| Since the theory of free radicals was proposed, people found that many human diseases are related to the production of free radicals by oxidation in the body, so the antioxidant peptides become a research hotspot. Antioxidant peptides can scavenge free radicals and prevent oxidation reaction, which help the body against disease. Antioxidant peptides derived from food protein have higher safety, lower side effect and strong antioxidant activity, so more and more get the attention of people. At present, the study of milk-derived antioxidant peptide mainly concentrated in the milk, research the goat milk is very little. Shaanxi province has rich resources of goat milk, but the types of products are fewer, likely the goat milk powder with low value added. Therefore, the study of antioxidant peptides from goat milk casein can develop goat milk products with antioxidant activity, which will increase product categories of goat milk, improve the functionality and value-added of goat milk products.In this study, the goat milk was used as raw material, and the goat milk casein was used as the substrate. The casein was hydrolyzed by seven proteases. Degree of hydrolysis, DPPH radical-scavenging activity, Fe2+ chelation activity and O2·-scavenging activity were used evaluation index, to select a protease with high antioxidant activity. Useing single factor experiment, factorial design and response surface experiments to optimize process condition. The enzymolysis liquid was separated and purified by using ultrafiltration, macroporous resin desalting and gel filtration chromatography. The main results of the study are as follows:1. Degree of hydrolysis and DPPH radical-scavenging activity were used evaluation index, the goat milk casein was hydrolyzed by proteinase K, Alcalase enzyme, trypsin, flavourzyme, bromelain, papain and neutral protease, under their optimal condition, respectively. The experimental results show that Alcalase was selected as the single enzymatic proteinase, Alcalase and papain was used in the compound enzymatic hydrolysate conditions.2. Single factor experiment to investigate the different hydrolysis conditions on the effects of production of antioxidant peptides from goat milk casein hydrolysated with Alcalase. Then, selected the main factors by the factorial experimental. The experiment results showed that the pH, substrate concentration and time were the main factors, which had a significant impact on the antioxidant peptides. The optimum hydrolysis conditions were determined by steepest ascent experiment and response surface experiment:temperature 69℃, pH 8.9, substrate concentration 4.4%, E/S ratio 2%, and hydrolysis time 172min under the conditions, DPPH radical-scavenging activity was (68.37±1.31)%, Fe2+chelation activity was (89.04±0.77)% and O2·-scavenging activity was (46.58±1.08)%.3. The different hydrolysis conditions on the effects of antioxidant peptides during goat milk casein hydrolysated with compound enzyme were investigated by single factor test. Then, selected the main factors by the factorial experimental. The results of the experiment showed that the temperature, E/S and the ratio of compound enzyme were the main factors, which had a significant impact on the antioxidant peptides. Finally, the compound enzyme hydrolysate conditions was optimized by response surface experiment: temperature 61℃, pH 9, substrate concentration 4%, E/S 5.6%, the ratio of Alcalase and papain 11:6 and hydrolysis time 180min, under the conditions, DPPH radical-scavenging activity was (73.81±0.93)%, Fe2+chelation activity was (93.44±1.46)% and O2·-scavenging activity was (51.69±1.17)%.4. The enzymolysis liquid was separated by ultrafiltration membrane with different molecular weight. The results showed that the DPPH radical-scavenging activity of the permeate which molecule weight less than 1 kDa was highest and it was 81.25%, the value of IC50 was 0.842mg/mL. This study provides the molecule weight of antioxidant peptides was mostly less than 1kDa. The enzymolysis liquid which molecule weight less than 1 kDa was separated by macroporous resin DA201-C. The adsorption liquid was adjusted to pH 4, with a concentration of 70% ethanol as eluant. At this moment, the DPPH radical-scavenging activity and the value of IC50 were 83.62% and 0.753 mg/mL, respectively.5. Sephadex G-15 gel chromatography was adapted to purification of macroporous resin desalting samples, distilled water as eluant, sample concentration was 80mg/mL, elution velocity was 0.8mL/min. Three components were obtained under this condition. Three fragments Ⅰ, Ⅱ and Ⅲ were obtained in this condition. The fragments Ⅱ had the DPPH radical-scavenging activity was 85.61% and IC50 was 0.543mg/mL. Then, using RP-HPLC analysis component Ⅱ found that separation of the sample is not a single component, so still need to further purification.Antioxidant peptides prepared in this research which derived from goat’s milk casein hydrolysated have a higher antioxidant activity which can be used as additive in the development of antioxidative peptide products functionality of goat milk products. At the same time, the research results of this study can promote the healthy and rapid development of goat’s milk industry in our province. |
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