Analysis of Beijing Douzhir Microbiota by High-Throughput Sequencing and Isolation of Acidogenic, Starch-Flocculating Strains

Abstract

Beijing Douzhir is a traditional Chinese fermented drink produced by the natural fermentation of mung beans as the raw material. Ma tofu is an edible by-product of Douzhir processing. Douzhir microbiota, particularly bacteria involved in the natural fermentation process, has not been clearly established, resulting in limited industrial Douzhir production. Here, three uncooked Douzhir samples (D group) and three uncooked Ma tofu samples (M group) (two replicates per sample) were collected from three manufacturers in different locations in Beijing. The composition and diversity of the bacterial communities in each sample were analyzed by high-throughput sequencing. In total, 637 operational taxonomic units (OTUs) were revealed in the D group through database alignment, and 656 OTUs were found in the M group. The Chao, ACE, and Shannon indices were not significantly different in Douzhir samples from different manufacturers (p > 0.05). Representatives of six phyla were found in all 12 samples. Dominant bacteria were isolated and identified using mung bean juice as the growth medium. In both Douzhir and Ma tofu samples, dominant bacteria belonging to Firmicutes and Proteobacteria comprised > 94% of the total microbiota. The dominant bacteria included members of the Lactococcus, Acetobacter, Streptococcus, and Lactobacillus genera. Considering the dominant-microbiota information, we employed a plate-separation technique and isolated two strains of acid-producing bacteria from the Douzhir and Ma tofu samples with starch-flocculating activity: Acetobacter indonesiensis and Lactococcus lactis subsp. lactis. Such strains can serve as a foundation for the standardized industrial production of Douzhir.

Keywords: Douzhir; Ma tofu; acidogenic; high-throughput sequencing; isolation; starch flocculation.

FIGURE 1

Production of mung bean starch by sour liquid flocculation.

FIGURE 2

Analysis of operational taxonomic units (OTUs) shared by different groups. (A) We identified 428, 449, and 391 OTUs in the D-DZ, D-LFS, and D-LCQK groups, respectively. The data showed that 297 OTUs were shared between the D-DZ and D-LFS groups; 278 were shared between the D-DZ and D-LCQK groups, and 274 were shared between the D-LFS and D-LCQK groups. The total shared richness was 218 OTUs, and the total richness of all was 637 OTUs. (B) We identified 450, 406, and 327 OTUs in the M-DZ, M-LFS, and M-LCQK groups, respectively. The data showed that 269 OTUs were shared between the M-DZ and M-LFS groups, 191 were shared between the M-DZ and M-LCQK, and 214 were shared between the M-LFS and M-LCQK groups. The total shared richness was 147 OTUs. The total richness of all groups was 656 OTUs.

FIGURE 3

Distributions of the Douzhir and Ma tofu microbiota compositions at the phylum level. (A) D-DZ. (B) D-LFS. (C) D-LCQK. (D) M-DZ. (E) M-LFS. (F) M-LCQK. The respective proportions of each phylum in the D-DZ, D-LFS, and D-LCQK groups were as follows: Bacteroidetes: 1.59, 1.72, and 3.55%; Firmicutes: 67.4, 77.12, and 87.17%; Proteobacteria: 30.55, 20.67, and 9.11%; others: 0.13, 0.24, and 0.05%. The respective proportions of each phylum in the M-DZ, M-LFS, and M-LCQK groups were as follows: Bacteroidetes: 5.15, 2.73, and 0.11%; Firmicutes: 77.31, 52.23, and 17.9%; Proteobacteria: 17.3, 44.15, and 81.95%; others: 0.08, 0.04, and 0.02%.

FIGURE 4

Distributions of the Douzhir and Ma tofu microbiota compositions at the genus level. (A) D-DZ. (B) D-LFS. (C) D-LCQK. (D) M-DZ. (E) M-LFS. (F) M-LCQK. The respective proportions of each genus in the D-DZ, D-LFS, and D-LCQK groups were as follows: Lactobacillus: 4.98, 4.86, and 5.02%; Lactococcus: 37.60, 45.31, and 54.53%; Acetobacter: 1.12, 0.89, and 0.59%; Streptococcus: 22.03, 23.76, and 18.25%; Klebsiella: 24.23, 13.18, and 4.86%; Pseudomonas: 2.22, 2.59, and 1.69%; Acinetobacter: 1.54, 2.92, and 1.28%; Clostridium: 0.15, 0.15, and 0.04%; Megasphaera: 0.55, 0.35, and 1.85%; others: 5.59, 5.99, and 11.90%. The respective proportions of each genus in the M-DZ, M-LFS, and M-LCQK group were as follows: Lactobacillus: 17.22, 24.79, and 12.82%; Lactococcus: 47.66, 12.54, and 1.24%; Acetobacter: 1.56, 31.09, and 80.67%; Streptococcus: 4.34, 3.08, and 3.47%; Klebsiella: 8.79, 2.18, and 0.50%; Pseudomonas: 2.76, 5.17, and 0.65%; Acinetobacter: 2.09, 4.02, and 0.06%; Clostridium: 1.78, 5.89, and 0.03%; Megasphaera: 3.26, 3.44, and 0.10%; others: 10.54, 7.81, and 0.46%.

FIGURE 5

Non-metric multidimensional scaling (NMDS) ordination. NMDS plots demonstrating that Douzhir and Ma tofu harbored different bacterial communities.

FIGURE 6

Phylogenetic relationships of D-23 and M-10 with related species based on partial 16S rDNA gene-sequence analysis. The phylogenetic tree was constructed using the neighbor-joining method (MEGA 5.0). The numbers at the nodes are bootstrap confidence levels (expressed as the percentage) from 1,000 replicates. The scale bar represents 0.05 substitutions per nucleotide position. Reference sequences were obtained from the GenBank nucleotide sequence database.

FIGURE 7

Optical micrograph of starch granule aggregation. (A) Un-inoculated mung bean starch milk. (B) Mung bean starch milk after the addition of D-23 strain fermentation liquid. (C) Mung bean starch milk after the addition of M-10 strain fermentation liquid.

FIGURE 8

Scanning electron micrograph of starch granules with attached D-23 and M-10 cells. (A) D-23 strain (10,000×). (B) M-10 strain (10,000×).