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 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

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"

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

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

Microarray based gene expression analysis of murine brown and subcutaneous adipose tissue: significance with human.

Two types of adipose tissues, white (WAT) and brown (BAT) are found in mammals. Increasingly novel strategies are being proposed for the treatment of obesity and its associated complications by altering amount and/or activity of BAT using mouse models.The present study was designed to: (a) investigate the differential expression of genes in LACA mice subcutaneous WAT (sWAT) and BAT using mouse DNA microarray, (b) to compare mouse differential gene expression with previously published human data; to understand any inter- species differences between the two and (c) to make a comparative assessment with C57BL/6 mouse strain. In mouse microarray studies, over 7003, 1176 and 401 probe sets showed more than two-fold, five-fold and ten-fold change respectively in differential expression between murine BAT and WAT. Microarray data was validated using quantitative RT-PCR of key genes showing high expression in BAT (Fabp3, Ucp1, Slc27a1) and sWAT (Ms4a1, H2-Ob, Bank1) or showing relatively low expression in BAT (Pgk1, Cox6b1) and sWAT (Slc20a1, Cd74). Multi-omic pathway analysis was employed to understand possible links between the organisms. When murine two fold data was compared with published human BAT and sWAT data, 90 genes showed parallel differential expression in both mouse and human. Out of these 90 genes, 46 showed same pattern of differential expression whereas the pattern was opposite for the remaining 44 genes. Based on our microarray results and its comparison with human data, we were able to identify genes (targets) (a) which can be studied in mouse model systems to extrapolate results to human (b) where caution should be exercised before extrapolation of murine data to human.Our study provides evidence for inter species (mouse vs human) differences in differential gene expression between sWAT and BAT. Critical understanding of this data may help in development of novel ways to engineer one form of adipose tissue to another using murine model with focus on human

Bioavailable Menthol (Transient Receptor Potential Melastatin-8 Agonist) Induces Energy Expending Phenotype in Differentiating Adipocytes

Recent evidence supports the role of menthol, a TRPM8 agonist, in enhanced energy expenditure, thermogenesis and BAT-like activity in classical WAT depots in a TRPM8 dependent and independent manner. The present study was designed to analyse whether oral and topical administration of menthol is bioavailable at subcutaneous adipose tissue and is sufficient to directlyinduce desired energy expenditure effects. GC-FID was performed to study menthol bioavailability in serum and subcutaneous white adipose tissue following oral and topical administration. Further, 3T3L1 adipocytes were treated with bioavailable menthol doses and different parameters (lipid accumulation, &#8220;browning/brite&#8221; and energy expenditure gene expression, metal analysis, mitochondrial complex&#8217;s gene expression) were studied. No difference was observed in serum levels but significant difference was seen in the menthol concentration on subcutaneous adipose tissues after oral and topical application. Menthol administration at bioavailable doses significantly increased &#8220;browning/brite&#8221; and energy expenditure phenotype, enhanced mitochondrial activity related gene expression, increased metal concentration during adipogenesis but did not alter the lipid accumulation as well as acute experiments were performed with lower dose of menthol on mature adipocytes In conclusion, the present study provides evidence that bioavailable menthol after single oral and topical administration is sufficient to induce &#8220;brite&#8221; phenotype in subcutaneous adipose tissue However, critical dose characterization for its clinical utility is required

Intrarectal Capsazepine Administration Modulates Colonic Mucosal Health in Mice

Antagonism of transient receptor potential vanniloid-1 (TRPV1) and desensitization of transient receptor potential ankyrin-1 (TRPA1) nociceptors alleviate inflammatory bowel diseases (IBD)-associated chronic pain. However, there is limited literature available about their role in regulating the mucosal layer, its interaction with host physiology, and luminal microbial community. The present study focuses on the effects&rsquo; intra rectal administration of capsazepine (modulator of TRPA1/TRPV1 expressing peptidergic sensory neurons) on colonic mucus production and gut health. We performed histological analysis, gut permeability alteration, gene expression changes, metabolite profiling, and gut microbial abundance in the ileum, colon, and cecum content of these animals. Intra rectal administration of capsazepine modulates TRPA1/TRPV1-positive nociceptors (behavioral pain assays) and resulted in damaged mucosal lining, increased gut permeability, and altered transcriptional profile of genes for goblet cell markers, mucus regulation, immune response, and tight junction proteins. The damage to mucosal lining prevented its role in enterosyne (short chain fatty acids) actions. These results suggest that caution must be exercised before employing TRPA1/TRPV1 modulation as a therapeutic option to alleviate pain caused due to IBD

Common genes expressed in both species while showing opposite trend of expression.

<p>Common genes that show opposite expression in human and mouse along with absolute fold change in both organisms.</p><p>Common genes expressed in both species while showing opposite trend of expression.</p