Widely targeted metabolomic analysis reveals that volatile metabolites in cigar tobacco leaves dynamically change during fermentation

Abstract

Changes in volatile metabolites during cigar tobacco leaves fermentation as well as the metabolic pathways of metabolites with significant differences were investigated to determine the influence of cigar tobacco leaves fermentation on its flavor. The volatile substances in cigar tobacco leaves at different stages were detected by headspace-solid phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS), and the main differences in volatile substances in cigar tobacco leaves at different fermentation stages of Yunxue1 in Yuxi production area were analyzed by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). The results show that in the process of cigar tobacco leaves fermentation (YXF0, YXF1, YXF2, YXF3, YXF4, YXF5), a total of 613 volatile metabolites were detected, and a significant difference was found in 263 kinds of metabolites. Among them, the main upregulated differential metabolites were 1,3,6,10-Cyclotetradecatetraene, 3,7,11-trimethyl-14-(1-methylethyl)-, [S-(E,Z,E,E)]-, Benzoic acid, Benzaldehyde, etc. While the main downregulated differential metabolites included beta.-Myrcene, trans-Farnesol, etc. The metabolites with significant differences are mainly concentrated in the biosynthesis of monoterpenes, diterpenes, sesquiterpenes and triterpenes, the degradation metabolism of amino acids, such as valine, leucine and isoleucine, and the biosynthesis of phenylpropyl. There were 8 different metabolites in 5 groups, including 4- (1-methylethyl) -1-cyclohexene-1-formaldehyde、2, 4-dihydroxyacetophenone、2-methylbutyl 3-methylbutyrate and methylpyrazine, all of which showed upregulation trend during fermentation. In the fermentation process, volatile metabolites participate in various synthesis and degradation pathways. The biosynthesis pathway of terpenes and amino acid synthesis and degradation pathway are connected to produce various terpenes, aldehydes and other substances, such as 1,3,6,10-Cyclotetradecatetraene, 3,7,11-trimethyl-14-(1-methylethyl)-, [S-(E,Z,E,E)]-、benzaldehyde and 4-hydroxybenzaldehyde, which are conducive to the overall flavor and quality of cigar tobacco leaves.

Keywords: Cigar tobacco leaves; Fermentation; Flavor; Metabolic pathway; Volatile metabolites.

Fig. 1

Metabolite categories and PCA score Figure. (A)Type and proportion of volatile metabolites of cigar smoke. Each color represents a metabolite category, and the color block area indicates the high or low proportion of that category. (B)Principal component analysis (PCA) scores of samples and quality control samples at different stages of fermentation. PC1 represents the first principal component, PC2 represents the second principal component, and percentage represents the interpretation rate of this principal component to the data set. Each point in the diagram represents a sample, and samples in the same Group are represented by the same color, and group is the grouping. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

OPLS-DA model generated fermentation in different periods of the score map and volcano map. (A)YXF0 vs. YXF1 OPLS-DA score chart. (B) YXF0 vs. YXF3 OPLS-DA score chart. (C)YXF0 vs. YXF5 OPLS-DA score chart. Each point in the diagram represents a sample, and samples in the same Group are represented by the same color. (D) YXF0 vs. YXF1 OPLS-DA S-plot chart. (E) YXF0 vs. YXF3 OPLS-DA S-plot chart. (F) YXF0 vs. YXF5 OPLS-DA S-plot chart. Red dots indicate that these metabolites have VIP values greater than or equal to 1, and green dots indicate that these metabolites have VIP values less than 1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Analysis of cluster heat map and Venn diagram for different fermentation periods. (A) Sample population clustering diagram. Horizontal is the sample name, vertical is the metabolite information, Group is the group, and different colors are the colors filled with different values obtained after standardized treatment of different relative contents (red represents high content, green represents low content). Where, Class is the first-class classification of substances; The cluster line on the left of the figure is the metabolite cluster line, and the cluster line on the top of the figure is the sample cluster line. (B) Overall sample Venn diagram. (C) YXF0 vs. YXF1, YXF0 vs. YXF3, YXF0 vs. YXF5 group difference Venn diagram. Each circle in the figure represents a comparison group, and the numbers in the overlapping part of the circle and the overlapping part represent the number of different metabolites shared between the comparison groups, while the numbers in the non-overlapping part represent the number of different metabolites unique to the comparison group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

The top 20 metabolites bar chart in the comparison of the difference multiples in each group. (A)YXF0 vs. YXF1 group. (B)YXF0 vs. YXF3 group. (C)YXF0 vs. YXF5 group. The horizontal coordinate is the log₂FC of the differential metabolite, that is, the logarithmic value of the differential multiple of the differential metabolite is taken as the base 2, and the vertical coordinate is the differential metabolite. Red represents upregulated metabolite content and green represents downregulated metabolite content. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

KEGG enrichment pathway map of different metabolites in different fermentation cycles. (A) YXF0 vs. YXF1 enrichment map. (B) YXF0 vs. YXF3 enrichment map. (C) YXF0 vs. YXF5 enrichment map. The horizontal coordinate represents the Rich Factor corresponding to each path, the vertical coordinate is the path name (sorted by P-value), the color of the points reflects the P-value size, and the redder indicates the more significant enrichment. The size of the dots represents the number of differentiated metabolites enriched. (D) Integration map of major metabolic pathways during fermentation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)