Isolation, Purification, And Identification Of Vasoactive Small Peptides From Soybean Meal

Food-derived foods with specific functions have been paid attention to by modern people due to their purely natural source properties,while supplementing basic nutrients,while also benefiting physical health,increasing resistance,lowering blood pressure,or relaxing blood vessels.As a readily available high-quality protein,soy protein products are often used to make nutraceutical products and are very popular,the market prospects are broad.For example,infant formula milk uses a large amount of fully hydrolyzed soybean decomposition amino acids.Soybean meal is a by-product of soybean crush oil.It is rich in soy protein and less fat.It is easy to obtain in China with abundant resources and low prices.Although its nutritional value is high,there is a low utilization rate.Therefore,the soybean vasoactive small peptides were prepared by using low cost natural soybean meal,which not only obtained high value-added soybean hydrolyzed protein products,but also achieved secondary recycling of soybean processing by-products,resulting in non-toxic side effects,high safety and biologically functional soybean active peptides,and circumvent long toxicological experiments in the development of functional foods.Soybean meal is degraded by fermentation through the action of microorganisms.The contained protein has a smaller molecular weight and nutrition is more easily absorbed.It is expected that it can also be used for advanced feed of aquatic products,and the market prospect is excellent.Therefore,this paper mainly uses soybean meal as raw material to ferment and specifically hydrolyze soybean meal protein with specific proteins,purifies the extracts of fermented soybean meal,and isolates peptides from small molecule proteins and peptide mixtures.The identification of sequence and the screening of its vasoactive activity aim at obtaining small vasoactive peptides of soybean,and its vasoactive effect is discussed,which provides a theoretical and technical basis for the industrial production of soy protein vasoactive small peptides.In this paper,protease-producing strains were firstly screened from Shanxi folk douchi,and 9 protease-producing strains were screened out by the comparison of transparent circle size,among which Y2,Y3,Y4,Y5,Y6,and Y7 were large.The 6strains were further screened,with the comparison of protease activity.The Y4 protease activity was the highest.The Y4 gene sequence was identified by 16S rRNA.The gene sequence was submitted to the GenBank database,accession number:KY419575.1,comparison by GenBank data and build phylogenetic tree,Y4 and Bacillus cereus were at the same branch,the similarity reached 98%.Soybean meal protein was decomposed with using microbial fermentation method.Soybean meal was fermented by the Y4 with the highest protease activity selected in this paper Chapter 2.After fermentation,the crude extract of soybean meal was obtained by water.The crude extract is filtered through two times 8 layers of gauze,static sedimentation,activated carbon and DA201-C macroporous resin adsorption,400mesh filter bags,plate filter to obtain a more clarified fermentation hydrolyzate,then filtered with the 10,000,1000,500 Da ultrafiltration membrane to obtain three polypeptide mixture components Fl（10000 Da）,F2（1000 Da）,and F3（500 Da）,which were concentrated and dried.The ACE inhibitory activity of the F1,F2,and F3 components were 28.78%,38.98%,and 40.40%,respectively.The highest was F3,was selected as the target component for isolation and purification.The F3 components were separated by gel filtration chromatography and four peaks P1,P2,P3,and P4 were separated and collected.The P1,P2,P3,and P4 were separated and purified again by two HPLC.A total of six single peak substances were isolated and their sequence structures were identified with PPSQ-21 and MALDI-TOF-TOF/MS.These small peptides were GPANV,PAIV,CQ,QC,CGAAP,HAGR.It can be seen that these peptides are 2⁵amino acid sequences.The vascular activity of experimental results showed that F1 and F3 components had vasodilating activity,EC₅₀ values were 5.22mmol/L,2.37mmol/L,F2 was without vasodilating activity,on the contrary with vasoconstriction activity,the same results were found in both high and low dose experiments.F1 and F3 components showed strong vasodilating activity regardless of dose level.The vasoactive assay results of 6small peptides isolated and purified from F3 showed different effects,GPANV and PANV showed higher vascular activity with EC₅₀ values of 0.42 mmol/L and 0.72mmol/L,respectively.The peptides showed no vasodilating activity of CQ and with vasoconstriction activity.The results of ACE inhibitory activity assay showed that these 6 small peptides showed different ACE inhibitory activities,which the ACE inhibitory activities of QC,CGAAP,and CQ were much higher than F3 components（40.40%）,the inhibition activities were 80.66%,79.77%,and 76.17%,respectively.Other small peptides showed lower ACE inhibition activities,the lowest was YN,the ACE inhibition activities was only 12.19%.From the data of vasoactive activity and ACE inhibition activity,small peptides with high ACE inhibitory activity do not necessarily show strong vasoactive activity,and the strength of vasoactive activity is not related to ACE inhibition activity.There is no direct correlation between them.perhaps,this has other potential mechanisms of action,and its structural-functional mechanism studies need to be confirmed by subsequent further experimental studies.