Effects Of Drying Processes On Bacillus Subtilis Microbial Ecological Agents And Its Mechanisms

Microbial ecological agent is a kind of green and effective substitutes for antibiotics.Bacillus subtilis can generate stress-resistant spores,which could survive in extreme environments and is one of important bacterial species for microbial ecological agent production.Effective biomass and spore properties determine the quality of B.subtilis microbial ecological agents significantly.Drying is an important unit operation for preparing microbial ecological agents,while its impacts on microbial cells and spores remain unclear.In this study,the influence of different drying methods on B.subtilis microbial ecological agents were systematically investigated.Hot air drying,vacuum freeze drying,and microwave drying were used to examine these influence on solid substrates,effective biomass,key genes for sporulation,and spore properties.Based on these results,the optimal drying conditions were determined.The main findings are as follows:1.Drying process affects water state in medium,which in turn affects sporulation.In the initial medium,lumen water accounted for 90%and bound water accounted for 7.7%.After hot air drying at 60°C for 4 h,lumen water accounted for12%and the bound water accounted for 88%.After hot air drying at 80°C for 2 h,the proportion of lumen water decreased to 5.9%while the proportion of bound water increased to 93.8%.After vacuum freeze drying for 4 h,lumen water accounted for7.2%while bound water accounted for 92.4%.After microwave drying for 20 min,lumen water accounted for 2.7%and bound water accounted for 97.4%.The bound water is closely combined to the solid substrate particles,which is difficult to access by microbial cells and thus facilitate sporulation.The decrease of glucose and xylose contents within mediums also benefits sporulation.2.The optimal drying condition is hot air drying at 60°C for 5 h.Effective biomass reached 6.18×10¹⁰CFU/g dry weight,which was 3.12 fold higher than the initial value before drying.Compared with 80°C hot air drying for 3 h,microwave low-fire drying,vacuum freeze drying,effective biomass under the optimal drying condition increased by 5.03 times,7.97 times,12.01 times,respectively.3.Drying process affects expression of key genes（e.g.,spo0A,sig F,and sig E）that regulating sporulation.Taking the gene expressions before drying as a control,spo0A,sig F,and sig E gene expressions during hot air drying at 60°C for 2 h were2.49,1.96,and 1.58 fold higher.spo0A,sig F,and sig E gene expressions were 1.49,1.19,and 1.48 fold higher after hot air drying at 80°C for 1 h.The expression levels of spo0A,sig F and sig E genes were 1.75,1.55,and 1.24 fold higher after vacuum freeze drying for 2 h.After microwave drying for 10 min,spo0A,sig F,and sig E gene expressions were 1.29,1.27,1.15 fold higher.During all drying processes,with the increase of drying time,expressions of spo0A,sig F,and sig E genes were higher than the initial level before drying,which significantly facilitate sporulation.The sporulation rate during hot air drying and microwave drying reached 100%,and that during vacuum freeze-drying was higher than 90%.4.Drying causes changes in the composition and content of fatty acids in spores.Fatty acids in spores mainly existed in the form of iso fatty acids and anteiso fatty acids.During all drying processes,spores contain four kinds of fatty acids:12:0,15:0anteiso,16:0iso,and 16:0.These fatty acid contents were 40%-70%.Because the stable fatty acid composition and content,the spore coat and cell membrane’s structure were stable,which resulted in the strong resistance of spores.The higher temperature during sporulation leaded to the stronger heat resistance of spores.In summary,the effective biomass was the largest after hot air drying at 60°C for 5 h,which reached 6.18×10¹⁰CFU/g.Expressions of key genes（e.g.,spo0A,sig F,and sig E）for sporulation increased during this drying process,of which sporulation rate reached 100%.Spore’s fatty acid composition and content were stable and shows stress resistance.Meanwhile,the medium reached a constant weight.Therefore,hot air drying at 60°C for 5 h is the optimal drying condition,which enhanced the effective biomass of B.subtilis microbial ecological agents.