Improvement in glucose tolerance and insulin sensitivity by probiotic strains of Indian gut origin in high-fat diet-fed C57BL/6J mice.

PURPOSE: Diabetes and obesity are characterized by glucose intolerance, fat deposition, inflammation, and dyslipidemia. Recent reports postulated that distinct gut microbiota alterations were observed in obese/diabetic subjects and modulating gut microbiota beneficially through specific probiotics could be a potential therapeutic option for type 2 diabetes/obesity. Therefore, we attempted to study the efficacy of probiotics of Indian gut origin (Lactobacillus plantarum MTCC5690 and Lactobacillus fermentum MTCC5689) along with a positive control, Lactobacillus rhamnosus (LGG) on glucose/lipid homeostasis in high-fat-diet-induced diabetic animal model. METHODS: C57BL/6J male mice were divided into seven groups (n = 6 per group) comprising feeding on: (1) Normal Pellet Diet (NPD), (2) High-Fat Diet (HFD), (3) HFD with LGG, (4) HFD with MTCC5690, (5) HFD with MTCC5689, (6) HFD with metformin, and 7) HFD with vildagliptin for a period of 6 months. Biochemical markers, glucose tolerance, insulin resistance, and GLP-1 and LPS levels were assessed by standard protocols. Gut integrity was measured by intestinal permeability test. Transcriptional levels of tight junction proteins (TJPs) were probed in small intestinal tissues while inflammatory signals and other pathway specific genes were profiled in liver, visceral adipose tissue, and skeletal muscle. RESULTS: Mice fed with HFD became insulin resistant, glucose intolerant, hyperglycemic, and dyslipidemic. Diabetic mice were characterized to exhibit decreased levels of GLP-1, increased gut permeability, increased circulatory levels of LPS, decrease in the gene expression patterns of intestinal tight junction markers (occludin and ZO-1), and increased proinflammatory gene markers (TNFα and IL6) in visceral fat along with decreased mRNA expression of FIAF and adiponectin. Diabetic mice also exhibited increased mRNA expression of ER stress markers in skeletal muscle. In addition, liver from HFD-fed diabetic mice showed increased gene expressions of proinflammation, lipogenesis, and gluconeogenesis. Probiotic interventions (most prominently the MTCC5689) resisted insulin resistance and development of diabetes in mice under HFD feeding and beneficially modulated all the biochemical and molecular alterations in a mechanistic way in several tissues. The metabolic benefits offered by the probiotics were also more or less similar to that of standard drugs such as metformin and vildagliptin. CONCLUSION: Native probiotic strains MTCC 5690 and MTCC 5689 appear to have potential against insulin resistance and type 2 diabetes with mechanistic, multiple tissue-specific mode of actions.

Adhesion of Lactobacilli and their anti-infectivity potential.

The probiotic potential of lactic acid bacteria primarily point toward colonizing ability of Lactobacilli as the most important attribute for endowing all the known beneficial effects in a host. Lactobacillus species exert health-promoting function in the gastrointestinal tract through various mechanisms such as pathogen exclusion, maintenance of microbial balance, immunomodulation, and other crucial functions. It has been seen that many surface layer proteins are involved in host adhesion, and play significant role in the modification of some signaling pathways within the host cells. Interaction between different bacterial cell surface proteins and host receptor has been imperative for a better understanding of the mechanism through which Lactobacilli exert their health-promoting functions.

Evaluation of casein & whey protein hydrolysates as well as milk fermentates from Lactobacillus helveticus for expression of gut hormones.

BACKGROUND & OBJECTIVES: Milk proteins play a beneficial role in the regulation of food intake, postprandial glycaemia and enteroendocrine hormone secretions and thus are receiving considerable attention for the management of metabolic inflammatory disorders such as type 2 diabetes mellitus (T2DM). The objective of this study was to evaluate the efficacy of peptide/s obtained from milk proteins (casein and whey) as well as from the milk fermented with Lactobacillus helveticus as secretagogues for gut hormones and to purify and characterize the active peptides. METHODS: Effect of hydrolysates of casein protein (CP) and whey protein (WP) and L. helveticus fermented milk on the expression of proglucagon, pro-gastric inhibitory peptide (GIP) and cholecystokinin (CCK) genes was monitored by real-time quantitative polymerase chain reaction. The active glucagon-like peptide-1 (GLP-1) secretion was also quantitatively measured using ELISA. RESULTS: Hydrolysates of CP and WP as well as fermentates of L. helveticus induced the proglucagon, pro-GIP and CCK expression and secretion of GLP-1 in STC-1 (pGIP/Neo) cells. However, intact casein exhibited maximum GLP-1 secretion and proglucagon expression. Two active peptides (F5 and F7) derived from CP1 and WP3 hydrolysates having the ability to upregulate the GLP-1 secretion by 1.6 and 1.8 folds were obtained, and the mass was found to be 786 and 824 Da, respectively, as determined by electrospray ionization-mass spectrometry. However, no single active peptide from L. helveticus fermented milk could be obtained. INTERPRETATION & CONCLUSIONS: Casein as well as fermentates obtained from L. helveticus fermented milk showed higher potential for GLP-1 induction. These can be explored as novel therapeutics to T2DM effectively after demonstrating their in vivo efficacy in appropriate animal models.

Synthetic Biology: Applications in the Food Sector.

Synthetic biology also termed as "genomic alchemy" represents a powerful area of science that is based on the convergence of biological sciences with systems engineering. It has been fittingly described as "moving from reading the genetic code to writing it" as it focuses on building, modeling, designing and fabricating novel biological systems using customized gene components that result in artificially created genetic circuitry. The scientifically compelling idea of the technological manipulation of life has been advocated since long time. Realization of this idea has gained momentum with development of high speed automation and the falling cost of gene sequencing and synthesis following the completion of the human genome project. Synthetic biology will certainly be instrumental in shaping the development of varying areas ranging from biomedicine, biopharmaceuticals, chemical production, food and dairy quality monitoring, packaging, and storage of food and dairy products, bioremediation and bioenergy production, etc. However, potential dangers of using synthetic life forms have to be acknowledged and adoption of policies by the scientific community to ensure safe practice while making important advancements in the ever expanding field of synthetic biology is to be fully supported and implemented.

Adhesion of indigenous Lactobacillus plantarum to gut extracellular matrix and its physicochemical characterization.

Adhesion to the human intestinal epithelial cell is considered as one of the important selection criteria of lactobacilli for probiotic attributes. Sixteen Lactobacillus plantarum strains from human origins were subjected for adhesion to extracellular matrix (ECM) components, and their physiochemical characterization, incubation time course and effect of different pH on bacterial adhesion in vitro were studied. Four strains showed significant binding to both fibronectin and mucin. After pretreatment with pepsin and trypsin, the bacterial adhesion to ECM reduced to the level of 50 % and with lysozyme significantly decreased by 65-70 %. Treatment with LiCl also strongly inhibited (90 %) the bacterial adhesion to ECM. Tested strains showed highest binding efficacy at time course of 120 and 180 min. Additionally, the binding of Lp91 to ECM was highest at pH 6 (155 ± 2.90 CFU/well). This study proved that surface layer components are proteinaceous in nature, which contributed in adhesion of lactobacillus strains. Further, the study can provide a better platform for introduction of new indigenous probiotic strains having strong adhesion potential for future use.

Probiotics as potential biotherapeutics in the management of type 2 diabetes - prospects and perspectives.

Diabetes mellitus is a looming epidemic worldwide, affecting almost all major sections of society, creating burdens on global health and economy. A large number of studies have identified a series of multiple risk factors such as genetic predisposition, epigenetic changes, unhealthy lifestyle, and altered gut microbiota that cause increased adiposity, ß-cell dysfunction, hyperglycemia, hypercholesterolemia, adiposity, dyslipidaemia, metabolic endotoxemia, systemic inflammation, intestinal permeability (leaky gut), defective secretion of incretins and oxidative stress associated with type 2 diabetes (T2D). Recent studies have proposed multifactorial interventions including dietary manipulation in the management of T2D. The same interventions have also been recommended by many national and international diabetes associations. These studies are aimed at deciphering the gut microbial influence on health and disease. Interestingly, results from several genomic, metagenomic and metabolomic studies have provided substantial information to target gut microbiota by dietary interventions for the management of T2D. Probiotics particularly lactobacilli and bifidobacteria have recently emerged as the prospective biotherapeutics with proven efficacy demonstrated in various in vitro and in vivo animal models adequately supported with their established multifunctional roles and mechanism of action for the prevention and disease treatment. The dietary interventions in conjunction with probiotics - a novel multifactorial strategy to abrogate progression and development of diabetes - hold considerable promise through improving the altered gut microbial composition and by targeting all the possible risk factors. This review will highlight the new developments in probiotic interventions and future prospects for exploring probiotic therapy in the prevention and control of lifestyle diseases like T2D.

Relative gene expression of bile salt hydrolase and surface proteins in two putative indigenous Lactobacillus plantarum strains under in vitro gut conditions.

Probiotic bacteria must overcome the toxicity of bile salts secreted in the gut and adhere to the epithelial cells to enable their better colonization with extended transit time. Expression of bile salt hydrolase and other proteins on the surface of probiotic bacteria can help in better survivability and optimal functionality in the gut. Two putative Lactobacillus plantarum isolates i.e., Lp9 and Lp91 along with standard strain CSCC5276 were used. A battery of six housekeeping genes viz. gapB, dnaG, gyrA, ldhD, rpoD and 16S rRNA were evaluated by using geNorm 3.4 excel based application for normalizing the expression of bile salt hydrolase (bsh), mucus-binding protein (mub), mucus adhesion promoting protein (mapA), and elongation factor thermo unstable (EF-Tu) in Lp9 and Lp91. The maximal level of relative bsh gene expression was recorded in Lp91 with 2.89 ± 0.14, 4.57 ± 0.37 and 6.38 ± 0.19 fold increase at 2% bile salt concentration after 1, 2 and 3 h, respectively. Similarly, mub and mapA genes were maximally expressed in Lp9 at the level of 20.07 ± 1.28 and 30.92 ± 1.51 fold, when MRS was supplemented with 0.05% mucin and 1% each of bile and pancreatin (pH 6.5). However, in case of EF-Tu, the maximal expression of 42.84 ± 5.64 fold was recorded in Lp91 in the presence of mucin alone (0.05%). Hence, the expression of bsh, mub, mapA and EF-Tu could be considered as prospective biomarkers for screening of novel probiotic lactobacillus strains for optimal functionality in the gut.

Anti-inflammatory and immunomodulatory efficacy of indigenous probiotic Lactobacillus plantarum Lp91 in colitis mouse model.

Probiotics can affect the immune homeostasis by altering the gut microbial balance and enhancing the immune system of gut, thus benefits in Inflammatory Bowel Disease, including Crohn's disease and Ulcerative colitis. Relative gene expression of pro, anti-inflammatory cytokines and other molecules in 2,4,6-trinitrobenzene sulfonic acid-induced colitis mouse model against Lactobacillus plantarum Lp91 (L. plantarum Lp91) was investigated by reverse transcription-quantitative PCR (RT-qPCR) using relative expression software tool (REST 2008 V2.0.7). L. plantarum Lp91 evoked significant down regulation of TNF-α and COX2 to 0.026 and 0.077 fold in colitis mouse model. No significant difference in expression of IL-12a cytokine in colitis mouse challenged with L. plantarum Lp91 was also observed. IL-10 was significantly up-regulated to 37.813 and 1.327 fold in colitis and non-colitis mouse challenged with L. plantarum Lp91. While, other anti-inflammatory markers i.e. COX1, IL-4 and IL-6 were significantly up regulated in colitis mouse challenged with L. plantarum Lp91. MUC2 gene was significantly up regulated to 2.216 fold in non-colitis group. L. plantarum Lp91, an indigenous probiotic culture, the main subject of this project exhibited strong immunemodulatory properties under in vivo conditions in mouse colitis model.

Antioxidative potential of lactobacilli isolated from the gut of Indian people.

Oxidative stress is one of the major causes of degenerative conditions occurring at cellular level with serious health implications. This study was aimed at investigating the antioxidative potentials of probiotic lactobacilli of Indian gut origin and their ability to augment antioxidant defense enzyme systems in the host cells under oxidative stress conditions. A total of 39 Lactobacillus cultures were assessed for their resistance against reactive oxygen species. Most of the cultures were moderately to strongly resistant towards 0.4 mM H(2)O(2). The Lactobacillus isolate CH4 was the most H(2)O(2) resistant culture with only 0.06 log cycle reduction. Majority of the cultures demonstrated high resistance towards hydroxyl ions and Lp21 was the most resistant with log count reduction of 0.20 fold only. Almost all the cultures were also quite resistant to superoxide anions. Lp21 also showed the highest superoxide dismutase content (0.8971 U). Amongst the 39 cultures, Lactobacillus spp. S3 showed the highest total antioxidative activity of 77.85 ± 0.13 % followed by Lp55 (56.1 ± 1.2 %) in terms of per cent inhibition of linolenic acid oxidation. Lp9 up-regulated the expression of superoxide dismutase 2 gene in HT-29 cells both at 0.1 mM (1.997 folds) and 1.0 mM H(2)O(2) (2.058 folds) concentrations. In case of glutathione peroxidase-1, Lp9, Lp91 and Lp55 showed significant (P < 0.001) up-regulation in the gene expression to the level of 5.451, 8.706 and 10.083 folds, respectively when HT-29 was challenged with 0.1 mM H(2)O(2). The expression of catalase gene was also significantly up-regulated by all the cultures at 0.1 mM H(2)O(2) conditions. It can be concluded that the antioxidative efficacy of the putative probiotic lactobacilli varied considerably between species and strains and the potential strains can be explored as prospective antioxidants to manage oxidative stress induced diseases.

Hypocholesterolaemic effect of dietary inclusion of two putative probiotic bile salt hydrolase-producing Lactobacillus plantarum strains in Sprague-Dawley rats.

The purpose of the present study was to evaluate the anti-hypercholesterolaemic effects of two putative probiotic bile salt hydrolase (Bsh)-producing Lactobacillus plantarum strains, i.e. Lp91 and Lp21, in rats. L. plantarum Lp91 exhibited excellent tolerance to low pH and high bile salt concentrations as well as showed potential Bsh activity, cholesterol assimilation and cholesterol co-precipitation ability along with L. plantarum Lp21 and NCDO82 strains. Furthermore, the potential effect of L. plantarum Lp91 on plasma cholesterol level was evaluated in Sprague-Dawley rats. Five treatment groups of rats (n 6) were fed experimental diets: normal diet, hypercholesterolaemic diet (HD), HD plus L. plantarum Lp91 (HD91) at ≥ 1·0 × 108 colony-forming units (cfu)/g, HD plus microencapsulated L. plantarum Lp91 (HDCap91) at ≥ 1·0 × 108 cfu/g and HD plus L. plantarum Lp21 (HD21) at ≥ 1·0 × 108 cfu/g for 3 weeks. Feed intake and feed efficiency differed significantly among the five groups. After 21 d of dietary treatment, comparative analysis revealed 23·26, 15·71 and 15·01 % reduction in total cholesterol, 21·09, 18·77 and 18·17 % reduction in TAG, 38·13, 23·22 and 21·42 % reduction in LDL-cholesterol, and the corresponding HDL-cholesterol values increased at the rate of 18·94, 10·30 and 7·78 % in treated groups HD91, HDCap91 and HD21, respectively. Faecal excretion of cholic acid and faecal lactobacilli counts were significantly higher in the probiotic treatment groups than in the control groups. In conclusion, these results suggest that the indigenous L. plantarum Lp91 strain has the potential to be explored as a probiotic in the management of hypercholesterolaemia.

Assessing the adhesion of putative indigenous probiotic lactobacilli to human colonic epithelial cells.

BACKGROUND & OBJECTIVES: Adherence of bacteria to epithelial cells and mucosal surfaces is a key criterion for selection of probiotic. We assessed the adhesion property of selected indigenous probiotic Lactobacillus strains based on their hydrophobicity and ability to adhere to human epithelial cells. METHODS: Five human faecal Lactobacillus isolates, one from buffalo milk and one from cheese were assessed for hydrophobicity following the microbial adhesion to hydrocarbons (MATH) method and colonization potentials based on their adherence to Caco2 and HT-29 colonic adenocarcinomal human intestinal epithelial cell lines. Lactobacillus strains that adhered to Caco2 and HT-29 cell lines were quantified by plating after trypsinization and simultaneously the adhered bacteria were also examined microscopically after staining with Geimsa stain and counted in different fields. RESULTS: Among the tested faecal isolates, L. plantarum Lp91 showed maximum percentage hydrophobicity (35.73±0.40 for n-hexadecane and 34.26±0.63 for toluene) closely followed by L. plantarum Lp9 (35.53±0.29 for n-hexadecane and 33.00±0.57 for toluene). Based on direct adhesion to epithelial cells, L. plantarum Lp91 was the most adhesive strain to HT-29 and Caco2 cell lines with per cent adhesion values of 12.8 ± 1.56 and 10.2 ± 1.09, respectively. L. delbrukeii CH4, was the least adhesive with corresponding figures of 2.5 ± 0.37 and 2.6 ± 0.20 per cent on HT-29 and Caco2 cell lines. Adhesion of the six isolated Lactobacillus strain to HT-29 cell and Caco2 lines as recorded under microscope varied between 131.0 ± 13.9 (Lp75) to 342.7 ± 50.52 (Lp91) and 44.7 ± 9.29 (CH4) to 315.7± 35.4 (Lp91), respectively. INTERPRETATION & CONCLUSIONS: Two Indigenous probiotic Lactobacillus strains (Lp9, Lp91) demonstrated their ability to adhere to epithelial cell and exhibited strong hydrophobicity under in vitro conditions, and thus could have better prospects to colonize the gut with extended transit.

Purification and characterization of milk clotting enzyme from goat (Capra hircus).

Chymosin, the major component of rennet (milk clotting enzyme), is an acid protease produced in the fourth stomach of milk-fed ruminants including goat and sheep in the form of an inactive precursor prochymosin. It is responsible for hydrolysis of kappa-casein chain in casein micelles of milk and therefore, used as milk coagulant in cheese preparation. The present investigation was undertaken to purify and characterize goat (Capra hircus) chymosin for its suitability as milk coagulant. The enzyme was extracted from abomasal tissue of kid and purified nearly 30-fold using anion exchanger and gel filtration chromatography. Goat chymosin resolved into three major active peaks, indicating possible heterogeneity when passed through DEAE-cellulose ion exchange column. The purified enzyme had a molecular mass of 36 kDa on SDS-PAGE, which was further confirmed by Western blot analysis. The purified enzyme preparation was stable up to 55 degrees C with maximum activity at 30 degrees C. The milk clotting activity was decreased steadily as pH is increased and indicated maximum activity at pH 5.5. Proteolytic activity of goat chymosin increased with incubation time at 37 degrees C. Goat chymosin was found to be more thermostable than cattle chymosin and equally stable to buffalo chymosin.

Modulation of intestinal barrier function to ameliorate Salmonella infection in mice by oral administration of fermented milks produced with Lactobacillus plantarum MTCC 5690 - a probiotic strain of Indian gut origin.

Probiotic Lactobacillus plantarum MTCC 5690, a probiotic strain of Indian gut origin, and milk formulations produced with the same were explored in this study as biotherapeutics by evaluating their functional efficacy against Salmonella infection in mice. The efficacy of milk formulations (fermented/unfermented) of MTCC 5690 for enhancement of intestinal barrier function was determined by monitoring the permeability and histopathology of the intestine. Infected mice fed with probiotic Dahi, fermented probiotic drink and sweetened fermented probiotic drink maintained the health and integrity of the intestinal epithelium as compared to those fed with PBS, milk, unfermented probiotic milk and Dahi. Our relative expression data revealed that the changes caused by MTCC 5690 in intestinal barrier function components were established through modulation of the key regulatory receptors Toll-like receptor 2 and Toll-like receptor 4. The results suggest that fermented milks of MTCC 5690 could enhance the defences of the intestinal barrier in enteric infection condition and, therefore, can be explored as a dietary-based strategy to reduce Salmonella infection in the human gut.

Live and heat-killed probiotic Lactobacillus casei Lbs2 protects from experimental colitis through Toll-like receptor 2-dependent induction of T-regulatory response.

Inflammatory bowel disease (IBD) is a group of inflammatory disorders of the intestine caused by dysregulated T-cell mediated immune response against commensal microflora. Probiotics are reported as therapeutically effective against IBD. However, variable efficacy of the live probiotic strains, difference in survival and persistence in the gut between the strains and the lack of insight into the mechanisms of probiotic action limit optimal therapeutic efficacy. Our aims were to evaluate the lactobacillus strains isolated from the North Indian population for the generation of regulatory cells and cytokines in the intestine, to study their effects on pro-inflammatory mediators in the mouse model of inflammatory bowel disease and to explore the underlying mechanisms of their actions. Among the selected lactobacillus strains, Lactobacillus casei Lbs2 (MTCC5953) significantly suppressed lipopolysaccharide-induced pro-inflammatory cytokine (TNF-alpha, IL-6) secretion. Both live and heat-killed Lbs2 polarized Th0 cells to T-regulatory (Treg) cells in vitro, increased the frequency of FoxP3(+) Treg cells in the mesenteric lymph nodes (MLNs) and alleviated macroscopic and histopathological features of colitis in probiotic-fed mice. Moreover, the levels of IL-12, TNF-alpha and IL-17A were suppressed, while IL-10 and TGF-beta levels were augmented in the colonic tissues of Lbs2-treated mice. The induced Treg (iTreg) cells secreted IL-10 and TGF-beta and exerted suppressive effects on the proliferation of effector T-cells. Adoptive transfer of iTreg cells ameliorated the disease manifestations of murine colitis and suppressed the levels of TNF-alpha and IL-17A. Finally, Lbs2 effects were mediated by Toll-like receptor 2 (TLR2) activation on the dendritic cells. This study identified live and heat-killed Lbs2 as putative therapeutic candidates against IBD and highlighted their Toll-like receptor 2-dependent immunomodulatory and regulatory function.

Effect of environmental factors on production of lichenin, a chromosomally encoded bacteriocin-like compound produced by Bacillus licheniformis 26L-10/3RA.

Effect of environmental factors on production of lichenin, a bacteriocin-like compound produced by Bacillus licheniformis 26L-10/3RA isolated from buffalo rumen was studied. Lichenin represents the first anaerobiosis-specific expression of broad-spectrum antibacterial compound effective only under anaerobic conditions. Production of lichenin by B. licheniformis 26L-10/3RA was found to be very high at 39 degrees C in L-10 medium supplemented with 0.5% glucose and 20% (w/v) inert thermocol beads. Lichenin production was highest at pH 6.8 after 72-96h of incubation. Our study also indicated that Lichenin is not a plasmid-linked characteristic and is encoded by chromosomal DNA. Results obtained can be used in large-scale production of Lichenin for potential application in manipulating rumen function intended for improving productivity of the ruminants.

Exploring specific primers targeted against different genes for a multiplex PCR for detection of Listeria monocytogenes.

The efficacy of six different sets of primers targeted against 16S rRNA and virulence genes such as 'iap', 'hly' and 'prf' was evaluated in separate PCR assays. The primer pairs targeted against 16S rRNA resulted into amplification of 1.2 kb PCR product. However, sets of primers targeted against different regions of 'iap' produced 371 and 660 bp PCR products, respectively. The primer pair targeted against 'prf' gene could produce 508 bp product. Three primer pairs targeted against different regions of 'hly', i.e., 'hly', 'hly A' and 'hly K9' were able to amplify 713, 276 and 384 bp products, respectively. The PCR conditions were also optimized in respect of two internal sets of primers falling within 'iap' and 'hly' genes that amplified 119 and 188 bp products to verify the PCR results obtained with respective external sets of primers. Three different combinations involving four sets of primers based on 16S rRNA, 'iap', 'hly' and 'prf' were explored in respective multiplex PCR assays in order to select a suitable combination. Combination 1 and 3 worked successfully as revealed by amplification of all the four bands of expected sizes on agarose gel. However, while optimizing the different parameters for developing a functional multiplex PCR, it was observed that in both these combinations, only two of the amplified products, i.e., 1.2 kb and 713 bp could be invariably detected. Hence, these two primers were combined in the multiplex PCR and the conditions were optimized for application in dairy foods for detection of Listeria monocytogenes.

Immunomodulatory activity of two potential probiotic strains in LPS-stimulated HT-29 cells.

The relative expression of mucin, pro- and anti-inflammatory genes besides other signaling molecules in HT-29 cells by two test probiotic strains of Lactobacillus plantarum Lp9 and Lp91 and the reference strain L. plantarum 5276 was evaluated by RT-qPCR using Relative Expression Software Tool qBase-Plus under in vitro simulated gut conditions. Ten house keeping genes were evaluated by using geNorm 3.4 excel based application. The most stable genes were RPL27, ACTB and B2M which were subsequently used for calculating the normalization factor. Under pretreatment conditions (4 h probiotic treatment, followed by lipopolysaccharide challenge for 3 h), all the three strains evoked downregulation of IL-8 expression by ~100 %, while in case of TNF-α, the downregulation of the relative gene expression was at the rate of 98.2, 93.8 and 98.0 % with Lp5276, Lp9 and Lp91, respectively, under the same set of conditions. Lp91 evoked maximum downregulation of IL12p35 and IFN-γ with corresponding fold reduction in relative expression of the two genes by 96.5 and 96.7 % during pre-treatment conditions. However, IL-10 and IFN-α were significantly upregulated to the extent of 8.13 ± 0.36 and 2.62 ± 0.14 fold by Lp91 under the same conditions. Lp9 and Lp91 were also quite effective in inducing the expression of Cox-1 and Cox-2 in HT-29 cells as can be reflected from their ratios, i.e., 5.90 and 6.50 (under pretreatment conditions); 3.79 and 4.36 (under co-culture conditions). Thus, the two putative indigenous L. plantarum strains Lp9 and Lp91 demonstrated immunomodulating functions in HT-29 cells at significant levels under different experimental conditions.

IS30-related transposon mediated insertional inactivation of bile salt hydrolase (bsh1) gene of Lactobacillus plantarum strain Lp20.

Lactobacillus plantarum is a flexible and versatile microorganism that inhabits a variety of niches, and its genome may express up to four bsh genes to maximize its survival in the mammalian gut. However, the ecological significance of multiple bsh genes in L. plantarum is still not clearly understood. Hence, this study demonstrated the disruption of bile salt hydrolase (bsh1) gene due to the insertion of a transposable element in L. plantarum Lp20 - a wild strain of human fecal origin. Surprisingly, L. plantarum strain Lp20 produced a ∼2.0 kb bsh1 amplicon against the normal size (∼1.0 kb) bsh1 amplicon of Bsh(+)L. plantarum Lp21. Strain Lp20 exhibited minimal Bsh activity in spite of having intact bsh2, bsh3 and bsh4 genes in its genome and hence had a Bsh(-) phenotype. Cloning and sequence characterization of Lp20 bsh1 gene predicted four individual open reading frames (ORFs) within this region. BLAST analysis of ORF1 and ORF2 revealed significant sequence similarity to the L. plantarum bsh1 gene while ORF3 and ORF4 showed high sequence homology to IS30-family transposases. Since, IS30-related transposon element was inserted within Lp20 bsh1 gene in reverse orientation (3'-5'), it introduced several stop codons and disrupted the protein reading frames of both Bsh1 and transposase. Inverted terminal repeats (GGCAGATTG) of transposon, mediated its insertion at 255-263 nt and 1301-1309 nt positions of Lp20 bsh1 gene. In conclusion, insertion of IS30 related-transposon within the bsh1 gene sequence of L. plantarum strain Lp20 demolished the integrity and functionality of Bsh1 enzyme. Additionally, this transposon DNA sequence remains active among various Lactobacillus spp. and hence harbors the potential to be explored in the development of efficient insertion mutagenesis system.

Amelioration of colitis in mouse model by exploring antioxidative potentials of an indigenous probiotic strain of Lactobacillus fermentum Lf1.

Based on the preliminary screening of eight indigenous putative probiotic Lactobacilli, Lactobacillus fermentum Lf1 was selected for assessing its antioxidative efficacy in DSS colitis mouse model based on its ability to enhance the expression of "Nrf2" by 6.43-fold and malondialdehyde (MDA) inhibition by 78.1 ± 0.24% in HT-29 cells under H2O2 stress. The Disease Activity Index and histological scores of Lf1-treated mice were lower than the control group. However, expression of "Nrf2" was not observed in Lf1-treated mice. A significant increase in the expression of antioxidative enzymes such as SOD2 and TrxR-1 was recorded in both of the groups. The expression of SOD2 was significantly downregulated in colitis-induced mice by -100.00-fold relative to control group, and the downregulation was considerably reduced to -37.04-fold in colitis Lf1 treatment group. Almost, a similar trend was recorded in case of "thioredoxin" expression, though "CAT" was refractile to expression. The Lf1-treated group had decreased malondialdehyde level as compared to colitis control (37.92 ± 6.31 versus 91.13 ± 5.76 µM/g). These results point towards Lf1-induced activation of the antioxidant enzyme system in the mouse model and its prospects to be explored as a new strategy for IBD management.

Relative expression of bacterial and host specific genes associated with probiotic survival and viability in the mice gut fed with Lactobacillus plantarum Lp91.

The present investigation was aimed at studying the relative expression of atpD (a key part of F1F0-ATPase operon), bsh (bile salt hydrolase), mub (mucus-binding protein) and MUC2 (mucin) genes in mouse model for establishing the in vivo functional efficacy of Lactobacillus plantarum Lp91 (MTCC5690) by reverse transcription-quantitative PCR (RT-qPCR). The atpD gene was significantly up-regulated to 2.0, 2.4 and 3.2 folds in Lp91 after 15, 30 and 60 min transit in the stomach of mice. The maximal significant (P<0.00) level of relative bsh gene expression was recorded in Lp91 with 41.6 fold in comparison to only 5.0 fold in reference strain Lp5276 after seven days of mice feeding. Simultaneously, mub gene expression increased to 12.8 and 22.7 fold in both Lp91 and Lp5276, respectively. The expression level of MUC2 was at the level of 1.6 and 2.1 fold in the host colon on administration with Lp91 and Lp5276 feeding, respectively. Hence, the expression of atpD, bsh, mub, MUC2 could be considered as prospective and potential biomarkers for screening of novel probiotic lactobacillus strains for optimal functionality in the gut.