Abstract
Research indicates that the gut microbiota (GM) plays an important role in the health of the host and during recent years the increase in the composition and functionality of the gut microbiota has become of increasing interest. Probiotics, prebiotics or combinations hereof, so-called synbiotics, may be used to change the composition and activity of the human GM and thereby potentially affect the host health beneficially. In this PhD study it was hypothesized that emerging synbiotics have the potential of modulating the human GM composition as well as the functionality. To gain the beneficial effects of both probiotics and prebiotics, they may be combined to synbiotics.
The definition of synbiotics implies that the prebiotic should be specific to the probiotic in question. Therefore, a library of 37 emerging prebiotics, comprising carbohydrates with a large range of degrees of polymerization (DP), monomeric units and glycosidic linkage, was investigated for their ability to stimulate the growth of the two probiotic bacteria Lactobacillus acidophilus NCFM (NCFM) and Bifidobacterium animalis subsp. lactis (Bl-04) in a mono culture screen. Four prebiotics stimulated growth of NCFM specifically, three stimulated growth of Bl-04 while five showed growth stimulation for both probiotics.
To investigate the impact of emerging prebiotics on protein levels of probiotic bacterium, NCFM was grown with cellobiose as carbohydrate source and changes in protein abundance as compared to glucose was investigated using two dimensional difference gel electrophoresis (2D-DIGE). This gave insight into the uptake and metabolism of cellobiose, but also into the potential effects on the function of NCFM as several proteins potentially involved in adhesion were affected.
Based on the screen, the most effective emerging prebiotics were selected for further investigation using a four-stage semi-continuous model system of the human colon. The selected combinations were NCFM with isomaltulose, cellobiose, raffinose and oat β-glucan hydrolyzed with endo-1,3-β-glucanase (OBGH), respectively and Bl-04 with melibiose, xylobiose, raffinose and maltotriose, respectively. Insoluble substrates, i.e. waxy maize starch granules, pectin-rich potato fiber and potato lintner starch, which were unable to pass through the tubing of the colonic model system, were investigated in combination with NCFM using batch type fermentation in the presence of human faecal inoculum. Growth of the probiotic bacteria and the influence on the microbiota was investigated using qPCR and the formation of short-chain fatty acids (SCFA) and branched-chain fatty acids were investigated by gas chromatography.
All emerging prebiotics were able to support growth of the probiotics in the colonic model system, which were increased by 10 3- 10 4 fold and 10–10 2 fold for NCFM and Bl-04 respectively. Interestingly, OBGH increased the levels of NCFM although no growth was found in mono culture screening. NCFM was increased by 10 5 fold for all combinations of the insoluble substrates. These findings indicate that the selected emerging prebiotics are able to provide a competitive advantage for NCFM and Bl-04. All the emerging synbiotics were able to induce changes in the predominant bacteria, observed as a decrease in the modified ratio of Bacteroidetes/Firmicutes (calculated using qPCR results for Bacteroides-Prevotella-Porphyromonas group, Clostridium perfringens cluster I, Clostridium coccoides - Eubacterium rectale group and Clostridial cluster XIV for the colonic
All the emerging synbiotics investigated in the colonic model system were able to increase levels of SCFA. Especially levels of acetate and butyrate were affected and a three to eight fold increase was observed as compared to control. In the batch fermentation however, levels of SCFA were decreased compared to control, but the emerging synbiotics increased levels of lactate, which were not detected in the control reaching concentrations of 35 – 58 mmol/L for the synbiotics. For BCFA, the emerging synbiotics investigated using the colonic model system decreased the levels of isobutyrate, 2-methylbutyrate and isovalerate, while production of BCFA in the batch type model was completely inhibited.
The findings from the model systems indicate that the emerging synbiotics investigated have the potential of inducing shifts in the composition and functionality of healthy human GM. Therefore the effects of NCFM in combination with cellobiose on the faecal microbiota of healthy humans was investigated in a clinical trial. The design of the trial was a randomized, double-blinded, placebo controlled, cross-over study using 16 volunteers. The volunteers consumed the synbiotic or the placebo combination daily for three weeks, after which they were crossed-over. Prior to the study, a baseline faecal sample was collected and faecal samples were collected at the end of each period. Numbers of lactobacilli were investigated using qPCR and the composition of the faecal microbiota was investigated using 454 tag-encoded amplicon pyrosequencing. Changes in the functionality of the microbiota were investigated by gas chromatography of SCFA and BCFA.
The product was well tolerated by all volunteers. Interestingly, NCFM was not detected after synbiotic intake, however numbers of lactobacilli were increased as compared to baseline (p=0.01) and placebo (p=0.04). Pyrosequencing revealed increase in the relative abundance of genera Bifidobacterium (P=0.04), Collinsella (p=0.02) and Eubacterium (p=0.04) and decrease in genus Dialister (p=0.03) as compared to placebo. Although this combination decreased the modified ratio of Bacteroidetes/Firmicutes in vitro, no effect was found in humans. Furthermore, no clear clustering was observed from principal coordinate analysis using the relative abundance of all genera detected. Analysis of enterotypes, i.e. the abundance of the genera Bacteroides, Prevotella and Ruminococcus, revealed that no changes were observed for six volunteers. However, changes were observed for the remaining volunteers and three of these volunteers appeared to shift enterotype dependent on placebo and synbiotic intake.
With regard to SCFA, no changes in acetate, propionate, butyrate nor total SCFA was observed for synbiotic intake compared to baseline and placebo. However, synbiotic intake increased 2-methylbutyrate as compared to both baseline (p=0.006) and placebo (p=0.01). Also total BCFA was increased as compared to baseline (p=0.02). Inverse correlations were found between the modified ratio of Bacteroidetes/Firmicutes and the production of acetate (p=0.04) and butyrate (p=0.03) for the emerging synbiotics investigated in the colonic model system. There was however not observed any correlation of Bacteroidetes/Firmicutes and acetate or butyrate in the human trial. Interestingly, genera known to harbor butyrate producing species were not correlated to the levels of faecal butyrate..
In conclusion, the results show that emerging prebiotics are able to induce changes on the protein level which may affect the activity of the probiotic. The results also indicate that emerging synbiotics have the potential of inducing changes in the composition and activity of the microbiota both in colonic models systems and in healthy humans. However, additional studies and larger human trials are necessary to clarify these effects. The findings of this work help gain insight into the interactions of probiotics and prebiotics and contribute to the understanding of the effects of synbiotics on the colonic microbiota of healthy humans.
The definition of synbiotics implies that the prebiotic should be specific to the probiotic in question. Therefore, a library of 37 emerging prebiotics, comprising carbohydrates with a large range of degrees of polymerization (DP), monomeric units and glycosidic linkage, was investigated for their ability to stimulate the growth of the two probiotic bacteria Lactobacillus acidophilus NCFM (NCFM) and Bifidobacterium animalis subsp. lactis (Bl-04) in a mono culture screen. Four prebiotics stimulated growth of NCFM specifically, three stimulated growth of Bl-04 while five showed growth stimulation for both probiotics.
To investigate the impact of emerging prebiotics on protein levels of probiotic bacterium, NCFM was grown with cellobiose as carbohydrate source and changes in protein abundance as compared to glucose was investigated using two dimensional difference gel electrophoresis (2D-DIGE). This gave insight into the uptake and metabolism of cellobiose, but also into the potential effects on the function of NCFM as several proteins potentially involved in adhesion were affected.
Based on the screen, the most effective emerging prebiotics were selected for further investigation using a four-stage semi-continuous model system of the human colon. The selected combinations were NCFM with isomaltulose, cellobiose, raffinose and oat β-glucan hydrolyzed with endo-1,3-β-glucanase (OBGH), respectively and Bl-04 with melibiose, xylobiose, raffinose and maltotriose, respectively. Insoluble substrates, i.e. waxy maize starch granules, pectin-rich potato fiber and potato lintner starch, which were unable to pass through the tubing of the colonic model system, were investigated in combination with NCFM using batch type fermentation in the presence of human faecal inoculum. Growth of the probiotic bacteria and the influence on the microbiota was investigated using qPCR and the formation of short-chain fatty acids (SCFA) and branched-chain fatty acids were investigated by gas chromatography.
All emerging prebiotics were able to support growth of the probiotics in the colonic model system, which were increased by 10 3- 10 4 fold and 10–10 2 fold for NCFM and Bl-04 respectively. Interestingly, OBGH increased the levels of NCFM although no growth was found in mono culture screening. NCFM was increased by 10 5 fold for all combinations of the insoluble substrates. These findings indicate that the selected emerging prebiotics are able to provide a competitive advantage for NCFM and Bl-04. All the emerging synbiotics were able to induce changes in the predominant bacteria, observed as a decrease in the modified ratio of Bacteroidetes/Firmicutes (calculated using qPCR results for Bacteroides-Prevotella-Porphyromonas group, Clostridium perfringens cluster I, Clostridium coccoides - Eubacterium rectale group and Clostridial cluster XIV for the colonic
All the emerging synbiotics investigated in the colonic model system were able to increase levels of SCFA. Especially levels of acetate and butyrate were affected and a three to eight fold increase was observed as compared to control. In the batch fermentation however, levels of SCFA were decreased compared to control, but the emerging synbiotics increased levels of lactate, which were not detected in the control reaching concentrations of 35 – 58 mmol/L for the synbiotics. For BCFA, the emerging synbiotics investigated using the colonic model system decreased the levels of isobutyrate, 2-methylbutyrate and isovalerate, while production of BCFA in the batch type model was completely inhibited.
The findings from the model systems indicate that the emerging synbiotics investigated have the potential of inducing shifts in the composition and functionality of healthy human GM. Therefore the effects of NCFM in combination with cellobiose on the faecal microbiota of healthy humans was investigated in a clinical trial. The design of the trial was a randomized, double-blinded, placebo controlled, cross-over study using 16 volunteers. The volunteers consumed the synbiotic or the placebo combination daily for three weeks, after which they were crossed-over. Prior to the study, a baseline faecal sample was collected and faecal samples were collected at the end of each period. Numbers of lactobacilli were investigated using qPCR and the composition of the faecal microbiota was investigated using 454 tag-encoded amplicon pyrosequencing. Changes in the functionality of the microbiota were investigated by gas chromatography of SCFA and BCFA.
The product was well tolerated by all volunteers. Interestingly, NCFM was not detected after synbiotic intake, however numbers of lactobacilli were increased as compared to baseline (p=0.01) and placebo (p=0.04). Pyrosequencing revealed increase in the relative abundance of genera Bifidobacterium (P=0.04), Collinsella (p=0.02) and Eubacterium (p=0.04) and decrease in genus Dialister (p=0.03) as compared to placebo. Although this combination decreased the modified ratio of Bacteroidetes/Firmicutes in vitro, no effect was found in humans. Furthermore, no clear clustering was observed from principal coordinate analysis using the relative abundance of all genera detected. Analysis of enterotypes, i.e. the abundance of the genera Bacteroides, Prevotella and Ruminococcus, revealed that no changes were observed for six volunteers. However, changes were observed for the remaining volunteers and three of these volunteers appeared to shift enterotype dependent on placebo and synbiotic intake.
With regard to SCFA, no changes in acetate, propionate, butyrate nor total SCFA was observed for synbiotic intake compared to baseline and placebo. However, synbiotic intake increased 2-methylbutyrate as compared to both baseline (p=0.006) and placebo (p=0.01). Also total BCFA was increased as compared to baseline (p=0.02). Inverse correlations were found between the modified ratio of Bacteroidetes/Firmicutes and the production of acetate (p=0.04) and butyrate (p=0.03) for the emerging synbiotics investigated in the colonic model system. There was however not observed any correlation of Bacteroidetes/Firmicutes and acetate or butyrate in the human trial. Interestingly, genera known to harbor butyrate producing species were not correlated to the levels of faecal butyrate..
In conclusion, the results show that emerging prebiotics are able to induce changes on the protein level which may affect the activity of the probiotic. The results also indicate that emerging synbiotics have the potential of inducing changes in the composition and activity of the microbiota both in colonic models systems and in healthy humans. However, additional studies and larger human trials are necessary to clarify these effects. The findings of this work help gain insight into the interactions of probiotics and prebiotics and contribute to the understanding of the effects of synbiotics on the colonic microbiota of healthy humans.
| Original language | English |
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| Publisher | Department of Food Science, University of Copenhagen |
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| Number of pages | 205 |
| Publication status | Published - 2012 |