Dietary intervention in vivo simulated food intake in normal human daily life with complex interactions between gut microbiota and hosts. It was concluded that the intake of nutrients was not significantly impacted by following an FHVD (Table S3), indicating that the variation of gut microbial composition and function were mainly driven by additional FHV in daily diet. Besides, the FHVD improved the taxa diversity after 8 weeks of intervention (Fig. 1b), and the result of principal coordinate analysis over the unweighted UniFrac distance (Fig. 1e) showed that the entire diversity was mainly driven by individual differences, because there was no overlap between paired samples with the individuals clustering together. Thus, it was found that there were significant differences in the diversity between pre- and post-intervention with the FHVD (horizontal and vertical), but interindividual variation seemed to contribute more to horizontal differences.
It was also obvious that the overall microbial structure showed significant alterations by the FHVD and NFHVD, however, their variation tendency was distinctly different. Although fewer significant change of Bacteroidetes was found during treatments, it was worth noting that the decrease of Bacteroidetes during the NFHVD and increase during the FHVD might jointly impact the Bacteroidetes/Firmicutes ratio (B/F ratio). Recently, a large-scale population study of the gut microbiota revealed that most OTUs from Bacteroidetes were more prevalent among healthy individuals in Guangdong Province 24. In addition, as the report described, a higher B/F ratio was related to decreased obesity risk and maintaining gut homeostasis 25; our results suggested that the additional FHV in the diet had the potential of healthcare by altering the gut microbiota composition. Moreover, the enterotype-determined Bacteroides and Prevotella were not significantly changed by FHVD intervention, suggesting that neither of the two medium diets could influence the individual enterotypes, while some low abundance clusters showed noteworthy differences. In the FHVD, the enriched Dialister spp. and Veillonella spp. are recognized to be propionate producers through the succinate pathway 26. Meanwhile, some species from Lachnospiraceae are known to utilize acetate and lactate to produce butyrate 27. Another butyrate producer, Coprococcus spp., was enriched in both diets, but it only showed significance in the NFHVD.
Faecalibacterium prausnitzii, a species from the Faecalibacterium genus which was listed as a live biotherapeutics 28, is beneficial to human health by generating immunoregulatory molecules such as butyrate 29 and negatively connected with a variety of diseases, such as T2DM 30, colitis 31, and gout 17. Interestingly, Faecalibacterium was dramatically reversed in FHVD-Post (compared with NFHVD-Post) (P < 0.05), suggesting a preservation of the probiotics by the FHVD. For the reducing taxa, genera from Proteobacteria, such as Shigella and Desulfovibrio, have been considered to contain opportunistic pathogens and proinflammatory bacteria 32,33. Simulated results were also found in an in vitro fermentation system of prebiotics by the gut microbiota 21. Overall, it was suggested that the addition of FHV enriched the abundance of Faecalibacterium and SCFA-producing bacteria and inhibited potential pathogens, thereby protecting the gut microbiome in healthy individuals.
Obviously, The gut microbiome is an ecosystem with complex interactions. It was suggested that microbial interactions are promoted in both diets (Fig. 2). Notably, the core taxa showed slight variation, with disappearing Shigella, a potential pathogen in Proteobacterium 33. Comparable results were also found in Sutterella and Desulfovibrio. The results indicated that the relative abundance and correlation of pathogenic bacteria were restrained by the FHVD. Meanwhile, Parabacteroides appeared to be a core node after the FHVD, which was reported to play an opposing role with diverse conditions in recent research 34. With the NFHVD intervention, the decrease in the positive correlation and increase in the negative correlation suggested that the microbial structure tended to be enriched in specific taxa, although the topological nodes showed an obvious improvement. Moreover, Faecalibacterium and Blautia had a positive correlation with the FHVD-Pre and -Post networks, reflecting a possible synergistic relationship between beneficial bacteria. A positive correlation was also found between Clostridium and Blautia. However, the underlying mechanism remains to be revealed because the functions of taxa in Clostridium are complex 35,36. In summary, these phenomena indicate that gut microbiota co-occurrence networks are structured to be healthier by an FHVD.
Furthermore, the effect of the FHVD on gut microbial metabolism showed distinct diverse in the two diets. In particular, the synthesis pathway of B vitamins were found specifically enriched in the FHVD. Previous studies have emphasized the importance of microbial water- and fat-soluble vitamins in modulating the gut microbiota and host immunoreactions in disease 37,38. Besides, The FHVD seems to modulate energy metabolism via strengthening the citrate cycle. Usually, the citrate cycle occurs in the mitochondrial matrix and is a core integration center for the carbohydrate, lipid, and protein metabolic pathways. As a critical component of the citrate cycle, citric acid exerts antioxidant, antiapoptotic, and anti-inflammatory actions in liver, brain, and cardiac tissues 39. The decrease in the sulfate reduction pathway was enhanced by the FHVD compared with the NFHVD, as the major product of this pathway, and it was reported that the effect of H2S on health was complex 40, further studies will need to assess with clinical phenotypes. Moreover, despite the same partial trends in the NFHVD, a global enrichment pathway of amino acid synthesis was found with the FHVD, such as threonine, methionine, and isoleucine, there is an increasing body of evidence indicating that the synthesis and metabolism of amino acids are crucial pathway to modulate host physiology. Amino acids can be synthesized by gut bacteria and are released into the intestinal tract for further entry into the circulatory system or utilized for the synthesis of bacterial cell components and functional metabolites such as SCFAs 41, meanwhile, they could further alter energy homeostasis, nutrition metabolism, gut health, and immunity 42. Other metabolic pathways also showed dispersive enrichment by intervention with the FHVD. However, most of them were less frequently reported to be connected with host physiology.
Widely targeted metabolic assay was used to evaluate the effect of the FHVD on the fecal metabolite profiles. Previous study reported that serine in the gut improved colonic morphology and alleviated inflammatory responses in mice with colitis 43. Glutamine, one of the most abundant amino acids in the human body, protects gut health by repairing intestinal barrier function 44. However, little evidence has been obtained to confirm whether these enriched metabolites were originated from the gut microbiota. In recent decades, tryptophan metabolites have been verified to be closely connected with human health by regulating neurotransmission and cytokine signaling 45. Tryptamine is mainly transferred from tryptophan by decarboxylases encoded in a variety of Firmicutes genomes, such as Blautia spp., Ruminococcus spp., Clostridium spp., and Lachnospiraceae spp. 46, and the latter pair was found to be significantly enriched in an FHVD. 5-Hydroxy tryptamine, another health-related tryptophan metabolite, was found to increase 1.38-fold, although the difference was not significant. Additionally, endogenous GABA was also considered to be a regulator of the nervous system through the gut-brain axis 47. The enhancement of neurotransmitter-benefit metabolites might be related to the function of the effect of supplying Qi and reinforcing deficiencies of FHV 4. However, more evidence is needed.
A consensus has been reached that beneficial bacteria can produce SCFAs, which lower the gut pH and inhibit the growth of pathogenic bacteria 48. In addition, SCFAs are also signal microbial metabolites to regulate host immunoreactions through G protein-coupled receptors (GPCRs) and fatty acid receptors (FFARs) 49. In our study, the dry weight of three predominate SCFAs in the feces, including acetate, propionate, and butyrate were promoted by the FHVD. There are two major pathways by which exogenous herbal nutrients of promote SCFA levels in the gut 50, one of which provides a fermentable carbon source that can only be metabolized by the gut microbiota. We considered that the most abundant carbohydrate could sever as available substrate for fermentation of gut microbiota, and the results of in vitro fermentation also confirmed the contribution of FHV extract on SCFAs. Our subsequent study will focus on the further functions of saccharides derived from FHV. In fact, by concluding the key enzyme gene abundance of the synthesis pathway of above SCFAs, the gene abundance of the butyrate synthesis pathway was found significantly increased in the FHVD. On the other hand, Acetate- and propionate-producing genera, including Lachnospiraceae and Dialister, were enriched in the FHVD, and the increased SCFA levels could also be related to enzyme activity in variety taxa 50.
Given the major metabolite differences driven by the FHVD, tripartite correlation analysis was performed to demonstrate the relationship between microbial pathway modules, taxa and metabolites (Fig. 6). The positive correlation between essential amino acids and FHVD-enriched Streptococcus spp., Blautia spp., and Lactobacillus kalixensis, rather than functional pathway, suggested that the increased amino acids were likely driven by interaction of these species. Additionally, GABA levels were positively associated with a wide range of enriched species, especially acid-production bacteria such as Blautia spp., Acidaminococcus spp. and Lactobacillus kalixensis. Another opinion is that GABA could be observed only in an acidic environment (pH < 5.5) in vitro 51, suggesting that an FHVD might promote these species to improve GABA concentrations by enhancing the SCFAs synthesis pathway. Despite the citrate cycle and vitamin synthesis pathways were co-enriched with several indistinctive and enriched bacteria (Bacteroides dorei, Ruminococcus bromii, and Blautia obeum), but few metabolites showed a distinct correlation with these pathways. This implied that the influence of an FHVD on the fecal metabolite profiles was more likely through alterations in the gut microbial consortium to modulate pathway abundance indirectly rather than individual species.