Effect Of Ultrasonic Pretreatment On Improving Lipid Lowering Activity Of Hydrolysates Of Buckwheat Globulin

Studies have found that excessive intake of high-fat diet can cause lipid metabolism disorders in the body,which can induce a variety of metabolic diseases.At present,the application of buckwheat protein and its active peptides in the prevention and treatment of metabolic diseases has become a research hotspot.However,there are still many problems,such as incomplete enzymatic hydrolysis,low efficiency of enzymatic hydrolysis and low activity of enzymatic products.In this study,buckwheat globulin was pretreated by ultrasound assisted alkaline protease hydrolysis to obtain high lipid-lowering activity of the enzymatic hydrolysate.Objective to study the lipid-lowering effect and mechanism of buckwheat globulin hydrolysate prepared by ultrasonic pretreatment on model cells at molecular and metabolic levels.The main conclusions are as follows:(1)Single factor experiment and response surface methodology were used to optimize the enzymatic hydrolysis of buckwheat globulin.The optimum conditions were obtained as follows: enzyme dosage 3.37%,p H=10,time 135 min and temperature 50℃.Furthermore,taking the degree of hydrolysis and the bile salt adsorption rate of the hydrolysate as the evaluation indexes,the buckwheat globulin was pretreated by ultrasonic wave,and the optimal conditions were obtained as follows: power 300 W,reaction time 15 min.The degree of hydrolysis of buckwheat globulin was 14.50% higher than that of the control group.The binding rate of sodium cholate and sodium taurocholate increased by 41.64% and 25.10%respectively,which indicated that ultrasonic pretreatment could effectively improve the degree of protein hydrolysis and the bile salt adsorption capacity of enzymatic hydrolysate,thus improving the lipid-lowering activity of enzymatic hydrolysate.(2)Compared with the enzymatic hydrolysate prepared by enzymatic hydrolysis alone,the enzymatic hydrolysate prepared by ultrasonic pretreatment had a better inhibitory effect on intracellular lipid accumulation,and could significantly reduce the contents of triglycerides(TG),total cholesterol(TC)and malonaldehyde(MDA)in HepG2 cells,Compared with the model group,it decreased by 51.42%,36.67% and 30.15% respectively.The change of lipid droplet content was also consistent with the decrease of TG content.In addition,the enzymatic hydrolysate after ultrasonic pretreatment could effectively improve the activity of superoxide dismutase(SOD)and total antioxidant capacity(T-AOC)in HepG2 cells,which were 35.82% and 34.93% higher than those in the model group,respectively.Moreover,the protective effect of enzymatic hydrolysate prepared by this method on oleic acid-induced oxidative damage is better than that without ultrasonic treatment.Furthermore,the transcriptional and protein levels of lipid metabolism genes in cells were measured.It was found that the hydrolysate of buckwheat globule could enhance LXRα activity,which can promote the transformation of triglyceride and reduce the synthesis of lipid.Enhancing PPARα expression could promote the growth of fatty acids β-Oxidation to enhance cell lipid metabolism.Inhibition of SREBP2 expression can promote the metabolic outflow of cholesterol and reduce the accumulation of cholesterol in cells.In conclusion,the effect of ultrasonic pretreatment followed by enzymatic hydrolysis is more obvious than that of enzymatic hydrolysis alone.(3)By multivariate statistical PLS-DA analysis,it can be seen that the normal group,model group,non-ultrasonic group and ultrasonic group are obviously distinguished,and the distribution of ultrasonic group is closer to the normal group,indicating that the intervention of buckwheat globulin hydrolysate after ultrasonic treatment can callback the changes of endogenous substances induced by oleic acid to a certain extent and make the cell state change to normal.21 kinds of different metabolites were screened out,including L-proline;O-aminobenzoic acid;L-tryptophan;Pantothenic acid,etc.The metabolites were uploaded to KEGG platform for metabolic pathway analysis.The metabolites involved 13 metabolic pathways,including tryptophan metabolism,the biosynthesis of pantothenic acid and coenzyme A and the metabolism of glycerophospholipids.Among them,tryptophan metabolism is the most important one and glycerophosphatidylcholine metabolism is the most important one.Therefore,the hydrolysate of buckwheat globulin prepared by ultrasonic pretreatment can improve the abnormal lipid metabolism of HepG2 cells by intervening the related pathways of amino acid metabolism,vitamin metabolism and phospholipid metabolism under the condition of fat accumulation.