3.1. Hot-pressure and 60Co-γ Treatment Eliminated the D. officinale Endophytes
To eliminate the D. officinale endophytes and decrease their interference on the gut microbiota in mice, D. officinale was treated with hot-pressure and 60Co-γ. After quality control, denoising, and removal of chimera sequences, 265,440 and 961,560 high-quality 16S and ITS1-ITS2 sequences were obtained. A total of 1,339 and 1,083 OTUs with 97% identity cutoffs of bacteria and fungi of D. officinale were found (Fig. 1). The number of bacterial and fungal OTUs of D. officinale was reduced significantly after 60Co-γ irradiation and hot pressure treatment; hot pressure treatment (121 ℃ for 40 min) decreased the OTUs most. Concurrently, the number of the phylum, class, order, family, genus, and species of bacteria (Fig. S1 A) and fungi (Fig. S1 B) of DO and ASDO40 decreased by 53.33%, 65.38%, 61.02%, 61.36%, 70.78%, and 64.97%, and 33.33%, 42.86%, 51.35%, 46.77%, 49.35%, and 44.68%, respectively. Overall, the sorts of D. officinale endophytic fungi and bacteria decreased the most significantly after 40 min hot pressure treatment.
Analysis of alpha diversity indicated that the community richness (Chao) and community diversity (Shannon) of endophytic bacteria and fungi in samples under hot pressure treatment (ASDO40) was lower than DO (Fig. S2 A-D). However, ASDO40 treatment did not change the dominant bacterial genera compared to DO samples (Fig. 2A). The top three dominant bacterial genera (Pseudomonas, Ochrobactrum, and Rhodococcus) in DO and ASDO samples were accounting for 69.45% and 95.48% of the relative abundance, respectively. However, in ASDO40, there was a decrease in the relative abundance of Burkholderia, Caballeronia, Paraburkholderia, Alloprevotella, Prevotella, Neisseria, and Streptococcus, compared with those in DO (Fig. 2A). In addition, ASDO40 treatment changed the dominant fungal genera. The top three dominant fungal genera in DO were Fusarium, unclassified-f-Didymellaceae, and Occultifur, accounting for 62.68% of the relative abundance, while those in ASDO were Fusarium, Cutaneotrichosporon, and Simplicillium, accounting for 63.65% of the relative abundance. Furthermore, ASDO reduced the relative abundance of Occultifur, Rhodotorula, Pyrenochaetopsis, Sporidiobolus, and Sordaria, compared with those in DO (Fig. 2B). Thus, ASDO40 treatment reduced the diversity and richness of D. officinale endophytes and participated in eliminating the interference of D. officinale endophytes.
3.2 Change in the Gut Microbiota Structure and Metabolite SCFAs after the Intake of D. officinale Juice
To analyze how D. officinale juice modulates the gut microbiota structure in mice, we carried out 16S and ITS sequencing on fecal samples of mice administered with sterile water (C), D. officinale juice (DO), and autoclaved D. officinale juice (ASDO) on days 0 and 28. In all fecal samples, Lactobacillus, Bifidobacterium, Desulfovibrio were the three top dominant bacterial genera, while Aspergillus, Penicillium, Acaulium were the three top dominant fungal genera. Our experiments showed that oral administration of D. officinale juice for 28 days (DO28) could effectively increase the diversity of gut microbiota and the relative abundance of beneficial endophytes and decrease the relative abundance of harmful endophytes. The results indicated that the number of bacterial and fungal OTUs in the C0 group and C28 group were not different but increased in the DO28 group in contrast to the DO0 group (Fig. S3 A-B). Meanwhile, the number of bacterial OTUs in the ASDO28 group increased compared with the ASDO0 in bacteria, but that of fungi decreased (Fig. S3 B). According to the Kruskal Wallis rank-sum test, there was no significant difference in the relative abundance of Lactobacillus, Ruminococcus, Alistipes, Aerococcus, Bacteroides, Lachnoclostridium, Anaerostipes, Parasutterella, Pyxidiophora, Cladosporium, Talaromyces, Rhodotorula, Filobasidium, Aspergillus, Mortierella, Penicillium, Cutaneotrichosporon, and Candida in C0, ASDO0 and DO0 groups (P < 0.05); in addition, the relative abundance of Bifidobacterium of DO0 was significantly lower than that of C0 and ASDO0 groups (P > 0.05). However, DO28 increased the relative abundance of bacterial genera, including Lactobacillus, Bifidobacterium, Ruminococcus, Alistipes, whereas decreased Aerococcus, Bacteroides, Lachnoclostridium, Anaerostipes, and Parasutterella compared with those in C28 and ASDO28 (Fig. 3A). DO28 increased the relative abundance of fungal genera, including Pyxidiophora, Cladosporium, Talaromyces, Rhodotorula, Filobasidium, and decreased Aspergillus, Mortierella, Penicillium, Cutaneotrichosporon, Candida, compared with those in C28 and ASDO28 groups (Fig. 3B). Our results also indicated that DO28 altered the gut microbiota structure in mice by increasing the beneficial bacteria Lactobacillus murinus (Wu et al., 2021 b), Lactobacillus johnsonii (Olnood et al., 2015), Bifidobacterium pseudolongum (Tatsuoka et al., 2021), and Lactobacillus reuteri (Singh et al., 2021), and reducing harmful bacteria Ochrobactrum anthropi (Grabowska-Markowska et al., 2019), Aerococcus urinaeequi (Heidemann et al., 2018), and Clostridium sp. cTPY-12 (Wang et al., 2019), compared with those in C28 and ASDO28 groups (Fig. S4 A).
SCFAs are one of the index components to evaluate gastrointestinal function (Shi et al., 2020); we found that the D. officinale juice could effectively increase the content of SCFAs in mouse guts. Compared with the C28 and ASDO28 groups, the concentration of total SCFAs in the DO28 group was increased (Fig. 4). Among them, acetic acid, propanoic acid, and butanoic acid contents of the DO28 group improved remarkably (P < 0.05), suggesting that DO28 may affect the intestinal environment to some extent by increasing SCFAs-producing bacteria, compared with those in C28 and ASDO28 groups. Taken together, D. officinale juice can effectively increase the contents of SCFAs.
These findings cumulatively suggest that D. officinale juice can effectively regulate gut microbiota by improving their diversity, increase the relative abundance of beneficial endophytes and the contents of SCFAs, and reduce the relative abundance of harmful endophytes while these were not observed in sterilized (autoclaved) D. officinale juice.