Bacterial Microbiota and Fatty Acids in the Faeces of Overweight and Obese Children.

The growing number of children with overweight and obesity constitutes a major health problem of the modern world and it has been suggested that intestinal microbiota may influence energy intake from food. The objectives of this study were to determine quantity and proportions of dominant genera of Bacteroides, Prevotella (phylum Bacteroidetes ); Clostridium , Lactobacillus (phylum Firmicutes ) and Bifidobacterium (phylum Actinobacteria ) in the intestines and to determine the content of short-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs) in the stool of 20 obese children and 20 children with normal body weight. Strains classified as Firmicutes ( Clostridium and Lactobacillus ) predominated in stool microbiota of obese children, while those of Bacteroidetes ( Prevotella and Bacteroides ) were in minority ( p < 0.001). Concentration of SCFAs in the stool of obese children was lower in comparison to the stool of normal weight children ( p = 0.04). However, these differences were significant only in obese children, not in overweight children in comparison with the lean ones. Therefore, in our study obesity was associated with intestinal dysbiosis and a predominance of phylum Firmicutes . Secondly, stool of obese children contained lower amounts of SCFAs.

Dietary resistant dextrins positively modulate fecal and cecal microbiota composition in young rats.

The objective of the present study was to demonstrate the effect of dietary resistant dextrins, as potential prebiotics, on the intestinal microflora of young rats. Enzyme-resistant dextrin, prepared by heating of potato starch in the presence of hydrochloric (0.1% dsb) and tartaric (40% dsb) acid at 130ºC for 2 h (CA-dextrin). The experiment was performed on 24 Wistar male rats at 3-wk of age, divided by analogues in three experimental groups (control, starch and dextrin). Analyses determined the overall bacterial counts and the counts of Lactobacillus, Bifidobacterium, Bacteroides and Clostridium strains within the feces and cecal contents of rats using fluorescence in situ hybridization method. CA-dextrin had no effect on primary growth indicators (body weight, body weight gain, dietary consumption) or the mass of the small intestine and the cecum, but dextrins caused a reduction in pH and the concentration of ammonia within the cecal contents. That supplementation of diet with resistant dextrins had a positive effect on composition of intestinal microflora in rats. It increased the counts of Bifidobacterium and Lactobacillus strains both in the feces and in the cecum. Moreover, it reduced the counts of Clostridium and Bacteroides strains. These results may suggest that resistant dextrins exerted a prebiotic-like effect in the large intestine.

Prebiotic properties of potato starch dextrins.

The objective of the present study was to compare the prebiotic properties of starch dextrins, that is, resistant dextrins obtained from potato starch in the process of simultaneous thermolysis and chemical modification, which were selected based on previous research. Both prepared dextrins met the definition criterion of dietary fiber and also the basic prebiotic criterion - they were not degraded by the digestive enzymes of the initial sections of the gastrointestinal tract. The growth of probiotic lactobacilli and bifidobacteria, as well as Escherichia coli, Enterococcus, Bacteroides, and Clostridium strains isolated from feces of healthy people, showed that both studied dextrins were utilized as a source of assimilable carbon and energy by the strains. Furthermore, better growth (higher numbers of cells) counts of probiotic bacteria than those of fecal isolates indicated that the studied resistant dextrins showed a selective effect. Both dextrins might be considered as substances with prebiotic properties due to their chemical and physical properties and selectivity towards the studied probiotic bacterial strains.

The influence of Lactobacillus casei DN 114 001 on the activity of faecal enzymes and genotoxicity of faecal water in the presence of heterocyclic aromatic amines.

High activity of bacterial enzymes in human colon and genotoxicity of faecal water (FW) are biomarkers of the harmful action of microbiota. The aim of the present study was to assess the activity of ß-glucuronidase and ß-glucosidase and the genotoxicity of FW in vitro after incubation with 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ) or 2-amino-1-methyl-6-phenyl-1H-imidazo[4,5-b]pyridine (PhIP) and probiotic Lactobacillus casei DN 114 001 (Actimel). Our results indicate, that IQ and PhIP greatly increased the activity of faecal enzymes (it was up to four times higher, as measured by spectrophotometric methods) and the genotoxicity of FW (% DNA in the tail was up to 3.2 times higher, as evaluated by the comet assay on Caco-2 cells) in 15 individuals from three age-dependent groups (breast-fed children, adults aged 30-40 years, elderly aged 75-85 years). Lb. casei DN 114 001 decreased the activity of faecal enzymes and the genotoxicity of FW exposed to PhIP and IQ mostly to control values. The activity of faecal enzymes after incubation with IQ was reduced by 71.8% in the FW of children, 37.5% in adults and 64.2% in elderly (ß-glucuronidase); as well as by 59.9% in children and 87.9% in elderly (ß-glucosidase). For PhIP the reduction was by 59.0% in the FW of children, 50.0% in adults and 81.2% in elderly (ß-glucuronidase) and by 20.2% in children, 20.7% in adults and 84.1% in elderly (ß-glucosidase). Lb. casei DN 114 001 also decreased the genotoxicity of FW to the greatest extent in adults after incubation with IQ (by 65.4%) and PhIP (by 69.6%) and it was found to correlate positively with the decrease in faecal enzymes activity. In conclusion, Lb. casei DN 114 001 may exert the protective effects against genotoxic and possibly pro-carcinogenic effects of food processing-derived chemicals present in faecal water.

Probiotic lactic acid bacteria detoxify N-nitrosodimethylamine.

Humans can be exposed to N-nitroso compounds (NOCs) due to many environmental sources, as well as endogenous formation. The main nitrosamine found in food products and also synthesised in vivo by intestinal microbiota is N-nitrosodimethylamine (NDMA). It can cause cancer of the stomach, kidney and colon. The effect of four probiotic Lactobacillus strains on NDMA was studied under different culture conditions (24 h in MRS, 168 h in modified MRS N, and 168 h in phosphate buffer). HPLC and GC-TEA methods were used for NDMA determination in supernatants. The influence of lactic acid bacteria on NDMA genotoxicity was investigated by means of the comet assay. Additionally, the effect of NDMA (2-100 µg ml⁻¹) on the growth and survival of the probiotic strains was studied. The results indicate that the bacteria decreased NDMA concentration by up to 50%, depending on the culture conditions, time of incubation, NDMA concentration, pH and bacterial strain. Lb. brevis 0945 lowered the concentration and genotoxicity of NDMA most effectively by up to 50%. This could be due to either adsorption or metabolism. The growth and survival of the bacteria was not affected by any of the tested NDMA concentrations.

Beta-glucuronidase and Beta-glucosidase activity in stool specimens of children with inflammatory bowel disease.

The aim of the study was to analyze the differences in the activity of beta-glucuronidase and beta-glucosidase in stool specimens of children with Inflammatory Bowel Diseases (IBD) and healthy subjects. The disease activity was determined according to the PCDAI scale (Crohn disease) and Truelove-Witts scale (Ulcerative colitis). Enzyme activity was determined by spectrophotometry. There was a correlation between the level of beta - glucosidase activity in stool and patient's age in the group of healthy controls, but not in the IBD group. beta-glucosidase activity in IBD and healthy subjects stool specimens did not differ significantly. The activity of beta-glucuronidase in children with IBD was two times lower than in the healthy group and was correlated with age in children with IBD, but not in the group of healthy ones.

Changes in gut microbiota in children with atopic dermatitis administered the bacteria Lactobacillus casei DN--114001.

Gut microbiota was analyzed in children, aged 6-18 months and suffering from atopic dermatitis before and after 3 month supplementation of their diet with Lactobacillus casei DN--114001 in a dose of 109 cells daily. On completion of this period the total number of fecal Lactobacillus sp. cells decreased from 7.86 Log10 CFU/g to 6.40 Log10 CFU/g. After the next 5 months (without dietary supplementation with the probiotic bacteria) the level of Lactobacillus sp. cells was maintained at the latter value. During the dietary supplementation with the probiotic strain, the level of Bifidobacterium cells was maintained at 6.15-6.89 Log10 CFU/g while after 5 months it decreased to 5.57 Log10 CFU/g. The population of Clostridium sp. was reduced after 3 months of dietary supplementation from 6.49 to 5.83 Log10 CFU/g and was maintained at the latter level during the next 5 months. The dietary supplementation had no effect on populations of Bacteroides sp., Enterococcus sp. and Enterobacteriaceae. Supplementation of children who developed atopic dermatitis with the preparation of Lactobacillus casei DN - 114001 positively affected their gut microbiota in terms of bifidobacteria and clostridia populations.

Probiotic preparation reduces the faecal water genotoxicity in chickens fed with aflatoxin B1 contaminated fodder.

The aim of the study was to evaluate the influence of a probiotic preparation on the genotoxicity of faecal water of broiler chickens fed with a fodder contaminated with aflatoxin B(1) (AFB(1)) at 1 or 5mg per kg. Human blood lymphocytes were exposed to chicken's faecal water samples and DNA damage was measured using the comet assay. Genotoxicity of faecal water did not depend on the AFB(1) concentration in the fodder. The mean DNA damage, measured as the percentage of DNA in the tail of the comets, for chickens fed with fodder with AFB(1) at 1 mg/kg was 16.80±0.66, at 5 mg/kg - 16.73±1.51 and in the controls - 12.79±0.66. The supplementation of fodder with the probiotic preparation decreased the extent of DNA damage to 10.02±0.39 for 1 mg/kg AFB(1) and to 11.89±0.72 for 5 mg/kg.

Beta-glucuronidase and beta-glucosidase activity of Lactobacillus and Enterococcus isolated from human feces.

The domination of microorganisms characterized by excessive activity of the so-called fecal enzymes may be one of the reasons of the large intestine cancers. These enzymes are mainly those that belong to the hydrolase and reductase classes and their excessive activity may lead to disorders in the functioning of the digestive tract. The aim of tise research was to determine the activity of beta-glucuronidase and beta-glucosidase of Lactobacillus and Enterococcus strains isolated from the feces of healthy children, aged 1 and 8, and adults, aged 30 and 80. The analysis included 10 strains isolated from the feces of individuals in each of the age groups. beta-glucuronidase activity in the case of the isolates from children, depending on the strain, equaled from about 0.15 mM/h/mg of protein to 0.26 mM/h/mg of protein and was lower, respectively, by 52.35% and 57.81%, than the beta-glucosidase activity. Simultaneously, the activity of the Lactobacillus enzymes from children was 2.4 times higher, and in case of the isolates obtained from adults they were 4.6 and 2.7 times higher than the activity of the Entercoccus enzymes. The highest beta-glucuronidase activity was observed in Lactobacillus isolates coming from an 80-year-old subject. The differences between the activity of Enterococcus beta-glucuronidase isolated from the feces of 1 and 8 year old children were statistically insignificant. On the other hand, in the case of the subjects aged 30 and 8 the isolates were characterized by activity lower by, respectively. 48% and 37% than the isolates coming from children. The highest beta-glucosidase activity was discovered in the case of Lactobacillus and Enterococcus coming from children, which was higher by 32% than the activity of the isolates from adult persons. Therefore, it was determined that the activity of beta-glucuronidase of Lactobacillus strains isolated from feces from people aged 80 was the highest, and the isolates of the examined microorganisms coming from children were characterized by the highest beta-glucosidase activity.

Ability of probiotic Lactobacillus casei DN 114001 to bind or/and metabolise heterocyclic aromatic amines in vitro.

INTRODUCTION: Heterocyclic aromatic amines (HCA) are compounds with high mutagenic potential, formed when meat is cooked at high temperatures of 150-300 degrees C. These compounds contribute to development of colon and gastric cancer. Western diet provides a lot of HCA and influences the available substrates for the intestinal microbiota which can activate HCA to direct acting mutagens. On the other hand, lactic acid bacteria existing in the colon and ingested with food including probiotics, may exert an anti-carcinogenic action, but the mechanism is still poorly understood. MATERIALS AND METHODS: In the present study we determined the ability of probiotic Lactobacillus casei DN 114001 (Actimel strain) to metabolise or adsorb three HCA: IQ, MelQx and PhIP in vitro. Lactobacilli were cultivated in MRS and in a modified MRS broth with reduced concentrations of nitrogen and carbon (MRS NC), with addition of 25 mug/ml of IQ, MelQx or PhIP. Their concentration after cultivation with L. casei DN 114001 was measured with high-performance liquid chromatography and the genotoxicity was evaluated by the alkaline comet assay. RESULTS AND CONCLUSIONS: It was measured, that after 24 h cultivation in MRS (cell density was 10(9) cfu/ml), rapid decrease of IQ and PhIP (98-99%) was observed, and the peaks on chromatograms were almost completely reduced. In case of MeIQx the decrease was about 27%. In a modified MRS broth (cell density was 10(8) cfu/ml), the ability to decrease HCA concentration during prolonged cultivation (to 168 h) depended on the growth phase of bacteria, and it was about 51.5% for IQ and at about 11.2% for MeIQx. Non-growing cells (in phosphate buffer), could reduce the content of IQ and PhIP from 72 h to the end of incubation. L. casei DN 114001 reduced genotoxicity of HCA (IQ from 46 to 48%; MeIQx from 35 to 65% and PhIP from 32 to 81%), and the degree depended on the incubation time, cell growth and the medium used. It may suggest that bacteria can metabolise or adsorb HCA.

Influence of phenol, p-cresol and indole on growth and survival of intestinal lactic acid bacteria.

Some intestinal bacteria can produce many genotoxic, mutagenic and carcinogenic substances. The major products of the bacterial aromatic amino acids fermentation-phenolic and indolic compounds which are responsible for colon cancer development are accumulated in the colon. The effect of phenol, p-cresol and indole (2, 20 and 100 microg/ml doses) on growth and survival of four strains of intestinal lactic acid bacteria was studied. Growth of bacteria was not affected by any of the concentrations of phenol and p-cresol tested. The growth of 2 strains was slightly inhibited by 100 microg/ml of indole. There was no influence of phenol and p-cresol on survival of lactic bacteria until 120 h and specific reaction to carcinogens depending on strain was observed after that incubation time. Indole concentrations 20 and 100 microg/ml appeared to be toxic for all tested strains but just after 24, 48 or 72 h of incubation depending on the strain. In total, 2 microg/ml of indole had a very little effect.