| Tobacco-Specific Nitrosamines(TSNAs)are N-nitroso-derivatives of pyridinealkaloids present only in tobacco and tobacco products and can cause serious health problems.Therefore,it is of great significance to reduce the content of TSNAs in tobacco and tobacco products.Currently,studies on the degradation of TSNAs have mainly focused on the utilization by microorganisms,and various bacteria have been shown to be able to degrade TSNAs,but the specific mechanisms are still unclear.In our previous study,a Bacillus pumilus strain 05-5402 was isolated from tobacco soil,which can efficiently degrade TSNAs in white burley.However,its degradation activity and mechanism on TSNAs in cured tobacco are still unknown.This study first analyze the degradation activity of strain 05-5402 on TSNAs in flue-cured tobacco,and then the changes of general metabolites and their release effects in the oral cavity.based on this,the combined transcriptomic and proteomic analysis will be carried out to reveal the underlying degradation mechanism of the TSNAs in flue-cured tobacco by the strain 05-5402,which will further lay the foundation for further theoretical research and practical industrial applications.We first analyzed the degradation activity of Bacillus pumilus 05-5402 on TSNAs in flue-cured tobacco at different temperatures(28 °C and 35 °C)and with different water content(30%,50%,100%).The results showed that,under the same water content,the degradation activity of 05-5402 strain on the TSNAs in tobacco leaves at 28 °C was significantly higher than that at 35 °C,and a higher water content exhibited a better degradation effect at 28 °C.The highest degradation rate of TSNAs at 100% water content at 28 °C reached 31.99 %.With the same condition,it was observed that the contents of general chemical components,such as total sugar,starch and reducing sugar,protein content and total nitrogen,in the flue-cured tobacco were significantly decreased,while the contents of nicotine,potassium ion and chloride ion did not change significantly.These results indicate that the 05-5402 strain might use the sugars in flue-cured tobacco as nutrient sources for growth during fermentation.As nitrogen sources,it prefered to use TSNAs as,but less nitrogen-containing substances such as proteins was used.In addition,analysis of in vitro simulation experiments showed that the release rate of nicotine in the flue-cured tobacco after fermentation by the 05-5402 strain reached more than 90 % after 8 minutes in the oral cavity,which was not significantly different from the control group,indicating that the degradation of TSNAs by the 05-5402 strain did not affect the release rate of nicotine.Based on the above analysis results,this study further the 05-5402 strain inoculated in the medium with TSNAs as the sole carbon and nitrogen source for fermentation,and the medium without TSNAs was used as a control.The mechanisms of the TSNAs degradation by the strain was analyzed by transcriptome sequencing.The results showed that 168 differentially expressed genes(DEGs)were obtained from total 3859 genes,of which 64 were up-regulated and 104 were down-regulated.The KEGG results showed that the DEGs were enriched into 46 pathways,indicating that the degradation of TSNAs by 05-5402 strain involved multiple mechanisms.Among these pathways,metabolic pathways are the most,including ABC transporters,microbial metabolism in different environments,amino acid biosynthesis,glycolysis/gluconeogenesis,carbon metabolism,fructose and mannose metabolism,pentose phosphate pathway,starch and sucrose metabolism,etc..At the same time,proteomics analysis was performed with the same samples.A total of 848 proteins were identified.Among them,42 were differential proteins,including 34 down-regulated proteins and 8 up-regulated proteins.Most differential expression proteins were also down-regulated,which was consistent with that of DEGs observed by transcriptomics.The KEGG enrichment results that the differential proteins were enriched into 46 metabolic pathways,including microbial metabolism in different environments,ribosome-related pathways,carbon metabolism,amino acid biosynthesis,glycolysis/gluconeogenesis,galactose metabolism,starch and sucrose metabolism and various amino acid metabolism.The results of proteomics analysis also showed that TSNAs was degraded by the 05-5402 strain through diverse mechanisms.Further conjoint omics analysis showed that carbon metabolism,glycolysis/gluconeogenesis,amino acid biosynthesis and metabolism of various amino acids may play a more important role in the degradation of TSNAs by 05-5402 strain.In summary,based on the previous work,in this study,the strain 05-5402 was further applied for the degradation of TSNAs in flue-cured tobacco,and the optimal fermentation conditions for the degradation and the changes of conventional chemical components was preliminarily determined.On this basis,cojointed transcriptomics and proteomics analysis further revealed that diverse mechanisms were involved in the degradation of TSNAs by the 05-5402 strain,among which amino acid biosynthesis,carbon metabolism,glycolysis / gluconeogenesis,starch and sucrose metabolism,and multiple amino acid metabolism might be more important.This study reveals the degradation mechanisms of TSNAs by Bacillus pumilus 05-5402,which lays a foundation for finally revealing the degradation mechanism of TSNAs by microorganisms,and provides a reference for the industrial application of TSNAs degradation by Bacillus fermentation. |
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