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.     

Effects of Enzyme Choice and Fractionation of Commercial Enzyme Preparations on Protein Recovery in Curd

Five different commercial milk clotting preparations (bovine rennet, calf rennet, calf rennet-porcine pepsin mixture, Mucor miebei protease, and modified Mucor miehei protease) were adjusted to equivalent milk clotting activities and then used to clot milk. Percentages of protein in the resulting wheys were compared. Calf rennet, bovine rennet, or modified Mucor miehei protease caused less loss of protein to the whey than Mucor miehei protease or calf rennet-porcine pepsin mixture. The five enzyme preparations were then fractionated by gel filtration. Fractions with milk clotting activity were pooled. Original enzyme preparations and the pooled fractions made from them were standardized to the same clotting activity, then used to coagulated milk to compare their effect on protein loss to the whey. Fractionation significantly improved protein recovery when bovine rennet and calf rennet-porcine pepsin mixture were used as coagulants but not when calf rennet, Mucor miehei protease, or modified Mucor miehei protease were used.     

Residual Milk Coagulants in Whey — Their Inactivation and Effects on Ultrafiltration Flux and Nitrogen Yields

Ten commercial milk coagulants were used to produce Cheddar cheese wheys. Wheys were then concentrated fourfold by ultrafiltration following pasteurization, separation, and prewarming. Fluxes were monitored. Wheys, final concentrates, and pooled permeates were tested for total nitrogen, nonprotein nitrogen, and fat. Wheys and concentrates were also tested for residual milk clotting activity. Residual activity was observed in the wheys and concentrates originating from the unmodified proteases of Mucor miehei and Mucor pusillus but not from the two modified proteases of Mucor miehei, an Endothia parasitica protease, two bovine pepsins, a bovine pepsin-swine pepsin blend, a Mucor pusillus-swine pepsin blend, or calf rennet, which served as the control. No significant differences in flux or nitrogen yields were observed.     

Inhibition of rennets by blood serum

Incubation of calf chymosin with the serum from blood of various mammalian species before addition to milk reduced its milk‐clotting activity at 30°C; the inhibitory potency of the sera was in the order horse > goat > cattle > donkey = human. For all species, inhibition of calf chymosin increased on increasing both the incubation time and the level of blood serum. Horse blood serum inhibited other milk coagulants in the order porcine pepsin > Cryphonectria parasitica proteinase = calf chymosin > Rhizomucor miehei proteinase = bovine pepsin. Methylamine‐treated blood serum did not inhibit calf chymosin, suggesting that α₂‐macroglobulin may be the principal inhibitor of rennet in blood serum.     

MILK CLOTTING AND CHEESE MAKING PROPERTIES OF A CHYMOSIN-LIKE ENZYME FROM HARP SEAL MUCOSA

Pepsin A was isolated from the gastric mucosa of two year old harp seal (Pagophilus groenlandicus). Seal pepsin A has a relatively high CU/PU ratio of 0.074, although it is lower than that of calf chymosin (0.170). Equivalent milk clotting units of chymosin and seal pepsin A catalyzed formation of the same amount of nonprotein nitrogen (NPN) when incubated with 2% casein and for both there was no appreciable increase in NPN formation in the second hour of incubation. Seal pepsin A was similar to chymosin in clotting milk substrate with respect to the influence of pH, dilution, calcium chloride and temperature. Cheddar cheeses prepared with either calf rennet or pepsin A as coagulating agent were similar in yield, chemical composition and taste as judged by sensory preference tests. Estimation of protein degradation in the aged cheeses by ultraviolet absorption of citrate-HC1 extracts, gel filtration chromatography of protein soluble in 6 N urea, and determination of amino acids indicated there was slightly more protein degradation in cheese prepared with calf rennet after 30 weeks aging. Sal pepsin A appears to make a major contribution to the excellent cheese-making characteristic of crude seal pepsin(s). However, other components of the seal mucosa extract are probably responsible for the accelerated aging of Cheddar cheese noted in previous reports.     

不同凝乳酶生产干酪成熟期间氨基酸的变化

以鲜奶为原料,以进口的EZAL、MAOLL作发酵剂(由乳油连球菌和乳酸链球菌组成),以微生物酶、羔羊皱胃酶、小牛皱胃酶和猪胃蛋白酶、木瓜蛋白酶、无花果蛋白酶作为凝乳酶生产硬质干酪,研究不同凝乳酶对干酪成熟期间氨基酸变化的影响。结果表明:不同凝乳酶生产干酪成熟期间氨基酸(FAA)含量的变化趋势:羔羊皱胃酶>小牛皱胃酶>微生物凝乳酶>无花果蛋白酶>猪胃蛋白酶>木瓜蛋白酶。     

Evaluation of Harp Seal Gastric Protease as a Rennet Substitute for Cheddar Cheese

A crude preparation of gastric proteases from Harp Seal (Pagophilus groenlandicus) was found to coagulate milk over a wider pH range than porcine pepsin and had a higher ratio of milk clotting to proteolytic activity with hemoglobin at pH 1.8. Cheddar cheese prepared with seal gastric protease (SGP) gave significantly higher sensory scores than cheese made with calf rennet. Chemical analysis of the cheeses revealed a lower concentration of citrate-HCl soluble nitrogen and less free and peptide-bound amino acids in SGP cheese than in the cheeses made with calf rennet and Mucor miehei protease.     

Heat-Stability of Milk-Clotting Enzymes in Conditions Encountered in Swiss Cheese Making

Chymosin is inactivated in gelled milk at pH 6.50 at 53°C according to a biphasic kinetic. Both phases appear to follow first-order kinetics. The D-value for the first phase is 30 min. In the same medium, the Mucor miehei and Mucor pusillus proteases are much more stable (D_53°C > 100 min) while bovine pepsin, a heat-labile Mucor miehei protease and the Endothia parasitica protease are rapidly inactivated (D_53°C < 10 min). pH appears to be the most important parameter for heat stability. Protein and calcium concentrations affect the resistance to heat treatment. A residual activity of chymosin in Swiss-type curd will be very weak at the most unless the curd is cooked at pH < 6.50.     

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.     

Use of Recombinant Chymosin in the Manufacture of Cheddar and Colby Cheese

Recombinant chymosin purified from E. coli K12 (Genencor Inc.) was examined for its suitability to manufacture Cheddar cheese. Commercial calf rennet or recombinant chymosin with and without Bacillus subtilis cellular homogenate was used to determine if enzyme sources affected Cheddar cheese quality or yield. Cheese was evaluated at 3 and 6 mo of age. Purified enzyme was mixed with cellular extracts from B. subtilis at approximately one and four times the protein concentration in the initial homogenates. Cellular materials did not affect clot times, cheese quality, or inhibit cheese acid development. No differences were noted between the yields of Colby cheese manufactured with calf rennet and recombinant chymosin. Initial Cheddar cheese manufactured with recombinant chymosin were excessively bitter, typical of cheese retaining excessive amounts of proteases. Dilution of purified prochymosin preparations increased total enzymic activity 2.7 times. Evidently, the purified recombinant preparations contained some oligomers that became active after being incorporated into the cheese mass, causing bitter flavors to develop during aging. Diluted enzyme preparations were used successfully in the manufacture of Cheddar cheese.     

混料设计优化牦牛“曲拉”凝乳酶干酪素的工艺及产品的性质分析

为提高牦牛产业的附加值,以牦牛乳提取酥油后进行凝固沉淀,再经自然发酵、风干而成的蛋白质含量丰富的产品“曲拉”为原料,采用胃蛋白酶、木瓜凝乳酶和酵母凝乳酶复配成混合凝乳酶,通过单因素实验和混料设计对凝乳干酪素的制备工艺进行研究,并对干酪素的理化性质、红外光谱特性、热力学性质进行分析。结果表明,混合酶质量分数1%(其中胃蛋白酶∶木瓜凝乳酶∶酵母凝乳酶的质量比为0.60∶0.18∶0.22),在pH 6.3、45℃、添加质量分数CaCl 21%条件下,凝乳30 min,出品率为80.35%。混合酶法制备干酪素的理化性质、红外光谱特性和热力学性质与小牛皱胃酶干酪素差异不显著,而且符合GB31638—2016要求。     

A novel electrochemical assay for chymosin determination using a label-free peptide as a substrate

Chymosin is a predominant enzyme in rennet and is used in cheese production because of its excellent milk-clotting activity. Herein, we proposed a facile and label-free electrochemical method for determining chymosin activity based on a peptide-based enzyme substrate. The synthesized substrate peptide for chymosin was assembled onto the surface of the Au-deposited grassy carbon electrode. The current was proportional to chymosin activity, and thus chymosin activity could be determined. The detection ranges of chymosin activity were 2.5 to 25 U mL^?1. The detection limit of chymosin activity was 0.8 U mL^?1. The sensing platform was used to quantify chymosin activity in commercial rennet with high selectivity, excellent stability, and satisfactory reproducibility. We developed a facile, fast, and effective electrochemical assay for detecting chymosin activity, which has potential applications in cheesemaking.     

Proteolytic and Milk Clotting Fractions in Milk Clotting Preparations

Five different milk clotting preparations were fractionated on Sephadex G-100 and then tested for milk clotting activity and for proteolysis of denatured hemoglobin. Two preparations were also tested for proteolytic activity on casein. Proteolytic activities on hemoglobin were correlated with clotting ability of bovine rennet, calf rennet, and rennin-pepsin mixture at pH 1.6 and with Mucor miehei protease at pH 5.2. Modified Mucor miehei protease activities on hemoglobin correlated equally well at pH 1.6 and 5.2. Gel filtration through Sephadex G-100 and elimination of nonclotting fractions reduced the proteolytic activities on hemoglobin at pH 5.2 of calf rennet, bovine rennet, Mucor miehei protease, modified Mucor miehei protease, and rennin-pepsin mixture by 68.6, 88.5, 3.7, 53.7, and 91.2%, respectively. At pH 1.6, proteolysis was reduced by 54.2, 41.2, 51.8, 59.5, and 60.8%. Proteolytic activities of bovine rennet and renninpepsin mixture on casein were reduced by 59.8 and 72%, respectively.     

Production and characterization of a milk-clotting protease in the crude enzymatic extract from the newly isolated Thermomucor indicae-seudaticae N31: (Milk-clotting protease from the newly isolated Thermomucor indicae-seudaticae N31)

Microbial rennet-like milk-clotting enzymes are aspartic proteinases that catalyze milk coagulation, substituting calf rennet. Crude enzymatic extract produced by the thermophilic fungus, Thermomucor indicae-seudaticae N31, on solid state fermentation (SSF) using wheat bran, exhibited high milk-clotting activity and low proteolytic activity after 24 h of fermentation. Highest milk-clotting activity (MCA) was at pH 5.7, at 70 °C and in 0.04 M CaCl₂; it was stable in the pH range 3.5–4.5 for 24 h and up to 45 °C for 1 h. MCA was highly inhibited by pepstatin A. Hydrolytic activity profile of the crude enzymatic extract on whole bovine casein, analyzed by gel electrophoresis (Urea–PAGE) and RP-HPLC revealed low proteolytic action towards casein fractions and a peptide profile similar to the one obtained with commercial Rhizomucor miehei protease (Hannilase).     

Purification and characterization of chymosin and pepsin from kid

The objective of this work was to study the characteristics of the gastric aspartic proteinases chymosin and pepsin which are constituents of the kid rennet. The two enzymes were extracted from abomasal tissue of one kid from a local indigenous breed, separated from each other by DEAE-cellulose chromatography and then were purified by gel filtration and anion-exchange chromatography. The molecular weights of the purified kid chymosin and pepsin as determined by gel filtration were 36 kDa and 40 kDa respectively. The isoelectric point of kid chymosin was as multiple forms of 3-6 zones at pH 4.6-5.1, while that of kid pepsin was at pH < or =3.0. Kid pepsin contained 0.37 molecules phosphorous per molecule and was totally inhibited by 5 muM pepstatin A, being more sensitive than kid chymosin. Both enzymes were almost equally as proteolytic as calf chymosin on total casein at pH 5.6. Kid pepsin activity was more pH and temperature dependent than kid chymosin activity. In comparison with the calf chymosin temperature sensitivity, the order of increased sensitivity was: calf chymosin 相似文献   

江米酒凝乳酶的纯化及凝乳机制初探

江米酒凝乳酶是扣碗酪生产中起关键作用的凝乳因子。文中采用纤维素离子交换柱从江米酒中得到分子质量为36·4ku的单一蛋白酶,其凝乳活力与蛋白酶水解活力的比值从纯化前的3·1提高到16·0。对纯化酶的基本特性研究表明,该酶在30~55℃,pH值3·0~7·0保持比较稳定的酶活性,而与胃蛋白酶、米黑毛霉凝乳酶及牛凝乳酶凝乳过程中的组织状态变化比较,初步认为江米酒凝乳酶的动力学或凝乳机制可能与牛凝乳酶等其他天冬氨酸蛋白酶不完全相同。     

Permeate Flux During Ultrafiltration of Whey: Influence of Milk Coagulant Used for Cheese Manufacture

A pilot-scale plate and frame UF system was used to fractionate Cheddar cheese whey and study the effects of different commercial milk coagulants on permeate flux. Coagulants used in this study were calf rennet, Mucor pusillus protease, and Mucor miebei protease. Whey UF performance studies were conducted at a commercial Cheddar cheese plant and at Cornell under controlled conditions. Ultrafiltration was done in a continuous mode and initial concentration factor was set at 2× to simulate the first stage of a multistage whey UF system.Permeate flux decline was rapid in the first 30 min of UF for all wheys studied. More important, the type of milk coagulant used in cheese making had a profound effect on permeate flux during whey UF. No differences in the gross composition of the various wheys were correlated with differences in permeate flux. The highest permeate flux was measured for UF of whey produced during manufacture of Cheddar cheese using coagulant derived from Mucor pusillus. Lowest permeate flux was measured for UF of whey produced during manufacture of Cheddar cheese using calf rennet. Whey from cheese manufactured using Mucor miebei coagulant had flux performance intermediate to Mucor pusillus and calf rennet. The impact of milk coagulants on whey UF process efficiency should be considered by cheese makers.     

Proteolytic activity of proteinases on macropeptide isolated from kappa-casein.

Proteolytic activities of chymosin, bovine pepsin, Mucor miehei rennet, Cryphonectria parasitica (formerly Endothia parasitica) rennet, trypsin, and chymotrypsin on kappa-casein macropeptide were measured. Macropeptide solutions (10 mg/ml of .05 M, pH 6.6 phosphate buffer) were incubated with the enzymes at 37 degrees C for various times, and their reactions were stopped by adding .025 ml of pepstatin (1 mg/ml of methanol). Peptides released from kappa-casein macropeptide were then fractionated using reverse-phase HPLC. At the pH of milk (pH 6.6), kappa-casein macropeptide was resistant to enzymic action by chymosin, bovine pepsin, and M. miehei and C. parasitica rennets. Bovine pepsin hydrolyzed kappa-casein macropeptide at pH 3. kappa-Casein macropeptide was readily hydrolyzed at pH 6.6 by trypsin and chymotrypsin. Possible physiological functions of the kappa-casein macropeptide are discussed in light of these findings.     

凝乳酶的研究进展

传统干酪制作使用的凝乳酶来源于小牛皱胃,目前小牛皱胃酶的供应量仅能满足世界干酪产量所需凝乳酶的20%～30%。小牛皱胃酶的供需矛盾和价格高昂等因素,使得寻求其替代物成为乳品领域科学研究的热点之一。本文首先介绍了小牛皱胃酶的研究现状,其次从动物、植物、基因重组以及微生物来源,综述了不同凝乳酶之间酶性质差异以及凝乳酶在干酪应用中的研究,旨在为凝乳酶的研究提供一定的理论参考,为寻找凝乳酶替代物提供思路。     

Compositional,biochemical and textural changes during ripening of Tulum cheese made with different coagulants

In the present study, biochemical, chemical and texture changes in Tulum cheeses made using calf rennet and microbial rennets (Aspergillus niger protease and Rhizomucor miehei protease) were compared during ripening for up to 90 days. A total of 15 free fatty acids (FFAs) were detected in the cheese samples. The peroxide values (PV) of the cheeses increased significantly (P < 0.05) during ripening and the cheese made with calf rennet had the highest PV. Proteolysis in the cheeses increased as the ripening time increased. α_s1‐casein and β‐casein degradation was higher in cheeses manufactured with R. miehei protease. Cheeses made with calf rennet were significantly (P < 0.05) harder, more adhesive, more cohesive and more resilient than those made with microbial rennet.