Optimization Of Fermentation Conditions Of Bacillus Amyloliquefaciens H6 And Evaluation Of Degradation Effect Of Degradation

Zearalenone(ZEN)is a mycotoxin widely found in food and feed.ZEN in contaminated agricultural and livestock products can pose a great threat to human health through the food chain,and there is no specific drug for the treatment of ZEN poisoning.Therefore,the prevention and control of mycotoxins has become one of the important research hotspots.Microbial detoxification has the advantages of high conversion efficiency,mild reaction conditions and no influence on feed quality,so it is the focus of research to solve the problem of mycotoxin.Bacillus amyloliquefaciens has the potential to degrade mycotoxins,secrete a variety of extracellular enzymes,and promote animal intestinal health and growth performance.It has been widely used in food,pharmaceutical,and agricultural fields.Bacillus amyloliquefaciens H6 isolated from laboratory has the function of inhibiting the growth of Fusarium graminearum and synthesizing thioesterase to efficiently degrade zearalenone.However,similar to other microorganisms,the original strains isolated from nature may have problems such as safety and low fermentation level,and whether Bacillus amyloliquefaciens H6 can reduce ZEN-induced toxic damage in mice and its mechanism remain unclear.In this study,Bacillus amyloliquefaciens H6 was used as the experimental strain,the safety of the strain was evaluated,the fermentation conditions of the strain were optimized,the detoxification effect of the strain on ZEN and ZEN derivatives in feed was verified,the optimal reaction conditions for degrading ZEN were explored,and the effect of degrading ZEN was evaluated in mice.Experiment 1:In vitro safety evaluation of Bacillus amyloliquefaciens H6The safety characteristics of Bacillus amyloliquefaciens H6 were determined in vitro.The results showed that Bacillus amyloliquefaciens H6 did not carry virulence genes bceT,cytK,ces,hblA,nheA,did not have nitrate reductase activity,did not have lysine,ornithine,arginine decarboxylase activity,and was sensitive to 10 antibiotics of penicillin G,ampicillin,cefazolin,amikacin,gentamicin,erythromycin,norfloxacin,ciprofloxacin,compound sulfamethoxazole,chloramphenicol.It showed good safety in virulence gene detection,metabolite toxicity test,drug resistance test and hemolysis test.Experiment 2:Optimization of fermentation process of Bacillus amyloliquefaciens H6 and thioesterase by response surface methodologyThe nutrient requirements and fermentation conditions of Bacillus amyloliquefaciens H6 were optimized by response surface methodology with viable bacteria count and spore count as indexes.Meanwhile,the mRNA expression level and the recombinant protein activity of thioesterase were also evaluated.[Results]The optimum carbon source,nitrogen source and inorganic salt screened by single factor experiment were soluble starch,yeast extract powder and MgSO4 respectively.Plackett-Burman test showed that soluble starch concentration,yeast leaching concentration and fermentation time were the key factors of fermentation.Box-Behnken test finally determined the optimal parameters of culture medium and fermentation process:soluble starch:32.16 g/L,yeast powder:16.27 g/L,MgSO4:10.00 g/L,fermentation time:At 39.28 h,the initial pH was 5.0,the inoculation amount was 1%,the rotation speed was 200 r/min,and the fermentation temperature was 37℃.The biological yield of Bacillus amyloliquefaciens H6 reached 6.33 × 109 CFU/mL by shaking flask and 1.09 × 1010 CFU/mL by 50 L fermenter.The fermentation process of Bacillus amyloliquefaciens H6 was optimized by response surface optimization.Compared with the initial culture-medium,the fermentation level was significantly increased(P<0.05),and the thioesterase activity was significantly increased(P<0.05),which laid a foundation for the industrial fermentation of Bacillus amyloliquefaciens H6..Experiment 3:Degradation of zearalenone and its derivatives by Bacillus amyloliquefaciens H6The moldy feed was detoxified by Bacillus amyloliquefaciens H6 fermentation.The results showed that the ZEN toxin content of the moldy feed with ZEN content of 436.74μg/Kg was reduced to 81.53 μg/Kg after detoxification by Bacillus amyloliquefaciens H6 fermentation,which could achieve the detoxification effect of moldy feed.Through Auto dock molecular docking prediction,ZEN derivative ZEN derivativeα-zearalenol,β-zearalenol,α-zearalenol ligand molecules and thioesterase protein can be targeted binding,further biodegradation verification,Bacillus amyloliquefaciens H6 has degradation effect on α-zearalenol,β-zearalenol and α-zearalenol.The optimum reaction temperature of ZEN degradation by thioesterase was 37℃,and the optimum reaction pH was 6.0.The addition of Fe2+,Mg2+and Mn2+could significantly increase the activity of thioesterase(P<0.05)and increase the degradation rate of ZEN.Experiment 4:Application evaluation of zearalenone degradation by Bacillus amyloliquefaciens H6 in vivoSpecific pathogen-free(SPF)Kunming mice were divided into 4 groups(Control group,ZEN group,H6 group,H6+ZEN group)for 28 days.Liver coefficient,serum biochemical indices(aspartate aminotransferase,alanine aminotransferase,alkaline phosphatase),liver oxidation index(malondialdehyde,total superoxide dismutase,catalase,glutathione catalase)were determined.Pathological changes of liver and small intestine were analyzed by HE staining,and the expression level of Nrf-2 oxidative stress signaling pathway gene in liver was detected,and microbial 16S rRNA in cecum contents was determined.The results showed that ZEN could reduce the growth performance of mice,induce oxidative damage in liver,change the morphological structure of liver and small intestine,produce pathological changes,and cause changes in cecal microorganisms.Bacillus amyloliquefaciens H6 can alleviate ZEN-induced weight loss in mice,reverse ZEN-induced oxidative stress-related serum biochemical indicators,liver oxidation index indicators,and Nrf-2 signaling pathway mRNA relative expression levels,and antagonize ZEN-induced oxidative damage in mouse liver.Compared with the ZEN group,the H6 strain reduced the pathological damage of the liver and small intestine.In the cecal microbial composition,Bacillus amyloliquefaciens H6 reshaped the cecal flora structure and had an application effect in mice.It can antagonize ZEN-induced liver and intestinal damage in mice and regulate the structure of intestinal flora.