Microbial and enzymatic changes in cigar tobacco leaves during air-curing and fermentation

Abstract

Metabolic enzyme activity and microbial composition of the air-curing and fermentation processes determine the quality of cigar tobacco leaves (CTLs). In this study, we reveal the evolution of the dominant microorganisms and microbial community structure at different stages of the air-curing and fermentation processes of CTLs. The results showed that the changes in metabolic enzymes occurred mainly during the air-curing phase, with polyphenol oxidase (PPO) being the most active at the browning phase. Pseudomonas, Bacteroides, Vibrio, Monographella, Bipolaris, and Aspergillus were the key microorganisms in the air-curing and fermentation processes. Principal coordinate analysis revealed significant separation of microbial communities between the air-curing and fermentation phases. Redundancy analysis showed that bacteria such as Proteobacteria, Firmicutes, Bacteroidota, and Acidobacteriota and fungi such as Ascomycota and Basidiomycota were correlated with enzyme activity and temperature and humidity. Bacteria mainly act in sugar metabolism, lipid metabolism, and amino acid metabolism, while fungi mainly degrade lignin, cellulose, and pectin through saprophytic action. Spearman correlation network analysis showed that Firmicutes, Proteobacteria, and Actinobacteria were the key bacterial taxa, while Dothideomycetes, Sordariomycetes, and Eurotiomycetes were the key fungal taxa. This research provides the basis for improving the quality of cigars by improving the air-curing and fermentation processes. KEY POINTS: • Changes in POD and PPO activity control the color change of CTLs at the air-curing stage. • Monographella, Aspergillus, Pseudomonas, and Vibrio play an important role in air-curing and fermentation. • Environmental temperature and humidity mainly affect the fermentation process, whereas bacteria such as Proteobacteria, Firmicutes, Bacteroidota, and Acidobacteriota and fungi such as Ascomycota and Basidiomycota are associated with enzyme activity and temperature and humidity.

Keywords: Air-curing; Cigar tobacco leaf; Enzyme activity; Fermentation; Microbial community.

Fig. 1

Appearance of CTLs at different periods of the air-curing and fermentation process

Fig. 2

Changes in the activities of phenylalanine deaminase (PAL, A), polyphenol oxidase (PPO, B), peroxidase (POD, C), amylase (AL, D), neutral transforming enzyme (NI, E), and alkaline protease (AKP, F) during air-curing and fermentation of CTLs. The data are expressed as the mean ± SEM with the ANOVA significance test, and changes with different lowercase letters were significant at the 0.05 level (p < 0.05). Curing represents the air-curing stage, and fermentation represents the fermentation stage

Fig. 3

Alpha diversity and beta diversity of microbial communities during modulation of CTLs. A Bacterial Shannon index during modulation. B Fungal Shannon index during modulation. Changes with different lowercase letters are significant at the 0.05 level (p < 0.05). C Principal coordinate analysis (PCoA) of bacteria during modulation. D PCoA of fungi during modulation

Fig. 4

Community dynamics of bacteria (A) and fungi (B) during modulation of CTLs. The graph represents the top 10 dominant microorganisms in terms of abundance at the genus level

Fig. 5

Heatmap of the functional relevance of microorganisms during modulation of CTLs. A Functional relevance of dominant bacteria at different modulation stages. B Functional correlation of dominant fungi at different stages of modulation

Fig. 6

Redundancy analysis (RDA) between enzyme activity and temperature and humidity during air-curing and fermentation of CTLs. The red arrows in the graph represent enzyme activity. The green arrows represent temperature (T) and humidity (H), and the blue arrows represent OTUs that have a contribution

Fig. 7

Spearman’s correlation analysis of bacteria (A) and fungi (B) during air-curing and fermentation of CTLs. The circles in the graph represent OTUs, with larger circles representing stronger correlations and different colors representing OTUs classified in different orders. The correlation R > 0.8, and the graph indicates a significant change at the 0.05 level (P < 0.05)

Fig. 8

Spearman’s correlation analysis plot between CTL microorganisms during air-curing and fermentation. The graphs indicate the correlation networks between the bacterial air-curing (A) and fermentation (B) stages and fungal air-curing (C) and fermentation (D) stages. The red line indicates a positive correlation, and the green line indicates a negative correlation. Circles indicate OTUs, text indicates OTU classification at the genus level, and circles of the same color indicate OTU annotations in the same genus. The correlation R > 0.9, and the graphs indicate a significant change at the 0.01 level (P < 0.01)