| The secondary antibacterial metabolites produced by Bacillus amyloliquefaciens have a certain broad-spectrum antibacterial effect.Therefore,Bacillus amyloliquefaciens can not only be used as probiotics in the feed industry,but also be used as biocontrol bacteria in the preservation of fruits and vegetables.In this study,probiotics with antibacterial effect on common pathogenic bacteria were screened.Firstly,it was confirmed by rescreening that they were antibacterial secondary metabolites produced by the strain.Then,the related physical and chemical properties and biological characteristics of antibacterial substances were studied.Finally,the fermentation conditions and nutrient components of medium for producing antibacterial substances were optimized,which was of great significance for future application in feed fermentation and fruit and vegetable preservation.Twelve lactic acid bacterias and nine Bacillus strains with inhibitory effect on Escherichia coli were preliminarily screened from the samples of traditional fermented foods in Guangxi,such as sour bamboo shoots,sour pepper,sour water,characteristic fruit mandarin,and intestinal tract of cultured animals.After eliminating the influence of organic acids,the antibacterial effect of lactic acid bacteria disappeared,and after eliminating the influence of hydrogen peroxide,the remaining 5 strains of Bacillus with strong antibacterial effect.A strain with the strongest inhibitory effect was selected for morphological observation,Gram staining,16 S r DNA molecular identification and named as Bacillus amyloliquefaciens A13.The physical and chemical analysis of the antibacterial substances produced by Bacillus amyloliquefaciens A13 showed that the antibacterial substances produced by the strain could maintain stable antibacterial activity after a variety of protease treatment,and could still maintain good antibacterial activity after strong acid,strong alkali,high temperature and various metal ions treatment.The fermentation supernatant produced by the strain not only had a significant inhibitory effect on common pathogenic bacteria such as Escherichia coli,Listeria monocytogenes and Salmonella enteritidis,but also had a significant inhibitory effect on pathogenic fungi such as Siraitia grosvenorii pathogen,Penicillium citrinum and Fusarium oxysporum.In addition,the fermentation supernatant could effectively inhibit the infection of Penicillium pericarpium and significantly reduce the degree of rotting of Wogan,showing good biological control effect.The antibacterial substances produced by Bacillus amyloliquefaciens A13 were isolated and purified.By comparing and optimizing ammonium sulfate precipitation method,acid precipitation method,methanol extraction method and ethyl acetate extraction method,80% saturation ammonium sulfate precipitation method was determined as the suitable method.The crude antibacterial products were further purified by gel filtration chromatography,and more antibacterial substances could be eluted.Tris-Tricine-SDS-PAGE electrophoresis and HPLC analysis showed that there were still seven protein substances in the eluent.The culture conditions and nutrient components of the medium for the production of antimicrobial substances by Bacillus amyloliquefaciens A13 were optimized by single factor and response surface methodology.The optimized fermentation conditions were as follows: seed age 4 h,culture temperature 37 °C,and culture time 12 h;the nutrient components of the medium were as follows: soybean peptone 10.5g/L,maltose 9.5 g/L,and sodium chloride 2 g/L;the diameter of inhibition zone produced by the optimized fermentation broth on Escherichia coli could reach 26.4 mm,which was 42.7% higher than that before optimization. |
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