| Reconstituted tobacco sheet（RTS）is of great economic importance to China.Compared to tobacco,RTS has the advantage of reducing harmful substances and improving quality indicators.The basic process is to extract the tobacco wastes with water,then the separated fibre material is put into a paper machine to make a substrate,then the extract is concentrated and configured into a coating solution to be added to the substrate,dried and slit into a finished product.RTS production lines have a wide and complex range of microorganisms,some of which can improve the quality of RTS smoking during the production process.In addition,spoilage often occurs in the concentrates after waste extraction.Therefore,this study was conducted to analyse the species in each material of the production line,to screen functional strains for enzyme and flavour production,and to find a biological preservative to inhibit spoilage bacteria in the production line.The main findings are as follows.（1）In this study,the microbial diversity of 25 materials in the RTS production line was analysed by high-throughput sequencing.393 bacterial genera were detected,with Lactobacillus and Bacillus as the dominant genera,and 165 fungal genera were detected,with Candida and Aspergillus as the dominant genera.The diversity and abundance of bacterial genera varied little between materials,while fungal genera varied relatively more.In the tobacco wastes extraction process,the relative abundance of Lactobacillus increased and the relative abundance of Candida generally decreased;in the paper making process,the relative abundance of the anaerobic fungus Veillonella increased significantly during the pulp copying process;while in the drying process of the manufactured substrates,the relative abundance of Lactobacillus,Veillonella,Trichosporon and Sphergillus increased.The relative abundance of heat-intolerant microorganisms such as Lactobacillus,Veillonella,Trichosporon and Sphingobacterium gradually decreased during the drying of the finished substrates,while the abundance of heat-tolerant Bacillus increased.From"raw material→process→finished product",the diversity of microbial species showed a trend of"less→more→less".（2）Thirty-one morphologically distinct strains were isolated and purified from tobacco stems,flakes and concentrates.A functional microorganism,Bacillus subtilis B-NS-7,was selected based on its enzyme and aroma production capacity,and was amplified by shaking flasks to produce 696.3 U·m L-1 protease activity,238.4 U·m L-1 amylase activity and 64.3U·m L-1 pectinase activity,100.0μg·m L-1of aroma.The fermentation conditions for aroma production were optimised by single-factor experiments:inoculum concentration of 1%,fermentation temperature of 37°C and fermentation time of 24 h.The aroma content of the tobacco concentrates were 116.3μg·m L-1.Neophytadiene was the main reason for the aroma enhancement and B.subtilis B-NS-7 was defined as an aldehydic and enolenic aromatic strain.Protein was reduced by 0.7 mg·L-1,starch by 2.1 mg·L-1 and pectin by 1.4 mg·L-1 during fermentation,while amino acid and reducing sugar contents did not change significantly.The results of the sensory evaluation showed that the fermented tobacco concentrates increased the aroma quality of the cigarettes,reduced the astringent and paper taste and enhanced the smoking quality of the cigarettes compared to the original liquid.（3）The minimum inhibitory concentration ofε-polylysine（ε-PL）was 50 mg·L-1to 100mg·L-1 for two strains of Leuconostoc sp.RS-1 and Lactobacillus formosensis RS-4 from tobacco concentrates.ε-PL showed strong inhibition at p H 4 to p H 8.The acidic conditions showed higher inhibition activity ofε-PL,and the total number of colonies in the stock solution was significantly higher than that in theε-PL.Therefore,ε-PL is suitable for use in high temperature acidic tobacco concentrates for antimicrobial inhibition. |
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