Martine Bensaada

Increased induction of apoptosis by Propionibacterium freudenreichii TL133 in colonic mucosal crypts of human microbiota-associated rats treated with 1,2-dimethylhydrazine

Propionibacterium freudenreichii, a food-grade bacterium able to kill colon cancer cell lines in vitro by apoptosis, may exert an anticarcinogenic effect in vivo. To assess this hypothesis, we administered daily 2 x 10(10) colony-forming units (CFU) of P. freudenreichii TL133 to human microbiota-associated (HMA) rats for 18 d. Either saline or 1,2-dimethylhydrazine (DMH) was also administered on days 13 and 17 and rats were killed on day 19. The levels of apoptosis and proliferation in the mid and distal colon were assessed by terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) and proliferating cell nuclear antigen (PCNA) immunolabelling, respectively. The administration of P. freudenreichii TL133 significantly increased the number of apoptotic cells in DMH-treated rats compared to those given DMH only (P < 0.01). Furthermore, propionibacteria were able to decrease the proliferation index in the distal colon after treatment with DMH (P < 0.01). Conversely, propionibacteria alone did not exert such an effect on healthy colonic mucosa. P. freudenreichii TL133 thus facilitated the elimination of damaged cells by apoptosis in the rat colon after genotoxic insult and may play a protective role against colon cancer.

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.

Ability of Lactobacillus fermentum to overcome host α-galactosidase deficiency, as evidenced by reduction of hydrogen excretion in rats consuming soya α-galacto-oligosaccharides

BackgroundSoya and its derivatives represent nutritionally high quality food products whose major drawback is their high content of α-galacto-oligosaccharides. These are not digested in the small intestine due to the natural absence of tissular α-galactosidase in mammals. The passage of these carbohydrates to the large intestine makes them available for fermentation by gas-producing bacteria leading to intestinal flatulence. The aim of the work reported here was to assess the ability of α-galactosidase-producing lactobacilli to improve the digestibility of α-galacto-oligosaccharides in situ.ResultsGnotobiotic rats were orally fed with soy milk and placed in respiratory chambers designed to monitor fermentative gas excretion. The validity of the animal model was first checked using gnotobiotic rats monoassociated with a Clostridium butyricum hydrogen (H2)-producing strain. Ingestion of native soy milk by these rats caused significant H2 emission while ingestion of α-galacto-oligosaccharide-free soy milk did not, thus validating the experimental system. When native soy milk was fermented using the α-galactosidase-producing Lactobacillus fermentum CRL722 strain, the resulting product failed to induce H2 emission in rats thus validating the bacterial model. When L. fermentum CRL722 was coadministered with native soy milk, a significant reduction (50 %, P = 0.019) in H2 emission was observed, showing that α-galactosidase from L. fermentum CRL722 remained active in situ, in the gastrointestinal tract of rats monoassociated with C. butyricum. In human-microbiota associated rats, L. fermentum CRL722 also induced a significant reduction of H2 emission (70 %, P = 0.004).ConclusionThese results strongly suggest that L. fermentum α-galactosidase is able to partially alleviate α-galactosidase deficiency in rats. This offers interesting perspectives in various applications in which lactic acid bacteria could be used as a vector for delivery of digestive enzymes in man and animals.

Ulva lactuca is poorly fermented but alters bacterial metabolism in rats inoculated with human faecal flora from methane and non‐methane producers

In vivo fermentation of Ulva lactuca was studied in previously germ-free rats inoculated with human flora obtained from non-, low- and high-methane producers (groups NMP, LMP and HMP, respectively), in comparison to germ-free rats. Rats were fed either a control diet or a diet containing 4% of dried Ulva lactuca. Production of metabolites varied according to the flora and diet. Ulva lactuca induced a specific high production of methane in the HMP group. With the three human flora, Ulva diet induced a similar increase in caecal pH. In the NMP and HMP groups, this increase was associated with a fall of lactic acid caecal concentration. In the LMP group it was related to a decrease in the concentration of short-chain fatty acids. Ulva lactuca appeared to be able to regulate the β-glucuronidase and β-glucosidase activities, reducing the relatively high levels observed in groups NMP and HMP and increasing the low levels obtained in the LMP group. Results show that, although it was poorly fermented, Ulva induced significant effects on the gut microflora metabolism. The methanogenic status of the human donor appeared to be an important factor.

Differential influence of butyrate concentration on proximal and distal colonic mucosa in rats born germ-free and associated with a strain of Clostridium paraputrificum.

In vivo influence of butyrate in colonic mucosa was studied using a model of gnotobiotic rats monoassociated with a Clostridium paraputrificum. Rats were fed a diet containing increasing amounts of non-digestible carbohydrates, the fermentation of which led to modulated amounts of butyrate in the large intestine. In the proximal colon, the increase in the butyrate concentration alters crypt depth and the number of mucus-containing cells; the increase in butyrate was highly correlated with the number of neutral-mucin-containing cells. Conversely, in the distal colon, no relation was found between the increase in butyrate concentration and crypt depth or number of mucin-containing cells. In both the proximal and distal colon, the mitotic index remained unchanged. In conclusion, in vivo production of physiological quantities of butyrate had a trophic effect on proximal colonic mucosa, but did not influence the distal epithelium.

Apple proanthocyanidins do not reduce the induction of preneoplastic lesions in the colon of rats associated with human microbiota.

Since the gut microbiota metabolizes various dietary constituents unabsorbed by the small intestine and modulates colon function, it plays an essential role in colon carcinogenesis. First, we have developed a model of human microbiota-associated rats (HMA), fed a human-type diet and injected with 1-2,dimethylhydrazine (DMH). We observed that the number and size of DMH-induced aberrant crypt foci (ACF) were significantly higher in HMA rats than in germ-free or conventional rats. Second, we used this model to assess the protective effect of an apple proanthocyanidin-rich extract (APE) on colon carcinogenesis. In this model, ACF number and multiplicity were not reduced by APE at 0.001% and 0.01% in drinking water. They were higher with APE 0.1% than with APE 0.01%. Therefore, the cross-talk between human microbiota and the colon epithelium should be taken into account in carcinogenesis models. Moreover, attention should be paid prior to using proanthocyanidin extracts as dietary supplements for humans.

Effects of chair‐restraint on gastrointestinal transit time and colonic fermentation in male rhesus monkey (Macaca mulatta)

Abstract: The incidence of an 18 day chair‐restraint on the digestive physiology of male rhesus monkey was investigated for space research purposes, comparing four trained restraint subjects with two vivarium controls. Chair‐restraint induced a 2.5‐fold acceleration of the gastrointestinal transit time, which persisted throughout the 7 day postrestraint period, and an increase of the fecal dry matter content, which mean value rose from 40.7% to 69.6%. Fecal pH remained unaltered throughout the experiment. Modifications of fermentative metabolites produced by the colonic microflora and excreted through the breath (hydrogen and methane) or in the feces (short chain fatty acids and ammonia) could not be reliably related to chair‐restraint and probably involved side‐stress factors. On the whole, alterations due to chair‐restraint are shown to be different from those reported in the literature, following a modification of the dietary composition. These data may help to predict the alterations of digestive physiology likely to occur in immobilized human patients.

Effects of ingestion of a green seaweed, Ulva lactuca, upon caecal and colonic mucosas in the germ-free rat and in the heteroxenic rat harbouring a human bacterial flora

Effect of ingestion of green seaweed, Ulva lactuca, (70 g kg−1) during a 6-week period on caecal and colonic mucosas was studied in germ-free (GF) rats and in heteroxenic (HE) rats harbouring a human bacterial flora (GF rats associated with a human flora). The pH and sulphide concentration of the caecal contents, crypt morphometry, mitotic index and mucin types in the caecal and distal colonic mucosas were determined. In the GF caecum, Ulva strongly increased crypt depth and mucin-containing cells irrespective of the mucin type studied (neutral, acidic or sulphated) compared to the control diet but had no significant effect on mitotic index. The crypt depth and mucin-containing cells in the caecum were higher in HE than in GF control rats. They were slightly but significantly increased by Ulva. In the distal colon mucosa of GF rats, Ulva decreased crypt depth and cell number as well as sulphomucin-containing cells. Conversely, in the HE rats, it increased crypt depth and reduced the number of neutral mucin-containing cells. These results show that Ulva lactuca exerts an intrinsic effect on mucosal morphometry and on mucin biosynthesis in GF rats. No pathological alteration was observed in the mucosas and no significant modification of the mitotic index or sulphide production was observed in HE rats. © 1999 Society of Chemical Industry