Preparation,Purification,Structural Identification And Mechanism Of Lipid-lowering And Anti-obesity Activities Of The Peptides Derived From Hazelnut (Corylus Heterophylla Fisch)

In recent years,with the continuous improvement of people’s living standards,the diet structure has undergone tremendous changes.Especially the excessive intake of various high-fat and high-suger foods,causing abnormal lipid metabolism,which leads to the incidence of obesity,atherosclerosis,cancer and cardiovascular diseases has increased year by year,seriously endangering human health.At present,the common diet pills in the market are not effective and have many side effects,causing a variety of adverse physiological reactions in the human body.Therefore,it is of great practical significance to explore a natural,safe and effective lipid-lowering and anti-obesity homologous of medicine and food product.Hazelnuts are abundant in China and Changbai Mountain area of Jilin is one of the major producing regions.Hazelnut have good edible and medicinal value,rich in nutrients,total protein content of about 20%,8 kinds of essential amino acids and it is a source of high quality natural vegetable protein active peptide.Based on this,this study used Changbai Mountain wild hazelnut protein as a raw material.Firstly,its enzymatic hydrolysis process was optimized by single factor and response surface method.Then,the separation,purification and structural identification of the active peptides were carried out through ultrafiltration,gel chromatography,reversed-phase high performance liquid chromatography and flight mass spectrometry.Finally,the lipid-lowering activity and the mechanism of action of the synthesized amino acid peptides were studied.The results are as follows:The five groups of single factor experiments and three factors and three levels of response surface test were carried out to determine the optimal conditions for the hydrolysis of hezalnut protein by Alcalase 2.4L.Optimal conditions: substrate concentration 2%,enzymatic hydrolysis temperature 54 ℃,enzyme amount 10422 U / g,pH 9.4,enzymatic hydrolysis time 150 min.The hazelnut protein was hydrolyzed under these conditions,and the degree of hydrolysis was 36.42%,and the pancreatic lipase inhibition rate was 42.97%,which was close to the predicted value.The pancreatic lipase inhibition rate,cholesterol micelle adsorption rate and lipid level after differentiation induced by 3T3-L1 preadipocytes were used as indicators.The separation,purification and structure identification of the lipid-lowering active peptides of hazelnut protein were carried out by ultrafiltration,Sephadex G-25,Sephadex G-15,reversed-phase high performance liquid chromatography(PR-HPLC)and mass spectrometry.The hazelnut protein hydrolysate was ultrafiltered to obtain the fraction A3(<3 kDa)with the best lipid-lowering activity.The C3 fraction was further purified by reversed-phase high performance liquid chromatography and mass spectrometry to screen 18 peptides for solidpjase synthesis.Three peptides,named Rrg-Leu-Leu-Pro-His(RLLPH)(38.16%)、Val-Leu-Leu-Rrg-Leu(VLLRL)(20.14%)、Gly-Val-Leu-Leu-Rrg-Leu(GVLLRL)(23.16%)and Phe-Leu-Leu-Pro-His(FLLPH)(26.31%),with more than 20% of total lipid formation were obtained by cellular assays.Based on the AMPK/ACC cell signaling pathway,we studied the lipid-lowering mechanism of the synthetic peptide RLLPH.The results showed that RLLPH attenuated adipogenesis via downregulating the mRNA expression levels of several adipogenesis-related factors and enzymes including PPARγ,C/EBPα,aP2,SREBP-1c,FAS,ACC1,and HMGCR in a dose-dependent manner.Moreover,RLLPH also up-regulated the levels of phosphorylated AMPK and its substrate ACC in 3T3-L1 adipocytes.This study clarified the lipid-lowering and anti-obesity activity of peptide origining from hazelnut protein and initially identified its possible mechanism system.It provides an important theoretical reference for the deep development and comprehensive utilization of wild hazelnut resources in Changbai Mountain of China.