Microbial metabolism during the thermophilic phase promotes the generation of aroma substances in nongxiangxing Daqu

Abstract

The thermophilic phase of Daqu fermentation is considered the key period for aroma production in Daqu, but little is known about the changes in substances during this phase. In this study, we combined a metabolomics approach with high-throughput sequencing to analyze the metabolic profiles and identify metabolism-associated microbes during the thermophilic phase of Daqu fermentation. The results revealed that the metabolic sets after 5 and 9 days of fermentation in the thermophilic phase were similar, and several amino acid and biosynthesis-related metabolic pathways were significantly enriched. In addition, pyrazines and alkanes increased and esters decreased significantly after the thermophilic phase. The metabolism of substances during the thermophilic phase involved 38 genera, and the main metabolic pathways involved were glycolysis, TCA cycle, butyric acid metabolism, and five amino acid metabolic pathways. In summary, this study points in the direction for unravelling the mechanism of aroma production in Daqu.

Keywords: Daqu; Key microbes; Metabolic mapping; Thermophilic phase; Untargeted metabolomics.

Fig. 1

Differential expression of NVOCs during the thermophilic phase. OPLS-DA on day 7 vs. day 12 of fermentation: positive ion mode (a) and negative ion mode (b); OPLS-DA analysis on day 7 vs. day 16 of fermentation: positive ion mode (c) and negative ion mode (d). Volcano plots of day 12 vs. day 7 (e) of fermentation and day 16 vs. day 7 (e) of fermentation. Red dots indicate significantly up-regulated compounds, blue dots indicate significantly down-regulated compounds, and grey dots represent compounds that were not significant between the two groups.

Fig. 2

Differential compound annotations during the thermophilic phase. Compound classification: (a) thermophilic phase 5-day group; (b) thermophilic phase 9-day group. Functional pathway: (c) thermophilic phase 5-day group and (d) thermophilic phase 9-day group; the upper right pathway is for first-level classification and the horizontal coordinates are for second-level classification.

Fig. 3

Differential metabolic set pathway enrichment in the thermophilic phase 5-day group (day 12 vs. day 7) versus the thermophilic phase 9-day group (day 16 vs. day 7). The vertical left coordinate displays a dendrogram of metabolic pathway clusters, categorised into six subclusters with different colours. The vertical right coordinate features the metabolic pathway ID. The closer the two pathway branches are, the more similar the metabolites involved in these two pathways are in the metabolic set. The darker the color, the higher the enrichment rate. (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 4

Differences in volatile organic compounds during the thermophilic phase. OPLS-DA on day 7 vs. day 12 of fermentation (a); OPLS-DA on day 7 vs. day 16 of fermentation (b). Clustering heatmap of differential volatile organic compounds (c). STAMP on day 7 vs. day 12 of fermentation (d), day 7 vs. day 16 of fermentation (e), and day 12 vs. day 16 of fermentation (f).

Fig. 5

Correlation analysis between differential compounds and microbial communities. Co-occurrence networks between NVOCs and bacteria (a) and fungi (b) and VOCs and bacteria (c) and fungi (d); r > 0.7, p < 0.01. Purple dots indicate compounds, green dots indicate microorganisms, the green solid line denotes a positive correlation, and the yellow dotted line denotes a negative correlation.

Fig. 6

Microbial metabolic mapping during the thermophilic phase. Pink boxes represent differential compounds; blue ovals represent correlative microbes.