Kinetics of pepsin-induced hydrolysis and the coagulation of milk proteins

Hydrolysis-induced coagulation of casein micelles by pepsin occurs during the digestion of milk. In this study, the effect of pH (6.7–5.3) and pepsin concentration (0.110–2.75 U/mL) on the hydrolysis of κ-casein and the coagulation of the casein micelles in bovine skim milk was investigated at 37°C using reverse-phase HPLC, oscillatory rheology, and confocal laser scanning microscopy. The hydrolysis of κ-casein followed a combined kinetic model of first-order hydrolysis and putative pepsin denaturation. The hydrolysis rate increased with increasing pepsin concentration at a given pH, was pH dependent, and reached a maximum at pH ～6.0. Both the increase in pepsin concentration and decrease in pH resulted in a shorter coagulation time. The extent of κ-casein hydrolysis required for coagulation was independent of the pepsin concentration at a given pH and, because of the lower electrostatic repulsion between para-casein micelles at lower pH, decreased markedly from ～73% to ～33% when pH decreased from 6.3 to 5.3. In addition, the rheological properties and the microstructures of the coagulum were markedly affected by the pH and the pepsin concentration. The knowledge obtained from this study provides further understanding on the mechanism of milk coagulation, occurring at the initial stage of transiting into gastric conditions with high pH and low pepsin concentration.     

Whey Protein Antigenicity Reduction by Fungal Proteinases and a Pepsin/Pancreatin Combination

Whey protein components were hydrolyzed with Corolase 7092? (peptidases from Aspergillus strains), pepsin and Corolase PP? (a mixture of pancreatic enzymes), either individually or in combination, in trials to eliminate protein allergenicity. The hydrolysates were characterized by physico-chemical and by immunological techniques using sera from patients allergic to milk proteins. Enzyme specificity rather than degree of hydrolysis or molecular mass distribution of hydrolysates determined the residual antigenicity of the whey proteins. Ultrafiltration was a prerequisite for obtaining hypoallergenic whey protein hydrolysates.     

大鲵胃蛋白酶分离纯化及其性质的研究

通过研究大鲵胃蛋白酶,可以了解大鲵消化机能,同时为食品加工提供新的材料。经80%硫酸铵沉淀、DEAE-52离子交换层析、SephadexG-100凝胶过滤层析以及HPLC,得到大鲵胃蛋白酶电泳纯制品。纯化倍数为239.87,回收率为4.4%,经SDS-PAGE测得该胃蛋白酶分子量为31ku,大鲵胃蛋白酶的最适温度是40℃,低于40℃稳定,当温度上升,活性迅速下降;最适pH为2,在pH2～6有很好的稳定性。EDTA、BrAc、NBS使该酶活性下降。以酪蛋白为底物,在pH2.0、40℃时测得米氏常数Km值为7.3×103mg/L,Vmax是2.674μg/min。      

超声波辅助酶法提取骨明胶研究

以脱脂脱矿骨粉为原料,利用超声波辅助胃蛋白酶法提取骨明胶。比较了骨素酶解前、酶解中以及酶解后经不同超声时间和功率处理对明胶得率的影响,单因素实验结果表明:骨素酶解前和酶解中经超声处理,明胶得率显著提高。在骨素酶解前和酶解中分别经250 W、20 min和250 W、10 min的超声处理,明胶的得率分别为81.35%和83.32%,与未经超声处理组相比,明胶得率分别提高了9.96%和11.93%,而骨素酶解后在300 W超声功率下处理10 min,明胶得率仅为71.59%,与未超声处理组71.39%接近。SEM图片显示,酶解前和酶解中经超声处理,会使骨素表面出现孔隙,而酶解后超声处理则会使骨素表面更加光滑,表明酶解前和酶解中超声处理对骨素具有疏松作用。SDS-PAGE结果显示,超声波辅助酶法提取明胶,对明胶分子量分布没有影响。超声波辅助酶法提取,提高了骨明胶的得率,可为工业上骨明胶的生产提供有益的参考和借鉴。      

Proteolysis of milk fat globule membrane proteins during in vitro gastric digestion of milk

The influence of gastric proteolysis on the physicochemical characteristics of milk fat globules and the proteins of the milk fat globule membrane (MFGM) in raw milk and cream was examined in vitro in simulated gastric fluid (SGF) containing various pepsin concentrations at pH 1.6 for up to 2 h. Apparent flocculation of the milk fat globules occurred in raw milk samples incubated in SGF containing pepsin, but no coalescence was observed in either raw milk samples or cream samples. The changes in the particle size of the fat globules as a result of the flocculation were dependent on the pepsin concentration. Correspondingly, the physical characteristics of the fat globules and the composition of the MFGM proteins in raw milk changed during incubation in SGF containing pepsin. The major MFGM proteins were hydrolyzed at different rates by the pepsin in the SGF; butyrophilin was more resistant than xanthine oxidase, PAS 6, or PAS 7. Peptides with various molecular weights, which altered with the time of incubation and the pepsin concentration, were present at the surfaces of the fat globules.     

磁性壳聚糖微球固定化胃蛋白酶的研究

利用反相悬浮交联法制备壳聚糖微球,然后采用化学共转化法制备了磁性壳聚糖微球(magnetic chitosan microspheres,M-CS),并对胃蛋白酶进行固定化研究。结果表明,制备的M-CS呈规则圆球形,有很好的磁响应性,并且在弱酸弱碱环境下能稳定保存。磁性壳聚糖微球对胃蛋白酶的吸附性实验表明,磁性壳聚糖微球能吸附胃蛋白酶,可是吸附胃蛋白酶的量受到载体与酶比例、溶液的离子浓度、溶液的pH值影响很大。胃蛋白酶动力学性质研究表明,相对于游离的胃蛋白酶,固定化后的酶的最适温度有所升高,最适温度在60℃、酸碱稳定性略有改善,最适pH4.0,故固定化后的酶的热稳定性和酸碱稳定性都得到明显改善。     

胃蛋白酶固定化条件的研究

采用壳聚糖吸附和戊二醛交联的方法,将胃蛋白酶固定于壳聚糖上.探讨了温度、交联剂用量、固定化时间以及pH值等条件对固定化效果的影响,并对固定化酶的理化性质进行了研究.确定了胃蛋白酶固定化的最佳条件:pH值3.5;戊二醛交联时间10 h;戊二醛含量5%;对酪蛋白的操作半衰期为50 d以上.     

响应面法优化胃蛋白酶制备花椒籽蛋白抗菌肽的研究

以花椒籽蛋白为原料,采用胃蛋白酶水解制备抗菌肽。在单因素实验结果的基础上,应用Box-Behnken中心组合方法进行四因素三水平的实验设计,以大肠杆菌（Escherichia coli）抑菌率为响应值建立数学模型,确定底物浓度4.9%、酶与底物比（g/g）0.9∶100、pH2.0、酶解温度32℃、酶解时间3h为最佳酶解条件。此条件下酶解产生的抗菌肽复合物的抑菌率可以达到60.96%。      

Kinetic Analysis of the Digestion of Bovine Type I Collagen Telopeptides with Porcine Pepsin

Collagen is frequently digested using pepsin in industries to produce a triple helical collagen without the N‐ and C‐terminal telopeptides. However, kinetic analysis of this reaction is difficult because several Lys residues in the N‐ and in the C‐terminal telopeptides form covalent bonds, leading to multiple substrates species, and pepsin cleaves collagen at various sites in the N‐terminal and in the C‐terminal telopeptides, yielding different products. Here we performed kinetic analysis of the digestion of bovine type I collagen with porcine pepsin. The reaction could be monitored by SDS‐PAGE by measuring the intensity of the protein bands corresponding to the variant β11 chain. We obtained kinetic parameters relative to the decrease in the variant β11 chain upon digestion. At pH 4.0, the K_m and k_cat values increased with increasing temperature (30 to 65 °C), although the k_cat/K_m values were stable. Additional cleavage at the helical region was detected at 45 to 65 °C. At 37 °C, the K_m and k_cat values increased with decreasing pH, and the k_cat/K_m values at pH 2.1 to 4.5 were stable and higher than those at pH 5.0 and 5.5. No additional cleavage was detected at the examined pH. Thus, the optimal pH and temperatures for selective digestion of collagen telopeptides with pepsin are 2.1 to 4.5 and 30 to 40 °C, respectively. These results suggest that the method might be useful for the kinetic analysis of the digestion of collagen telopeptides with pepsin.