Studies On The Optimization Of Preparation Of Selenium-enriched Walnut Protein And Its Antioxidant Activity

Walnut(Juglans regia L.)belongs to the Juglans family and has medicinal effects such as nourishing qi and blood,warming the lungs and kidneys,moistening the intestines and relieving asthma.Walnut has high nutritional value and is one of the "four major dried fruits".China’s walnut output ranks first in the world,and walnuts are grown in most areas of the country.Selenium is one of the eighteen trace elements necessary for the human body.Studies have shown that selenium is closely related to dozens of diseases such as Kashin-Beck disease,cancer,and enjoys an important position in the medical field.This article uses Hubei Enshi selenium-enriched walnuts as experimental materials to explore the optimal extraction process conditions,selenium content,selenium forms and in vitro antioxidant activities of selenium-enriched walnut proteins,and analyze the process conditions of walnut proteolysis and the resistance of enzymatic hydrolysis products.Oxidation activity,and preliminary estimation of the molecular weight distribution of walnut protein and enzymatic hydrolysis products.These results provide an important theoretical basis for the development of functional products of walnut protein and natural selenium supplements.The main experimental results are as follows:1 In this experiment,alkaline solution and acid precipitation method was used to extract selenium-enriched walnut protein.With walnut protein extraction rate as the dependent variable,the effects of lye p H,extraction temperature,and liquid-to-material ratio on the extraction rate were calculated by comparison.The optimal extraction process conditions of selenium-enriched walnut protein were explored,and on this basis,response surface methodology(RSM)was used to optimize the extraction method and process parameters of walnut protein.The results show that the isoelectric point p H of selenium-enriched walnut protein is 4.5.When the lye p H is 9.0,the extraction temperature is 55°C,and the liquid-to-material ratio is 20:1(m L/g),the maximum extraction rate of walnut protein reaches56.52%.The influence of various factors on the extraction rate of selenium-enriched walnut protein is as follows: extraction temperature>liquid-to-material ratio>ale p H.The optimal process parameters obtained after the optimization of RSM method are: lye p H 8.97,extraction temperature 53.64(℃),liquid-to-material ratio 22.27(m L/g).2 Determine the total selenium,inorganic selenium and selenium forms of three walnut proteins with different selenium content.According to the level of total selenium content,the numbers are YSX(1.3345±0.0090mg/kg),LCX(0.6219±0.0090mg/kg),SCX(0.4219±0.0221mg/kg).The selenium form in selenium-enriched walnut protein was determined by liquid phase-hydride atomic fluorescence spectrometry(LC-HG-AFS),and the in vitro antioxidant activity of walnut protein with different selenium content was compared.The results show that the selenium in the selenium-enriched walnut samples used in this experiment mainly exists in two forms: selenocystine(Se Cys2)and selenomethionine(Se Met).Within the concentration range set in the experiment,the removal rate of selenium-enriched walnut protein solution to DPPH· and·OH is proportional to the mass concentration of the sample,and the maximum removal rate can reach 69.57%(DPPH·)and46.54%(·OH),respectively.The in vitro antioxidant activity is as follows: Vc>high selenium walnut protein>medium selenium walnut protein>low selenium walnut protein,indicating that selenium enrichment can enhance the in vitro antioxidant activity of walnut protein to a certain extent.3 Using the degree of hydrolysis as an indicator,under the action of alkaline protease,the p H of the enzymatic hydrolysis,the temperature of the enzymatic hydrolysis,and the time of the enzymatic hydrolysis on the degree of hydrolysis(DH%)were explored,and the in vitro resistance of the enzymatic hydrolysate was compared.Oxidation activity.Using SDS-PAGE gel electrophoresis,the relative molecular weight distribution of selenium-enriched walnut protein was analyzed preliminarily.The results showed that the optimal process conditions for enzymatic hydrolysis of walnut protein were: enzyme addition 4000U/g,enzymatic hydrolysis p H 10.0,enzymatic hydrolysis temperature 45℃,enzymatic hydrolysis duration 4 hours.There are 7 electrophoresis strips of walnut protein extracted by alkaline solution,and the molecular weight is mainly distributed between 17-72 k Da.After enzymolysis,the content of components with molecular weight of 5-15 k Da increases.Within a certain concentration range,the removal rate of selenium-enriched walnut peptide solution to DPPH· and ·OH is proportional to the concentration of the sample,and the removal rate is as high as 70.67%(DPPH·)and 62.98%(·OH),indicating that the molecular weight is relatively high.Small walnut peptides have better antioxidant activity than walnut protein.