Optimization Of Fermentation Conditions And Isolation And Purification Of Bacillomycin D In Bacillus Subtilis G70

Bacillus subtilis,one of thermophilic,aerobic and spore-producing Gram-positive bacteria,grows faster,survives easier,and produces a variety of bioactive substances that are non-toxic to humans and animals.Antibacterial lipopeptide is a kind of low molecular peptide with high-efficiency broad-spectrum antibacterial activity synthesized.Antibacterial lipopetide has broad application prospects in food processing and storage,agricultural biological control and drug development.Therefore,strengthening the research on increasing the production of antimicrobial peptides and reducing the cost of separation and purification of products have important practical significance for the industrial application of antimicrobial peptides.Bacillomycin D is an antibacterial lipopeptide catalyzed by Bacillus subtilis non-ribosomal polypetide synthetase,which has a strong inhibitory effect on pathogenic fungi such as Aspergillus flavus,Fusarium graminearum,Alternaria and Candida.Therefore,Bacillomycin D has great potential in food or food processing,antisepsis and preservation.However,the yield of Bacillomycin D is low,while the cost of industrial separation and purification of Bacillomycin D is high.Therefore,optimizing the fermentation conditions of Bacillomycin D to increase the yield of Bacillomycin D production,and establishing an efficient and simple industrial separation and purification method,is the key problem to be solved in the current research of Bacillomycin D.In this study,the production of Bacillomycin D was improved by optimizing the medium and conditions of fermentation in Bacillus subtilis G70.Further,the industrial separation and purification process of Bacillomycin D was set up.The main results are as following:1?Optimization of the medium and conditions of fermentation for producing Bacillomycin D in Bacillus subtilis G70.The medium composition and fermentation conditions of Bacillus subtilis G70 were optimized by single factor test,Plackett-Burman test and Box-Behnken response surface test.The optimal fermentation medium and fermentation conditions were as follows:23.01 g/L sucrose,10.38 g/L Sodium glutamate,4.00 g/L corn syrup,1.0 g/L Potassium dihydrogen phosphate,0.32 g/L magnesium sulfate heptahydrate,initial medium pH was 7.0.The optimal fermentation conditions were as follows:inoculation amount 4%,fermentation temperature 33?,shaking speed 180 rpm,fermentation time 72 h.Under this condition,the yield of Bacillomycin D was 935.18 mg/L,and the predicted value was 929.30 mg/L,indicating that the regression model could predict the yield of Bacillomycin D.By optimizing the medium and conditions of fermentation,the production of Bacillomycin D was doubled compared with the original Landy medium.The yield of Bacillomycin D in fermentor was 1271.34 mg/L,which was 1.36 times larger than the yield in the shake flask.2?Isolation and purification of Bacillomycin D.First,we diluted the fermentation broth three times and adjusted pH to 4.0.Then we added 0.6 g/L chitosan and 0.3 g/L sodium alginate.After stayed for 30 minutes,2 g diatomite was added for suction filtration.At the end of filtering,we cleaned filter cake by 50%alcohol and repeated the filtration process three times.Under these conditions,the Bacillomycin D recovery rate was 92.12±0.34%,the purity of Bacillomycin D was 72.3±0.53%.