Effects Of Different Treatments On Functional Activity And Desensitization Of Buckwheat Protein

Many studies have found that excessive intake of high-fat diet will lead to lipid accumulation in the body,causing lipid metabolism disorder,and increasing the risk of suffering from atherosclerosis,ketosis,obesity,hypertension and other metabolic diseases.Buckwheat is a fake grain that is rich in nutrients such as starch,protein,vitamins,and dietary fiber.Buckwheat protein has potential biological activities such as lowering blood sugar and regulating blood lipids,and can enhance the body’s immune system.However,current research has shown that buckwheat protein may cause allergic reactions,which has become a major constraint on the development of functional buckwheat products.The existing research shows that fermentation,enzymolysis and other methods can effectively degrade the protein to form small molecular peptides,so that the protein can obtain higher biological activity,and can also degrade the target allergic protein to achieve the purpose of desensitization.However,due to the problems of incomplete protease hydrolysis,low efficiency and many other problems,it is appropriate to use physical methods to carry out auxiliary enzymolysis treatment,modify the buckwheat protein,and expose more sites,Thus improving the hydrolysis effect of proteases.This article takes untreated buckwheat protein as the research object,and utilizes fermentation of buckwheat powder by Streptococcus pentosus,alkaline protease enzymatic hydrolysis,ultrasound,and ultra-high pressure physical technology to assist enzymatic hydrolysis to obtain buckwheat protein degradation products without obvious allergens.A Hep G2 hyperlipidemic cell model is established using a certain concentration of oleic acid induction,and its lipid-lowering effect is verified at the cellular level.The main research conclusions are as follows:(1)Using a single factor experiment and using the degree of hydrolysis of buckwheat protein as an evaluation index,the optimal enzymatic hydrolysis conditions for alkaline protease were obtained: time 120 minutes,enzyme addition of 3.0%,temperature 50 ℃,and p H=10;Subsequently,buckwheat protein was treated with ultra-high pressure and ultrasonic assisted enzymatic hydrolysis,and the optimal process conditions were obtained: ultrasonic power of 300 W,working time of 15 minutes,high pressure of 500 Mpa,and working time of15 minutes.At this time,the hydrolysis degree of buckwheat protein was significantly increased compared to the single enzyme hydrolysis treatment group.Using molecular weight distribution as an evaluation indicator for fermentation time,the optimal fermentation time was determined to be 16 hours.This indicates that pre-treatment can degrade buckwheat protein,and physical assisted enzymatic hydrolysis has a stronger protein degradation effect than single enzymatic hydrolysis or single bacterial fermentation.Subsequently,the in vitro lipid-lowering ability of buckwheat protein degradation products was evaluated through bile salt adsorption ability.Finally,Western blot experiments were used to evaluate the desensitization level of buckwheat,and it was found that the combination of ultra-high pressure and enzymatic hydrolysis of buckwheat protein has a better desensitization effect.(2)Using low concentration oleic acid to establish a high-fat Hep G2 cell model,compared to protein products prepared without pretreatment,the degradation products prepared with auxiliary treatment have better lipid-lowering activity,and the combination of ultra-high pressure and enzymatic hydrolysis has the best lipid-lowering activity,which can significantly reduce the content of triglycerides(TG)and cholesterol(TC)in high-fat cells,and can effectively protect and repair oxidative damage in high-fat cells,It can significantly inhibit the generation of malondialdehyde(MDA);Promote the increase of superoxide dismutase(SOD)activity and total antioxidant capacity(T-AOC).Subsequently,through oil red O staining experiments,changes in lipid droplet aggregation content in different groups of cells can be observed,and the results of these changes are consistent with the reduction in lipid indicators mentioned above.Using molecular biology techniques to further evaluate the transcription and protein levels of lipid metabolism genes in cell models,the results showed that buckwheat protein degradation products obtained through high-pressure assisted enzymatic hydrolysis can better enhance LXR α,PPAR α Gene expression and inhibition of SREBP2 expression can reduce fat aggregation,accelerate fat degradation,and achieve the goal of regulating lipid metabolism disorders.(3)Using liquid chromatography(LC-MS)for experimental design and cell metabolomics analysis,the experimental results were compared between different groups.It was found that the normal group,model group,untreated group,fermentation group,enzymatic hydrolysis group,ultrasound group,and ultra-high pressure combined enzymatic hydrolysis group were clearly distinguished,and the ultra-high pressure combined enzymatic hydrolysis group was closer to the normal group.This result indicates that the ultra-high pressure combined enzymatic hydrolysis group can make the cell state more normal.Among them,there are 24 landmark differential metabolites: niacin,linoleic acid,phospholipids;Various substances such as amino acids.Subsequently,KEGG platform validation analysis was conducted on relevant lipid metabolism pathways,and it was found that Hep G2 cells mainly involve 12 lipid metabolism pathways,among which nicotinic acid and nicotinamide metabolism have the highest metabolic contribution values.Therefore,the buckwheat protein degradation product prepared by ultra-high pressure combined with enzymatic hydrolysis can effectively regulate the lipid metabolism disorder in Hep G2 cells by regulating the amino acid,vitamin,phospholipid and other related lipid metabolism pathways,thereby exerting its functional activity and achieving the effect of lipid-lowering.