Claude Andrieux

Intestinal mucin distribution in the germ-free rat and in the heteroxenic rat harbouring a human bacterial flora: effect of inulin in the diet.

A colorimetric method was used on water-soluble mucin extracted from mucosal scrapings and contents of the caecum and the colon of five germ-free (GF) rats and five heteroxenic (HE) rats harbouring a human flora (GF rats associated with a human flora). These rats were fed on a diet containing either 100 g sucrose/kg or 100 g inulin/kg. Histological stains, periodic acid-Schiff, alcian blue pH 2.5 and alcian blue pH 0.5 were used to discriminate between neutral, acidic and acidic sulphated mucins respectively. Spectrocolorimetric assays led to a calculated absorbance value for 1 mg of the initial mucin extract. Each mucin type was compared between treatments. The caecal contents of GF rats contained more acidic mucin than sulphomucin, which was present in the same proportion as neutral mucin. Their colonic contents contained more acidic mucins than sulphomucin, which in turn was more abundant than neutral mucin. Their caecal mucosa mucin distribution differed from that of the contents: very little acidic mucin was present and neutral and sulphomucin proportions were of the same order of magnitude. Inulin increased the amount of neutral mucin in the caecal contents and of sulphated mucins in the colonic contents and increased the amounts of neutral and acidic mucins in the caecal mucosa. Mucin distribution in the HE rats was very different from that in the GF rats: the caecal contents contained a high proportion of acidic mucins and very little sulphomucin. The same distribution of mucins was observed in the colonic contents. The caecal mucosa contained less acidic mucin and more sulphomucin than the caecal contents. Inulin decreased acidic mucins and increased sulphated mucins in the caecal contents and increased neutral and sulphated mucins in the colonic contents. Inulin increased sulphomucin in the caecal mucosa and decreased acidic mucin in the caecal and colonic mucosas. The very low amount of mucin that was recovered in the colonic mucosa suggests that, in the presence of the bacterial flora and associated with inulin in the diet, mucin was extensively released from the mucosa to the colonic lumen. This might be related to the bacterial metabolites produced.

Comparative study of the fermentative characteristics of inulin and different types of fibre in rats inoculated with a human whole faecal flora

It is known that the physico-chemical characteristics of fibre modify their fermentation characteristics in the colon. Previously we showed the varying effects of inulin and different types of fibre on the hepatic and intestinal xenobiotic-metabolizing enzymes (XME) in initially germ-free rats inoculated with a human, methanogenic, whole-faecal flora (Roland et al. 1994). The aim of the present work was to assess whether or not these effects could be related to differences in production of fermentation metabolites (gases excreted in vivo and caecal metabolites) due to the different compositions of fibre. The different types of fibres were analysed with regard to their solubility and their composition of neutral monomers and uronic acids. Inulin was totally soluble, carrot (Daucus carota), cocoa (Theobroma cacao) and wheat bran were partially soluble; pea (Pisum sativum) and oat were nearly totally insoluble. Uronic acids were found mostly in carrot and cocoa fibre. Glucose was present as the main neutral monomer in each fibre type. Xylose was found also in wheat bran, pea and oat fibres, and arabinose was found in wheat bran. Inulin consumption led to high levels of H2 production but no CH4 production, to a 4-fold greater caecal concentration of butyrate than with the other fibres and to a decrease in caecal pH. Conversely, rats fed on carrot or cocoa fibre produced a large amount of CH4 but no H2 and generated a different profile of short-chain fatty acids (SCFA). The lowest amounts of gases and SCFA were found in rats fed on wheat bran, pea and oat fibre. We observed a relationship between the caecal concentration of SCFA and the activity of hepatic glutathione-S-transferase (EC 2.5.1.18) but no direct link was shown between the other XME and the fermentation profile.

Effects of orally administered Lactobacillus casei DN-114 001 on the composition or activities of the dominant faecal microbiota in healthy humans

The composition and activities of the faecal microbiota in twelve healthy subjects analysed in a single open study were monitored before (1-week baseline step), during (10 d supplementation step) and after (10 d follow-up step) the ingestion of a fermented milk containing Lactobacillus casei DN-114 001. Fluorescent in situ hybridisation with group-specific DNA probes, real-time PCR using L. paracasei group-specific primers and temporal temperature gradient gel electrophoresis (TTGE) using group-specific primers were carried out, together with bacterial enzyme activity and metabolite analyses to monitor the structure and activities of the faecal microbiota. L. casei DNA was detected in the faeces of all of the subjects by TTGE after 10 d supplementation. Its quantification by real-time PCR showed a 1000-fold increase during the test step compared with initial levels. No major modification in either the dominant members of the faecal microbiota or their activities was observed during the trial. In conclusion, the short-term consumption of a milk product containing L. casei DN-114 001 was accompanied by a high, transient increase in the quantity of this strain in the faeces of all of the subjects without markedly affecting biochemical or bacteriological factors.

Gnotobiotic rats harboring human intestinal microbiota as a model for studying cholesterol-to-coprostanol conversion

The efficiency of microbial reduction of cholesterol to coprostanol in human gut is highly variable among population and mechanisms remain unexplored. In the present study, we investigated whether microbial communities and their cholesterol metabolism characteristics can be transferred to germ-free rats. Two groups of six, initially germ-free rats were associated with two different human microbiota, exhibiting high and low cholesterol-reducing activities. Four months after inoculation, enumeration of coprostanoligenic bacteria, fecal coprostanol levels and composition of the fecal microbial communities were studied in gnotobiotic rats and compared with those of the human donors. Combination of culture (most probable number enumeration of active bacteria) and biochemical approaches (extraction followed by gas chromatography of sterols) showed that gnotobiotic rats harbored a coprostanoligenic bacterial population level and exhibited coprostanoligenic activities similar to those of the corresponding human donor. On the other hand, molecular approaches (whole-cell hybridization with fluorescently labeled 16S rRNA-targeted oligonucleotide probes, and temporal temperature gradient gel electrophoresis of bacterial 16S rRNA gene amplicons) demonstrated that gnotobiotic rats reproduced a stable microbial community, close to the human donor microbiota at the group or genus levels but different at the dominant species level. These results suggest that the gnotobiotic rat model can be used to explore the still unknown human intestinal microbiota involved in luminal cholesterol metabolism, including regulation of expression of its activity and impact on health.

Composition and metabolism of the intestinal microbiota in consumers and non-consumers of yogurt

The objective of the present study was to evaluate the impact of a regular consumption of yogurt on the composition and metabolism of the human intestinal microbiota. Adult subjects were selected on the basis of daily food records and divided into two groups: yogurt consumers (at least 200 g yogurt consumed per d, n 30); non-consumers (no yogurt, n 21). Their faecal microbiota was analysed using molecular methods (in situ hybridisation and PCR amplification combined with separation by denaturing gel electrophoresis) and its metabolic characteristics were assessed by measuring glycosidase, P-glucuronidase and reductase activities and profiling SCFA, neutral sterols and bile acids. The yogurt starter Lactobacillus delbrueckii ssp. bulgaricus (identity confirmed by 16S rRNA sequencing) was detected in 73% of faecal samples from fermented milk consumers v. 28% from non-consumers (P=0.003). In yogurt consumers, the level of Enterobacteriaceae was significantly lower (P=0.006) and 13-galactosidase activity was significantly increased (P=0.048). In addition, within this group, 3-galactosidase activity and the Bifidobacterium population were both positively correlated with the amount of fermented milk ingested (r 0.66, P<0.0001 and r 0.43, P=0.018, respectively). Apart from these effects, which can be considered beneficial to the host, no other major differences could be detected regarding the composition and metabolic activity of intestinal microbiota.

Effects of some poorly digestible carbohydrates on bile acid bacterial transformations in the rat

The effects of ingestion of poorly digestible carbohydrates on bacterial transformations of cholic acid and beta-muricholic acid were studied in rats fed on increasing levels of lactose, lactulose, amylomaize or potato starches. Each level was given for 3 weeks and, at the end of each dietary treatment, bile acid faecal composition was analysed and a group of six rats was killed every 4 h during 24 h to determine the amounts of fermented carbohydrate and fermentation characteristics (caecal pH, volatile fatty acids (VFA) and lactic acid concentrations). Fermentation of carbohydrates decreased caecal pH and enhanced caecal VFA and lactic acid concentrations. Irrespective of the poorly digestible carbohydrate, the variation of bacterial transformation always occurred in the same way: the bacterial transformation of beta-muricholic acid into hyodeoxycholic acid was the first to disappear, while omega-muricholic acid formation increased; second, cholic acid transformation decreased and finally all bile acid transformations were strongly affected. There was a significant correlation between bile acid transfer and the minimal caecal pH in vivo. This effect of pH was similar in vitro. To determine whether the levels of bacteria which transformed bile acids were modified, rats fed on the highest amounts of poorly digestible carbohydrates were introduced into isolators and carbohydrate feeding was stopped. Caecal pH recovered its initial value but bile acid transformations remained changed, suggesting that the intestinal microflora were modified by ingestion of fermentable carbohydrates.

Variation of mucin distribution in the rat intestine, caecum and colon: effect of the bacterial flora.

The influence of the intestinal microflora on mucin types was studied in the small intestine, caecum and colon of conventional (CV) rats as compared to germ-free (GF) rats. A colorimetric method was used on purified water-soluble mucin extracted from mucosal scrapings and contents. Variations occurred between the three anatomical sites both in the mucosas and intestinal contents of GF rats. In CV rats, the presence of the bacterial flora led to different effects depending on the intestinal site: in the small intestinal mucosa, neutral and sulphomucins values were higher whereas sialomucin was much lower. Conversely, sialomucin was higher in the caecal and colonic mucosas and contents whereas sulphated mucins were decreased significantly in caecal contents and caecal and colonic mucosas. These variations in the contents may reflect the bacterial mucolytic activity and the effect of bacterial metabolites on the mucosa.

Effects of galacto-oligosaccharide and bacterial status on mucin distribution in mucosa and on large intestine fermentation in rats

The purpose of the present paper was to study the effects of a dietary undigestible carbohydrate and intestinal microflora on mucin distribution (neutral, acid, sulphonated), glycolytic activities: beta-D-galactosidase (EC 3.2.1.23), N-acetyl-beta-D-galactosaminidase (EC 3.2.1.43), N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30), alpha-L-fucosidase (EC 3.2.1.51) and bacterial metabolism (gas production, short-chain fatty acids (SCFA) and lactic acid caecal concentration) in germ-free (GF), conventional (CV) and heteroxenic (HE) rats (GF rats associated with a human flora). Rats were fed on either a control diet or a diet containing 40 g trans-galactosylated oligosaccharide (TOS)/kg. In GF rats fed on the control diet caecal pH was almost neutral and glycolytic activities negligible. The number of mucus-containing cells increased from the caecum to the colon for the three types of mucin. TOS had no effect in the caecum but it modified mucin cell repartition in the colon. In CV and HE rats fed on the control diet caecal pH was similar (6.8), but caecal SCFA and lactic acid concentrations (mumol/g) and gas production (ml/24 h) were higher in CV (70, 5.9 and 2.3 respectively) than in HE rats (32, 4.6 and 0.4 respectively). In CV, as in HE rats, acid-mucin-containing cells increased from the caecum to the colon and glycolytic activities were similar. TOS reduced acid-mucin-containing cells in the caecum of CV rats by twofold but had no effect in either the caecum or the colon of HE rats. TOS strongly increased beta-galactosidase activity and slightly modified the other glycolytic activities. Its effect on bacterial metabolites depended on bacterial status. However, comparison between CV and HE rats showed no evident relationship between the number of mucus-containing cells and measured bacterial metabolites. Differences between CV and HE rats might be due to bacterial microflora specificity. TOS had an intrinsic effect on mucus cell distribution in the colon of GF rats. In CV and HE rats the presence of the flora abolished this effect.

Contribution of the digestive tract microflora to amylomaize starch degradation in the rat

To study in vivo the contribution of the bacterial flora to amylomaize starch degradation in the rat, germ-free and conventional rats were fed on a diet containing either a normal maize starch or an amylomaize starch. In germ-free rats maize starch was almost totally digested in the small intestine, whereas 40% of the ingested amylomaize starch reached the caecum and 30% was excreted, despite the very high endogenous amylase activity. Study by transmission electron microscopy of germ-free caecal contents showed an endocorrosion of the starch granule. In conventional rats, as in germ-free rats, digestibility of maize starch reached 98% in the small intestine, whereas that of amylomaize starch was only 60%. In the caecum of these rats amylomaize starch was fermented, and this led to a decrease in caecal pH and to formation of short-chain fatty acids (SCFA), especially propionate. Comparison between conventional rats fed on maize starch or amylomaize starch showed that caecal SCFA concentrations during a circadian cycle varied in the same way whereas total SCFA and lactic acid concentrations were much higher in rats fed on amylomaize starch. Amylase (EC 3.2.1.1) activity was similar in the caecal contents of conventional rats whatever the ingested starch. It was lower in conventional than in germ-free rats, but no starch granule remained in the caecum of conventional rats. These results showed that bacterial amylase was more efficient at degrading resistant amylomaize starch than endogenous amylase.

Survival of Bifidobacterium animalis DN-173 010 in the Faecal Microbiota after Administration in Lyophilised Form or in Fermented Product – A Randomised Study in Healthy Adults

The survival of Bifidobacterium animalis strain DN-173 010 was assessed after its ingestion in a fermented product or in a lyophilised form. Twelve healthy subjects were included in a randomised, open study with 2 parallel groups. The composition and activities of the faecal microbiota were monitored before (10-day baseline step), during (1-week product administration step) and after (10-day follow-up step) the ingestion of 1 of the 2 products. A colony immunoblotting method, fluorescent in situ hybridisation with group-specific DNA probes, and temporal temperature gradient gel electrophoresis using group-specific primers were carried out to compare survival of B. animalis strain DN-173 010 after ingestion of the 2 products, together with analyses of enzyme activities and faecal metabolites. At the end of the supplementation step, the mean number of B. animalis DN-173 010 quantified by immunodetection in the faeces of 5 of 6 subjects in each treatment group was ≧108 colony-forming units/g faeces. These numbers corresponded to an average survival of 22% for the lyophilised form and 20% for the fermented product. At the same step, the PCR temporal temperature gradient gel electrophoresis profiles showed a double band corresponding to the B. animalis DN-173 010 pattern for 11 subjects. No major modification was observed during the trial in either the dominant members of the faecal microbiota assessed by fluorescent in situ hybridisation or their activities. In conclusion, we show that the lyophilised form of B. animalis DN-173 010 survives transit and could represent a more convenient form to administer for long-term clinical trials.