Studies On The Purification,identification,and Hypoglycemic Activity Of Peptides From Chinese Rice Wine

Type 2 diabetes is one of the most common and serious metabolic diseases.Diet plays an important role in the occurrence and development of type 2 diabetes.Chinese rice wine(CRW),also called rice wine or yellow wine,is widely distributed in various regions of China and is the brewed wine with the highest peptide content.This thesis analyzed the physical and chemical properties of the peptides in CRW from different regions;the CRW peptides were separated and purified by cation exchange chromatography and reverse high performance liquid chromatography,and the amino acids sequence of peptides were identify by liquid chromatography tandem-mass spectrometry(LC-MS/MS);glucose uptake promoting activity of CRW peptides was assessed using rat L6 myoblast model,and Western blot was used to reveal the mechanism of the hypoglycemic activity of CRW peptide from the protein level.The main findings are as follows:(1)Studies on the physicochemical properties of rice wine peptides from different regions and evaluation of the glucose uptake promoting activityUsing peptide content,molecular weight distribution and amino acid composition as indicators,the physical and chemical properties of rice wine peptides from different regions were studied.It was found that the peptide content of rice wine was 4.53～9.06g/L,and the peptide molecular weight is mainly distributed in 200～1500 Da,and contained a comprehensive range of amino acids.Using L6 myoblast model,it was found that the Shaoxing rice wine peptide extract had the strongest glucose uptake promoting activity in L6 cells.(2)The establishment of peptide profiles of rice wine from different regionsPeptomics technology was used to identify the amino acid sequences of rice wine peptides and analyze the sources of peptides,and then comparing the unique peptides and common peptides of rice wine from different regions.The results showed that 325,172,149 and 62 peptides were identified from Shaoxing,Fujian,Hubei,and Guangdong rice wine peptide extracts.More than 75% of the rice wine peptides were derived from proteins of rice,wheat,and yeast.Rice wine peptides in different regions crossed each other,DIVALPAGVAH,GVLRPGQL,VLRPGQL,IGGIGTVPVGR,VNIENPSR were the common peptides of rice wine in Shaoxing,Fujian,Hubei and Guangdong.(3)Studies on separation,purification and hypoglycemic activity of Shaoxing Rice wine peptidesThe peptide extracts of Shaoxing rice wine were separated and purified by cation exchange chromatography and reversed high performance liquid chromatography to obtain the fractions F1-2,F1-6 and F1-7 with the strongest ability to promote glucose uptake in L6 cells.Western blot was further used to detect the effect of peptide fractions F1-2,F1-6 and F1-7 on the activation of Akt,AMPK,MAPK signal pathways and GLU4 translocation.The results showed that the components F1-2,F1-6 and F1-7 F1-7 significantly activated AMPK pathway,p38 MAPK pathway and p44/42 MAPK pathway,and F1-2 significantly promoted GLUT4 translocation.(4)Studies on the sequence identification and hypoglycemic mechanism of Shaoxing rice wine peptidesMass spectrometry technology was used to identify the peptide sequences of the active fractions.The results showed that a total of 108 peptides were identified from fractions F1-2,F1-6 and F1-7.In the base peak chromatogram,there are 17 peptides with a relative intensity higher than 40%.Peptides with the strongest glucose uptake promoting activity among the 17 peptides were found to be VYVPPE and LQPGQGQPGYD.Western blot was further used to detect the effects of peptides VYVPPE and LQPGQGQPGYD on the activation of Akt,AMPK,and MAPK signaling pathways and GLU4 translocation,and pathway inhibitors were used to clarify the key signaling pathways.Studies showed that VYVPPE and LQPGQGQPGYD activated AMPK and MAPK signaling pathways by increasing the phosphorylation level of AMPK protein,p38 protein and/or p44/42 protein,and promote the translocation of GLUT4,thereby promoting glucose uptake in L6 cells.In summary,the rice wine peptide extracts and its main single peptide had the ability to promote glucose uptake in L6 myoblasts.Activating the phosphorylation of key proteins in AMPK and MAPK signaling pathways and promoting the translocation of GLUT4 were the key signaling pathways for rice wine peptides to promote glucose uptake.The results of this thesis confirmed that rice wine peptides had potential hypoglycemic effects,which not only enriched the activity of rice wine peptides,but also provided a theoretical basis for the research of peptides in rice wine from different regions.