Purification Of Soybean Paste Oligopeptides And Transport In Cell Model

During the fermentation of soy sauce, small molecule proteins(called soy sauceoligopeptide) obtained by soy protein enzymatic hydrolysis by microbial effect, which had avariety of biological activity. In this study, the oligopeptides which have higher antioxidantactivity were extracted and purified from soy sauce by the method of ultrafiltration separation,gel chromatography, ion chromatography and other separation methods. And its structure wasdetermined by mass spectrometry. According to the result of determination, low poly peptideswere being synthesis. Research the differences of the synthetic oligopeptides activity betweenbefore and after digestion in simulated conditions of human intestinal tract. And in Caco-2cellmodel, the mechanism of absorption of low poly peptide in the human body was studied. Themain research contents were as follows.Firstly, this study separated the oligopeptide from the soy sauce mixture with ultrafiltrationprocess and freeze-dried. Obtain the optimum conditions were sample DEAE ion exchange resinon the optimum pH being8.6, dextran gel G-15separate optimal flow rate being2mL min-1, thesample concentration being1.2mg· mL-1, and the high oxidation activity of two groups of soysauce low poly peptide molecular weight ranging from925Da to1124Da, and from580Da to925Da.Secondly, the sequence of oligopeptide components with highly activity was determined byMALDI mass spectrometry, the sequence of three groups were GV-9(GVPSRMSSK), RN-9(RNGSDAGGV) and VR-7(VRGSPSS), respectively.Thirdly, under the body temperature environment which was based mimic humangastrointestinal digestion as external environment, three kinds of synthetic peptide which wereGV-9, RN-9, VR-7respectively were through simulation of the digestive juice processing, andthen were determined the antioxidant index. Comparing them to the activity of synthetic peptidewhich were not dealed with the simulation of digestive juice processing, it was found that tjerewas no obvious differences between hydrolysis activity of soy sauce low poly peptide dealingwitn digestive juice processing and hydrolysis activity of soy sauce low poly peptide not dealingwitn digestive juice processing, the formen was slightly high.This shows that three peptides canbe used as antioxidant compounds used by human because of not concerning the digestive damaging structure.in the bodyFourthly, the model of human cells was establishment and validation.The growth of formswhich was Caco-2cell cultured on the poly ester of carbon membrane after21d were similar toon the small intestinal epithelial cells. The transepithelial electrical resistance value conformed tothe requirements. The cells could produce polarization which could be used as a simulation ofthe small intestine absorption in vitro model for synthesis of peptide transport experimentresearch.Lastly, research the absorption of the three synthetic peptides (GV-9, RN-9and VR-7)from soy sauce by Caco-2cell model. The transhipment amount of GV-9, RN-9and VR-7showed linearly increase under the condition of same concentration before120min. After theGV-9, RN-9and VR-7were transported, the values of Papp were all greater than1x10-6cms-1. So the three peptides in the small intestine had higher bioavailability and better oralabsorption effect. The Pratio of GV-9, RN-9and VR-7were smaller than1.5which couldpreliminary judge that the main transfer process was passive transport,and may also were activetransport.