Sequencing metadata
After getting rid of low-quality reads and chimeras, 1,923,284 high-quality 16S rRNA gene sequences (V4-533-786 bp) without chimera were obtained, with an average of 80136.83 ± 86.63 sequences per sample, ranging from 80034 to 80344. The average length of these sequences is 411 bp, and they belong to 1703 operational taxonomic units (OTUs) on the basis of 97% similarity. Each sample had an average of 567.67 ± 7.09 OTUs. After calculation, the effective data accounted for 98%, while the average Q20 was 80.67, and the average GC ratio was 52.79% (Table 1).
Microbial diversity in the gastrointestinal tracts of the mice
The microbial community richness (alpha diversity) was measured using the Shannon index, Chao1, and observed species. No significant differences were observed between the P and R groups. According to the Chao1 and observed species analysis, significant differences were found in the bacterial community structure of P vs. W and R vs. W (Fig. 1, P<0.05).
To examine beta diversity among mice in W, P, and R groups, weighted and unweighted UniFrac distances were both calculated to estimate dissimilarities in the community membership. PCoA was applied to visualize distances, showing that mice treated with water, Pu-erh, and Dian Hong black tea harbored distinct microbial taxa (Wang et al. 2020) (Fig. 2). Based on membership, the microbial communities of mice treated with water, Pu-erh, and Dian Hong black tea, clustered together and were separated from each other along the principal coordinate axis. Differences in community memberships between the different groups were also proven to be significant by ANOSIM (P < 0.01), indicating distinct microbial community structures among mice treated with different teas.
Differences in the microbial communities of mice treated with water, black tea, and Pu-erh
The specific taxonomic groups of species (e.g., kingdom, phylum, class, order, family, genus, and species) were classified (Fig. 3). Two groups of bacteria dominated the intestinal microbiota of different groups of mice, namely Firmicutes and Bacteroidetes, which accounted for more than 97% of reads. The relative abundance of these two predominant microbial divisions (phylum level) differed between the mice treated with water, Pu-erh, and Dian Hong black tea. The mice in the W, P, and R groups contained 67.48%, 70.11%, and 77.65% Firmicutes, and 28.41%, 26.65%, and 19.44% Bacteroidetes. At the genus level, Lactobacillus, Lachnospiraceae, Ruminococcaceae, Alistipes, Alloprevotella, Turicibacter, Bacteroides, Desulfovibrio, Faecalibaculum, and Parasutterella were dominant.
According to the Venn diagrams, a total of 620 OTUs are obtained via OTU clustering, consisting of three groups. The cluster numbers of OTUs in the Dian Hong black tea group, the control group, and the Pu-erh tea group were 569, 560, and 574. Of these, the Dian Hong black tea group and the control group shared 528 OTUs in the overlapping portion, the Pu-erh tea group and the control group shared 531 OTUs, and the Pu-erh tea group and the Dian Hong black tea group shared 532 OTUs. Moreover, 19 species were unique to the Pu-erh tea group, nine species were unique to the control group, and 17 species were unique to the Dian Hong black tea group (Fig. 4).
LEfSe was employed to identify specific genera that were differentially distributed among the mice in the W, P, and R groups, presenting a potent tool that focuses on the significance of differences, as well as biological relevance. The LEfSe results are shown in Fig. 5. Furthermore, 22 genera were differentially represented among the three groups, with 12 being more abundant in the mice of the P group (e.g., Lactobacillaceae, Lactobacillus spp., Bacilli, Lactobacillales, Ruminococcaceae, Clostridium papyrosolvens, Ruminococcaceae spp., Erysipelotrichales, Erysipelotrichia, Alloprevotella spp. and Prevotellaceae) and six being more abundant in the mice of the R group (e.g., Lachnospiraceae, Lachnospiraceae spp., Clostridiales, Clostridia, Firmicutes, and Clostridiales bacterium CIEAF 020).