Preparation,Separation,Purification And Structural Identification Of Mung Bean Antioxidant Peptides

Mung bean(Vigna radiata)is an economic crop with high nutritional value and medicinal value.It has the characteristics of low fat,medium starch,high protein and homology of medical and food.Mung bean is a kind of high-quality plant protein,which has abundant amino acids and balanced ratio.Bioactive peptides have unique absorption mechanism in the body,low sensitization,no toxic effects,and have multiple biological activities such as anti-oxidation,lowering blood pressure and immune-regulation,so they are widely concerned.In this study,mung bean protein was extracted by alkaline extraction and acid precipitation method,and then hydrolyzed through enzymatic technology.The enzymatic parameters were optimized by single factor and response surface methodology.Subsequently,the antioxidant activity of mung bean peptides(MBPs)was evaluated by different mechanisms of chemical antioxidation in vitro and H₂O₂ induced oxidative damage model of HepG2cells.Finally,the MBPs was separated and purified by chromatography,and the target components was identified by mass spectrometry.The specific results are as follows:(1)The alkaline protease Alcalase 2.4L FG was used to hydrolyze mung bean protein to prepare MBPs,and the degree of hydrolysis(DH)was used as an indicator to study the influence of substrate concentration,enzymatic hydrolysis temperature,pH,enzyme dosage and enzymatic hydrolysis time on DH.Design Expert.V8.0.6.1software determined the optimal enzymatic hydrolysis process parameters of mung bean protein as follows:substrate mass concentration was 4.90%,pH value was 9.0,enzyme dosage was 5.06%,enzymolysis temperature was 50?,enzymolysis time was 4 h,The obtained degree of hydrolysis was 30.24%.(2)The prepared MBPs had a small molecular weight,high content of basic and hydrophobic amino acids,and showed a certain degree of in vitro antioxidant capacity.MBPs contained 17 kinds of amino acids,of which 8 kinds of essential amino acids in human body accounted for 34.18%.The proportions of non-polar amino acids,polar amino acids,basic amino acids and acidic amino acids were 35.27%,11.91%,19.40%and 33.43%,respectively.High performance liquid chromatography showed that MBPHs were mainly composed of peptides with a molecular weight of less than 3000Da(95.36%),and the proportion of peptides with a molecular weight of less than2000 Da was 85.98%.The FI-TR spectrum showed that MBPs contained?-helix and?-sheet secondary structures,in addition to the presence of a variety of unsaturated groups.The vitro free radical scavenging test showed that MBPs had strong scavenging ability on DPPH and ABTS free radicals.When the concentrations were10.0 mg/mL and 2.0 mg/mL,the scavenging rate reached 60.47±4.41%and 77.76±1.42%,respectively.(3)MBPs had protective effect against H₂O₂ induced oxidative damage in HepG2 cells.The results showed that MBPs had no effect on the normal proliferation of HepG2 cells.And compared with the model group,the survival rate of cells pre-protected by MBPs increased significantly(P<0.01).In addition,MBPs could protect HepG2 against oxidative damage through scavenging ROS,increasing the SOD activity and decreasing the MDA content.(4)Gel chromatography(Sephadex G-25)and UPLC-Q-TOF-MS/MS were used for separation,purification and structure identification of MBPs.The results showed that Sephadex G-25 separated MBPs into 6 components:F1,F2,F3,F4,F5,F6,among them,the smallest molecular weight component F6 had the strongest DPPH free radical scavenging activity.When the concentration was 2.0 mg/mL,the scavenging rate of DPPH free radical was 55.40±3.97%.A total of 8 peptides in component F6 were identified by mass spectrometry,and their amino acid sequences were:Cys-Val-Val-Gly-Phe-His(846.37 Da),Ala-Ser-Phe-Ala-Ser-Met-Pro-His(846.37 Da),Arg-Pro-Lys-Pro-Pro-His(730.42 Da),Gly-Asn-Trp-Gly-Pro-Leu(642.3Da),Gln-Pro-Ala-Asn-Val-Phe(675.32 Da),Ala-Pro-Ala-Pro-Ile-Tyr(630.33 Da),Ala-Arg-Phe-Pro-Ala(560.31 Da),Leu-Ala-Ala-Cys-Gly-Glu-Phe-His(846.37 Da).The molecular weights of the 8 peptides were all less than 1000 Da,and the hydrophobic and basic amino acids related to antioxidant peptides accounted for a relatively high proportion of the peptides.