Isolation,Purification And Structural Identification And Application Of Antioxidant Peptides Derived From Goat Milk

Milk source protein is one of the important sources of antioxidant peptides.It is one of the natural antioxidants and has no side effects.The antioxidant peptides in milk protein can be released by enzymatic hydrolysis or fermentation.Purification can improve the activity of peptides,develop healthy dairy products,and improve the nutritional value and added value of dairy products.This project will use goat milk protein single enzyme?Alcalase enzyme?enzymatic hydrolysate?GCH-A?and goat milk protein dual enzyme?Alcalase enzyme and papain?enzymatic hydrolysate?GCH-AP?,Lactobacillus plantarum LP60 fermented sheep milk?FGM-LP?and Lactobacillus casei L61 fermented sheep milk?FGM-LC?as raw materials.Antioxidation peptides were separated and purified by ultrafiltration and gel chromatography.Amino acid sequence was identified by reverse high performance liquid chromatography?RP-HPLC?and mass spectrometry.Furthermore,the stability of antioxidant peptides under simulated gastrointestinal fluid,bile salt,temperature and pH was studied,and the antioxidant effects of antioxidant peptides in 8 kinds of foods were investigated.The main findings are as follows:1.Three kinds of ultrafiltration membranes with different truncated molecular weights can be used for the separation of antioxidant peptides from goat milk source.The highest active antioxidant peptides were isolated from the transmissible liquid with molecular weight less than 3000 Da.Ultrafiltration can enrich antioxidative peptide components,improve antioxidant activity and decrease IC₅₀0 value.The lowest of the IC₅₀0 of the ultrafiltration component VII of the GCH-A,GCH-AP,FGM-LP,FGM-LCwere respectively 0.631±0.014mg/mL,0.647±0.016 mg/mL,0.657±0.013 mg/mL,and 0.652±0.009mg/mL.2.Sephadex G-25 and G-15 was used to separate and purify the ultrafiltration component VII of the GCH-A,GCH-AP,FGM-LP,FGM-LC.The highest activity was obtained from the separation of A₂,B₂,D₃,E_3.Purified by Sephadex G-15,the highest activity of A_2-2,B_2-2,D_3-1,E_2-1-1 was obtained,and theIC₅₀0 of each component was 0.372 mg/mL 0.379 mg/mL 0.507 mg/mL0.403mg/mLrespectively,and The activity of component A_2-2,B_2-2-2 was higher than that of component D_3-1,E_2-1.3.RP-HPLC analysis of components A_2-2,B_2-2,D_3-1,E_2-1-1 revealed that the isolated component was not a single peptide.LC/MS/MS analysis showed that the 3 peptides of component A_2-2-2 was mainly derived from?-casein;the 14peptides of component B_2-2-2 mainly came from?-casein,?_S11 casein,?_S2-casein and?-casein;the 10 peptides of component D_3-1-1 mainly came from?-lactalbumin,?-lactoglobulin and?_S2-casein;the 28 peptides of component E_2-1are mainly derived from?-lactalbumin,?-lactoglobulin,?_S1-casein,?_S2-casein and?-casein.4.The effects of simulated gastrointestinal fluid on the activities of four kinds of antioxidant peptides were significant.After simulated gastrointestinal digestion,the activities of components A_2-2,B_2-2,D_3-1-1 and E_2-1-1 decreased 37.87-21.21.The order of the decrease of the four antioxidative peptides was A_2-2>B_2-2>D_3-1>E_2-1.The effects of simulated gastrointestinal fluid on the activities of four antioxidant peptides were significant.After simulated gastrointestinal digestion,the activities of components A_2-2,B_2-2,D_3-1-1 and E_2-1-1 decreased 37.87-21.21.The descending order of the four antioxidative peptides was A_2-2>B_2-2>D_3-1>E_2-1.The effect of bile salts on the four anti-oxidative peptides was less than that of gastrointestinal fluids,and the antioxidative activity decreased less than 10%.The order of decline was A_2-2>D_3-1>E_2-1>B_2-2.5.The four antioxidation peptides are easily inactivated in high temperature,strong acid environment and strong alkali environment.The activity of component D_3-1,E_2-1-1 was stable at pH 4.5-6.5,the activity of component A_2-2,B_2-2was stable at pH6.5-8.5 and the activity of four components was below 42?.The heat resistance of component A_2-2-2 was better than that of B_2-2,D_3-1-1 and E_2-1,and the activity of A_2-2-2 was decreased 27.86-34.68.The order of resistance to acid and alkalinity of the four components was E_2-1>D_3-1>B_2-2>A_2-2-2 and D_3-1>E_2-1>B_2-2>A_2-2,respectively.The activity decreased 6.22-30.01.6.The four anti-oxidation peptides prepared were added to eight foods respectively.The results showed that the anti-oxidation activity of the four anti-oxidation peptides in solid foods was superior to that of liquid foods.The activity of four antioxidant peptides decreased by 16%in solid foods and 25.4%in liquid foods.The four antioxidant peptides prepared in this study have high antioxidant activity and good stability,and can provide functional factors for the development of antioxidant foods or health products.The identification of sheep milk-derived antioxidant peptides is a follow-up health food product.Development provides a theoretical basis and technical support.