Comparison of the principal proteins in bovine, caprine, buffalo, equine and camel milk

Proteomic analysis of bovine, caprine, buffalo, equine and camel milk highlighted significant interspecies differences. Camel milk was found to be devoid of β-lactoglobulin, whereas β-lactoglobulin was the major whey protein in bovine, buffalo, caprine, and equine milk. Five different isoforms of κ-casein were found in camel milk, analogous to the micro-heterogeneity observed for bovine κ-casein. Several spots observed in 2D-electrophoretograms of milk of all species could tentatively be identified as polypeptides arising from the enzymatic hydrolysis of caseins. The understanding gained from the proteomic comparison of these milks may be of relevance both in terms of identifying sources of hypoallergenic alternatives to bovine milk and detection of adulteration of milk samples and products.     

The effect of concentration of chymosin on the yield and sensory properties of camel cheese and on its microbiological quality

The transformation of camel milk into soft cheese by using chymosin and yoghurt starter culture (Streptococcus thermophilus and Lactobacillus bulgaricus) was investigated. The cheese yield and sensory properties were related to the concentration of chymosin. A yield of 16.74 g/100 mL of milk was obtained with a chymosin concentration of 1.7 mL/L of milk. The cheeses obtained with concentrations ranging between 1.0 mL and 2.9 mL of chymosin/L of milk scored highly regarding their sensory properties and had an acceptable microbiological quality. This study demonstrated that cheesemaking from camel milk can be made successfully providing that the appropriate chymosin concentration is used; and that 1.7 mL of chymosin/L of milk was optimal.     

Influence of pH on retention of camel chymosin in curd

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 coagulant activity of each chymosin and centrifuged. Chymosin activity in whey was determined using the synthetic peptide Pro-Thr-Glu-Phe-(NO₂-Phe)-Arg-Leu as substrate and HPLC analysis of the resulting product. Camel chymosin had 2.7 times lower activity in milk than bovine chymosin at the 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 of the camel chymosin molecule.     

Factors influencing the gelation and rennetability of camel milk using camel chymosin

The effects of temperature, pH, concentration of camel chymosin and addition of CaCl₂ on the hydrolysis of κ-casein (κ−CN) and the coagulation kinetics of camel milk were investigated. The rate of κ−CN hydrolysis was higher at 40 °C than at 30 °C and with increasing addition of chymosin and decreasing pH. For all samples gelation was initiated at levels of camel milk κ−CN hydrolysis >95%. The gelation time (T_g) of camel milk was significantly reduced (from 717 to 526 s) at 30 °C when the concentration of chymosin was increased, but was independent of chymosin concentration at 40 °C. Reducing pH also reduced T_g. The gel firmness increased at 40 °C (58 Pa) compared with 30 °C (44 Pa) and effect of CaCl₂ addition on the gelation properties of camel milk was found to be dependent on pH; a significant improvement was only found at pH 6.3.     

Characterization of the non-coagulating enzyme fraction of different milk-clotting preparations

Proteolytic milk-clotting enzymes are extracted from various sources (animals, plants, fungi) and processed according to various methods that are specific to each manufacturer or cheese-maker. Chemical composition and polypeptide patterns of 24 milk-clotting preparations from animal and fungal sources: 10 commercial rennets, 9 artisanal calf rennets, 2 recombinant chymosin preparations and 3 microbial preparations, were compared in order to identify differences according to both their manufacturing process and their source. The preparation from Cryphonectria parasitica had the highest ammonia and small peptide content. Commercial rennets and preparations from Rhizomucor miehei had the highest NaCl and pH values while artisanal rennets had the lowest and recombinant chymosins were intermediate. In comparison with the other commercial preparations, commercial rennets had the highest variability in chemical composition and their polypeptide profiles showed numerous protein bands ranging from 15 kDa to 197 kDa, like artisanal rennets. Protein composition of commercial rennets revealed the presence of bovine serum albumin, either native or degraded, and degraded chymosin. The results indicated that the source of coagulating enzymes and the conditions applied for enzyme extraction led to specific chemical compositions, polypeptide patterns and protein composition which are described in this article.     

Influence of milk-clotting enzymes on acidification rate of natural whey starter culture

The aim of this work was to study the influence of milk-clotting enzymes on whey starter culture and hard cheesemaking. Four cheeses were prepared simultaneously per cheesemaking day, and the experiment repeated on eight consecutive days. Adult bovine rennet was used in the control cheeses, and three formulations of fermentation-produced chymosin (FPC) in the experimental cheeses. Whey cultures were obtained from the whey of the preceding cheeses, incubated individually for 24 h at 42°C. pH values of the control and experimental whey did not differ significantly before incubation, but after 24 h incubation the pH of the FPC whey was significantly higher than that of the control. Soluble nitrogen content in trichloroacetic acid 2% and 12% and in phosphotungstic acid 2.5% was significantly lower for all experimental whey samples than for control whey. This probably explains the decrease in the acidification rate of the whey cultures when bovine coagulant is replaced by FPC in hard cheesemaking.     

Natural heterogeneity of chymosin and pepsin in extracts of bovine stomachs

The two main components, chymosin and pepsin, in calf and adult bovine rennet show heterogeneity with respect to their charge and activity. A large number of single stomachs have been extracted and their isoenzymes have been analysed by HPLC. The results showed three genetic forms of chymosin A, B and C and these are present in individual stomachs in the following combinations: AA, BB, CC, AB, AC and BC. The C variant, which can be distinguished from the degradation product of chymosin A, has never been characterised before. All three chymosins have been purified and partially characterised. Also bovine pepsins show a natural variability: the ratio between the main pepsin fractions varies according to the age of the animal and it therefore provides a criterion to distinguish between pepsin from calf and adult bovine animals.     

牛凝乳酶原基因在大肠杆菌中的高效表达及活性检测

以实验室保存的携带凝乳酶原前体基因的重组载体pMD 19-T/bPPC为模板克隆凝乳酶原基因,经双酶切后与载体pET-30a连接得到重组载体pET-30a/bPC,转化大肠杆菌BL21(DE3),经IPTG诱导后,采用SDS-PAGE检测目的蛋白表达情况。重组蛋白经变性/复性、DEAE-Sepharose Fast Flow纯化和自催化后检测凝乳活性。结果表明,重组凝乳酶原基因在大肠杆菌中高效表达,表达量占菌体总蛋白的68%,采用Arima K方法检测,其凝乳活力达到80 SU/mL。因此,通过大肠杆菌表达系统大量制备具有生物活性的重组牛凝乳酶原的策略是可行的,研究结果为弥补国内天然牛凝乳酶的短缺提供一种途径。     

Interaction between sodium chloride and texture in semi-hard Danish cheese as affected by brining time,dl-starter culture,chymosin type and cheese ripening

Reduced NaCl in semi-hard cheeses greatly affects textural and sensory properties. The interaction between cheese NaCl concentration and texture was affected by brining time (0–28 h), dl-starter cultures (C1, C2, and C3), chymosin type (bovine or camel), and ripening time (1–12 weeks). Cheese NaCl levels ranged from <0.15 to 1.90% (w/w). NaCl distribution changed during ripening; migration from cheese edge to core led to a more homogeneous NaCl distribution after 12 weeks. As ripening time increased, cheese firmness decreased. Cheeses with reduced NaCl were less firm and more compressible. Cheeses produced with C2 were significantly firmer than those produced with C1; cheeses produced with C3 had higher firmness and compressibility. In NaCl reduced cheese, use of camel chymosin as coagulant resulted in significantly higher firmness than that given using bovine chymosin. Overall, cheese NaCl content is reducible without significant textural impact using well-defined starter cultures and camel chymosin.     

Isolation,purification and characterization of chymosin from riverine buffalo (Bubalos bubalis)

Chymosin, an aspartyl proteinase, is used for curdling of milk and manufacture of cheese. We report the purification and the physicochemical properties of chymosin isolated from the abomasal tissue of buffalo calves. The enzyme preparation extracted from buffalo abomasal tissues could be purified 29-fold using anion exchange and gel filtration chromatography. The molecular weight of the purified enzyme was 35.6 kDa on SDS-PAGE. Partial N-terminal amino acid sequence of the first eight amino acid sequences of buffalo chymosin was identical to the first eight amino acid sequences of cattle chymosin. Buffalo chymosin exhibited a skewed bell-shaped stability profile as a function of temperature with maximum activity near 55 degrees C. Milk clotting activity decreased gradually as pH increased. The enzyme became completely inactive, however, above pH 7.0. The ratio of milk clotting to proteolytic activity was 3.03. When compared with cattle chymosin, there were subtle differences in the stability and relative proteolytic activity of buffalo chymosin.     

辣木凝乳酶水解酪蛋白位点及其酪蛋白磷酸肽、酪蛋白糖巨肽

基于十二烷基硫酸钠-聚丙烯酰胺凝胶电泳,液相色谱-串联质谱法研究辣木凝乳酶对酪蛋白的酶切位点和水解特性,并分析该凝乳酶水解产生的酪蛋白糖巨肽和酪蛋白磷酸肽。结果表明,辣木凝乳酶酶切κ-酪蛋白的Arg 93-His 94位点导致凝乳,区别于其他已报道的凝乳酶,具有明显特异性。酶作用下κ-酪蛋白的米氏常数Km=0.49 mg/mL,最大反应速率Vm=45.2 U/min,有效促进酪蛋白胶束的聚集和凝乳。该酶对α-、β-、κ-酪蛋白产生不同程度的水解,对α-酪蛋白的水解速度最快,说明其适用于软质奶酪的加工。以酪蛋白为底物,通过钡-乙醇沉淀法得到酪蛋白磷酸肽（casein phosphopeptides,CPPs）,产率为15.87%,得到的CPPs可以使钙沉淀推迟23 min,有利于钙离子被肠道有效吸收。以水牛乳为底物时,乳凝固后析出的乳清中含酪蛋白糖巨肽6.61 mg/mL,糖基化程度477.527 μg/mg。研究为新型植物凝乳酶的研发,及其在奶酪、酪蛋白活性肽等产品的应用提供科学依据。     

Effects of blends of camel and calf chymosin on proteolysis,residual coagulant activity,microstructure, and sensory characteristics of Beyaz peynir

Beyaz peynir, a white brined cheese, was manufactured using different blends of camel chymosin (100, 75, 50, 25, and 0%) with calf chymosin and ripened for 90 d. The purpose of this study was to determine the best mixture of coagulant for Beyaz peynir, in terms of proteolysis, texture, and melting characteristics. The cheeses were evaluated in terms of chemical composition, levels of proteolysis, total free amino acids, texture, meltability, residual coagulant activity, microstructure, and sensory properties during 90 d of ripening. Differences in the gross chemical composition were statistically significant for all types of cheeses. Levels of proteolysis were highly dependent on the blends of the coagulants. Higher proteolysis was observed in cheeses that used a higher ratio of calf chymosin. Differences in urea-PAGE and peptide profiles of each cheese were observed as well. Meltability values proportionally increased with the higher increasing levels of calf chymosin in the blend formula. These coagulants had a slight effect on the microstructure of cheeses. The cheese made with camel chymosin had a harder texture than calf chymosin cheese, and hardness values of all cheese samples decreased during ripening. The cheeses with a high ratio of calf chymosin had higher residual enzyme activity than those made with camel chymosin. No significant difference in sensory properties was observed among the cheeses. In conclusion, cheeses made with a high level of calf chymosin had a higher level of proteolysis, residual coagulant activity, and meltability. The cheeses also had a softer texture than cheeses made with a high content of camel chymosin. Camel chymosin may be used as a coagulant alone if low or limited levels of proteolysis are desired in cheese.     

Proteolysis and antioxidant activity of peptic,tryptic and chymotryptic hydrolysates of cow,buffalo, goat and camel caseins

Caseins of cow, buffalo, goat and camel milks were hydrolysed using pepsin, trypsin and chymotrypsin. The rate and degree of casein hydrolysis and the antioxidant activity (AA) of the casein hydrolysates (CH) were followed. Camel casein showed the highest rate and degree of hydrolysis with pepsin and trypsin, while cow casein was more rapidly hydrolysed with chymotrypsin than other caseins studied. The AA of all CH increased to a maximum after 24 h of hydrolysis. The AA of tryptic hydrolysates was higher (P < 0.002) than that of peptic hydrolysates. Camel CH exhibited higher AA than hydrolysates of other caseins.     

牛凝乳酶原基因在食品级乳酸乳球菌中的重组表达

将牛凝乳酶原基因连接pNZ8149载体,并转化乳酸乳球菌NZ3900,经乳酸链球菌素Nisin诱导,测得重组菌株胞内凝乳酶活力达到0.7 SU/mL,培养基中检测不到凝乳酶活力,实现了牛凝乳酶原基因在乳酸链球菌Nisin诱导基因表达系统（nisin controlled gene expression system,NICE）中活性表达。在此基础上,将分泌信号肽SPusp45连接于pNZ8149,构建了分泌型表达载体pNZ8149s,并实现牛凝乳酶原基因在NICE系统中分泌表达。当使用1 ng/mLNisin诱导5 h后,重组菌株胞内检测不到凝乳酶活力,培养基中凝乳酶活力为1.2 SU/mL,说明pNZ8149s能够促使凝乳酶原从乳酸乳球菌中分泌。该方法为重组牛凝乳酶在食品级菌株中重组表达提供了一种可行的方案。     

Effect of pepsin-treated bovine and goat caseinomacropeptide on Escherichia coli and Lactobacillus rhamnosus in acidic conditions

Caseinomacropeptide (CMP) is a 7-kDa phosphoglycopolypeptide released from κ-casein during milk digestion and in the cheesemaking process. The objective of the study was to analyze the effect of pepsin-treated CMP from cow and goat milk on the resistance of Escherichia coli and Lactobacillus rhamnosus during acid stress. Bacterial cells in the exponential growth phase were suspended in acidified phosphate buffered saline with or without pepsin-treated CMP. Viability was determined during a 90-min incubation period. Pepsin-treated CMP exhibited bactericidal activity at pH 3.5 when added in a dose-dependent manner to E. coli, decreasing survival by more than 90% within 15 min at 0.25 mg/mL. At pH >4.5, the bactericidal activity disappeared, indicating that pepsin-treated CMP was efficient at low pH only. The effectiveness of pepsin-treated CMP at pH 3.5 was not affected by the presence of glycoconjugates linked to CMP or by the bovine or caprine origin of milk. In contrast, L. rhamnosus, a probiotic, was more resistant to acid stress when pepsin-treated bovine or caprine CMP was added to the media. Viability reached 50% after 60 min of incubation at pH 3 compared with 5% survival in the media without added pepsin-treated CMP. Neither glycosylation extent nor sequence variations between CMP from bovine milk and caprine milk affected the protective activity of hydrolyzed CMP toward L. rhamnosus. This suggests that encrypted bioactive peptides released by the pepsin treatment of CMP had an antibacterial effect on E. coli in acidic media, but improved the resistance of L. rhamnosus to acid stress. The peptide fragment accountable for bactericidal activity is the N-terminal region κ-casein f(106-124).     

Effects of Ultrasound Treatment on the Properties of Chymosin

When chymosin was extracted by ultrasound curd tension and syneresis were significantly lower for Berridge substrate coagulated by the ultrasound-treated chymosin than by the control. Experimental chymosin had a shorter induction period and was more heat-sensitive than the control. Activation energy of chymosin obtained by ultrasound treatment was significantly lower than that of the control. Ultrasound treatment did not significantly change the chromatographic patterns of chymosin. Only two distinct enzymatically active proteins were observed with DEAE-cellulose chromatography. Electrophoretic properties were similar for chymosins obtained by ultrasound and control methods.     

凝乳酶蛋白质工程的研究

由于微生物凝乳酶过多的非专一性水解和热稳定性高导致干酪产量降低和成熟中出现苦味。应用重组技术和蛋白质工程技术，使酶的一级结构发生可选择的和系统的变化，得到的蛋白质便具有所期望的功能特性。应用蛋白质工程对酶改性涉及到改变亚基结构和更换残基，改变酶的动力学参数和热稳定性，改变底物特异性以及最适pH等。     

Suitability of recombinant camel (Camelus dromedarius) chymosin as a coagulant for Cheddar cheese

Cheddar-type cheeses were manufactured using fermentation-produced camel or calf chymosin. There were no significant differences in the composition and pH between the cheeses made with either coagulant. The extent of primary proteolysis was significantly lower in cheeses made with camel chymosin than in cheeses made with calf chymosin. There were large quantitative differences between the peptide profiles of cheeses; however, the levels of amino acids were similar except for isoleucine, histidine and lysine. The cheeses made with camel chymosin were characterized by lower intensities of sulphur and brothy flavours and showed less bitter taste; however, the cheeses made with calf chymosin had greater breakdown of texture, higher smoothness and mouthcoating and were more cohesive and adhesive. The results of this study suggest that camel chymosin appears to be suitable for making Cheddar cheese with lower levels of proteolysis but with good flavour.     

Relative Proteolytic Action of Milk-Clotting Enzyme Preparations on Bovine α-, β- and β-casein

Concentrations of seven milk-clotting enzyme preparations were standardized to equal clot times. Portions of bovine α_s-, β- and κ-casein were treated with enzymes. Proteolytic activity of the coagulants on each casein fraction was determined using the TNBS (2,4,6-trinitrobenzene-sulfonic acid) procedure. Recombinant chymosin showed the lowest degree of proteolysis on α_s- and β-caseins. Excessive proteolysis of calf rennet appeared to be due to the pepsin fraction. M. miehei and M. pusillus var Lindt proteases showed similar degradation of caseins, but M. pusillus var Lindt was more proteolytic when β-casein was the substrate. C. parasitica protease showed the highest degree of proteolysis on α_s- and β-caseins but was the least proteolytic on κ-casein.     

Comparative milk fatty acid analysis of different dairy species

This study compared the fatty acid (FA) profile of milk from different dairy mammals: buffalo, camel, cow, goat and yak. Data from milk samples and reports in the literature were processed using principal component analysis. The results showed that camel milk contained the lowest levels of C4:0–C12:0 and highest levels of unsaturated fatty acids. Goat milk had the highest level of C8:0–C14:0 FA. Characteristic differences were observed for yak and buffalo milk, which differed from cow milk by their higher levels of saturated fatty acids and lower levels of polyunsaturated fatty acids. It is therefore suggested that each animal species’ milk has its own specific milk FA profile.