Brigitta Kleessen

Oligofructose and long-chain inulin: influence on the gut microbial ecology of rats associated with a human faecal flora

Dietary incorporation of fermentable, indigestible fructans may be of benefit to gastrointestinal health by providing short-chain fatty acids, stimulating the proliferation of bifidobacteria or lactobacilli and suppressing potential pathogenic organisms in the gut. We tested the hypothesis that the effects of fructans on caecal, colonic and faecal short-chain fatty acid concentration and microflora composition depend on their chain length. Germ-free rats associated with a human faecal flora were randomly assigned to one of four treatments as follows: (1) commercial standard diet as a control (Con); (2) Con+50 g short-chain oligofructose/kg (OF); (3) C+50 g long-chain inulin/kg (lcIN); or (4) Con+50 g OF-lcIN/kg (Mix OF-lcIN). Changes in bacterial population groups in response to feeding these diets were investigated with 16S rRNA-targeted probes applied in in situ hybridization. Mix OF-lcIN- and lcIN-containing diets resulted in larger numbers of caecal, colonic and faecal bacteria of the Clostridium coccoides-Eubacterium rectale cluster than Con (10.6 and 10.3 v. 9.5 log10/g wet wt), whereas OF alone did not affect this bacterial group in caecum, colon or faeces. A bifidogenic effect was only observed in the colon and faeces of OF-treated rats. More lactobacilli were found in caecal and colonic contents of Mix OF-lcIN-fed rats and in faeces of OF-fed rats compared with Con. Mix OF-lcIN and OF led to significantly smaller numbers of caecal, colonic and faecal bacteria belonging to the Clostridium histolyticum and C. lituseburense groups than Con (6.8 and 6.9 v. 7.9 log10/g wet wt). Counts of total bacteria, Bacteroides-Prevotella and Enterobacteriaceae did not differ between the groups. OF and/or lcIN-containing diets significantly increased the caecal and colonic concentration of butyrate and its relative molar proportion. Only lcIN-containing diets resulted in a higher faecal concentration of butyrate than Con. Higher molar proportions of faecal butyrate were observed with all diets that had been supplemented with OF and/or lcIN. Stimulation of butyrate production could be of interest for the prevention of ulcerative colitis and colon cancer.

Effects of inulin on faecal bifidobacteria in human subjects.

A controlled study with eight healthy free-living subjects was carried out, in which energy intake was adjusted to the individual energy requirements. On administration of inulin, blood lipids, the faecal microflora, short-chain fatty acids and accompanying gastrointestinal symptoms were characterized in order to investigate the long-term effect of inulin. During the run-in phase (8 d), subjects received a typical Western diet providing 45% energy as fat and 40% energy as carbohydrate. Subsequently, the subjects consumed a fat-reduced diet which provided 30% energy as fat and 55% energy as carbohydrate for a period of 64 d using inulin as a fat replacer. The amounts of inulin consumed by the subjects (up to 34 g/d) were based on individual energy requirements with the aim to keep the diet isoenergetic with that used in the run-in period. To assess the effects of inulin administration, a control study (run-in and intervention) was carried out in which subjects consumed the same diet but devoid of inulin during the whole course of the study. To investigate the effect of inulin on faecal flora composition total bacteria and bifidobacteria in the faeces were enumerated by in situ hybridization with 16S rRNA targeted oligonucleotide probes. Inulin significantly increased bifidobacteria from 9.8 to 11.0 log10/g dry faeces and caused a moderate increase in gastrointestinal symptoms such as flatulence and bloatedness, whereas blood lipids and short-chain fatty acids remained essentially unaffected.

Mucosal and invading bacteria in patients with inflammatory bowel disease compared with controls.

Background: Endogenous intestinal bacteria and/or specific bacterial pathogens are suspected of being involved in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to investigate IBD tissues for different bacterial population groups harbouring the mucosal surface and/or invading the mucosa. Methods: Tissue sections from surgical resections from the terminal ileum and/or the colon from 24 IBD patients (12 active ulcerative colitis (UC), 12 active Crohn disease (CD)) and 14 non-IBD controls were studied by fluorescent in situ hybridization on a quantifiable basis. Results: More bacteria were detected on the mucosal surface of IBD patients than on those of non-IBD controls ( P < 0.05). Bacterial invasion of the mucosa was evident in 83.3% of colonic specimens from the UC patients, in 55.6% of the ileal and in 25% of the colonic specimens from the CD patients, but no bacteria were detected in the tissues of the controls. Colonic UC specimens were colonized by a variety of organisms, such as bacteria belonging to the gamma subdivision of Proteobacteria , the Enterobacteriaceae , the Bacteroides/Prevotella cluster, the Clostridium histolyticum/Clostridium lituseburense group, the Clostridium coccoides/Eubacterium rectale group, high G + C Gram-positive bacteria, or sulphate-reducing bacteria, while CD samples harboured mainly bacteria belonging to the former three groups. Conclusion: Pathogenic events in CD and UC may be associated with different alterations in the mucosal flora of the ileum and colon.

Bacterial and fungal microbiota in relation to probiotic therapy (VSL#3) in pouchitis

Background: The intestinal microbiota plays a critical role in the pathophysiology of pouchitis, a major complication after ileal pouch anal anastomosis in patients with ulcerative colitis. Recently, controlled trials have demonstrated that probiotics are effective in maintenance of remission in pouchitis patients. However, the mechanism by which therapy with probiotics works remains elusive. This study explores the role of the bacterial and fungal flora in a controlled trial for maintenance of remission in pouchitis patients with the probiotic VSL#3 compound. Methods: The mucosa associated pouch microbiota was investigated before and after therapy with VSL#3 by analysis of endoscopic biopsies using ribosomal DNA/RNA based community fingerprint analysis, clone libraries, real time polymerase chain reaction (PCR), and fluorescence in situ hybridisation. Patients were recruited from a placebo controlled remission maintenance trial with VSL#3. Results: Patients who developed pouchitis while treated with placebo had low bacterial and high fungal diversity. Bacterial diversity was increased and fungal diversity was reduced in patients in remission maintained with VSL#3 (p = 0.001). Real time PCR experiments demonstrated that VSL#3 increased the total number of bacterial cells (p = 0.002) and modified the spectrum of bacteria towards anaerobic species. Taxa specific clone libraries for Lactobacilli and Bifidobacteria showed that the richness and spectrum of these bacteria were altered under probiotic therapy. Conclusions: Probiotic therapy with VSL#3 increases the total number of intestinal bacterial cells as well as the richness and diversity of the bacterial microbiota, especially the anaerobic flora. The diversity of the fungal flora is repressed. Restoration of the integrity of a “protective” intestinal mucosa related microbiota could therefore be a potential mechanism of probiotic bacteria in inflammatory barrier diseases of the lower gastrointestinal tract.

Fructans in the diet cause alterations of intestinal mucosal architecture, released mucins and mucosa-associated bifidobacteria in gnotobiotic rats

The effects of fructans in the diet on the mucosal morphometry (height of villi, depth of the crypts, number of goblet cells), the thickness of the epithelial mucus layer and the histochemical composition of intestinal mucosubstances in the distal jejunum and the distal colon were investigated by comparing germ-free (GF) rats, rats harbouring Bacteroides vulgatus and Bifidobacterium longum (diassociated (DA) rats), and rats with a human faecal flora (HFA). The rats were fed either a commercial standard diet (ST) or ST + (50 g oligofructose (OF)-long-chain inulin (lcIN))/kg. Changes in total bacteria, bifidobacteria and Bacteroides-Prevotella in response to feeding these diets were investigated by fluorescent in situ hybridization with 16S rRNA-targeted probes both in intestinal contents (lumen bacteria) and tissue sections (mucosa-associated bacteria). The OF-lcIN-containing diet resulted in higher villi and deeper crypts in bacteria-associated, but not in GF rats. In DA and HFA rats, the colonic epithelial mucus layer was thicker and the numbers of the goblet cells were greater than in GF rats. These effects were enhanced by the OF-lcIN-containing diet. In both dietary groups, bacterial colonization of GF rats caused an increase in neutral mucins in the distal jejunum and colon. Bacteria-associated rats had more acidic mucins in the colon than GF rats, and the OF-lcIN-containing diet stimulated sulfomucins as the predominant type of acidic mucins, while sialomucins dominated in the ST-fed groups. The number of mucosa-associated bifidobacteria detected in the colon of DA and HFA rats was greater with OF-lcIN than ST (4.9 and 5.4 v. 3.5 and 4.0 log10/mm2 mucosal surface respectively), whereas the number of luminal bifidobacteria was only affected by fructans in DA rats. Bacteroides did not differ between the groups. The stabilisation of the gut mucosal barrier, either by changes in the mucosal architecture itself, in released mucins or by stimulation of mucosal bifidobacteria with fructans, could become an important topic in the treatment and prophylaxis of gastrointestinal disorders and health maintenance.

Jerusalem artichoke and chicory inulin in bakery products affect faecal microbiota of healthy volunteers.

A study was conducted to test the effects of Jerusalem artichoke inulin (JA) or chicory inulin (CH) in snack bars on composition of faecal microbiota, concentration of faecal SCFA, bowel habit and gastrointestinal symptoms. Forty-five volunteers participated in a double-blind, randomized, placebo-controlled, parallel-group study. At the end of a 7 d run-in period, subjects were randomly assigned to three groups of fifteen subjects each, consuming either snack bars with CH or JA, or snack bars without fructans (placebo); for 7 d (adaptation period), they ingested one snack bar per day (7.7 g fructan/d) and continued for 14 d with two snack bars per day. The composition of the microbiota was monitored weekly. The consumption of CH or JA increased counts of bifidobacteria (+1.2 log10 in 21 d) and reduced Bacteroides/Prevotella in number and the Clostridium histolyticum/C. lituseburense group in frequency at the end of intervention (P < 0.05). No changes in concentration of faecal SCFA were observed. Consumption of snack bars resulted in a slight increase in stool frequency. Stool consistency was slightly affected in subjects consuming two snack bars containing CH or JA per day (P < 0.05). Consumption of CH or JA resulted in mild and sometimes moderate flatulence in a few subjects compared to placebo (P < 0.05). No structural differences were detected between CH and JA before and after processing. In conclusion, adaptation on increased doses of CH or JA in bakery products stimulates the growth of bifidobacteria and may contribute to the suppression of potential pathogenic bacteria.

Modulation of gut mucosal biofilms

Non-digestible inulin-type fructans, such as oligofructose and high-molecular-weight inulin, have been shown to have the ability to alter the intestinal microbiota composition in such a way that members of the microbial community, generally considered as health-promoting, are stimulated. Bifidobacteria and lactobacilli are the most frequently targeted organisms. Less information exists on effects of inulin-type fructans on the composition, metabolism and health-related significance of bacteria at or near the mucosa surface or in the mucus layer forming mucosa-associated biofilms. Using rats inoculated with a human faecal flora as an experimental model we have found that inulin-type fructans in the diet modulated the gut microbiota by stimulation of mucosa-associated bifidobacteria as well as by partial reduction of pathogenic Salmonella enterica subsp. enterica serovar Typhimurium and thereby benefit health. In addition to changes in mucosal biofilms, inulin-type fructans also induced changes in the colonic mucosa stimulating proliferation in the crypts, increasing the release of mucins, and altering the profile of mucin components in the goblet cells and epithelial mucus layer. These results indicate that inulin-type fructans may stabilise the gut mucosal barrier. Dietary supplementation with these prebiotics could offer a new approach to supporting the barrier function of the mucosa.

Effect of isomalt consumption on faecal microflora and colonic metabolism in healthy volunteers

Due to its low digestibility in the small intestine, a major fraction of the polyol isomalt reaches the colon. However, little is known about effects on the intestinal microflora. During two 4-week periods in a double-blind, placebo-controlled, cross-over design, nineteen healthy volunteers consumed a controlled basal diet enriched with either 30 g isomalt or 30 g sucrose daily. Stools were collected at the end of each test phase and various microbiological and luminal markers were analysed. Fermentation characteristics of isomalt were also investigated in vitro. Microbiological analyses of faecal samples indicated a shift of the gut flora towards an increase of bifidobacteria following consumption of the isomalt diet compared with the sucrose diet (P<0.05). During the isomalt phase, the activity of bacterial beta-glucosidase decreased (P<0.05) whereas beta-glucuronidase, sulfatase, nitroreductase and urease remained unchanged. Faecal polyamines were not different between test periods with the exception of cadaverine, which showed a trend towards a lower concentration following isomalt (P=0.055). Faecal SCFA, lactate, bile acids, neutral sterols, N, NH3, phenol and p-cresol were not affected by isomalt consumption. In vitro, isomalt was metabolized in several bifidobacteria strains and yielded high butyrate concentrations. Isomalt, which is used widely as a low-glycaemic and low-energy sweetener, has to be considered a prebiotic carbohydrate that might contribute to a healthy luminal environment of the colonic mucosa.

Effect of inulin supplementation on selected gastric, duodenal, and caecal microbiota and short chain fatty acid pattern in growing piglets

Abstract We explored whether bifidobacteria and lactobacilli numbers and other selected bacteria in the upper intestine and the caecum of growing pigs were affected by diet and intake of inulin. Starting at two weeks after weaning (28 d) 72 pigs were fed two types of diets (wheat/barley (WB) or maize/gluten (MG)), without or with 3% inulin (WB + I, MG + I) for three and six weeks. Intestinal bacteria were quantified by fluorescence-in-situ-hybridization (n = 8/group). Duration of feeding had no effect on the variables tested, so data for both periods were pooled. Gastric total bacteria amounted to log10 7.4/g digesta. Bifidobacteria were detected in stomach and duodenum two weeks after weaning and disappeared thereafter. In jejunum and caecum bifidobacteria were present at a level of log10 7.0/g digesta. Inulin did not alter numbers of lactobacilli, bifidobacteria, enterococci, enterobacteria and bacteria of the Clostridium coccoides/Eubacterium rectale-group. Inulin disappearance in stomach plus jejunum was higher with the MG diet (73.7 vs. 60.7%, p = 0.013). Caecal acetate was lower in inulin-supplemented diets (p < 0.05) whereas propionate and butyrate were higher in pigs fed the WB diets (p < 0.05). With the WB diet total caecal short chain fatty acids concentration was higher which resulted in a lower pH value (p < 0.05).

The effect of alimentary polyamine depletion on germ-free and conventional rats

Abstract Polyamine-deficient semisynthetic diet or polyamine-rich standard rat chow (Altromin 1320) were fed to germ-free and conventional rats to study the influence of alimentary polyamine intake on the endogenous polyamine content and the polyamine formation by the intestinal microflora. Putrescine was the major polyamine in the intestinal contents or feces of germ-free rats. In contrast, the intestinal contents and feces of conventional rats contained mainly spermidine, but only low concentrations of putrescine and spermine. Cadaverine was not detected at all. These polyamine patterns were not affected by the dietary polyamine intake. The polyamine patterns of blood plasma and colonic tissue were similar in germ-free and conventional rats: putrescine was the major polyamine in plasma, whereas spermidine and spermine predominated in colonic tissue. The concentrations of putrescine in plasma and of spermidine and spermine in colonic tissue were lower in rats fed the polyamine-deficient semisynthetic diet than in rats fed the Altromin diet. This difference was greater in germ-free than in conventional rats. Bacteroides, Eubacterium, and Lactobacillus were the predominant organisms of the fecal flora found in conventional rats. The composition of the microflora differed only slightly in response to the diet. In conclusion, (1) putrescine is the main endogenously generated polyamine secreted into the gut lumen, (2) the high spermidine content in the luminal content of all intestinal segments of the conventional rats was independent of the diet and therefore must be of microbial origin, and (3) the intraluminal microbial polyamine formation seems to be inversely related to the alimentary polyamine supply.