Study On The Separation, Purification And Stability Of Anti-oxidant Peptides From Purple Kidney Bean

In this experiment,the purple bean protein was used as the research object.Two enzymes with higher hydrolysis effect were screened by single enzyme hydrolysis.The hydrolysis was used as the reference index to carry out the double enzyme mixed hydrolysis experiment.The single factor combined response surface experiment was used for data analysis.Obtain optimal hydrolysis conditions in order to obtain better hydrolysis and antioxidant activity,separate and purify by macroporous resin technology,obtain high purity active peptide,and analyze DPPH clearance by high performance liquid chromatography and first-order mass spectrometry.Rate and primary structure to determine the optimal purification process,and finally explore the effects of external conditions(pH,temperature,NaCl and simulated gastrointestinal digestion)on the antioxidant activity of scutellarin.The results are as follows:1.Enzymatic preparation of anthocyanin antioxidant peptides.Through the single-enzymatic hydrolysis of the alfalfa protein,the two enzymes with the best hydrolysis effect were selected as neutral protease and alkaline protease,and the double-enzyme simultaneous complex hydrolysis experiment was carried out.The single-factor combined orthogonal experiment was used to optimize the simultaneous hydrolysis of the two enzymes.The optimal hydrolysis conditions were as follows: temperature 55 ?,pH = 10,alkaline protease: neutral protease = 1: 2,mixed enzyme addition amount 5 mL,under which conditions the protein hydrolysis degree of the purple kidney bean was 42.2 % and the antioxidant activity was obtained under these conditions.2.Separation and preparation experiments of purple kidney bean antioxidant peptides.In this study,the following conclusions were obtained by studying the static adsorption capacity of different types of macroporous adsorption resins and the dynamic adsorption test: the optimal macroporous adsorption resin model XAD-7HP was identified,and the macroporous resin was adsorbed and desorbed on this basis.The conditions were optimized.The results showed that the optimal pH was 7.0,the optimal desorbent was 60 % ethanol solution,the optimal desorption time was 30 min,the optimal feed rate was 1 mL/min,and the feed mass was 30 mL.The elution and enrichment time of antioxidant peptides was concentrated at 10~30 min.Three high-activity antioxidant peptides were obtained by reversed-phase high performance liquid chromatography.The amino acid sequence of the three components obtained by mass spectrometry was Phe-Leu-Val-Asp-Arg-Ile,Phe-Leu-Val-Ala-Pro-Asp-Asp and Lys-Asp-Arg-Val-Ile-Ser-Glu-Leu,a significant increase in antioxidant activity compared to before purification.3.Stability test of alfalfa antioxidant peptides.The solubility of the antioxidant peptide of Alfalfa was increased first and then decreased with the increase of pH value,and the solubility reached the highest at pH 10.The DPPH scavenging ability of the antioxidant peptide of Alfalfa was greatly affected by temperature.With the increase of temperature,its DPPH scavenging ability is gradually reduced,and its antioxidant activity is stable in the environment of pH 7-8,while the anti-oxidation activity is reduced in the peracid or over-alkali environment,during storage,processing and taking.The high temperature and extreme pH environment should be avoided.With the increase of NaCl mass fraction,the DPPH scavenging ability of the purple kidney bean antioxidant peptide also showed an upward trend.In vitro simulated digestion showed that gastric juice digestion can improve the antioxidant activity of the purple bean kidney antioxidant peptide.Activity,while artificial intestinal fluid and gastrointestinal digestion alone resulted in a significant decrease in its antioxidant activity,but also maintained high biological activity.In this study,the complex enzyme system was used to hydrolyze the alfalfa protein.The macroporous resin and reverse high performance liquid chromatography were used to separate and purify the cowpea protein.The antioxidant properties,structure and amino acid sequence of the obtained cowpea protein peptide were analyzed.The stability of the antioxidant peptide of Alfalfa Bean was studied,which provided a new way for the intensive processing of the antioxidant peptide of Cowpea,which provided a reference for the production and industrial application of the antioxidant peptide of Alfalfa.