| High pressure treatment is a research focus in the field of meat science. The high pressureenhancement of enzymes is the recent development of high hydrostatic pressure technology.Pepsin is the important byproduct of meat processing and widely used in the enzymatic hydrolysisof meat protein. It was found that the high pressure treatment could induce stabilization andactivation of pepsin, which suggested that it could be used to improve the functional propertiesand increase the enzymatic hydrolysis rate and degree of protein. But this mechanism hasn’t beenreported. Based on the previous study, the optimum enzymatic hydrolysis conditions of casein wasfirstly optimized, and then the experimental system of casein and pepsin under high hydrostaticpressure will be established to investigate the effects of the environmental factors on theenzymatic property, structure and catalytic activity of pepsin in this study. The relationshipbetween its physicochemical changes and catalytic activity under high hydrostatic pressure will beclarified. And the degree of hydrolysis and antioxidant activity of thehydrolysis from theexperimental system of casein and pepsin was also analyzed.The obtained results will be helpful toprovide important basises for developing the high-pressure processing technology of food,prompting the application of enzyme in food processing and developing/utilizing the functionalbioactive peptides of protein from milk. The main contents and results of this study was got asfollows:1. Selection of hydrolase and optimization of the hydrolyzate reactionPepsin, papain, trypsin, neutrase and alcalase was used for casein hydrolysis in this study,pepsin was the best enzyme for the progress from the degree of hydrolysis (DH) determination. Insingle factor experiment, hydrolysis temperature, pH, enzyme concentration and time was selectedto deduce the conditions of pepsin hydrolysis with casein. Based on the results, response surfacemethodology (RSM) was employed and the mathematical model for hydrolysis conditions wasbuilt through the regression analysis and model evaluation. The optimal hydrolysis conditionswere pH1.8, enzyme concentration21.2%and50℃. Under this conditions, degree of hydrolysiswas28.15%.2. Internal mechanism of enzyme activity treated by high pressure treatmentPepsin was processed by pressure, the intrinsic effect of high pressure treatment on enzymeactivity of pepsin was studied. The enzyme activity of pepsin was increased from16.50×104u to18.53×104u, improved12.30%under100MPa,15min. Through the analysis of pepsin’ thiol,disulfide bonds, hydrophobic-the index characteristic of protein tertiary structure as the activecenter of the enzyme, and subunit composition of discovery under high-pressure treatment, we gotthat the enzyme active site was changed under100MPa,15min from the changing of secondarykey of enzyme structure, transformating of pepsin’ subunit between37.0KDa,21.0KDa and15.0KDa. GPC analysis showed that, the pressure of50~100MPa for10min had an importantinfluence on the hydrodynamic radii and intrinsic viscosity of the enzyme. The contacted probability of enzyme active site and the substrate was increased by the influence of high-pressurehydration interaction between protein subunits.3. Effect of high-pressure treatment on enzymatic properties of pepsinPepsin was treated by high-pressure in order to measured the change of the degree ofhydrolysis, the presence form and conten of nitrogen when hydrolyzed casein. And the inherentmechanism of high-pressure treatment improving the enzymatic process of casein by pepsin wasstudied through the analysis of subunit composition and molecular weight distribution of peptide.The results show that: the enzyme hydrolysis degree of casein hydrolyzed by pepsin treated byhigh-pressure had the same trend with high-pressure-enzyme. the degree of hydrolysis had a bettertreatment effect in the100MPa, about15min, which was increased from8.32%to11.1%,improved33.41%. And under this pressure-treated condition, some insoluble hydrophobicpeptides changed into soluble nitrogen and amino acid nitrogen which more easily digested andabsorbed; and the amino nitrogen generated was more than the amount of transformed solublenitrogen, resulting in a reduced of peptide-based nitrogen content. Effect of high-pressuretreatment on subunits of the hydrolysates were not significant, the molecular weight of thehydrolysates concentrated at500-2000Da, compared with casein’ molecular weight of25-35KDa,can illustrated that casein was hydrolyzed in a higher degree. Pressure-treatment at100MPa,15min prompted hydrolysates’ large molecular weight peptides of2000-3000Da changed into smallpeptide fragments of100-2000Da and <500Da through the analysis of molecular weightdistribution of hydrolysates.4. Effect of high pressure treatment on antioxidant activity of the hydrolysatesThe effect of high pressure treatment on enzymatic hydrolysis of casein hydrolyzed by pepsinand antioxidant activity of the hydrolysates was investigated in this article. Based on degree ofhydrolysis (DH), the effect of high pressure treatment on enzymatic hydrolysis of casein wasstudied. Then the DPPH, superoxide anion radicals, hydroxyl radical scavenging activities andreducing power of casein hydrolysates were measured in order to assess the influence of differentpressure and presuure-holding time on antioxidant activity of the hydrolysates. The results showedthat pepsin (E), casein (S) and a combination of both (E+S)’s degree of hydrolysis weresignificantly increase (p<0.05) under100~200MPa,10~20min. The DPPH and hydroxyl radicalscavenging activities were significantly improved under a certain pressure at about150MPa,10min. The effect of high pressure treatment on E and S’ superoxide anion radicals scavengingactivities was better in50MPa,10min and100MPa,5min, respectively. For the reducing power,E+S and S also had better influence under150MPa,15min and200MPa,15min. And due to thedifferent pressure treatments and handling objects, the enzymatic characteristics and antioxidantproperties of hydrolysates were differently. |
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