Survival and synergistic growth of mixed cultures of bifidobacteria and lactobacilli combined with prebiotic oligosaccharides in a gastrointestinal tract simulator

Background: Probiotics, especially in combination with non-digestible oligosaccharides, may balance the gut microflora while multistrain preparations may express an improved functionality over single strain cultures. In vitro gastrointestinal models enable to test survival and growth dynamics of mixed strain probiotics in a controlled, replicable manner. Methods: The robustness and compatibility of multistrain probiotics composed of bifidobacteria and lactobacilli combined with mixed prebiotics (galacto-, fructo- and xylo-oligosaccharides or galactooligosaccharides and soluble starch) were studied using a dynamic gastrointestinal tract simulator (GITS). The exposure to acid and bile of the upper gastrointestinal tract was followed by dilution with a continuous decrease of the dilution rate (de-celerostat) to simulate the descending nutrient availability of the large intestine. The bacterial numbers and metabolic products were analyzed and the growth parameters determined. Results: The most acid- and bile-resistant strains were Lactobacillus plantarum F44 and L. paracasei F8. Bifidobacterium breve 46 had the highest specific growth rate and, although sensitive to bile exposure, recovered during the dilution phase in most experiments. B. breve 46, L. plantarum F44, and L. paracasei F8 were selected as the most promising strains for further studies. Conclusions: De-celerostat cultivation can be applied to study the mixed bacterial cultures under defined conditions of decreasing nutrient availability to select a compatible set of strains

Prebiotic-non-digestible oligosaccharides preference of probiotic bifidobacteria and antimicrobial activity against Clostridium difficile.

Bifidobacterium breve 46, Bifidobacteriumlactis 8:8 and Bifidobacteriumlongum 6:18 and three reference strains B. breve CCUG 24611, B. lactis JCM 10602, and Bifidobacteriumpseudocatenulatum JCM 1200 were examined for acid and bile tolerance, prebiotic utilization and antimicrobial activity against four Clostridium difficile (CD) strains including the hypervirulent strain, PCR ribotype NAP1/027. B. lactis 8:8 and B. lactis JCM 10602 exhibited a high tolerance in MRSC broth with pH 2.5 for 30 min. B. breve 46 and B. lactis 8:8 remained 100% viable in MRSC broth with 5% porcine bile after 4 h. All six strains showed a high prebiotic degrading ability (prebiotic score) with galactooligosaccharides (GOS), isomaltooligosaccharides (IMOS) and lactulose as carbon sources and moderate degradation of fructooligosaccharides (FOS). Xylooligosaccharides (XOS) was metabolized to a greater extent by B. lactis 8:8, B. lactis JCM 10602, B. pseudocatenulatum JCM 1200 and B. longum 6:18 (prebiotic score >50%). All strains exhibited extracellular antimicrobial activity (AMA) against four CD strains including the CD NAP1/027. AMA of B. breve 46, B. lactis 8:8 and B. lactis JCM 10602 strains was mainly ascribed to a combined action of organic acids and heat stable, protease sensitive antimicrobial peptides when cells were grown in MRSC broth with glucose and by acids when grown with five different prebiotic-non-digestible oligosaccharides (NDOs). None of C. difficile strains degraded five prebiotic-NDOs. Whole cells of B. breve 46 and B. lactis 8:8 and their supernatants inhibited the growth and toxin production of the CD NAP1/027 strain

Bile Enhances Cell Surface Hydrophobicity and Biofilm Formation of Bifidobacteria.

Twenty-four human bifidobacterial strains were analysed for cell surface hydrophobicity (CSH) using a salt aggregation test (SAT) and a Congo red binding (CRB) assay. Three strains were selected for a systematic study on the CSH and biofilm formation: Bifidobacterium breve 46, Bifidobacterium animalis ssp. lactis 8:8 and a reference strain B. animalis ssp. lactis JCM 10602. CRB of the B. breve 46 and B. animalis ssp. lactis JCM 10602 was significantly enhanced (P < 0.05) when grown in deMan-Rogosa-Sharpe cysteine (MRSC) broth supplemented with taurocholic acid (TA) or native porcine bile (PB). An enhanced CSH of the strains grown with PB and gastric mucin correlated with an increased mucin binding and an enhanced biofilm formation in prebiotic oligosaccharide-supplemented cultures. The three strains showed late bile-induced biofilm (72 h) under an anaerobic growth condition, and both B. animalis ssp. lactis strains showed a late bile-induced biofilm formation under aerobic conditions shown by crystal violet staining. These two strains were thus considered to be oxygen tolerant and more robust. Furthermore, enhanced biofilm formation of these robust bifidobacterial strains in the presence of prebiotics may allow for strong colonisation in the gastrointestinal tract when administered to in vivo models as a "synbiotic supplement"

Bile stimulates Cell Surface Hydrophobicity, Congo Red Binding and Biofilm Formation of Lactobacillus strains.

Seventeen Lactobacillus strains were tested for cell surface hydrophobicity (CSH) using the salt aggregation test (SAT) and Congo red binding (CRB) assay. CRB was pH and ionic strength dependent and protease sensitive and in the presence of 100 μg/ml cholesterol, the CRB was significantly reduced. Autoaggregating (AA) L. crispatus strains showed 50% more CRB than the reference strain, the curli-producing E.coli MC4100. CRB of L. crispatus 12005, L. paracasei F8, L. plantarum F44 and L. paracasei F19 was enhanced when grown in MRS broth with 0.5% taurocholic acid (TA) or 5% porcine bile (PB) (P<0.05). CSH was also enhanced for the non-AA strains, L. plantarum F44, L. paracasei F19 and L. rhamnosus GG when grown in MRS broth with 0.5% TA, 5% PB or 0.25% mucin with enhanced biofilm formation in MRS broth with bile (P<0.05). Two AA strains, L. crispatus 12005 and L. paracasei F8, developed biofilm independent of bile or mucin. In summary, under bile stressed growth conditions, early (24 h cultures) biofilm formation is associated with an increase in hydrophobic cell surface proteins and high CRB. Late mature (72 h culture) biofilm contained more carbohydrates as shown by crystal violet staining. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.All rights reserved

In vitro Mutagen binding and antimutagenic activity of human Lactobacillus rhamnosus 231

In vitro mutagen binding ability of human Lactobacillus rhamnosus 231 (Lr 231) was evaluated against acridine orange (AO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 2-amino-3, 8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx) and 4-nitro-o-phenylenediamine (NPD). Binding of AO by Lr 231 is due to adsorption, thereby leading to removal of mutagen in solution and is instantaneous, pH- and concentration-dependent. Whereas, binding of MNNG and MeIQx by Lr 231 results into biotransformation leading to detoxification with subsequent loss of mutagenicity as determined by spectral analysis, thin layer chromatography and Ames test. Binding of mutagen by Lr 231 was dependent on culture age and optimum binding of AO, MNNG and MeIQx was observed to occur with 24 h old culture. Cells of Lr 231 were subjected to different chemical treatments prior to binding studies. Results indicated cell wall component such as cell wall polysaccharide, peptidoglycan, carbohydrates and proteins plays an important role in adsorption of AO, also involving hydrophilic and ionic interactions. Binding, biotransformation and detoxification of MNNG and MeIQx by Lr 231 was dependent on cell surface characteristics mainly involving carbohydrates, proteins, teichoic acid/lipoteichoic acid, hydrophobic interaction and presence of thiol group. L rhamnosus 231 bound MNNG instantaneously. More than 96 (p < 0.01) and 70% (p < 0.05) cells remained viable after mutagen binding and various pretreatments respectively. This study shows Lr 231 exhibits ability to bind and detoxify potent mutagens, and this property can be useful in formulating fermented foods for removal of potent mutagens. (C) 2011 Elsevier Ltd. All rights reserved

Determination of an antimicrobial activity of Weissella confusa, Lactobacillus fermentum, and Lactobacillus plantarum against clinical pathogenic strains of Escherichia coli and Staphylococcus aureus in co-culture

Lactic acid bacteria (LAB) have long been used to produce safe and high quality products as they are potential producers of a wide range of antimicrobial compounds that exert either narrow or wide spectrum antimicrobial activity towards spoilage or disease-causing organisms. The present investigation aimed to study the antimicrobial effect of three LAB strains, viz., Lactobacillus plantarum (86), Lactobacillus fermentum (AI2) and Weissella confusa (AI10), against two clinical pathogenic strains viz., Escherichia coli NG 502121 and Staphylococcus aureus AY 507047 in co-culture. Effects of change in inoculum size, and growth measurement at different time intervals were also studied. The pH and viable count were measured for initial as well as 24 h incubated samples. A significant (P < 0.05) reduction (2–3 log cycles) in growth of both pathogens while co-cultured with LAB strains was observed. The nonsignificant (P < 0.05) pH difference revealed the action of other metabolites apart from organic acids. LAB strains overruled the growth of E. coli and S. aureus within 10 and 6 h of the initial growth stage, respectively, compared to controls. These results led us to further characterize and purify the antimicrobial compound produced by the studied strains, so that they can be exploited in the production of safe foods with longer shelf life

Prebiotic preferences of human lactobacilli strains in co-culture with bifidobacteria and antimicrobial activity against Clostridium difficile.

To evaluate robustness, prebiotic utilisation of Lactobacillus paracasei F8 and Lact. plantarum F44 in mono- and co-cultures with Bifidobacterium breve 46 and B. animalis sub sp. lactis 8:8 and antimicrobial activity of co-culture against Clostridium difficile

A novel multi-strain probiotic and synbiotic supplement for prevention of Clostridium difficile infection in a murine model.

The protective effect of a multi-strain probiotic and synbiotic formulation was evaluated in C57BL/6 mice infected with Clostridium difficile (CD) NAP1/027. Antibiotic treated mice were divided into four groups. Group 1, fed with a synbiotic formulation consisting of Lactobacillus plantarum F44, L. paracasei F8, Bifidobacterium breve 46, B. lactis 8:8, galacto-oligosaccharides (GOS), isomalto-oligosaccharides (IMOS) and resistant starch (RS); group 2, fed with the same four probiotic strains as in group 1; group 3, fed with the same prebiotic supplements as mentioned in group 1 for seven days before CD infection and group 4, the control group, was antibiotic treated and infected with NAP1/027 strain. Faeces and caecal contents were collected for microbial cell viability, quantitative PCR (qPCR), toxin analyses and histopathology. Synbiotics and probiotics fed mice showed a significant increase of total bifidobacteria (P < 0.05). Total lactobacilli count was increased in group 1. The caecal toxins were negative in group 2 mice, and one sample each from group 1 and 3 was positive. qPCR of caecal content showed significant reduction in NAP1/027 DNA copies in group 1-2 and significantly higher numbers of B. breve 46, L. plantarum F44 and L. paracasei F8 in group 1 and 2 (P < 0.05) but much less pronounced in group 3-4. This study demonstrated that the newly developed synbiotic or multi-strain probiotic formulation conferred protection against NAP1/027 infection in C57BL/6 mice. This holds promising to conduct future human studies