Gut Microbiota in Older People: Metabolism and Composition
Gut Microbiota in Older People: Metabolism and Composition
Until quite recently, the general belief was that, while food intake may regulate certain metabolic activities associated with intestinal microorganisms, changing diet has little effect on overall gut microecology. However, the introduction of functional foods into the diet has raised a major challenge to this concept, and it is increasingly being recognised that species composition of the microbiota, as well as many of its physiological traits can be modified by relatively simple changes in food intake, particularly probiotics, prebiotics and synbiotics. Probiotics are live microbial food supplements that benefit health through, for example, stimulation of immune function. Prebiotics are nondigestible food ingredients, for example, inulins, fructo-oligosaccharides and galacto-oligosaccharides, that beneficially affect the host by selectively stimulating the growth and/or activities of beneficial bacteria such as lactobacilli and bifidobacteria in the colon, and they have been used prophylactically and to treat a number of gastrointestinal conditions Prebiotics have been shown to stimulate bifidobacteria, with the suppression of other organisms such as enterobacteria, and to induce changes in the production of large intestinal metabolites such as SCFA. A synbiotic is a synergistic combination of a probiotic and prebiotic, where the aim is to stimulate growth of the probiotic in the gut by providing a preferred carbon and energy source for the bacteria.
In previous studies, we have demonstrated that marked changes occur in the composition and metabolic capabilities of the microbiota in older people, in both health and disease. This was most obviously manifested as qualitative and quantitative variations in SCFA excretion, together with marked reductions in bifidobacterial communities and increased enterobacterial prevalence. Age-related reductions in bifidobacteria and increases in facultative anaerobes, including enterobacteria, have been reported in other studies on older people.
Evidence for the usefulness of synbiotic therapy on bifidobacterial populations was shown in a small DBRCT in our laboratory, in which nine older people fed with a synbiotic composed of B. lactis BL-01 and B. bifidum BB-02 and inulin, showed significant increases in B. bifidum and total bifidobacteria during synbiotic consumption, due not only to increases in the probiotics, but also in endogenous bifidobacteria. We had previously shown that the synbiotic used in the present work has therapeutic effects in patients with active IBD, and that it increased mucosal bifidobacteria and reduced mucosal pro-inflammatory TNF-α. There were no safety issues with synbiotic use in the studies.
The current investigation demonstrated that synbiotic consumption was effective in achieving its primary objective of introducing beneficial microorganisms into the gastrointestinal tract in older people, thereby modulating colonic species composition, and changing microbial community structure in the large bowel. While there were no obvious differences in total bacterial numbers and Bacteroidetes during the study, significant increases in Actinobacteria (mainly bifidobacteria) occurred in volunteers receiving the synbiotic. Faecal bifidobacteria, at the start of the investigation, were in the range reported previously in one other prebiotic study in older people, and no significant differences were detected in bifidobacteria baseline concentrations in people taking the synbiotic before the placebo, indicating that synbiotic did not lead to a sustained increase in indigenous bifidobacterial populations. A difference of log 1.2 was observed in total bifidobacteria from the start and end of synbiotic feeding period, while there were no changes in the placebos. It was not possible to distinguish the probiotic strain from other B. longum strains detected in faecal material, as it has the same genetic attributes as indigenous B. longum. Increased colonisation with B. longum, and other bifidobacterial species were found in the majority of subjects receiving the synbiotic.
Species belonging to the phylum Firmicutes were also shown to increase significantly during synbiotic consumption, while Proteobacteria, which includes the family Enterobacteriaceae, decreased. A significant increase in butyrate along with an increased butyrate molar ratio occurred with synbiotic intake (Table 7). The phylum Firmicutes contains members that play a role in carbohydrate breakdown and fermentation in the gut, and produce butyrate. This SCFA is the principal energy source for intestinal colonocytes. It may also be protective against colon cancer, while acting on the immune system, and maintaining mucosal barrier integrity by stimulating mucin production. Faecalibacterium prausnitzii and species associated with Clostridium cluster XIVa, which are butyrate-producing firmicutes, have been found in previous studies in old people, and growth of F. praunitzii has been shown to be stimulated by inulin. The Firmicutes/Bacteroidetes ratio increased from 1.3 to 6.6 during synbiotic feeding, while it maintained baseline values during the placebo period. A reduction in the ratio has been implicated in conditions such as obesity, and, in two previous studies in older subjects, the Firmicutes/Bacteroidetes ratio was shown to decrease with age, and to be lower than in young adults. Therefore, the synbiotic used in this study may be beneficial in maintaining the ratio closer to that of a younger adult.
SCFA formation is one of the most important processes mediated by colonic microorganisms, and they have a multiplicity of effects on host metabolism. In a previous study, an overall reduction in faecal excretion of SCFA was shown to occur with age and, recently, older people have been shown to have fewer copies of the butyryl CoA transferase gene compared with young adults. As the majority of SCFA are absorbed from the gut or undergo further metabolism in the microbiota itself, higher faecal SCFA could indicate large increases in production. Changes in acetate excretion during synbiotic feeding may be linked to increased production by bifidobacteria. Lactate formed by bifidobacteria is further metabolised by cross-feeding, resulting in elevated faecal butyrate seen with the synbiotic. The increase in SCFA may also be responsible for some of the other effects observed in the study, as acetate and butyrate manifest anti-inflammatory and immune-modulatory effects, and, recently, it has been shown that acetate produced by bifidobacteria improves host epithelial defences, and protects mice from infection by enteropathogens.
The secondary objective of this investigation was to assess the impact of the synbiotic on health parameters and the immune system. Health effects were determined throughout the study by measuring changes in weight, bowel habit, blood lipid, cholesterol, glucose and insulin concentrations. Although there was a trend towards reduced insulin and abdominal pain at 2 weeks during intake of the synbiotic, this was not significant. Neither were any of the changes with the other parameters tested. This is not surprising, as baseline bowel habit values indicated that the volunteers did not have constipation or excess stool production. This may be due to the individuals in the study being relatively healthy and mainly recruited from the community. It has been reported that several markers of inflammation and health parameters are higher in people in long-stay residential care, compared with those living in the community.
Ageing is associated with variable changes in immune responses (immunosenescence), such as blood IgG or IgA levels, while TH1 responses decline as people get older, and others, such as the TH2 response, increase. Elevated production of pro-inflammatory cytokines is associated with ageing, and the low-grade inflammation that exists in older people influences the onset of several age-associated diseases, such as cardiovascular disease, type 2 diabetes and arthritis. Probiotics have been shown to modulate the immune system by increasing phagocytosis, TH1 responses and regulating production of pro-inflammatory cytokines through IL-10. Reductions in pro-inflammatory cytokine IL-6 in PMBC have been observed in other prebiotic studies in older people, and a prebiotic galacto-oligosaccharide mixture in healthy older volunteers was shown to decrease TNF-α, IL-1β and IL-6, and increase production of IL-10 and natural killer cells. Evidence of systemic inflammation can be indicated by increased serum CRP, which has been shown to correlate closely with circulating IL-6. Therefore, we quantified the effects of the synbiotic on the immune system by measuring serum IgA, IgG, CRP and a range of cytokines (IL-1β, IL-6, TNF-α, IL-10, IFN-γ, IL-8, IL-4) in fasting bloods. While no significant differences were found regarding IL-10, IL-1β, IgA, IgM or CRP, IL-4 and INF-γ concentrations decreased during synbiotic feeding, although this was not statistically significant. Importantly, however, significant reductions in TNF-α occurred at 2 and 4 weeks of synbiotic consumption compared with the placebo group. Significant reductions in IL-6, IL-8 and MCP-1 occurred after 2 weeks of synbiotic intake, but this was not maintained at 4 weeks. Greater numbers of enterobacteria in older people have previously been linked with increased IL-6 and IL-8 production, and significant reductions in Proteobacteria were detected at 2 and 4 weeks in the synbiotic group compared with the placebos.
Significant increases in bifidobacterial species, other than B. longum, occurred in the volunteers when they were receiving the synbiotic. This indicates the advantage of using synbiotics compared with either probiotics or prebiotics alone, whereby the two components of the synbiotic may act in synergy to alleviate inflammation, by providing a substrate for the probiotic, increasing autochthonous bifidobacteria and stimulating SCFA production.
In conclusion, this short-term placebo-controlled investigation has provided evidence that synbiotics have the potential to be developed into acceptable therapies for use in older populations to maintain a healthy gut microbiota, and significantly reduce natural pro-inflammatory responses associated with ageing.
Discussion
Until quite recently, the general belief was that, while food intake may regulate certain metabolic activities associated with intestinal microorganisms, changing diet has little effect on overall gut microecology. However, the introduction of functional foods into the diet has raised a major challenge to this concept, and it is increasingly being recognised that species composition of the microbiota, as well as many of its physiological traits can be modified by relatively simple changes in food intake, particularly probiotics, prebiotics and synbiotics. Probiotics are live microbial food supplements that benefit health through, for example, stimulation of immune function. Prebiotics are nondigestible food ingredients, for example, inulins, fructo-oligosaccharides and galacto-oligosaccharides, that beneficially affect the host by selectively stimulating the growth and/or activities of beneficial bacteria such as lactobacilli and bifidobacteria in the colon, and they have been used prophylactically and to treat a number of gastrointestinal conditions Prebiotics have been shown to stimulate bifidobacteria, with the suppression of other organisms such as enterobacteria, and to induce changes in the production of large intestinal metabolites such as SCFA. A synbiotic is a synergistic combination of a probiotic and prebiotic, where the aim is to stimulate growth of the probiotic in the gut by providing a preferred carbon and energy source for the bacteria.
In previous studies, we have demonstrated that marked changes occur in the composition and metabolic capabilities of the microbiota in older people, in both health and disease. This was most obviously manifested as qualitative and quantitative variations in SCFA excretion, together with marked reductions in bifidobacterial communities and increased enterobacterial prevalence. Age-related reductions in bifidobacteria and increases in facultative anaerobes, including enterobacteria, have been reported in other studies on older people.
Evidence for the usefulness of synbiotic therapy on bifidobacterial populations was shown in a small DBRCT in our laboratory, in which nine older people fed with a synbiotic composed of B. lactis BL-01 and B. bifidum BB-02 and inulin, showed significant increases in B. bifidum and total bifidobacteria during synbiotic consumption, due not only to increases in the probiotics, but also in endogenous bifidobacteria. We had previously shown that the synbiotic used in the present work has therapeutic effects in patients with active IBD, and that it increased mucosal bifidobacteria and reduced mucosal pro-inflammatory TNF-α. There were no safety issues with synbiotic use in the studies.
The current investigation demonstrated that synbiotic consumption was effective in achieving its primary objective of introducing beneficial microorganisms into the gastrointestinal tract in older people, thereby modulating colonic species composition, and changing microbial community structure in the large bowel. While there were no obvious differences in total bacterial numbers and Bacteroidetes during the study, significant increases in Actinobacteria (mainly bifidobacteria) occurred in volunteers receiving the synbiotic. Faecal bifidobacteria, at the start of the investigation, were in the range reported previously in one other prebiotic study in older people, and no significant differences were detected in bifidobacteria baseline concentrations in people taking the synbiotic before the placebo, indicating that synbiotic did not lead to a sustained increase in indigenous bifidobacterial populations. A difference of log 1.2 was observed in total bifidobacteria from the start and end of synbiotic feeding period, while there were no changes in the placebos. It was not possible to distinguish the probiotic strain from other B. longum strains detected in faecal material, as it has the same genetic attributes as indigenous B. longum. Increased colonisation with B. longum, and other bifidobacterial species were found in the majority of subjects receiving the synbiotic.
Species belonging to the phylum Firmicutes were also shown to increase significantly during synbiotic consumption, while Proteobacteria, which includes the family Enterobacteriaceae, decreased. A significant increase in butyrate along with an increased butyrate molar ratio occurred with synbiotic intake (Table 7). The phylum Firmicutes contains members that play a role in carbohydrate breakdown and fermentation in the gut, and produce butyrate. This SCFA is the principal energy source for intestinal colonocytes. It may also be protective against colon cancer, while acting on the immune system, and maintaining mucosal barrier integrity by stimulating mucin production. Faecalibacterium prausnitzii and species associated with Clostridium cluster XIVa, which are butyrate-producing firmicutes, have been found in previous studies in old people, and growth of F. praunitzii has been shown to be stimulated by inulin. The Firmicutes/Bacteroidetes ratio increased from 1.3 to 6.6 during synbiotic feeding, while it maintained baseline values during the placebo period. A reduction in the ratio has been implicated in conditions such as obesity, and, in two previous studies in older subjects, the Firmicutes/Bacteroidetes ratio was shown to decrease with age, and to be lower than in young adults. Therefore, the synbiotic used in this study may be beneficial in maintaining the ratio closer to that of a younger adult.
SCFA formation is one of the most important processes mediated by colonic microorganisms, and they have a multiplicity of effects on host metabolism. In a previous study, an overall reduction in faecal excretion of SCFA was shown to occur with age and, recently, older people have been shown to have fewer copies of the butyryl CoA transferase gene compared with young adults. As the majority of SCFA are absorbed from the gut or undergo further metabolism in the microbiota itself, higher faecal SCFA could indicate large increases in production. Changes in acetate excretion during synbiotic feeding may be linked to increased production by bifidobacteria. Lactate formed by bifidobacteria is further metabolised by cross-feeding, resulting in elevated faecal butyrate seen with the synbiotic. The increase in SCFA may also be responsible for some of the other effects observed in the study, as acetate and butyrate manifest anti-inflammatory and immune-modulatory effects, and, recently, it has been shown that acetate produced by bifidobacteria improves host epithelial defences, and protects mice from infection by enteropathogens.
The secondary objective of this investigation was to assess the impact of the synbiotic on health parameters and the immune system. Health effects were determined throughout the study by measuring changes in weight, bowel habit, blood lipid, cholesterol, glucose and insulin concentrations. Although there was a trend towards reduced insulin and abdominal pain at 2 weeks during intake of the synbiotic, this was not significant. Neither were any of the changes with the other parameters tested. This is not surprising, as baseline bowel habit values indicated that the volunteers did not have constipation or excess stool production. This may be due to the individuals in the study being relatively healthy and mainly recruited from the community. It has been reported that several markers of inflammation and health parameters are higher in people in long-stay residential care, compared with those living in the community.
Ageing is associated with variable changes in immune responses (immunosenescence), such as blood IgG or IgA levels, while TH1 responses decline as people get older, and others, such as the TH2 response, increase. Elevated production of pro-inflammatory cytokines is associated with ageing, and the low-grade inflammation that exists in older people influences the onset of several age-associated diseases, such as cardiovascular disease, type 2 diabetes and arthritis. Probiotics have been shown to modulate the immune system by increasing phagocytosis, TH1 responses and regulating production of pro-inflammatory cytokines through IL-10. Reductions in pro-inflammatory cytokine IL-6 in PMBC have been observed in other prebiotic studies in older people, and a prebiotic galacto-oligosaccharide mixture in healthy older volunteers was shown to decrease TNF-α, IL-1β and IL-6, and increase production of IL-10 and natural killer cells. Evidence of systemic inflammation can be indicated by increased serum CRP, which has been shown to correlate closely with circulating IL-6. Therefore, we quantified the effects of the synbiotic on the immune system by measuring serum IgA, IgG, CRP and a range of cytokines (IL-1β, IL-6, TNF-α, IL-10, IFN-γ, IL-8, IL-4) in fasting bloods. While no significant differences were found regarding IL-10, IL-1β, IgA, IgM or CRP, IL-4 and INF-γ concentrations decreased during synbiotic feeding, although this was not statistically significant. Importantly, however, significant reductions in TNF-α occurred at 2 and 4 weeks of synbiotic consumption compared with the placebo group. Significant reductions in IL-6, IL-8 and MCP-1 occurred after 2 weeks of synbiotic intake, but this was not maintained at 4 weeks. Greater numbers of enterobacteria in older people have previously been linked with increased IL-6 and IL-8 production, and significant reductions in Proteobacteria were detected at 2 and 4 weeks in the synbiotic group compared with the placebos.
Significant increases in bifidobacterial species, other than B. longum, occurred in the volunteers when they were receiving the synbiotic. This indicates the advantage of using synbiotics compared with either probiotics or prebiotics alone, whereby the two components of the synbiotic may act in synergy to alleviate inflammation, by providing a substrate for the probiotic, increasing autochthonous bifidobacteria and stimulating SCFA production.
In conclusion, this short-term placebo-controlled investigation has provided evidence that synbiotics have the potential to be developed into acceptable therapies for use in older populations to maintain a healthy gut microbiota, and significantly reduce natural pro-inflammatory responses associated with ageing.
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