| The problems of bacterial resistance and antibiotic residues have brought enormous hidden dangers to human health and public health safety,and also pose a serious threat to the healthy development of the aquaculture industry.In 2020,China will also fully implement the prohibition on the addition and application of antibiotics in livestock and poultry breeding.The research and development of new green feed additive products that replace antibiotics is a major demand in the current industry.Probiotics have become one of the important new green anti-microbial products due to their non-toxic,harmless,no drug residue,and environmentally friendly characteristics.Bacillus subtilis is a probiotic that can be directly added to feed in the current feed additive license catalog in China.As a frequently added probiotic,it is added to animal feed in the form of spores,commonly used to improve animal intestinal function,promote animal growth,and prevent disease.The key to the development and application of Bacillus subtilis feed additives lies in the excavation of strains,including the isolation,identification,original biological characteristics,stress resistance,genetic stability,strain quality control,and identification of functional components of the strains.In this study,four strains of Bacillus subtilis and four strains of Bacillus amyloliquefaciens were obtained from the feces of healthy breeding pigs in the quarantine farm of imported breeding pigs in Jilin Province by isolation and 16S r DNA sequencing.Studies have been conducted on the resistance of isolated strains to temperature,artificial gastric juice,artificial intestinal juice,bile salt solution tolerance tests,and antibiotic susceptibility tests.The results indicate that strains such as Bacillus subtilis G2B9-Q have good potential for the development and application of probiotics.The fermentation medium composition and culture conditions of the strain were optimized using Plackett Burman experiment and Box-Bchnken response surface method.The optimized culture scheme was validated in shake flask culture.The results showed that the spore yield of G2B9-Q strain could reach 6.97×10~8 CFU/m L,the optimal solution of the spore quantity response value that conforms to the response surface derivation.The antimicrobial spectrum of eight isolated strains against 68indicator bacteria was determined,with the broadest antimicrobial spectrum being Bacillus amyloliquefaciens Y106-A1-3,Bacillus subtilis G2B9-Q,and Bacillus subtilis G2B9-1.The antibacterial substance of Bacillus subtilis G2B9-Q is present in the culture supernatant.The active components are isolated using Tris-Tricine-SDS-PAGE and Native-PAGE methods.Establishing a separation and concentration method for antibacterial active ingredients,and study the resistance of antibacterial active ingredients.The results show that it has good antibacterial performance and stability,and has the potential to further develop antibacterial agents.Establish a mouse infection model with Clostridium perfringens,and conduct therapeutic efficacy studies using Bacillus subtilis G2B9-Q and its antibacterial active components.The results show that it can improve the pathological damage caused by Clostridium perfringens infection,reduce the expression of pro-inflammatory factors,increase the expression of anti-inflammatory factors,and improve intestinal microbiota,with the potential to replace antibiotics in the treatment of Clostridium perfringens.To sum up,this study conducted a study on the probiotic potential of Bacillus subtilis and Bacillus amyloliquefaciens from healthy pig feces,optimized the fermentation medium formula and fermentation conditions of G2B9-Q strain,clarified the properties of antibacterial active substances produced by G2B9-Q and their potential in preventing and treating Clostridium perfringens infection,laying a foundation for further development of Bacillus subtilis preparations with good application prospects. |
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