Optimization Of Fermentation Conditions And Separation And Purification Of Exopolysaccharides From Bacillus Subtilis

Microbial exopolysaccharides(EPS)are water-soluble polysaccharides that are secreted to the outside of the cell wall(environment)during the process of growth and metabolism,and are easily separated from the bacteria.They belong to the secondary metabolites of microorganisms and can be dissolved or dispersed.In water,it has thickening effect and colloidal properties,and can be used in combination with various salts to form a useful film.It exhibits high viscosity,pseudoplasticity,and thermal stability under low concentrations.It has unique physics and rheological properties and can be used as a microbial flocculant,film former,preservative,emulsifier,antioxidant,etc.,and is widely used in food,cosmetics,chemical and petroleum extraction and other fields.The Bacillus subtilis is a food-grade industrial production strain,which is safer than other bacteria,but its production is still low,which is a key factor restricting its industrial production.Therefore,it is hoped that by solving the problem of efficient production of extracellular polysaccharides of Bacillus subtilis,Bacillus subtilis exopolysaccharides will become a new source of future food-grade polysaccharides.The aim of this study was to optimize the fermentation conditions of Bacillus subtilis exopolysaccharides,and to isolate and purify extracellular polysaccharides with higher purity,and to provide a feasible process route for the final industrial production of Bacillus subtilis exopolysaccharides.In this study,EPS fermentation technology was systematically studied for the purpose of increasing EPS production.A Bacillus subtilis strain NS-18 preserved in the laboratory was used as the starting strain.The optimum medium composition and culture conditions were obtained by single factor optimization experiments;the important factors influencing EPS production were screened by PB experiments;and the response surface was evaluated by Box-Behnken.The experiment optimized the optimal combination of fermentation process;the feeding process and fermentation method were further optimized.Finally,the EPS was separated and purified,and a high-purity EPS product was obtained by freeze-drying and identified.The main results of this study are summarized below:1.Through the single factor optimization experiments,PB design and response surface optimization experiments,the optimum fermentation medium and fermentation conditions for the high-yield strains were determined on the shaker level.This technology was used to incubate at the level of 250 ml shake flasks for 168 h,EPS.The actual output can reach 3.48 g/L.2.After the fermentation feeding process was finally optimized,it was cultured on a 2.5 L shake flask level for 120 h.The EPS yield could reach 8.26 g/L.3.The EPS was isolated,purified and freeze-dried to obtain a pale yellow viscous polysaccharide with a final yield of 22.64%and a purity of 90.6%.Samples were analyzed by thin layer chromatography and found in the four monosaccharide components of the standard(glucose,galactose,xylose,arabinose,rhamnose,mannose,fructose):glucose,arabinose,mannose With galactose almost at the same level,it is speculated that the polysaccharide may contain the above four monosaccharide components,which is basically consistent with the literature reports.The production of microbial exopolysaccharides is a major problem that hinders its industrial application.Increasing the yield of extracellular polysaccharides from Bacillus subtilis is of reference significance for the industrial production of more microbial exopolysaccharides.Compared with other similar studies,the yield of Bacillus subtilis NS-18 strain after system optimization is about 100%higher,reaching 8.26 g/L,showing a high yield advantage,which is of great significance for industrial production.