Preparation Of Goat Whey Protein High Fisher Ratio Oligopeptide And Study On Its Functional Activity

As a by-product of cheese production in the food industry,whey has the advantages of high branched chain amino acids and low aromatic amino acids,and it is an ideal source of high F value oligopeptides.The continuous development of protein processing by-products with abundant sources and low prices as raw materials is also the key to the research of high F-value oligopeptides.In this study,goat whey protein was used to prepare high-F-value oligopeptides,and their antioxidant activity was evaluated.The liver-protecting mechanism of high-F-value oligopeptides was investigated for the first time.This study provided a theoretical basis for the application of high Fisher ratio oligopeptide as the functional ingredients in formula foods for special medical purposes related to liver disease.In this study,pepsin and flavor protease were used to perform stepwise enzymatic hydrolysis of goat whey protein.Taking the F value and degree of hydrolysis as reference indicators,the optimized enzymatic hydrolysis conditions were obtained by orthogonal tests.Firstly,pepsin(1%,w/w)was used to hydrolysis the whey protein at p H 2 and 50?for 2 h.Afterwards,flavor protease(0.5%,w/w)was used to hydrolysis the former hydrolysates at p H 7 and 45?for 3 h.The dearomatization process,that the aromatic amino acids in hydrolysate were adsorbed by activated carbon,was optimized according to the Fisher ratio.The optimized condition were that:dearomatization p H is 2 and carbon to liquid the ratio is 1:5 for 2h.The Fisher ratio is 26.32 under the optimal process conditions.Amino acid sequencing results showed that the branched chain amino acids in the system were101.568 ng/m L derived by amino acid sequencing,which accounted for 17.88%of the total amino acids.According to gel chromatography,the peptides in the dearomatization solution were distributed below 2000 Da,which was preliminarily determined to be high F-value oligopeptide solution(HFO).Free radical scavenging experiments were conducted to test its antioxidant activity in vitro with ascorbic acid as a positive control.The results showed that the scavenging rates of 1,1-diphenyl-2-pyridylhydrazino(DPPH)and3-ethylbenzothiazolin-6-sulfonate(ABTS)were 77.27%and 99.63%at the concentration of 2.0 mg/m L and 0.50 mg/m L,respectively.Furthermore,the scavenging rate of hydroxyl radicals reached 92.31%at a concentration of 0.25mg/m L.We also found that the scavenging rates of three free radicals were concentration-dependent(P<0.05).Moreover,A CCl₄-induced mouse oxidative stress model was established to verify the antioxidant properties of HFO in vivo.The results showed that,the experimental group after the intervention of HFO significantly reduced the content of malondialdehyde(MDA)in the serum of mice(P<0.05),while,the superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)vitality was significantly higher than that of the model group(P<0.05)compared with the model group.The mouse models of acute liver injury induced by CCl₄were established to verify the ability of HFO to repair liver injury.The results showed that the liver index and serum transaminase levels of mice in the experimental group treated with HFO were significantly increased(P<0.05)comparing with the model group.In addition,the pathological observation of mouse liver tissue showed that HFO can improve liver tissue diseases'physiological changes.Western-blot tests showed that HFO can increase the expression level of the inflammation-suppressing factor inhibitor a of NF-?B(I?B?)and decrese the expression level of Phosphorylated p65 to reduce the inflammatory response.The results of quantitative real-time Polymerase Chain Reaction(q RT-PCR)showed that HFO down-regulated the relative expression of tumor necrosis factor-?(TNF-?),Interleukin-6(IL-6)and Interleukin-1?(IL-1?)to achieve anti-inflammation and protect liver cells.In summary,the HFO obtained from goat whey protein by hydrolyzing and dearomatizing showed great physiological activities of anti-oxidation and liver protection.This study not only expands the high-value processing and utilization of whey,but also has important significance for the diversification and deeper development and application of whey high Fisher ratio oligopeptide products.Especially in the context of fighting the new coronavirus epidemic,it provides a theoretical basis for the precision manufacturing of special foods related to improving immunity and liver disease.