Study On Peptide Preparation By Bacillus Subtilis 10160 Liquid Fermentation Of Soybean Meal Strengthened By Auxiliary Factors

Soy bean meal?SBM?,a by-product after extraction of soybean oil,contains a high level of protein?45%-55%?.However its utilization rate is very low.SBM is a good quality plant protein resource with high cost performance.Making full use of SBM rationally to prepare functional proteins and peptides can greatly improve its value.Based on the content and yield of peptides as well as the previous studies on seed culture and fermentation media,a single factor design combined with orthogonal optimization tests were performed in this study.The liquid fermentation performances of SBM strengthened by the exogenous enzyme and mixed strains were studied.Furthermore,the atmospheric and room temperature plasma?ARTP?mutagenesis technology was used to improve the ability of protease by mutagenizing Bacillus subtilis 10160.The main conclusions are shown as follows:?1?The objective of this study was to prepare high content peptides from SBM.The optimum conditions of SBM fermentation were as follows:20%of soybean meal,0.5%of KH₂PO₄.1%of corn flour,10%of inoculum,32? of fermentation temperature,initial pH of 5.8,and 24 h of fermentation time.Under this condition,the yield and content of peptides were 12.15%and 56.26%,respectively.The analysis results of the fermentation product showed that neutral protease was the main hydrolase when the peptide was produced by SBM liquid fermentation,and the fermentation products contained a high ACE inhibition rate.Meanwhile,the proportion of peptides below 1000 Da was as high as 67.15%.It was concluded that high-content and high-activity peptides can be prepared by SBM liquid fermentation by Bacillus subtilis 10160 and the utilization value of soybean meal was greatly increased.?2?The optimum conditions of SBM liquid fermentation strengthened by strain and exogenous enzyme synergistic fermentation were that the Bacillus subtilis was fermented for 12 h and then alkaline protease was added and fermented for 12 h?enzyme content of 2,600 U/g and fermentation temperature of 36??.Under this condition,the yield and content peptides were 18.89%and 75.21%,respectively.Compared to the result of the non-enzymatic single strain fermentation in the Chapter2,the peptide yield,content,ACE inhibition rate,and neutral protease and alkaline protease activities were significantly increased by 55.47%,33.68%,11.27%,39.44%,and 128.06%,respectively.In addition,the proportion of high-activity peptide fragments?molecular weight less than 1000 Da?was significantly increased by13.83%.?3?The optimum conditions of SBM liquid fermentation strengthened by mixed strains were that the Bacillus subtilis 10160 was fermented for 12 h and then Bacillus subtilis 20030 was added and fermented for 12 h under the inoculum of 15%,inoculation ratio of 2:1,and fermentation temperature of 36?.Under this condition,the yield and content peptides were 12.35%and 58.32%,respectively.Compared to the result of the single strain fermentation in the Chapter 2,the peptide content and alkaline protease activity were significantly increased by 3.66%and 19.02%,respectively.However the peptide yield,neutral protease activity,and ACE inhibition rate did not change significantly.The proportion of peptides?molecular weight less than 5000 Da?of the mixed strain fermentation was almost the same as that of the single bacteria fermentation.?4?Bacillus subtilis 10160 was treated by ARTP.Under the optimum mutagenesis dose of lethality 80%,the ARTP mutagenesis time was 10 s.A stable mutant strain of C12 was screened.Compared with the original strain,the neutral protease activity of C12 strain was increased by 85.0%.The proteins extracted from C12 and the original strains were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis?SDS-PAGE?.It was observed that the number of protein bands did not change,but the protein concentration increased after mutagenesis treatment.Two-dimensional electrophoresis?2-DE?results showed that eight differential protein spots were identified in which there were six up-regulated proteins and two down-regulated proteins.After the identification of differential proteins by MALDI-TOF-TOF MS and KEGG pathway analysis,three up-regulated proteins?pyruvate dehydrogenase,butanediol dehydrogenase,and fructose-bisphosphate aldolase,class II?were involved in the tricarboxylic acid cycle,butanoate metabolism,and glycolysis metabolim.It was concluded that the metabolism of Bacillus subtilis 10160 was accelerated by the ARTP mutagenesis,resulting in the increased protease activity.Compared to the result of the non-enzymatic single strain fermentation in the Chapter 2,the yield and content of peptides and the neutral protease activity in the fermentation broths of C12 strain were significantly increased by 11.77%,13.24%,and 29.84%,respectively.However there were no significant differences in the molecular weight distribution and ACE inhibition rate.It was concluded that ARTP treatment of Bacillus subtilis 10160 can significantly increase the yield and content of peptides produced by liquid fermentation of soybean meal.