Microbial Expression And High-Density Fermentation Optimization Of Umami Peptides

Umami peptides endow food with the characteristic taste and bring the pleasant feeling for people.Due to the advantages of"green,nutrition,health",umami peptides have been widely concerned by people and show the great potential of market.In recent years,the focus of research on umami peptides are mainly in extraction and identification,sequence structure and taste mechanism,while the high density fermentation of umami peptides are rarely reported.At present,the production methods of umami peptides are three methods:chemical synthesis,enzymatic extraction and microbial expression,the former two methods belong to the traditional method of preparing umami peptides,which are not suitable for industrial production due to their high costs,low yields,complicated operations,potential toxicity of reagents and other factors.The production of umami peptides by microbial expression method is more obvious than traditional methods,which has low cost and high yield.While there is less research on how to prepare umami peptides efficiently and safely by using this method,especially the production of high cell density fermentation is still in the preliminary exploration stage.In this study,Beefy Meaty Peptide?BMP?obtained from digesting beef hydrolysate is used as the research object to construct the microbial expression system that meets food safety,while exploring the culture conditions and control conditions of high cell density fermentation to obtain an efficient and safe preparation process of umami peptides.The specific research processes are as follows:Firstly,the“generally recognized as safe”Bacillus subtilis?B.subtilis 168?and the cell density-dependent high-efficiency expression promoter P_srfA were chosen as the host expression bacteria and the expression promoter of the umami peptide respectively,which to avoid the inducers in the production of umami peptides potential risks.The 6×His tags is added to the C-terminal of the umami peptide chain to obtain high-purity multi-copy tandem umami peptide BMP?8BMP?.Finally,the self-induced expression host strain of B.subtilis168/pMA09srfA-8BMP was constructed and efficiently expressed 8BMP,which is in line with food safety.The fermentation medium and fermentation control conditions were investigated at the shake flask level in order to increase the expression level of umami peptide 8BMP,and the optimal culture conditions for fermentation were determined as:the carbon source is glucose with a concentration of 20 g/L,the concentration of nitrogen source?NH₄⁺?is 0.25 mol/L,the pH value is 7,the inoculation amount is 6%,and the culture temperature is 37?.Under this optimal culture conditions,the dry cell weight of bacteria and the yield of umami peptide8BMP reached 3.32 g/L and 0.172 g/L,respectively.Finally,based on the optimized culture conditions of the shake flask,the high cell density fermentation mode and control conditions of 8BMP were explored.Firstly,the batch fermentation experiment was carried out,and the yield of the umami peptide was 6 times that of the shake flask level.While in the late fermentation period,there was an unfavorable situation that the glucose content in the fermentation broth was completely consumed,resulting in bacterial growth and product expression stop.In order to further increase the expression of umami taste peptides and solve the problems of batch fermentation,two different feeding modes of constant-rate feeding cultivation and two-stage feeding cultivation?exponential feeding cultivation and constant-rate feeding cultivation?are used to further control the feeding of high-density fermentation.The results showed that the yield of umami peptides was significantly improved,which were 1.6 times and 2.4 times of batch fermentation,respectively.However,in the middle-late stage of feeding,the excessively high glucose concentration in the medium produces substrate inhibition,which affects the growth of the cells and the expression of the product.Therefore,the Pontryagin maximum principle is used to optimize the feeding process and established a more in line with the actual growth of the bacterial cells feeding model to solve the situation of excessive feeding.During the entire fermentation process,the accumulation of residual sugar is low,and the final yield of umami peptide is 3.16 g/L,which is 2.8 times that of batch fermentation.The results show that the optimized feeding process control based on the Pontryagin maximum principle is conducive to the formation of umami peptides,and provides a reference for the efficient and safe production of other taste peptides.