Analysis Of Microbial Communities Of Hongda And K326 Tobacco During Aging

Tobacco aging is an important step in the production and processing of tobacco products and is closely linked to chemical,enzymatic and microbial actions.Microorganisms influence the entire tobacco aging process and are a key factor in enhancing the fermentation of tobacco leaves.Through their own metabolic activities,microorganisms can not only degrade harmful substances such as nicotine,which are detrimental to the quality of tobacco,but also break down large molecules such as proteins and starch into small molecules,to improve the safety and aroma quality of the tobacco leaves.Previous research on tobacco microorganisms has mainly focused on the isolation of a small number of culturable microorganisms in the aging process using traditional methods and the effect of relevant microbial agents or enzyme preparations on the fermentation effect of tobacco.The important role played by many nonculturable or difficult to culture microorganisms in tobacco aging has been neglected,and a comprehensive and systematic study of the microbial community in the whole tobacco aging process is lacking.Therefore,it is necessary to further investigate the microbial community succession during tobacco aging and reveal its change patterns and driving mechanisms,to clarify the main dominant microorganisms and their biological functions during tobacco aging,to provide a basis for the development and utilization of microbial resources for aroma production and harm reduction during tobacco aging,and to provide a scientific basis for research on the mechanism and artificial control of tobacco aging.To address the above issues,this study investigated the microbial community changes and functional succession of two tobacco samples,Hongda and K326,during different periods of tobacco aging by high-throughput sequencing.The community structure,distribution and succession of dominant microbial groups were investigated,and the main dominant species and their microbial functions during tobacco aging were studied.The relationship between microbial community succession and environmental factors was also investigated by measuring changes in the content of chemical indicators related to tobacco quality and by correlation analysis.Based on the results of high-throughput sequencing,the culturable bacteria in aged tobacco were screened and a resource bank of aged tobacco microbial strains was established,which further improved the microbial fermentation process of aged tobacco and provided strain resources and certain theoretical guidance for the artificial aging control of tobacco.The main research results are as follows.1.Analysis of microbial community structure and diversity showed that:(1)1119 OTUs were obtained from the bacterial community after 16 S r RNA high-throughput sequencing,which were annotated and assigned to 20 phyla,39 classes,87 orders,164 families and 376 genera.At the genus level,Pseudomonas,Sphingomonas,Methylobacterium,Massilia,Pantoea and Acinetobacter and Stenotrophomonas were the dominant groups,all belonging to Proteobacteria.(2)1356 OTUs were obtained from the fungal community by ITS gene high-throughput,belonging to 9 phyla,25 classes,74 orders,177 families and 287 genera.The main dominant groups are Alternaria,Aspergillus,Symmetrospora,Cladosporium and Phoma.Except Symmetrospora belong to Basidiomycota,all others belong to Ascomycota.(3)The diversity analysis showed there were significant differences in the diversity of the microbial communities in the different samples,and the structure and diversity of the microbial communities in the same sample also changed with aging time.The richness and diversity of the microbial community in Hongda tobacco decreased in the later stages of aging(15-21months)compared to the pre-aging period(3-12months),while the diversity decreased but richness increased in the K326 tobacco sample.(4)Microbial network analysis showed that the aging process enhanced the complexity and interactions of bacterial and fungal community networks in tobacco samples.The key interrelated taxa in the early stages of aging were dominated by bacterial communities,while the role of fungal communities increased in the later stages of aging.2.Results of microbial community functional succession.(1)The FAPROTAX results show that the microbial functions of the bacterial community are mainly related to chemoheterotrophy,where microorganisms use the tobacco leaf as a substrate to break down the organic matter in it during aging to obtain the materials and energy needed for growth and metabolism.Other functions related to the nitrogen cycle,pathogens,aromatic compound degradation,fermentation,etc.Changes in abundance are associated with the involvement of microorganisms in the nitrogen cycle to degrade tobacco-specific nitrosamines,the fermentation process to kill most bacterial pathogens and an increase in aromatic substances.(2)The PICRUSt annotation results indicate that the bacterial community is very rich in material and energy metabolic functions during tobacco aging,suggesting that the bacterial community is involved in many material transformation and metabolic processes during tobacco aging,providing the material and energy basis for the whole aging process.(3)The results of FUNGuild indicate a change in the main nutritional mode of the fungi with a decrease in the common nutritional mode dominated by the pathogenic-rotten-symbiotic nutritional type and an increase in the specialized nutritional mode of the saprophytic and symbiotic nutritional types.The relative abundance of functions associated with pathogenic fungi at the guild level also decreased with aging,while the later stages of aging were dominated by saprophytes.(4)PCA of the predicted microbial community functions showed differences in the microbial functions of the samples at different stages of aging.The microbial functions of the bacterial and fungal communities underwent successional changes at different aging times.3.Environmental factor correlation analysis showed that:(1)During the aging of the tobacco leaves,the content of soluble sugars,nicotine,cembretriene,total nitrogen,total alkaloids,chloride,moisture,starch,pectin,and lipids all decreased to varying degrees,except for an increase in potassium content in the red large sample and phenolic content in both samples.(2)The results of the quality evaluation showed that with increasing aging time,the irritation and dryness of the tobacco leaves decreased,the aroma quality and sweetness increased,and the quality of the tobacco leaves was significantly improved.(3)Environmental factors were significantly correlated with dominant species at the microbial genus level,with total nitrogen and lipid content being the most significant factors correlated with bacterial and fungal communities during tobacco aging.4.The results of the screening for culturable bacteria in tobacco showed that the 345 bacterial strains were isolated from tobacco samples at different aging times,mainly composed of Proteobacteria and Firmicutes,w which were consistent with the results of highthroughput sequencing.The dominant taxa were mainly Bacillus and Enterobacteriaceae,accounting for 54.78% and 21.74% of the total number of culturable bacteria;the abundance and diversity of culturable bacteria decreased with the aging process.In summary,this study investigated the structure and functional succession of microbial communities during the whole aging process of tobacco leaves by high-throughput sequencing.The study clarified the dynamic changes in the composition of the dominant species of bacterial and fungal communities and the functional succession of microorganisms during the aging process,analyzed the relationship between microbial environment factors and microbial communities during the aging process,promoted the establishment of a microbial strain resource base for aging tobacco leaves,provided certain strain resources and scientific basis for regulating and promoting the artificial aging of tobacco leaves,It not only provides a scientific basis for regulating and promoting the artificial aging of tobacco leaves,but also lays the foundation for the development and utilization of functional microbial resources,and provides a reference methods for revealing the succession of microbial communities in other similar tobacco fermentation habitats.