Analysis Of Microbial Community Structure And Function During Fermentation Of Cigar Tobacco Leaves

Fermentation of cigar tobacco leaves is a key step to improve improve its appearance color,physical properties,aroma concentration and aroma quality.During the fermentation process,microbial communities play an important role in driving macromolecular substances(cellulose,starch,protein,etc.)to degrade and transform into various aroma compounds.Due to the immature cultivation level and fermentation technology of domestic cigar tobacco,the quality of domestic cigar tobacco is low,so it is imperative to accelerate the research on cigar tobacco fermentation technology.In this paper,systematic analysis and research were carried out on cigar tobacco leaves from different places of origin and at different fermentation stages.The microbial community structure and function in the fermentation process were analyzed,and functional microorganisms in cigar tobacco leaves were excavated to improve the aroma and quality of domestic cigar tobacco leaves.The main findings are as follows:(1)To understand the core microbiotas of high-quality cigar tobacco leaves and explore the impact of microbial community differences on the aroma compounds and aroma of cigar tobacco leaves,the high-quality cigar tobacco leaves from four producing areas at home and abroad were compared and analyzed horizontally through the omics technology.The results showed that the dominant microbial genera in high-quality cigar tobacco leaves at home and abroad were Staphylococcus,Pseudomonas,Sphingomonas,Aspergillus,Sampaiozyma,Alternaria,Wallemia and Thermoascus,and the main aroma compounds are aldehydes and ketones.The microbial communities in cigar tobacco leaves from different production areas are quite different,and the differences in microbial communities lead to different aromatic compounds and aroma in cigar tobacco leaves from different production areas.(2)To understand the dynamic succession and function of microbial community in the process of cigar tobacco fermentation,the domestic cigar tobacco leaves at different fermentation stages were compared and analyzed longitudinally by using omics technology.The results showed that the dominant microbiotas of domestic cigar tobacco leaves,for example,Aspergillus gradually increased with the fermentation,Sampaiozyma and Chloroplast gradually decreased with the fermentation,Pseudomonas and Alternaria first increased and then decreased.Their functions in the fermentation process of cigar tobacco leaves are mainly divided into impurity removal and aroma generation.In the agricultural fermentation stage,dominant microbiotas,such as Pseudomonas,Chloroplast,Sampaiozyma,and Alternaria,act on the degradation of macromolecular compounds and remove the impurities of cigar tobacco leaves.In the Industrial fermentation stage,dominant microbiotas,such as Acinetobacter,Staphylococcus,and Aspergillus,mainly promote the production of aroma compounds and enhance the aroma of cigar tobacco leaves.(3)Due to the important contribution of aldehyde and ketone aroma substances to the aroma of cigar tobacco leaves,a high-throughput sorting method targeting the microorganisms producing aldehydes or ketones has been established.The aldehyde and ketone producing microorganisms sorted by flow cytometry will be identified and analyzed by high-throughput sequencing and microbial culture.The results showed that the main microorganisms producing aldehydes or ketones in cigar tobacco leaves were Acinetobacter,Sphingomonas,Solibacillus,Lysinibacillus,and Bacillus.106 strains with high yield of aldehyde or ketone were obtained through pure culture.They can synthesize aromatic substances such as benzaldehyde,isophorone,2,4-dimethyl benzaldehyde,hydroxyacetone,benzeneacetaldehyde,4-hydroxy-3-ethoxybenzaldehyde and 3-methyl-butanal in synthetic medium.(4)The strains with high production of aldehyde or ketone aroma compounds have been applied to domestic cigar tobacco leaves for intensive fermentation,and the strains Acinetobacter sp.1H8 and A.indicus 3B2 could significantly improve the aroma and quality of domestic cigar tobacco leaves.The addition of the two strains could significantly change the original microbial community of cigar tobacco leaves,Acinetobacter sp.1H8 accelerated the succession of the microbial community,while A.indicus 3B2 reconstructed the microbial community structure.Inoculation with Acinetobacter sp.1H8 significantly increased the content of 3,5,5-trimethyl-2-cyclohexen-1-one,heptanal,solanone,megastigmatrienone 4,octanoic acid,ethyl ester,3,5-octadien-2-one,and 6-methyl-5-hepten-2-one in domestic cigar tobacco leaves.Inoculation of A.indicus 3B2 coud significantly increase the content of megastigmatrienone,2-ethyl-1-hexanol,tetramethyl-pyrazine,trimethyl-pyrazine,neophytadiene,and 2,6-dimethyl-pyrazine in domestic cigar tobacco leaves.