed the highest milk-clotting activity was selected. Cell growth and recombinant bovine chymosin production were optimized in flask cultures during methanol induction phase achieving the highest coagulant activity with low pH values, a temperature of 25°C and with the addition of sorbitol and ascorbic acid at the beginning of this period. The scaling up of the fermentation process to lab-scale stirred bioreactor using optimized conditions, allowed to reach 240 g DCW/L of biomass level and 96 IMCU/ml of milk-clotting activity. The enzyme activity corresponded to 53 mg/L of recombinant bovine chymosin production after 120 h of methanol induction. Western blot analysis of the culture supernatant showed that recombinant chymosin did not suffer degradation during the protein production phase. By a procedure that included high performance gel filtration chromatography and 3 kDa fast ultrafiltration, the recombinant bovine chymosin was purified and concentrated from fermentation cultures, generating a specific activity of 800 IMCU/Total Abs(280 nm) and a total activity recovery of 56%. This study indicated that P. pastoris is a suitable expression system for bioreactor based fed-batch fermentation process for the efficient production of recombinant bovine chymosin under methanol-inducible AOX1 promoter.
Børsting, Mette Winther
need of an extended ripening period to reach a similar cheese structure as in cheeses produced with BC. The aim of this project was to compensate for the lower proteolytic activity in cheese produced with CC compared to BC. Selection of dairy lactic acid bacteria (LAB) for cheese production with high...... ability to influence proteolysis and structure during cheese ripening. In an attempt to improve the screening methods and contribute to the development of a new classification system of Latcococcus lactic strains, the peptide profile formed by selected strains after growth in milk was analyzed and...... culture mediated an increase in the total amount of amino acids as well as a shorter structure. A model system, used to study the retention of chymosin in a curd, showed that the retention of CC was less dependent on pH compared to BC, and the retention of CC was higher than BC in the pH interval 6...
Kingston, Diego; Novelli, Guido F; Cerrutti, Patricia; Recupero, Matias N; Blasco, Martin; Galvagno, Miguel A
The use of agroindustrial wastes not only decreases bioprocesses and disposal costs but also contributes to the upgrading of the residues. An active recombinant methanol-inducible bovine chymosin has been expressed in our laboratory in the yeastKomagataella pastoris, and grape pomace extracts (GRE) were proposed as a convenient C-energy source for the biomass production of the genetically engineered strain. Carbon and nitrogen sources, growth factors, and initial pH conditions were selected b...
Kingston, Diego; Novelli, Guido F; Cerrutti, Patricia; Recupero, Matias N; Blasco, Martin; Galvagno, Miguel A
The use of agroindustrial wastes not only decreases bioprocesses and disposal costs but also contributes to the upgrading of the residues. An active recombinant methanol-inducible bovine chymosin has been expressed in our laboratory in the yeastKomagataella pastoris, and grape pomace extracts (GRE) were proposed as a convenient C-energy source for the biomass production of the genetically engineered strain. Carbon and nitrogen sources, growth factors, and initial pH conditions were selected by classical methodology; thereafter, growth conditions optimization was performed using statistical designed experiments (DoEs). In the presence of (in g·L(-1)) 67.0 monosaccharides (glucose and fructose) from GRE, 5.0 (NH4)2SO4, and 10.0 sugar cane molasses (CMz), a yield of 20.0 g·L(-1) cell dry weight (CDW) was obtained aerobically after 60 h incubation at 28°C and pH 4.0. Applying a fed-batch strategy with methanol:sorbitol as the enzyme inducers, a chymosin production of 8.53 International Milk Clotting Units (IMCU) per mg protein was obtained in the supernatant. The results presented show that through a statistical design, a simple, cheap, and easy to prepare culture medium could be developed using two agroindustrial derivatives (GRE and CMz) to obtain a higher value added product.
Kingston, Diego; Novelli, Guido F; Cerrutti, Patricia; Recupero, Matias N; Blasco, Martin; Galvagno, Miguel A
The use of agroindustrial wastes not only decreases bioprocesses and disposal costs but also contributes to the upgrading of the residues. An active recombinant methanol-inducible bovine chymosin has been expressed in our laboratory in the yeastKomagataella pastoris, and grape pomace extracts (GRE) were proposed as a convenient C-energy source for the biomass production of the genetically engineered strain. Carbon and nitrogen sources, growth factors, and initial pH conditions were selected by classical methodology; thereafter, growth conditions optimization was performed using statistical designed experiments (DoEs). In the presence of (in g·L(-1)) 67.0 monosaccharides (glucose and fructose) from GRE, 5.0 (NH4)2SO4, and 10.0 sugar cane molasses (CMz), a yield of 20.0 g·L(-1) cell dry weight (CDW) was obtained aerobically after 60 h incubation at 28°C and pH 4.0. Applying a fed-batch strategy with methanol:sorbitol as the enzyme inducers, a chymosin production of 8.53 International Milk Clotting Units (IMCU) per mg protein was obtained in the supernatant. The results presented show that through a statistical design, a simple, cheap, and easy to prepare culture medium could be developed using two agroindustrial derivatives (GRE and CMz) to obtain a higher value added product. PMID:25493192
Moynihan, A C; Govindasamy-Lucey, S; Jaeggi, J J; Johnson, M E; Lucey, J A; McSweeney, P L H
The objective of this study was to compare the effect of coagulant (bovine calf chymosin, BCC, or camel chymosin, CC), on the functional and sensory properties and performance shelf-life of low-moisture, part-skim (LMPS) Mozzarella. Both chymosins were used at 2 levels [0.05 and 0.037 international milk clotting units (IMCU)/mL], and clotting temperature was varied to achieve similar gelation times for each treatment (as this also affects cheese properties). Functionality was assessed at various cheese ages using dynamic low-amplitude oscillatory rheology and performance of baked cheese on pizza. Cheese composition was not significantly different between treatments. The level of total calcium or insoluble (INSOL) calcium did not differ significantly among the cheeses initially or during ripening. Proteolysis in cheese made with BCC was higher than in cheeses made with CC. At 84 d of ripening, maximum loss tangent values were not significantly different in the cheeses, suggesting that these cheeses had similar melt characteristics. After 14 d of cheese ripening, the crossover temperature (loss tangent = 1 or melting temperature) was higher when CC was used as coagulant. This was due to lower proteolysis in the CC cheeses compared with those made with BCC because the pH and INSOL calcium levels were similar in all cheeses. Cheeses made with CC maintained higher hardness values over 84 d of ripening compared with BCC and maintained higher sensory firmness values and adhesiveness of mass scores during ripening. When melted on pizzas, cheese made with CC had lower blister quantity and the cheeses were firmer and chewier. Because the 2 types of cheeses had similar moisture contents, pH values, and INSOL Ca levels, differences in proteolysis were responsible for the firmer and chewier texture of CC cheeses. When cheese performance on baked pizza was analyzed, properties such as blister quantity, strand thickness, hardness, and chewiness were maintained for a longer
Ansari, Samiul M; Coletta, Andrea; Skeby, Katrine Kirkeby;
-inhibited conformation in which the side chain of Tyr77 occludes the binding site. On the basis of kinetic, mutagenesis and crystallographic data, it has been widely reported that a HPHPH sequence in the P8-P4 residues of the natural substrate κ-casein acts as the allosteric activator, but the mechanism by which...... to vacate a pocket that may then be occupied by the side chain of Tyr77. The free energy surface for the self-inhibited to open transition is significantly altered by the presence of the HPHPH sequence of κ-casein....... and to compute the free energy surface for the process. The simulations reveal that allosteric activation is initiated by interactions between the HPHPH sequence of κ-casein and a small α-helical region of chymosin (residues 112-116). A small conformational change in the α-helix causes the side chain of Phe114...
张超垒; 王成忠; 张志国; 张玲梅
In terms of natural screening,mutation breeding and the breeding of gene engineering, this paper reviewed the rearch advancement of the strain breeding which can produce chymosin in China.%综述了目前我国在凝乳酶产生菌在自然选育、诱变育种和基因工程育种这3方面的研究进展情况.
Baum, Andreas; Hansen, P. W.; Nørgaard, Lars;
In this study, we introduce enzymatic perturbation combined with Fourier transform infrared (FTIR) spectroscopy as a concept for quantifying casein in subcritical heated skim milk using chemometric multiway analysis. Chymosin is a protease that cleaves specifically caseins. As a result...... of hydrolysis, all casein proteins clot to form a creamy precipitate, and whey proteins remain in the supernatant. We monitored the cheese-clotting reaction in real time using FTIR and analyzed the resulting evolution profiles to establish calibration models using parallel factor analysis and multiway partial...
张健; 张莉; 李玉秋; 杨贞耐
The optimal conditions for producing chymosin in 5L fermenter by recombinant Pichia pastoris Gsl 15/pPICZaA-Prochy were determined in order to offer technical bases for pilot scale production. After induction for 84h, the highest enzyme activity (152SU/ml) was achieved with batch culture, in which, lOg/L sorbitol was added at the beginning of induction phase, methanol concentration was kept at 0.1%, 10g/l peptone was added every 12h, pH value was kept at 3.0 and the concentration of oleic acid was 0.2%. The purification of chymosin was analyzed by sodium dodecyl sul-fete benzene agarose gel electrophoresis (SDS-PAGE). Under the concentration of salt ions at 2mol/L and flow rate 2.5mL/min, the recovery rate of chymosin was 82.33% and the enzyme sample was concentrated by 20 folds.%以重组毕赤酵母GS115/pPICZaA-Prochy为凝乳酶生产菌种,研究其在5L发酵罐水平的最佳产酶条件,为中试生产提供依据.采用批量发酵,甲醇浓度0.1％,诱导阶段每12h添加10g/L蛋白胨,发酵液pH值为3.0,油酸浓度0.2％,诱导初期批量加入10g/L山梨醇共基培养,诱导84h可获得152SU/mL的酶活力.采用十二烷基磺酸钠-苯琼脂糖凝胶电泳分析,在发酵液(去菌体后)样品盐离子浓度2mol/L,流速2.5mL/min,酶回收率达到82％,浓缩倍数20倍.
王艳萍; 张殿伟; 程巧玲; 白小佳
Chymosin is a key enzyme in cheese production which causes milk curd.It plays a very important role in the formation of texture and flavor in cheese.With the development of cheese industry, chymosin made by traditional method is not sufficient for its production, so the researchers are looking for the new sources of chymosin.MJ229 can produce chymosin, that was isolated from glutinous rice wine which was a traditional food in south of China.This paper studied the extraction process of chymosin from the Mucor MJ229 solid state fermentation broth, using single factor analysis of the extraction time, salt concentration, extraction temperature, extraction times which affect extraction of chymosin.The results showed that the optimized extraction conditions were: the temperature is 40℃, NaCl concentration is 0.3 mol/L, extracted 20min for 3 times.%凝乳酶是干酪生产中的关键性酶.它对干酪的质构形成及特有风味的形成有非常重要的作用.随着干酪工业的不断发展,传统来源的凝乳酶已远不能满足生产需要,各国研究者不断地寻找新的来源.霉菌MJ229是从我国传统食品江米酒酒醪中筛选得到的一株凝乳酶高产菌株.本论文对霉菌MJ229固态发酵物中凝乳酶的提取工艺进行了研究,分别研究了提取时间、食盐浓度、提取温度、次数对固态发酵液中凝乳酶提取效果的影响.结果表明,霉菌MJ229固态发酵中凝乳酶在温度40℃条件下,用0.3mol/L的NaCl溶液提取20min,提取3次的效果为最好.
Baum, A; Hansen, P W; Nørgaard, L; Sørensen, John; Mikkelsen, J D
In this study, we introduce enzymatic perturbation combined with Fourier transform infrared (FTIR) spectroscopy as a concept for quantifying casein in subcritical heated skim milk using chemometric multiway analysis. Chymosin is a protease that cleaves specifically caseins. As a result of hydrolysis, all casein proteins clot to form a creamy precipitate, and whey proteins remain in the supernatant. We monitored the cheese-clotting reaction in real time using FTIR and analyzed the resulting evolution profiles to establish calibration models using parallel factor analysis and multiway partial least squares regression. Because we observed casein-specific kinetic changes, the retrieved models were independent of the chemical background matrix and were therefore robust against possible covariance effects. We tested the robustness of the models by spiking the milk solutions with whey, calcium, and cream. This method can be used at different stages in the dairy production chain to ensure the quality of the delivered milk. In particular, the cheese-making industry can benefit from such methods to optimize production control. PMID:27265175
A Study on Relationships between the Activities of Repsin,Chymosin, Lactase and Their mRNA Relative Expressional Volumes and Development of the Stomach and Intestines of 7-35 Day Old Lambs%7~35日龄羔羊胃肠胃蛋白酶、凝乳酶、乳糖酶活性与其 mRNA 相对表达量和胃肠发育的关系研究
牛越峰; 雒秋江; 陈勇; 李凤鸣; 张慧玲
Fifty six head of Small-tail Han lamb with body weight of (3 360±338)g were selected in ex-periment and were divided randomly into 14 groups (n =4,in each group).From 4 day old the lambs were fed with milk powder substitute (MPS)(n =28)or fish meal substitute (FMS)(n =28)respectively,and from 21 day old,8 of lamb fed with MPS or FMS were turned to be fed with starter ration respectively. Each group of lamb at 7,14,21,28 and 35 day old was sacrificed to take the abomasum and duodenum tis-sue,measuring the activities of the pepsin,chymosin and lactase and the corresponding relative expression of mRNA,for studying the development of 3 digestive enzymes of lamb fed with MPS,FMS and starter ra-tion respectively.The result showed that in abomasum or duodenum tissue of newborn lamb the activities of the chymosin and lactase were decreased with the age of lamb by 1.3 U/g (r 2 = 0.92,P 0.05)respectively,but the pepsin activity was weakly related with the age.The relationships be-tween the activities of the pepsin,chymosin and lactase and thier corresponding relative expression of mR-NA was poor,the mRNA expressions of the pepsin and chymosin were fluctuated by high-low-high with the age,however the activity of lactose was not significantly changed with the age of lamb.When FMS was fed,the activities of pepsin,and chymosinwere decreased,the weight of abomasum was (36.0 ±3.0)g at 7 day old of lamb,and was (38.3±5.4)g at 35 day old,suggesting a growth retardation.Turning to starter ration at 21 days of lamb fed priviously with MPS did not significantly affect the activities of the pepsin, chymosin and lactase.But the abomasum and duodenum and daily weight gains of the lambs reduced.Fur-thermore,the activities of pepsin,chymosin and lactase in the milk substitutes,which were fed priviously to the lambs,tended to declining which affected obviously abomasum and duodenum and daily weight gains of the lambs.It is concluded that the pepsin activity is weakly related with the age
The manufacture of (semi-)hard cheese from ultrafiltered milk (UF-cheese) enables the partial incorporation of whey proteins in the cheese, thereby increasing its yield. The transfer of whey proteins in curd from (UF-)milk was studied in relation to the degree of ultrafiltration of the milk and the
朱仁俊; 石振兴; 甘伯中; 胡永金
Roos, de A.
The proteolytic action of proteases present in cheese plays a major role in the ripening of cheese. These proteases originate from the rennet, the starter cultures and from the milk itself. The proteolysis in cheese results in the degradation of the casein proteins into smaller peptides and free ami
Børsting, Mette Winther; Qvist, Karsten Bruun; Rasmussen, Morten;
Despite substantial research, it is still difficult to produce high quality reduced-fat Cheddar cheese. The objective of this study was to investigate how two coagulants, bovine chymosin (BC) and camel chymosin (CC) having different proteolytic activities and two starter cultures, an O-culture (O...
Efeito da hidrólise enzimática sobre propriedades funcionais de caseína bovina coagulada pela ação da quimosina Effect of enzimatic hydrolysis on functional properties of bovine casein coagulated by the action of chymosin
Janesca Alban Roman; Valdemiro Carlos Sgarbieri
Este trabalho teve como objetivo verificar a influência do grau de hidrólise sobre as propriedades funcionais hidrofílicas (capacidade de absorção espontânea de água-CAA, capacidade de retenção de água-CRA e solubilidade da proteína-SP) e de superfície (capacidade de formação e estabilização de espumas e de emulsões) da caseína bovina obtida por coagulação enzimática. Os graus de hidrólise (GH) estudados foram: 5,7; 12,8; 20,1 e 35,8%. Observou-se que quanto maior o GH maior a SP e menor a CR...
Mikkelsen, T.L.; Frokiaer, H.; Topp, C.;
dodecyl sulfate-PAGE, N-terminal sequencing, and mass spectrometry. The hydrophobicity of CMP was studied by means of 1-anilino-naphthalene-8-sulfonic acid binding experiments. Four CMP products prepared by different methods were studied: CMP produced by enzymatic (chymosin or pepsin) hydrolysis of kappa...
ABREU DA SILVA, Alexandra
La chymosine, enzyme utilisée dans l´industrie fromagère pour coaguler le lait, est traditionnellement extraite à partir du quatrième estomac de veaux non sevrés. La préparation ainsi obtenue dénommée présure a été depuis un certain nombre d´années substituée par divers coagulants d´origine microbienne et de plantes car la production de cette enzyme seule n´était plus capable de répondre à la demande mondiale du fait notamment de l´augmentation de la production fromagère. La chymosine est cep...
牛凝乳酶具有高凝乳活性和低非特异蛋白水解活性而广泛用于干酪制备.随着干酪市场的逐年增长,牛凝乳酶出现供不应求,其替代品应运而生,包括其他动物凝乳酶、植物凝乳酶、微生物凝乳酶、重组凝乳酶.在这些替代品中,重组牛凝乳酶显示出与天然牛凝乳酶相似的酶学特性,并且纯度更高,制作的干酪品质更好.目前已经有重组牛凝乳酶上市出售,成功替代了天然牛凝乳酶,但关于提高重组牛凝乳酶表达量与催化活性的研究远未止步.文中主要综述了凝乳酶结构、牛凝乳酶的替代品和重组凝乳酶的研究进展,详细综述了一些提高重组牛凝乳酶的表达方法,为外源蛋白在宿主中的高水平表达提供了有效参考.%Calf rennet is widely used in the process of cheese-making due to its high milk-clotting activity and low nonspecific proteolytic activity.As the cheese market increases in recent years,calf rennet supply falls short of demand and its substitutes are studied extensively,such as animal rennet,plant coagulant,microbial coagulant and recombinant chymosin.Among these substitutes,the recombinant bovine chymosin shows high similar enzymic properties to the natural calf rennet,and possesses high purity,thus the quality of the cheese made with it is better.Now the recombinant bovine chymosins have been listed for sale and the recombinant bovine chymosin have replaced the natural calf rennet successfully.But the studies on the improvement of expression quantity and catalytic activity of recombinant bovine chymosin never stop.In this paper,the research progress on the structure of chymosin,the calf rennet substitutes and the recombinant chymosin,and particularly the methods used to improve the expression quantity of the recombinant bovine chymosin is reviewed.These methods can offer certain reference for expression of foreign proteins at a high level in hosts.
Gama Salgado, Jose Antonio; Kangwa, Martin; Fernandez-Lahore, Marcelo
Background Extracellular aspartic proteinase (MCAP) produced by Mucor circinelloides in solid state fermentations has been shown to possess milk clotting activity and represents a potential replacement for bovine chymosin in cheese manufacturing. Despite its prospects in the dairy industry, the molecular characteristics of this enzyme remain unknown. This work focuses on MCAP cloning and optimization of heterologous expression in Pichia pastoris, and characterization of the enzyme. Results Th...
A bacteriocin-producing strain of Lactobacillus paracasei DPC 4715 was used as an adjunct culture in Cheddar cheese in order to control the growth of “wild” nonstarter lactic acid bacteria. No suppression of growth of the indicator strain was observed in the experimental cheese. The bacteriocin produced by Lactobacillus paracasei DPC 4715 was sensitive to chymosin and cathepsin D and it may have been cleaved by the rennet used for the cheese manufactured or by indigenous milk proteases. A ser...
Full Text Available Abstract Background The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Results Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold in cells grown with casein or casein phosphopeptides (CPPs. CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs, whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase, which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. Conclusions In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.
Yetişmeyen, Atilla; Çimer, Atilla; Özer, Melike; Odabaşı, Sabiha; Deveci, Orgun
In this study, a microbial enzyme and calf rennet were used to manufacture Turkish White Pickled cheese from ultrafiltrated milk. The ultrafiltration of the skim milk was achieved with volume reduction of 65.2% by using Dow Denmark A/S, Lab-Anlage M20 UF(1) equipment. The calf rennet at a strength of 1/15.000 was composed of a mixture of chymosin/pepsin (90:10) while the microbial enzyme at a strength of 1/196.000 was obtained from Mucor miehei. The physical, chemical and organoleptic propert...
Koutina, Glykeria; Knudsen, Jes Christian; Andersen, Ulf;
lactose, to obtain varying levels of micellar calcium and phosphorus but constant value of pH, serum and free calcium, and serum phosphorus. Bovine chymosin was added to the skim milk samples after dialysis and microstructural and rheological properties during gel formation were recorded at 30°C. Samples......Colloidal calcium phosphate is an essential part of casein micelles and being responsible for their stability. Different mineralization of casein micelles was obtained by acidification of skim milk to pH 6.5, 6.0 or 5.5, followed by a dialysis method, using simulated milk ultrafiltrate without...
7; ,; Sharma, Rajan; Rajput, Yudhishthir; Mann, Bimlesh
International audience Glycomacropeptide (GMP) is a C-terminal part (f 106–169) of kappa-casein which is released in whey during cheese making by the action of chymosin. GMP being a biologically active component has gained much attention in the past decade. It also has unique chemical and functional properties. Many of the biological properties have been ascribed to the carbohydrate moieties attached to the peptide. The unique set of amino acids in GMP makes it a sought-after ingredient wi...
Morgan, Alison J; Riley, Lisa G; Sheehy, Paul A; Wynn, Peter C
Colostrum consists of a number of biologically active proteins and peptides that influence physiological function and development of a neonate. The present study investigated the biological activity of peptides released from first day bovine colostrum through in vitro and in vivo enzymatic digestion. This was assessed for proliferative activity using a human intestinal epithelial cell line, T84. Digestion of the protein fraction of bovine colostrum in vitro was conducted with the enzymes pepsin, chymosin and trypsin. Pepsin and chymosin digests yielded protein fractions with proliferative activity similar to that observed with undigested colostrum and the positive control foetal calf serum (FCS). In contrast trypsin digestion significantly (P<0·05) decreased colostral proliferative activity when co-cultured with cells when compared with undigested colostrum. The proliferative activity of undigested colostrum protein and abomasal whey protein digesta significantly increased (P<0·05) epithelial cell proliferation in comparison to a synthetic peptide mix. Bovine colostrum protein digested in vivo was collected from different regions of the gastrointestinal tract (GIT) in newborn calves fed either once (n=3 calves) or three times at 12-h intervals (n=3 calves). Digesta collected from the distal duodenum, jejunum and colon of calves fed once, significantly (P<0·05) stimulated cell proliferation in comparison with comparable samples collected from calves fed multiple times. These peptide enriched fractions are likely to yield candidate peptides with potential application for gastrointestinal repair in mammalian species. PMID:24433585
Petrella, G; Pati, S; Gagliardi, R; Rizzuti, A; Mastrorilli, P; la Gatta, B; Di Luccia, A
The guarantee of the origin and quality of raw material is essential for the protection and valorization of Campana buffalo mozzarella cheese. The risk of utilization of semifinished products and stored milk in substitution for fresh milk is increasing, due to the continuous desire to reduce production costs. A proteomics approach and electrophoresis survey of retail mozzarella cheeses indicated different rates of proteolysis in the production of dairy industries. The use of fresh milk and correct cheesemaking protocol yielded only γ-caseins, which are derived from β-casein by plasmin, and para-κ-casein, which is derived from κ-casein by chymosin. The detection of abnormal hydrolysis resulting in β- and αS1-casein fragments, identified by mass spectrometry, indicates the use of stored milk or stored and pressed curd, or the reuse of unsold mozzarella cheese, to produce mozzarella. The formation of γ-caseins and other fragments during a long storage of raw materials at room or refrigeration temperature was ascribed to plasmin (endogenous milk enzyme), whereas formation of αS1-casein fragments, mainly αS1-I(6P)- and αS1-I(7P)-casein during the storage of curd was ascribed to the action of chymosin (exogenous enzyme) from rennet. Sodium dodecyl sulfate-PAGE and alkaline urea-PAGE permitted us to evaluate the freshness of the raw materials used in the manufacturing of buffalo mozzarella cheese and to reveal possible inappropriate preservation. PMID:26364106
Nawal S. Ahmed
Full Text Available Background. Mozzarella is one of several pasta filata or stretched curd cheeses that originated in Italy. The name pasta filata refers to a unique plasticizing and texturing treatments of the fresh curd in hot water that imparts to the finished cheese its characteristic fibrous structure and melting properties. Mozzarella cheese made from standardized buffalo milk with 3 and 1.5% fat. The effect of coagulant types (calf rennet, chymosin and Mucor miehei rennet on the cheese properties was carried out. Material and methods. Fresh raw buffalo milk and starter cultures of Streptococcus salvarius ssp. thermophilus and Lactobacillus delbrueckii ssp. bulgaricus were used. The coagulants were calf rennet powder (HA-LA, microbial rennet powder (formase ISOTL from Mucor miehei and chymosin derived by fermentation (CDF. Milk, curd, whey,kneading water and cheese were analysed. The slab gel electrophoresis patterns of Mozzarella cheese were also applied. Statistical analyses were also applied on the obtained data. Results. Recovery of DM of both curd and cheese decreased in case of using Mucor miehei rennet while the recovery of TP and fat content in both curd and cheese and their loss to whey and kneading water were nearly the same. Soluble nitrogen and soluble tyrosine and tryptophan contents elevated with increasing the storage period. Increasing rate of the soluble nitrogen in case of using Mucor miehei rennet was higher compared to that made with the other types of coagulant. TVFA content increased with advancing the storage period, also increased with increasing the fat content of the original milk fat used. No effect can be seen due to the coagulant types. The meltability increased with storage period progress. While the effect of the type of coagulant enzyme hade neglect effect on meltability fat leakage and oiling off. Mozzarella cheese made with Mucor miehei rennet obtained the highest firmness compared with those made using calf rennet, or
Full Text Available Rennet composition varies along with several factors such as source (animal species, herbage, microbial and genetic, physical state (liquid, powder and paste, enzymatic composition (chymosin/ pepsin ratio, lipolitic enzymes. Main factors influencing rennet paste are diet (milk feeding, weaning and slaughtering age. Rennet paste composition influences cheese-making and ripening processes in cheese. Little is known about the effects of probiotic addition to milk substitute on the microbial status and enzyme profile of rennet, and on ripening characteristics of cheese. The aim of the study was to evaluate the effects of rearing systems and of lamb slaughtering age on microbial profile and time of coagulation of rennet pastes used for Pecorino Foggiano cheese-making.
C. P. Sánchez Henao
Full Text Available The Mucor miehei zygomycete produces an acid protease (EC:126.96.36.199 resembling calf rennet chymosin characteristics. It has been suggested that low glucose concentration levels could be why enzyme synthesis, co-mes to an end even though enzyme production is still great (Escobar and Barnett, 1993, 1995. To overcome this possible limitation, a two stage research process was designed; the relationship between protease production and sugar consumption was studied initially to determine the periods of time when enzyme production is still high and glucose concentration close to zero. The following stage concentrated on developing a glucose fed-batch process during the afore mentioned time periods to observe any increase or decrease in enzyme production. During the batch studies, it was found that maximum enzyme activity (EA was 165 UC/ml for an average glucose consumption rate of 0.1813 g/1 h. Based on the previous.
Timotijević Gordana S.
Full Text Available Aspartic proteinases from buckwheat seeds are analyzed. Three forms of 47 kDa, 40 kDa and 28 kDa, were purified from mature buckwheat seeds, while two forms of 47 kDa and 28 kDa were detected in developing buckwheat seeds using pepstatin A affinity chromatography. A form of 47 kDa was selectively precipitated from other forms by ammonium sulfate precipitation. This enzyme resembles the chymosin-like pattern of proteolytic activity, as it was shown using BSA and k-casein as substrates, clarifying its ability for milk-clotting. The 47 kDa aspartic proteinase form is localized in the membrane fraction. .
Full Text Available Antimicrobial peptide is commonly present in all species as a component of their innate immune defense against infection. Antimicrobial peptides derived from milk such as isracidin, casocidin, casecidin and other fragments with variety of amino acid sequence are released upon enzymatic hydrolysis from milk protein К-casein, α-casein, β-casein, α-lactalbumin and β- lactoglobulin. These peptides were produced by the activity of digestive or microbial protease such as trypsin, pepsin, chymosin or alcalase. The mode of action of these peptides is by interaction of their positive with negative charge of target cell membrane leading to disruption of membrane associated with physiological event such as cell division or translocation of peptide across the membrane to interact with cytoplasmic target. Modification of charged or nonpolar aliphatic residues within peptides can enhance or reduce the activities of the peptides against a number of microbial strains and it seems to be strain dependent. Several peptides act not only as an antimicrobial but also as an angiotensin-converting enzyme inhibitor, antioxidant, immunomodulator, antiinflamation, food and feed preservative. Although the commercial production of these peptides is still limited due to lack of suitable large-scale technologies, fast development of some methods for peptide production will hopefully increase the possibility for mass production.
Feijoo-Siota, Lucía; Blasco, Lucía; Rodríguez-Rama, José Luis; Barros-Velázquez, Jorge; Miguel, Trinidad de; Sánchez-Pérez, Angeles; Villa, Tomás G
This paper reviews the general characteristics of exo and endopeptidases of microbial origin currently used in the milk industry. It also includes recent patents developed either to potentiate the enzymatic activity or to improve the resulting milk derivatives. The main application of these proteases is in the cheese-making industry. Although this industry preferentially uses animal rennets, and in particular genetically engineered chymosins, it also utilizes milk coagulants of microbial origin. Enzymes derived from Rhizomucor miehei, Rhizomucor pusillus and Cryphonectria parasitica are currently used to replace the conventional milk-clotting enzymes. In addition, the dairy industry uses microbial endo and exoproteases for relatively new applications, such as debittering and flavor generation in cheese, accelerated cheese ripening, manufacture of protein hydrolysates with improved functional properties, and production of enzyme-modified cheeses. Lactic acid bacteria play an essential role in these processes, hence these bacteria and the proteases they produce are currently being investigated by the dairy industry and are the subject of many of their patent applications.
Bruna S. D. R. Aranha
Full Text Available The Mannich reaction is extremely useful to form molecules that contain nitrogen groups. Currently, there is a great interest turned to this class of compounds due to its biological properties. The recent literature reports that the utilization of enzymes can be an adequate methodology on this type of reaction. According to the exposed above and with the intention of synthesizing the -aminoketones, some catalysts like Chymosin and also some microorganisms such as Saccharomyces cerevisiae, (sourdough bread and Lactobacilos (ATCC 90827LA10N19.10-E.U.A were applied. These reactions were carried out by using water as solvent at room temperature excelling for the Green Chemistry principles. Figure 1 shows the reaction scheme involving 1 eq.mol of aniline, 1,3 eq.mol of formaldehyde, 10 ml of water and 0,25g of the catalyst during four days. The reaction furnished satisfactory yields which are in accordance with the literature data.
Anna Carolina da Silva
Full Text Available There are several obstacles to the use of chymosin in cheese production. Consequently, plant proteases have been studied as possible rennet substitutes, but most of these enzymes are unsuitable for the manufacture of cheese. The aim of this study was to evaluate the potential of latex from Sideroxylon obtusifolium as a source of milk-clotting proteases and to partially characterize the enzyme. The enzyme extract showed high protease and coagulant activities, with an optimal pH of 8.0 and temperature of 55 °C. The enzyme was stable in wide ranges of temperature and pH. Its activity was not affected by any metal ions tested; but was inhibited by phenylmethanesulfonyl fluoride and pepstatin. For the coagulant activity, the optimal concentration of CaCl2 was 10 µmol L- 1. Polyacrylamide gel electrophoresis showed four bands, with molecular weights between 17 and 64 kDa. These results indicate that the enzyme can be applied to the cheese industry.
Johnson, M E; Lucey, J A
Over the last 25 yr, cheese production in the United States has more than doubled with most of the increase due to production in the western states. Processing large volumes of milk into cheese has necessitated changes in vat size and design, reliance on computer software, and milk standardization, including use of membrane concentration of milk either at the cheese plant or on the farm. There has been increased interest in specialty cheeses including cheese made from sheep, goat, and organic milks. In addition, membrane processing of whey into various value-added components has become routine. Changes in cheese manufacturing protocols have resulted in a reduction of the manufacturing time and the necessity for consistent and reliable starter activity. Major advances in the genetics of microorganisms have not only resulted in widespread use of fermentation-produced chymosin but also in starter bacteria with improved resistance to bacteriophage infection. Genomics and proteomics have increased the likelihood of the development of nonstarter adjuncts with specific enzymatic activity. Indeed, the use of adjunct microorganisms to produce cheese with a unique flavor profile or to produce cheese with more consistent or better quality flavor has gained almost universal acceptance.
Campos Motta, T M; Hoff, R B; Barreto, F; Andrade, R B S; Lorenzini, D M; Meneghini, L Z; Pizzolato, T M
Caseinomacropeptide (CMP) is a peptide released by chymosin in cheese production, remaining in whey. Thus, CMP can be used as a biomarker to fluid milk adulteration through whey addition. Commonly, CMP is analyzed by reversed phase (RP-HPLC) or size-exclusion chromatography (SEC). However, some psychrotropic microorganisms - specially Pseudomonas fluorescens - when present in storaged milk, can produce, by enzymatic pathway, a CMP-like peptide generally called pseudo-CMP. These two peptides differ from each other only by one amino acid. RP-HPLC and SEC methods are unable to distinguish these two peptides, which demand development of a confirmatory method with high selectivity. Considering the several degrees of glycosilation and phosphorylation sites in CMP, allied with possible genetic variation (CMP A and CMP B), analytical methods able to differentiate these peptides are extremely complex. In the present work, we developed a proteomic-like technique for separation and characterization of these peptides, using liquid chromatography coupled to mass spectrometry with electrospray ionization able to differentiate and subsequently quantify CMP and pseudo-CMP in milk samples in order to identify adulteration or contamination of these products. The method shows satisfactory precision (<11%) with a detection limit of 1.0 µg mL(-1) and quantification limit of 5.0 µg mL(-1). Specificity, matrix effects and applicability to real samples analysis were also performed and discussed.
Dahal, Yuba; Schmit, Jeremy
Salt is one of the major factors that effects protein solubility. Often, at low salt concentration regime, protein solubility increases with the salt concentration(salting in) whereas at high salt concentration regime, solubility decreases with the increase in salt concentration(salting out). There are no quantitative theories to explain salting in and salting out. We have developed a model to describe the salting in and salting out. Our model accounts for the electrostatic Coulomb energy, salt entropy and non-electrostatic interaction between proteins. We analytically solve the linearized Poisson Boltzmann equation modelling the protein charge by a first order multipole expansion. In our model, protein charges are modulated by the anion binding. Consideration of only the zeroth order term in protein charge doesn't help to describe salting in phenomenon because of the repulsive interaction. To capture the salting in behaviour, it requires an attractive electrostatic interaction in low salt regime. Our work shows that at low salt concentration, dipole interaction is the cause for salting in and at high salt concentration a salt-dependent depletion interaction dominates and gives the salting out. Our theoretical result is consistent with the experimental result for Chymosin protein NIH Grant No R01GM107487.
The deep development and utilization of aquatic waste is summarized briefly, these aquatic waste can be used in food natural fungicide, chymosin, thioguanine, chondroitin sulfate, dennatan sulfate, oligomeric peptides, small peptides oral solution, CMC, meaty flavor, fish enzyme, glyeopruteins and taurine, which provided a reference for further development and utilization of fish waste resources.%简单综述了水产品下脚料深度开发利用的研究情况,主要可应用在食品天然防霉剂、凝乳酶、硫鸟嘌呤(6 -TG)、硫酸软骨素、硫酸皮肤素、寡聚肽、小分子肽口服液、高吸收性钙剂(CMC)、热反应型肉香调味基料、鱼酶、糖蛋白、牛磺酸中,为进一步开发利用鱼下脚料资源提供参考.
Full Text Available In this study, Örgü cheese has been produced by using different coagulating enzymes (calf rennet, microbial enzymes, recombinant chymosin. The effects of different coagulating enzymes which are used on the characteristic of mineral material and cheese has been observed during 90 days ripening time. Mineral material contents of Örgü cheese have been determined with ICP-OES (inductively coupled plasma optical emission spectroscopy. Proteolysis levels of cheese have been observed with chemical analysis and help of SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis. The determined difference between analysis results, titratable acidity, total nitrogen, water soluble nitrogen, ripening index, total solid, fat, fat in total solid, salt, salt in total solid, ash, texture, mineral material (Ca, Fe, Cu, Al, Mg, Mn of Örgü cheese’s analysis result haven’t been regarded as significant statistically. Each of enzymes which are used effects similarly on α-casein and β-casein during the ripening time and each of the ratios which are gained have been closely determined.
ZHENG Yu-cai; ZHONG Guang-hui; WANG Yong; PENG Xian-wen; MAO Yong-jiang; ZOU Si-xiang; CHEN Wei-hua; CHEN Jie
Yaks of two lactating types, the half-lactating yak and the total lactating yak, were investigated in their milk compositions, milk coagulation properties, and contents of three kinds of hormones, glucose,and protein in plasma. The half-lactating Maiwa yak and Jiulong yak contained significantly higher contents of protein, fat, and activities of alkaline phosphatase and γ-glutamyl transpeptidase in milk than that of the corresponding total-lactating yak breeds, with reduced milk yield and similar lactose level and relative percentages of main milk protein components. The half-lactatin yak resembled yaks in a late stage of lactation in their biochemical composition of milk; however, significant differences were also observed, which indicated that the former was in a special stage of lactation quite different from dairy cows. Milk of the total-lactating or halflactating yaks could be coagulated normally by adding chymosin, with a similar coagulation time. No significant difference was observed between prolactin and progesterone concentrations in plasma of the half-lactating yaks and total-lactating yaks; however, half-lactating yaks had significantly lower level of oestradiol-17 β in plasma than total-lactating yaks.
Full Text Available The aim of the work was compared of two different methods (the visual method and the nephelo turbidimetry method for determination of rennet coagulation time. It was observed the effect of heat treatment of milk; types of rennet and addition of different amount of CaCl2 into the pasteurized milk. It was used two different chymosin rennet. For the visual method was milk sample (100 mL equilibrated at 35 °C, 1 mL of rennet was added into milk and was measured the time required for the first visible flakes (visual method. For the determination rennet coagulation time by nephelo-turbidimetry was removed part of milk with rennet and placed into nephelo-turbidimetry. Milk had a titratable acidity in the range from 6.5 to 7.0 °SH, average pH of milk was 6.68. Dry matter content was in range from 12.351 to 13.142%. The average content of protein by Kjeldahl was 3.14%, fat by Gerber 4.34%, lactose by polarimetry 4.68% and calcium content 1.1%. The pasteurized milk had the worst rennet coagulation time about 32 s compared to the raw milk. The difference coagulation time between milk with addition of 20 µL CaCl2 and 40 µL CaCl2 was in range 21 s to 26 s by visual method. The difference coagulation time between milk with addition of 20 µL CaCl2 and 40 µL CaCl2 was 15 s by nephelo-turbidimetry method. There occurred statistically non-significant differences in most of the measurements, comparing the visual and the nephelo-turbidimetric method. The heat treatment, addition of CaCl2 and using of different rennet had an influence on the curd category. It was obtained, that using nephelo-turbidimetry shown objective results for measuring the rennet coagulation time contrary the subjective visual method. Further, the results obtained by nephelo-turbidimetry are accurate and determined with the lower variation.
Ana Claudia S. Lima
: lipase, Lipozyme® and chymosin. The reactions were kept under stirring at room temperature from 24 to 48 hours in those which the enzymes were usedlike catalysts. All the reactions were monitored by thin layer chromatography. The crude of the reaction was purified on a silica gel column (70-230 mesh and the obtained compounds were characterized by infrared spectroscopy, gas chromatography and nuclear magnetic resonance. All the reactions biocatalyzed by enzymes showed biocatalytic potential, but their concentration, the basicity of the amine and the reaction medium interfered in the reaction yield. The both three enzymes presented a higher affinity as for the less polar solvents (THF as for the higher polar solvents(DMSO. However it was proposed the introduction of new low cost biocatalysts and environmentally safe on Mannich reactions.
王景会; 李玉秋; 郑丽; 李倬琳; 高岩; 杨贞耐
The rennet from Mucor pusillus is one of the major sources of tnicrobial rennet, but it has some defects compared with the bovine chymosin. Aspartic proteinases from fungi are usually more heat resistant . When these enzymes are used for cheese making, the residue of enzymatic activity after heat treatment (55 ℃) following milk coagulation may still exist and cause proteolysis during cheese storage, bitter taste, softness and lower yield of the cheese products. In order to develop a rennet product with suitable milk clotting properties, the gene of rennet was cloned from Mucor pusillus, and the expression vector pPIC9K/M was constructed. The plasmids of pPIC9K/M was linearized with Sac I , and transformed into Pichia pastoris GS115 competent cells. The rennet was secretory expressed in Pichia pastoris successfully , and a strong band at about 46 kD was shown by SDS - PAGE. Activity tests showed that the rennet activity in the culture supernatant was 311.8 U/mL. The purity of recombinant rennet reached 92% with a 51.89% activity recovery.%采用基因工程的方法对微小毛霉(Mucor pusillus)凝乳酶进行分子改造，获得适于乳品工业生产用的凝乳酶，克隆到了微小毛霉凝乳酶基因，构建了酵母表达质粒pPIC9K/M。线性化后电击转入毕赤酵母GS115，在甲醇诱导下进行凝乳酶的初步发酵试验，通过pH反应及酶抑制反应试验证明，重组凝乳酶获得了分泌表达。SDS-PAGE分析表明重组凝乳酶的分子量约为46kD，与理论值(45.3 kD)基本相符，培养基上清液中凝乳酶的活性为311.8 U/mL，纯化的总回收率为51.89％，纯度达到92％。