Traditional fermentation processes, representing the synergistic interaction between human practices and natural microorganism, have evolved over millennia and become deeply embedded in culinary traditions worldwide. From lactic acid bacteria metabolism in yogurt (Chen et al., 2017) to the saccharification and fermentation processes involving jiuqu (a traditional fermentation starter), microbial communities shape food flavor profiles and drive the diversity of human diets through distinct biochemical transformation pathways. This transformation process is particularly complex in the fermentation of cigar tobacco leaves, where quality development depends on both physicochemical changes and intricate microbial interactions with tobacco substrates (Yao et al., 2022b). Microorganisms colonizing cigar surfaces could degrade macromolecules such as proteins and polysaccharides, thereby facilitating aroma precursor formation and contributing to the cigar’s characteristic aroma through secondary metabolite production (Yao et al., 2023). These multi-layered biotransformation processes render microbial community succession a key factor in regulating cigar quality (Jia et al., 2023).

Traditional fermentation processes are often limited by long durations and poor controllability. Current research explores optimization strategies using exogenous additives. The use of plant extracts, microbial inoculants (Yao et al., 2022a), and other exogenous compounds can enhance fermentation efficiency by modifying the microenvironment, guiding microbial community structure, and accelerating functional metabolism (Hu et al., 2023). Notable examples include the addition of Fangxian huangjiu (a traditional Chinese yellow rice wine), which significantly improves microbial composition and product quality (Yao et al., 2024). The intervention of humi (parched rice) promotes aroma precursor formation and optimizes tobacco chemical composition (Ren et al., 2024). The combined use of plant extracts and microbial agents by Cai et al. (2024) successfully reshaped microbial communities, markedly enhancing cigar aroma richness and smoke freshness (Cai et al., 2024). These results confirm that exogenous additives influence fermentation via two mechanisms: microecological remodeling and metabolic network regulation.

Tobacco production generates substantial organic waste, such as tobacco flower buds, whose utilization remains limited. Improper disposal of these residues not only causes environmental pollution but also results in considerable resource waste (Shen et al., 2024). Tobacco flower buds, a byproduct of tobacco topping, are rich in aromatic compounds and nutrients, representing untapped potential (Xu et al., 2016). Although existing studies have demonstrated the positive impact of tobacco flower bud extract on the fermentation of cigar tobacco leaves (Zheng et al., 2023), critical gaps remain in understanding its underlying mechanisms. This study introduces tobacco flower bud extract into the cigar fermentation system, employing metagenomic sequencing to systematically elucidate its regulatory effects on microbial community composition, functional gene expression, and aroma compound biosynthesis. This microbial ecology-driven research strategy provides theoretical insights into improving fermentation efficiency and optimizing product flavor, while also proposing innovative approaches for utilization tobacco waste, thereby promoting circular economy principles and contributing to sustainable development goals, as graphically summarized in Figure 1.

Tobacco flower buds, as a typical agricultural by-product, have traditionally been regarded as processing waste. In recent years, they have been found to be rich in natural aroma precursors and nutrients, and to possess unique potential for application in cigar tobacco leaves fermentation (Ding et al., 2024). Compared with the study by Yao et al. (2024) using Fangxian huangjiu (a traditional Chinese yellow rice wine), the aroma precursors of tobacco flower buds (e.g., β-ionone, phenylacetaldehyde, etc.) exhibit greater structural similarity to the tobacco leaves matrix, making them more favorable for aroma formation. Previous studies have shown that the addition of tobacco flower buds or their extracts can significantly improve tobacco quality (Nong, 2015). However, the mechanisms by which they influence microbial community dynamics and metabolic network remain unclear. Fermentation, as a key link in the cigar production process, centers on the conversion of macromolecules (e.g., proteins, sugars) into small molecules and volatile compounds under the synergistic action of microorganisms and enzymes. Traditional fermentation largely relies on natural processes but is often limited by long durations and poor controllability. In recent years, strategy involving the exogenous addition of fermentation media to enhance efficiency by modulating microbial activity have attracted gained increasing attention (Fang et al., 2024), although most studies remain limited to single-strain applications or chemical index analyses (Guo et al., 2024). In this study, we systematically revealed the multilevel regulation of microbial communities, chemical components and aroma quality of tobacco bud extracts in cigar fermentation by integrating macrogenomics and flavoromics techniques, providing a theoretical basis for the application of plant resources in fermentation engineering.

The addition of tobacco flower bud extract significantly altered the chemical profile of fermented leaves. In the FE group, nicotine content decreased markedly compared with the natural fermentation group (P < 0.001), with a 35% greater reduction than that reported by Qiao et al. (2024) for Saccharomyces cerevisiae-treated groups. This reduction mitigates nicotine’s harshness and associated health risks (Abrams et al., 2018), likely mediated by Staphylococcus-driven nicotine degradation (Pei et al., 2025). Total sugar and reducing sugar contents was increased by 12.5%–18.75% and 13.04%–27.27%, respectively. These elevated sugar levels provided sufficient substrates for Maillard reactions, helping to neutralize nicotine-induced irritation, improve smoke smoothness, and promote the generation of acids and other compounds that help stabilize smoke pH and reduce harshness. Additionally, total nitrogen content decreased markedly (P < 0.01) compared with that before fermentation (UW group). Protein and starch are macromolecules in tobacco, high levels of which are detrimental to the smoking characteristics of tobacco (Fang et al., 2024). In addition, the potassium-to-chloride (K/Cl) ratio was optimized to an optimal range of 4-10, which significantly improved the combustibility and softness of the tobacco.

GC-MS analysis results showed pronounced shifts in aroma composition. Unfermented leaves treated with flower bud extract (UFE group) exhibited the most total aroma content (1,135.03 μg/g), dominated by neophytadiene, β-ionone, phenylacetaldehyde, and 5-methyl-2-furaldehyde. Flower bud extract enriched total aroma by 106%, directly supplementing key compounds like beta-Ionone, D-Solanone, 5-methyl-2-furaldehyde, phenylacetaldehyde and sclareol (from 53.84 μg/g to 161.94 μg/g). Post-fermentation (FE group), total aroma decreased by 20.5% compared with UFE, but compositional optimization occurred: carotenoid degradation products (e.g., farnesyl acetone, citronellal) increased by 6.05%, Maillard reaction products (e.g., furfuryl alcohol) by 21.16%, and Cembranoid degradation products (e.g., D-solanone) by 26.64%, alongside a 24.6% reduction in neophytadiene to minimize grassy off-flavors. Notably, FE retained a 12.3% aroma advantage over FW, likely due to microbial metabolic shifts and accelerated chlorophyll derivative conversion during high-temperature fermentation (Wu et al., 2023). Similar to Staphylococcus nepalensis-enhanced carotenoid degradation (Pei et al., 2025), Staphylococcus dominance in FE likely drove aroma enrichment. Carotenoid derivatives (e.g., farnesylacetone’s fruity notes) and indole’s floral tones synergistically crafted the cigar’s complex aroma (Jia et al., 2025), while citronellal’s freshness enhanced aromatic complexity. Tobacco flower buds, rich in Maillard reaction precursors, have been used to develop flavor-enhancing agents (Bai et al., 2018). Cembranoid degradation products, particularly solanone-a major neutral aroma compound in tobacco-impart a fresh carrot-like sweetness that refines smoke smoothness (Yao et al., 2022b). These results underscore the necessity of synergizing tobacco flower bud extract with fermentation. The unfermented stage establishes a foundation for precursor accumulation through the flower buds’ nutrient richness, while functional microbial metabolism during fermentation achieves final aroma transformation and balance. The increased levels of key aroma compounds and optimized compositional ratios demonstrate that integrating flower bud extract with fermentation significantly enhances tobacco leaves quality.

Changes in chemical and aroma compounds were closely associated with the structure of microbial communities and their metabolic activities. Macrogenomic sequencing results showed that the introduction of the bud flower extract triggered a colony remodeling—relative abundance of Aspergillus surged from 5% to 63% during the unfermented stage (UFE group), while Staphylococcus decreased abruptly from 63% to 2%. This initial flora dominance may stem from sugars and amino acids in the extract being more compatible with the metabolic requirements of Aspergillus. Although rapid proliferation of Aspergillus may increase the risk of mold (Wu et al., 2024), its amylase activity reserves reducing sugars for subsequent fermentation (Wu et al., 2023). Its abundance was effectively suppressed to 1% by temperature and humidity regulation in the subsequent fermentation stage (FE group), achieving a balance between aroma persistence and safety. Bacterial dominance was further confirmed through LEfSe analysis, Fourteen characteristic genera were identified in the UW group (e.g., Sphingomonas, Pseudomonas, Methylobacterium), while 26 genera were found in the UFE group, including Aspergillus, Aureimonas, Escherichia, Methylorubrum, Leucosporidium. Leucosporidium is a psychrophilic yeast that is not adapted to the high-temperature conditions of cigar fermentation (Glushakova et al., 2021; Pan et al., 2024). Its presence in the UFE group likely originates from environmental sources or microbial residues in the flower bud extract, rather than from active fermentation. As it has no known role in aroma formation or tobacco quality improvement, Leucosporidium is not considered a beneficial genus in this context. After fermentation, the number of microbial genera declined from 26 species in the UFE group to 5 species in the FE group, including Staphylococcus and Pseudoalteromonas, The LDA score of Staphylococcus reached 4.8, indicating a shift toward microbial community specialization. Staphylococcus has been shown to play a dominant role in the process of tobacco fermentation, and is capable of generating aroma precursors by degrading proteins and polysaccharides, and facilitating the conversion of sugars into volatile aroma components (Hu et al., 2019). Pseudoalteromonas complements the metabolism of Staphylococcus through the secretion of lipase, amylase, and protease (Odeyemi et al., 2018) to jointly enhance the efficiency of substrate conversion. In contrast, only Pantoea was present in the FW group. Previous studies have shown that Pantoea is widely present in the brewing environment of traditional fermented foods due to its good environmental resistance. Furthermore, it has been associated with the formation of various aroma substances (Zhang et al., 2025). The predominance of Sphingomonas in the pre fermentation UW group suggests its potential importance at this stage. Previous studies have shown that Sphingomonas can be used for biodegradation of aromatic compounds (Pinyakong et al., 2003). Aspergillus was the dominant genus in the UFE group, and its relative abundance decreased dramatically as fermentation progressed, consistent with the results of Zhao et al. (2024).

Metabolic profiling revealed enhanced carbohydrate metabolism and membrane transport post-fermentation, underpinning substrate conversion and energy supply (Ding et al., 2023). Increased Maillard reaction intermediates (e.g., 5-Methyl-2-furaldehyde) (Abrams et al., 2018) aligned with aroma optimization. Crucially, the flower bud extract suppressed harmful metabolite pathways (e.g., carcinogens reduced by 47.0%), enhancing product safety. However, (1) Aspergillus suppression requires stringent humidity and temperature control, and (2) excessive Staphylococcus dominance may compromise diversity. Future studies should explore synergistic applications of flower bud extract with multifunctional strains (e.g., sugar-metabolizing, antimicrobial species) to refine efficiency and stability.

In conclusion, the incorporation of tobacco bud extract led to microbial community structure remodeling, metabolic network and regulation of aroma synthesis pathways, which significantly enhanced the fermentation efficiency and quality of cigar tobacco leaves. These findings offer a promising strategy for the high-value utilization of agricultural byproducts and provide a foundation for optimizing fermentation processes across diverse agricultural products.