Exploration of microbiome diversity of stacked fermented grains by flow cytometry and cell sorting

Abstract

Sauce-flavor baijiu is one of the twelve flavor types of Chinese distilled fermented product. Microbial composition plays a key role in the stacked fermentation of Baijiu, which uses grains as raw materials and produces flavor compounds, however, the active microbial community and its relationship remain unclear. Here, we investigated the total and active microbial communities of stacked fermented grains of sauce-flavored Baijiu using flow cytometry and high-throughput sequencing technology, respectively. By using traditional high-throughput sequencing technology, a total of 24 bacterial and 14 fungal genera were identified as the core microbiota, the core bacteria were Lactobacillus (0.08-39.05%), Acetobacter (0.25-81.92%), Weissella (0.03-29.61%), etc. The core fungi were Issatchenkia (23.11-98.21%), Monascus (0.02-26.36%), Pichia (0.33-37.56%), etc. In contrast, using flow cytometry combined with high-throughput sequencing, the active dominant bacterial genera after cell sorting were found to be Herbaspirillum, Chitinophaga, Ralstonia, Phenylobacterium, Mucilaginibacter, and Bradyrhizobium, etc., whereas the active dominant fungal genera detected were Aspergillus, Pichia, Exophiala, Candelabrochaete, Italiomyces, and Papiliotrema, etc. These results indicate that although the abundance of Acetobacter, Monascus, and Issatchenkia was high after stacked fermentation, they may have little biological activity. Flow cytometry and cell sorting techniques have been used in the study of beer and wine, but exploring the microbiome in such a complex environment as Chinese baijiu has not been reported. The results also reveal that flow cytometry and cell sorting are convenient methods for rapidly monitoring complex microbial flora and can assist in exploring complex environmental samples.

Keywords: cell sorting; flow cytometry; high-throughput sequencing; microbiome diversity; stacked fermented grains.

FIGURE 1

Results of flow cytometry analysis of model microorganisms. (A) Metabolically active bacteria distributed in the region after flow cytometry analysis. (B) Metabolically active mold in the region after flow cytometry analysis. (C) Metabolically active yeast distributed in the region after flow cytometry analysis.

FIGURE 2

Microbial changes during rounds 1–3 of stacking by flow cytometry analysis. (A) Flow cytometric analysis of the first round of stacked grain activity staining. (B) Flow cytometric analysis of the second round of stacked grain activity staining. (C) Flow cytometric analysis of the third round of stacked grain activity staining. (D) Changes in the number of microorganisms in 1–3 rounds of stacked fermented grains.

FIGURE 3

Flow cytometry analysis of different extraction times on stacked fermented grains. (A) Effect of different shaking times on the internal density of cell. (B–G) The effect of shaking time of 0, 5, 10, 15, 20, and 25 min on the microbial morphology of stacked fermented grains by flow cytometry. (b–g) Counted microspheres were detected at different shaking times.

FIGURE 4

Microbial abundance in 1–3 rounds of stacked fermented grains based on high-throughput sequencing data. (A) Bacterial abundance map for stacking rounds 1–3 of stacked fermented grains. (B) Fungal abundance map for stacking rounds 1–3 of stacked fermented grains.

FIGURE 5

Map of microbial abundance in different areas after sorting samples at the end of the third round of stacking.