Mechanism Of Microbial Removal Of Sterols In Tobacco

Tobacco is a special product that has attracted consumers’ attention.Not only because of its sensory appeal to smokers,but also because of its safety issues.There are more than 100 kinds of clearly harmful and carcinogenic components in the smoke,which are produced by the burning of tobacco leaves.Among them,polycyclic aromatic hydrocarbons(PAH)are the most famous carcinogens in tobacco smoke.Sterols are a kind of plant sterols ubiquitous in tobacco.Although they have almost no contribution to the quality of tobacco leaves,they can increase the release of fused ring aromatic hydrocarbons after burning.Therefore,the removal of sterols is very important for reducing the harm of tobacco leaves.At present,the published studies on the removal of sterols in tobacco leaves are very limited Based on this,this research mainly explores the effect and mechanism of screening strains on the removal of sterols from tobacco leaves from the perspective of microbial adsorption and degradation,with the hope that microbial methods can reduce tobacco harm.In particular,the removal of sterols provides a certain theoretical basis and data support.The main results obtained in this experiment are as follows:1.The contents and differences of five sterols in four different types of tobacco leaves were analyzed by using the triple quadrupole liquid-mass spectrometry technology(UPLC-TriQ-MS/MS).Due to the differences in the growth conditions and environment of different types of tobacco leaves,there are significant differences in the content of five sterols in four different types of tobacco leaves.Specifically,among the four different types of tobacco leaves,stigmasterol,campesterol and cholesterol in cigar tobacco leaves are the highest,and the content of five sterols in oriental tobacco is the lowest.For sun-cured tobacco,in addition to the cholesterol content,the content of the other four sterols is second only to cigar tobacco.Interestingly,flue-cured tobacco does not contain ergosterol,and the content of the other four sterols is significantly lower than that of sun-cured tobacco and cigars,but higher than that of oriental tobacco.The ranking results of the total sterol content are as follows:cigar tobacco>sun-cured tobacco>flue-cured tobacco>oriental tobacco.In addition,for four different types of tobacco leaves,stigmasterol is the highest content of the five sterols.In summary,the content of sterols in cigar tobacco leaves,especially stigmasterol,is the highest among the four different types of tobacco leaves.2.Based on the high content of sterols in cigars,with cigars as the representative,a comprehensive analysis of the microbial community and metabolic functions in the fermentation process of cigar tobacco leaves.Chemical composition analysis showed that after artificial fermentation,the content of most substances in cigar tobacco leaves decreased,especially sterols.Fermentation changes the community structure of bacteria and fungi.RDA shows that sterol content is most susceptible to the influence of fungal communities.FAPROTAX and FUNGuild analysis results show that the dominant bacteria in cigars have important application prospects in degrading aromatic compounds,hydrocarbons,aliphatic compounds and lignin.Through the comprehensive analysis of the microbial community and metabolic function during the fermentation process of cigar tobacco,it points out the direction for the targeted screening of microbial colonies that can remove sterols in the later stage.3.A fungus Aspergillus fumigatus LSD-1 that can effectively remove sterols was selected from cigar tobacco leaves.Taking cigar tobacco leaves as the research object,aiming at the fungi on the tobacco leaves,using phytosterols as the sole carbon source,the strain obtained was morphologically and molecularly identified as Aspergillus fumigatus,named LSD-1.Taking stigmasterol as the representative of the five sterols,it is found through verification that the mycelial pellets of Aspergillus fumigatus LSD-1 can quickly remove most of the stigmasterol in the aqueous solution,and after analysis,the mycelial pellets of Aspergillus fumigatus LSD-1 are removed from the aqueous solution.The stigmasterol is mainly adsorption.4.Both live and inactivated LSD-1 can effectively remove stigmasterol,P-sitosterol,campesterol,cholesterol and ergosterol.The mechanism of its effect is proved to be a physical process of biosorption.Both the live and inactivated hyphal pellets of LSD-1 can effectively remove stigmasterol,β-sitosterol,rapeseed sterol,cholesterol and ergosterol in aqueous solutions and tobacco extracts,among which the removal rate of live hyphal pellets It is as high as 90%,and the inactivated mycelial pellet is over 80%.Although the adsorption efficiency of live mycelium pellets is better than that of inactivated mycelial pellets,it was found to promote the production of ergosterol when the live mycelial pellets removed the tobacco extract.However,inactivated mycelium pellets will not produce ergosterol while maintaining a high removal rate.Therefore,like the live mycelial pellets of LSD-1,the inactivated LSD-1 mycelial pellets should be a better choice for removing sterols from the tobacco extract.In addition,the results of BET and FTIR analysis indicated that the adsorption of sterols by the active and inactivated hypha pellets of LSD-1 may be a physical process.In general,the application of LSD-1 in tobacco extract provides theoretical support for reducing harmful components in the tobacco industry.5.Based on the non-targeted metabolomics analysis,it was found that mycobacteria may mainly enhance the permeability of cell membranes through unsaturated fatty acids,and then metabolize sterol molecules.The analysis of the metabolites of mycobacteria degrading sterols using non-targeted metabolomics methods showed that both the PCA and PLS score maps showed obvious discrete phenomena,indicating that the intracellular metabolites of mycobacteria had undergone significant changes.The main biomarkers are docosahexaenoic acid,luteolin,elegans lactone,farnesal,4-allylphenol,as well as acid urea,N-acetyl-D-tryptophan,Shikimic acid pyridoxal,L-methionine.They belong to unsaturated fatty acids,terpenoids and amino acids,and energy metabolites.The degradation of stigmasterol by mycobacteria may be a process that requires energy.When ingesting sterol molecules,unsaturated fatty acids are needed to enhance the permeability of cell membranes to facilitate the entry of sterol molecules into cells,and terpenoids may be mycobacteria Metabolites produced during the degradation of sterols.