To explore the optimum fermentation conditions for tobacco leaves and also screen the microbiota and metabolites that are beneficial for fermentation. Tobacco leaves were fermented at 25 °C, 35 °C, and 45 °C for 2, 4, and 6 weeks, respectively. For identification of the best fermentation temperature, physicochemical properties and sensory quality of fermented tobacco were investigated. Subsequently, based on the appropriate temperature, 16 s rRNA sequencing and metabolomics analysis of tobacco were performed to monitor the change of microbes and metabolites during fermentation process (from 2 to 6 weeks). Sensory quality analysis indicated that fermentation at 45 °C for 6 weeks represented the optimum condition. Metabolomics analysis showed that a total of 415 metabolites were annotated. The increase of fermentation period led to significant changes of metabolites. Results revealed an increase in concentration of L-phenylalanine and sphingosine as well as decreased concentration of betaine and phytosphingosine with the prolongation of fermentation period (2 to 6 weeks). Distinct changes in the microbiota were also observed with prolongation of the fermentation time. Results revealed that Pseudomonas, Pantoea, and Burkholderia were dominant bacteria in fermentation at 45 °C for 6 weeks. With the extension of the fermentation time, the abundance of Pseudomonas increased, while that of Sphingomonas and Methylobacterium decreased. Furthermore, microbiota profiles were tightly relevant to the altered metabolites, especially compounds involved in the sphingolipid metabolism. Suitable fermentation conditions were 45 °C for 6 weeks; phytosphingosine and sphingosine might affect tobacco fermentation via the sphingolipid metabolism pathway. This study provides a theoretical basis for guiding tobacco fermentation and gives insights into reducing harmful substances during tobacco fermentation.

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发酵过程中微生物群和代谢物对烟草品质的影响

探索烟叶的最佳发酵条件，筛选有利于发酵的微生物群和代谢产物。烟叶分别在25°C、35°C和45°C下发酵2、4和6周。为了确定最佳发酵温度，对发酵烟草的理化性质和感官质量进行了研究。随后，基于适宜的温度，对烟草进行16s rRNA测序和代谢组学分析，监测发酵过程（2至6周）微生物和代谢物的变化。感官品质分析表明，45℃发酵6周是最佳条件。代谢组学分析显示，总共注释了 415 个代谢物。发酵时间的延长导致代谢物发生显着变化。结果表明，随着发酵时间（2～6周）的延长，L-苯丙氨酸和鞘氨醇的浓度增加，而甜菜碱和植物鞘氨醇的浓度下降。随着发酵时间的延长，还观察到微生物群的明显变化。结果显示，45℃发酵6周时，假单胞菌、泛菌属和伯克霍尔德氏菌为优势菌。随着发酵时间的延长，假单胞菌的丰度增加，而鞘氨醇单胞菌和甲基杆菌的丰度减少。此外，微生物群谱与改变的代谢物密切相关，尤其是参与鞘脂代谢的化合物。适宜的发酵条件为45℃，6周；植物鞘氨醇和鞘氨醇可能通过鞘脂代谢途径影响烟草发酵。该研究为指导烟草发酵提供了理论依据，并对减少烟草发酵过程中的有害物质提出了见解。