Investigation on dynamic changes of volatile components, non-volatile constituents and microorganisms in cigar tobacco leaves fermented with nobiletin at different stages based on multi-omics analysis

Flavonoid monomers from Citri Reticulatae Pericarpium, particularly nobiletin, can enhance the aroma quality of cigar tobacco leaves (CTLs) during fermentation. However, the nobiletin (herein abbreviated as CCPS)-mediated changes in chemical components and microorganisms of cigar tobacco leaves at different fermentation days remain unclear.CCPS was added to Dexue No. 1 CTLs at 0.3% (w/w), and the CTLs was fermented at 35 °C and 75% relative humidity for 30 days. To elucidate the changes of volatile and non-volatile constituents and microorganisms in CCPS-treated samples at different fermentation days, this study integrated multi-omics technologies consisting of metagenomics, HS-GC-IMS, HS-SPME-GC-MS and untargeted metabolomics for investigation. Results indicated that the total sugar, reducing sugar, protein, and pectin content of CTLs significantly decreased by 22.36–60.99% during fermentation. CCPS significantly altered leaf physicochemical indices and enzyme activities. The amino acid content presented a significant tendency of initial increase followed by decrease. The total amount of volatile organic compounds (VOCs) showed increasing trend. Alcohols, ketones, and aldehydes were the main small molecule flavor compounds, and heterocyclic aroma-producing substances were significantly enriched. Core genera (Alternaria, Aspergillus, Staphylococcus) strongly correlated with flavor metabolites. Metabolomics combined with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that amino acid metabolism may serve as the core regulatory pathway during CTL fermentation. This study provides theoretical evidence for elucidating the changes in chemical components and microbial communities of CTLs across different fermentation days mediated by CCPS.