| Antioxidant peptide which is derived from natural proteins is a low-molecular-weight polypeptide containing 2 to 20 amino acids embodying the nature of containing antioxidant properties,scavenging free radicals,chelating metal ions as well as the reducing ability.It has a very wide range of applications in the food industry,cosmetics industry,health products and pharmaceutical industry because of its extended sources,high safety,good antioxidant effect,good stability and other advantages.This project mainly explored the best hydrolysis technology for extracting 5% casein by combining lactic acid bacteria with protease.First,from 9 strains of lactic acid bacteria deposited in the laboratory,a strain of lactic acid bacteria with high protease-producing activity and good casein degradation ability was screened.Second,this lactic acid bacteria was combined with 6 kinds of proteases,and the protease combination with lactic acid bacteria to produce the best antioxidant peptide was determined from the antioxidant capacity and the degree of hydrolysis of the fermented hydrolysis,and the fermentation and hydrolysis of lactic acid bacteria and protease combination were followed.The process of casein preparation of antioxidant peptides was optimized by response surface.The best process was used to ferment and hydrolyze casein to obtain antioxidant peptides.Finally,the changes of the antioxidant peptides during the simulated gastrointestinal digestion were measured.The simulated gastric juice digestion at 0,30,60,90,120 min and the simulated intestinal digestion at 1,2,3,and 4 hours were measured Changes in oxidation resistance,degree of hydrolysis,and molecular weight of peptides.The main results of this experiment are as follows.(1)Based on the study of the properties of 9 lactic acid bacteria preserved in the laboratory and their ability to decompose casein,it is concluded that when the lactic acid bacteria are cultured to a stable period,the maximum protease production activity of Lactobacillus rhamnosus GG is 103.78 ± 7.06 U/ml,which is higher than other strains significantly(P<0.05).The other strains with higher protease activity were Lactobacillus plantarum,Bifidobacterium animalis BB-12,and Streptococcus thermophilus.The protease activities of the three lactic acid bacteria were 88.11±5.18 U/m L,70.49±1.96 U/m L,68.53±5.18 U/m L,the lowest protease activity of Lactobacillus fermentation Q6 is 21.53 ± 2.99 U / m L(P <0.05).The antioxidant activity of each strain's fermented casein product and the degree of hydrolysis gradually increased as the fermentation time.According to the research for 9 strains of lactic acid bacteria,the antioxidant activity of LGG casein product was significantly higher than that of other strains(P<0.05),and the degree of hydrolysis was also significantly higher than other strains(P<0.05).The analysis of the growth of 9 lactic acid bacteria in the casein solution system,it was concluded that as the increasing of the culturing time,the number of viable bacteria in the casein solution system continued to increase,and the growth of LGG was better than other strains(P<0.05).By measuring the protease activity of 9 strains of lactic acid bacteria in the casein solution system,it was concluded that the protease activity gradually increases before 8h of fermentation,and the protease activity of some strains will decrease after 8 h,and the protease activity of LGG still performance better than other strains(P<0.05).(2)By analyzing the oxidation resistance and hydrolysis degree of lactic acid bacteria-bound protease fermentation hydrolysis,the best combination degree of protease with its ratio was obtained.The best combination of lactic acid bacteria and protease is: LGG + papain + flavor protease,the ratio of protease is 1: 1.At this time,the DPPH free radical scavenging rate of the fermentation hydrolysis is 84.51 ± 0.50%,and the maximum hydroxyl radical is 79.37 ± 1.17%.The degree is 22.37 ± 0.36%.(3)The single-factor analysis and the response surface Box-Benhnken test design were used to determine the optimal fermentation and hydrolysis process for the combined fermentation with hydrolysis of bacterial enzymes.Response surface optimization analysis of the major and minor sequence of each factor was: lactic acid bacteria inoculum amount> temperature> enzyme addition amount> fermentation and hydrolysis time.Finally,the optimal fermentation and hydrolysis conditions for casein antioxidant peptides were determined: LGG inoculation amount was 3%(v/w),the fermentation hydrolysis temperature is 37°C,the fermentation hydrolysis time is 12 hours,and the enzyme addition amount is 0.25%(w/w).Under these circumstances,the maximum value of DPPH free and removal rate of the fermentation hydrolysis product is 92.12±0.02%.(4)According to the simulation of gastrointestinal digestion by antioxidant peptides.It was concluded that within 2 hours' simulated gastric juice digestion,antioxidant properties of the antioxidant peptides decreased and the DPPH free radical scavenging rate decreased from the initial 88.52±0.99% to 49.62±2.84%.From the initial 79.37±1.17% to 40.35±1.11%,the DPPH free radical scavenging rate of antioxidant peptides increased to 90.45±1.78% within 4 hours' simulating intestinal digestion.At the same time,the hydroxyl radical scavenging rate increased to 76.49±0.62%.During the simulated gastric juice digestion,the degree of hydrolysis of the digestive product slowly increased from 23.03±0.13% to 24.51±0.17%,and the degree of hydrolysis increased significantly during the simulated intestinal digestion to 33.31±0.13%.Tricine-SDS-PAGE gel electrophoresis results showed that the content of lower molecular weight peptides gradually increased throughout the simulated digestion process. |
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