We investigated the microbial and metabolic features of the faecal microbiota in both young and midlife subjects using in vitro batch fermentation model. Soluble starch, a representative of digestible carbohydrate, and inulin, a representative of non-digestible carbohydrate, were selected to study the metabolic responses. Both the feces and fermented broth samples were processed and subjected to 16S rRNA gene sequencing and SCFAs detection. We also determined gas production in the fermentation model as it is one of the most production metabolites beside SCFAs.
Difference in the structure of the microbiota community
Based on the results of the16S rRNA gene sequencing, the alpha-diversity indices, such as Shannon, Simpson and Chao1 index are similar between midlife and young in both faecal and fermented samples (Fig. 1A), suggesting the consistent diversity of the gut microbiota between the two age groups. This agrees with previous reports that the faecal microbiota structures of 20 to 60 years old are in a stable stage in this aspect.[35, 36] However, when the relative abundance was compared among the top ten genera, Bifidobacterium and Parasutterella in young group were found to be significantly higher than those in midlife subjects (Fig. 1C), suggesting that some differences started to appear in the faecal microbiota between the two age groups. Furthermore, PCA was performed to reveal the difference between young and midlife group. Despite the faecal scattered plots and starch broths could not differentiate the two groups (Fig. 2A and 2B), the broth samples after inulin fermentation can be obviously clustered on age (Fig. 2C), suggesting an age-related change in the microbial community structure of midlife.
Impact of environmental factors on microbiota
Canonical Correspondence Analysis (CCA) was performed to investigate the effects of age and carbon source on the variation in microbiota (Fig. 3). The scatter plot displayed the clustering on age and culture medium. The small percentage of the axis suggests that most of the variation in the system could not be explained by age and carbon source alone. However, the shift of clustering plot in the direction of both medium and age suggests that carbon source and age are the main drivers of the variation with carbon-source being the first and age second.
Difference in Taxonomic composition
Faecal samples
Linear discriminant analysis (LDA) effect size (LEfSe) was used to identify the age-related bacterial feature in the microbiota before and after fermentation (Fig. 4). As for the faecal samples, LEfSe revealed 27 taxa overrepresent in young group but none in midlife group (Fig. 4A). The taxa involve 11 genera in 4 phyla, suggesting a wide range of changes in relative abundance between youth and midlife. Among the 27 taxa, the top 5 were taxa within phylum Actinobacteria, including Actinobacteria (class), Actinobacteria (phylum), Bifidobacteriales (order), Bifidobacteriaceae (family) and Bifidobacterium (genus) in turn. Bifidobacteria dominates in infant gut microbiota and reduces with age.[37] Similar to the observation that Bifidobacterium disappears in middle-aged mice,[12] our data reveal that Bifidobacterium is the genus declined most in human midlife. Furthermore, 8 taxa, including Collinsella and Coriobacteriales (order) and Coriobacteriaceae (family), were observed to decline in midlife. This is also similar to the observation in mice that the relative abundance of both Bifidobacterium spp. and Coriobacteriaceae spp. decreases strongly and significantly in middle-aged mice compared to young subjects.[12]
Four genera, including Faecalibacterium, Butyricicoccus, Desulfovibrio and Parasutterella were abundant in young group but absent in midlife group. Faecalibacterium and Butyricicoccus are the main butyrate products found in the intestine.[38, 39] Thus, the relative abundance of butyrate-producing bacteria differs between young and midlife groups. Desulfovibrio is the dominant sulfate-reducing bacteria in the colon and reduces sulfate compounds to hydrogen sulfide (H2S)[40] which is one of the main microbiota-derived gases contributing to human colonic homeostasis.[41] Parasutterella was the most changed one in phylum Proteobacteria, a result also observed in aging mice.[12] Parasutterella is a core component of human gut microbiota and produces various metabolites including aromatic amino acid, bilirubin, purine, and bile acid derivatives.[42] Our results thus suggest the core composition in gut microbiota begins to reduce in midlife.
Beta-diversity analysis of 16S rRNA gene sequencing revealed that Faecalibacterium, Bifidobacterium and Parasutterella were in the top 10 genera with decreased relative abundance (Fig. 1C), a result consistent with LEfSe analysis. Furthermore, qPCR of Bifidobacterium also showed the significant reduction in feces of midlife group compared to young subjects (Fig. 5A). Bifidobacterium is thought to play pivotal roles in maintaining human health.[43, 44] Its decline in numbers is one of the most marked changes in the elderly gut.[45] Faecalibacterium is known to associate with the grip strength in elderly.[46] Hence, we conclude that microbial alterations associated with aging also occur in midlife, rather than the previous thought to begin in elderly.
Starch fermented samples
After starch fermentation (Fig. 4B), there were 11 taxa overrepresented in young group, predominated by Selenomonadales (order), Negativicutes (class), Veillonellaceae (family), Desulfovibrio (genus), Bifidobacterium (genus) and its belonging family and order, Comamonas (genus) and its belonging family, Catenibacterium (genus) and Bacillales (order). In contrast, only 6 taxa were overrepresented in midlife, including Proteobacteria (phylum), Peptoniphilus (genus), Lachnospiracea-incertae_sedis (genus) and Staphylococcus (genus). Veillonellaceae, belonging to class Negativicutes and further belonging to order Selenomonadales, is reported to play roles in host carbohydrates metabolism.[47] Veillonellaceae and its higher order taxa are the top taxa in starch fermentation indicates their roles seem to be more important for starch degradation compared to Bifidobacterium.
Inulin fermented samples
In term of the inulin fermentation (Fig. 4C), LEfSe revealed 8 taxa overrepresented in young group compared to midlife group. Five are taxa within phylum Actinobacteria, including Bifidobacteriumand, the remaining 3 taxa are Catenibacterium (genus), Comamonas (genus) and its belonging family. These taxa are all present in LEfSe results of both feces and starch broth, suggesting they are the key bacteria in young microbiota for saccharolytic process. On the other side, LEfSe identified 14 taxa overrepresented after inulin fermentation in midlife group. The top 5 taxa are Proteobacteria (phylum), Escherichia-Shigella (genus), Enterobacteriaceae (family), Enterobacteriales (order), Gama-proteobacteria (class), and the remaining taxa are Peptostreptococcus (genus), Delftia (genus), Streptococcaceae (family),Fusicatenibacter (genus), Parasutterella (genus), Phascolarctobacterium (genus), Acidaminococcareae (family), Enterococcus (genus), Enterococcaceae (family). The shared taxa number within young group after fermentation is 8, contrast to it however, only 1 taxa, Proteobacteria (phylum) is common in midlife group, suggesting that the reduction of bifidobacteria and increase of Proteobacteria can be taxonomic characteristics for midlife microbiota to ferment carbohydrates..
Difference in metabolic response
SCFAs in both feces and fermented broths were measured. As shown in Fig. 5C, most individual SCFAs in the feces in midlife group were lower than those in young group, except for propionate, but without significant difference. After starch fermentation, the most individual SCFAs in midlife were lower than those in young group (Fig. 6C-H), which is similar to the case in feces. However, after inulin fermentation, the reduction of acetate in midlife (Fig. 6C) was significant (p = 0.0002) compared to young group despite of similar level of butyrate and propionate in the two age groups. A similar result has been observed in aging mice that butyrate and propionate do not differ between two age groups, but acetate is significantly lower in old mice.[12] It is worth pointing out that the total SCFAs in midlife broth of inulin declines pronouncedly (p = 0.052). In addition, inulin fermentation also caused significant gas production increase in midlife group (p = 0.008) (Fig. 6I), and inulin degradation decrease (p = 0.002) compared to young group (Fig. 6J), a result comparable to the decline of saccharolytic capability in aging mice.[11] Hence, we can conclude that the age-related metabolic responses occur only under inulin fermentation, consistent with the microbiota changes revealed by PCA (Fig. 2C) and CCA (Fig. 3). This is also consistent with a previous report of age-related functional characteristics in metagenomes that midlife with age 38-43 differentiates from young and elderly, and constitutes a good watershed in data clustering between age groups.[48]
Correlation between the metabolites and the genera
To understand the contribution of genera to the altered metabolites, Spearman’s correlation coefficient was used to analyze the association between metabolites concentration and genus abundance. As for the starch fermentation, 3 genera, including Bifidobacterium, Desulfovibrio and Staphylococcus, show their correlation with six metabolic parameters (Fig. 7A). As for inulin fermentation, 8 genera, including Fusicatenibacter, Phascolarctobacterium, Peptostreptococcus, Delftia, Bifidobacterium, Parasutterella, Comamonas and Catenibacterium, correlate with seven metabolic parameters (Fig. 7B).
Based on metabolite measurement and relative to young group (Fig. 6), the acetate production in midlife group was significantly decreased after inulin fermentation. The heatmap of inulin fermentation (Fig. 7B) showed that two genera, Parasutterella and Bifidobacterium, have significant and positive correlation with acetate (p < 0.01). Bifidobacterium was the top abundant genus in young group, while Parasutterella was fifth abundant in midlife group (based on LEfSe, Fig. 4C). Therefore, the difference of acetate between the two age groups after inulin fermentation might be interpreted as a result of confrontation between Bifidobacterium and Parasutterella. The strong correlation with acetate and the higher abundance indicate that Bifidobacterium has a strong influence on the production of acetate compared with Parasutterella. Hence, the absence of Bifidobacterium might be responsible for the significant decrease of acetate in midlife group after inulin fermentation.
Contrary to acetate, gas production was significantly increased in midlife group. Based on Spearman’s correlation analysis (Fig. 7B), both Bifidobacterium and Peptostreptococcus were significantly correlated with gas production but in opposite directions. Peptostreptococcus showed a positive correlation resulting in a high gas production in midlife group, while Bifidobacterium had a negative correlation causing a low gas level in young group. The correlation between these two genera and gas production is beneficial to exacerbate the difference in gas production between midlife and young group. Thus, the absence of Bifidobacterium in midlife seems also contribute to the significant increase in gas production after inulin fermentation. This observation is supported by a recent report that Bifidobacterium genome lacks of hydrogenase genes [49] and uses acetate as its main metabolite.[50] In human intervention trial of resistant starch, some volunteers were found as “non-responders” with > 60 % of unfermented starch remaining in their stools while others with < 4%.[51] The difference in the initial composition of individual’s gut microbiota may be the cause.[52, 53] Therefore, the reduction of Bifidobacterium in midlife feces seems to be responsible for the significant drop in metabolic responses to inulin for midlife microbiota.
In vitro fermentation significantly caused reduction of inulin degradation rate and decline of Bifidobacterium, suggesting an attenuated saccharolytic capacity of inulin in midlife group. This is consistent with the previous study of aging. Bifidobacteria bears the capacity to ferment a variety of carbohydrate and fiber compounds.[54] The saccharolytic genes were found to decrease in elderly gut microbiota[21] and older age mice.[11] However, the age-related changes in microbial community structure and metabolites only occurred in the fermentation of inulin but not starch, suggesting that the microbial changes in midlife associated with aging are still at an early stage, corresponding to a partial decline of function.
In this study, we found that acetate was significantly reduced in midlife after inulin fermentation compared to young group. This may lead to health concerns in midlife as acetate is most productive acid among SCFAs and appears to stimulate leptin secretion in adipocytes, involving energy balance and appetite,[16] conditioning immune cell in response to protect against T2D[55] and regulating blood pressure.[56, 57] The impaired acetate production might also relate to weight gain in midlife.[58] Considering that approximately 44% acetate in plasma is microbiota-derived,[59] the reduced production of acetate by microbiota may further relate to other physiological changes in midlife, such as T2D.[8] and cardiovascular disease.[10]
Interestingly, contrast to the significant reduction of inulin degradation and acetate production, the reduction in number of Bifidobacterium is not significant after inulin fermentation. Concerning the diversity of inulin catabolism[60] and its niche- and strain-specific acetate production,[61] the loss of certain Bifidobacterium species or strains that produce large amounts of acetate might be the reason for the significant decrease of acetate in midlife group. This is supported by a recent study that changes in the composition of Bifidobacterium species occur with ageing.[37] Considering the benefits of bifidobacteria-produced acetate in intestinal defense,[62] the reduction mechanism of Bifidobacterium species in midlife is worthy of further study.