Raj Kumar Duary

Functional and Probiotic Attributes of an Indigenous Isolate of Lactobacillus plantarum

Background Probiotic microorganisms favorably alter the intestinal microflora balance, promote intestinal integrity and mobility, inhibit the growth of harmful bacteria and increase resistance to infection. Probiotics are increasingly used in nutraceuticals, functional foods or in microbial interference treatment. However, the effectiveness of probiotic organism is considered to be population-specific due to variation in gut microflora, food habits and specific host-microbial interactions. Most of the probiotic strains available in the market are of western or European origin, and a strong need for exploring new indigenous probiotic organisms is felt. Methods and Findings An indigenous isolate Lp9 identified as Lactobacillus plantarum by molecular-typing methods was studied extensively for its functional and probiotic attributes, viz., acid and bile salt tolerance, cell surface hydrophobicity, autoaggregation and Caco-2 cell-binding as well as antibacterial and antioxidative activities. Lp9 isolate could survive 2 h incubation at pH 1.5–2.0 and toxicity of 1.5–2.0% oxgall bile. Lp9 could deconjugate major bile salts like glycocholate and deoxytaurocholate, indicating its potential to cause hypocholesterolemia. The isolate exhibited cell-surface hydrophobicity of ∼37% and autoaggregation of ∼31%. Presence of putative probiotic marker genes like mucus-binding protein (mub), fibronectin-binding protein (fbp) and bile salt hydrolase (bsh) were confirmed by PCR. Presence of these genes suggested the possibility of specific interaction and colonization potential of Lp9 isolate in the gut, which was also suggested by a good adhesion ratio of 7.4±1.3% with Caco-2 cell line. The isolate demonstrated higher free radical scavenging activity than standard probiotics L. johnsonii LA1 and L. acidophilus LA7. Lp9 also exhibited antibacterial activity against E. coli, L. monocytogenes, S. typhi, S. aureus and B. cereus. Conclusion The indigenous Lactobacillus plantarum Lp9 exhibited high resistance against low pH and bile and possessed antibacterial, antioxidative and cholesterol lowering properties with a potential for exploitation in the development of indigenous functional food or nutraceuticals.

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

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.

Management of metabolic syndrome through probiotic and prebiotic interventions

Metabolic syndrome is a complex disorder caused by a cluster of interrelated factors that increases the risk of cardiovascular diseases and type 2 diabetes. Obesity is the main precursor for metabolic syndrome that can be targeted in developing various therapies. With this view, several physical, psychological, pharmaceutical and dietary therapies have been proposed for the management of obesity. However, dietary strategies found more appropriate without any adverse health effects. Application of probiotics and prebiotics as biotherapeutics is the new emerging area in developing dietary strategies and many people are interested in learning the facts behind these health claims. Recent studies established the role of probiotics and prebiotics in weight management with possible mechanisms of improved microbial balance, decreased food intake, decreased abdominal adiposity and increased mucosal integrity with decreased inflammatory tone. Hence, the above “Pharmaco-nutritional” approach has been selected and extensively reviewed to gain thorough knowledge on putative mechanisms of probiotic and prebiotic action in order to develop dietary strategies for the management of metabolic syndrome.

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.

Effect of repeated cycled crystallization on digestibility and molecular structure of glutinous Bora rice starch

The effects of repeated cycled crystallization on the digestibility and molecular structure of glutinous Bora rice starch were investigated. Temperature cycle 4/45°C; cycle duration 5d; time interval of cycles 24h; and starch to water ratio 1:2 were found to be optimum for SDS (slow digestible starch) product development. The SDS content increased from 18.01±2.11% to 82.81±2.34%. An increase in the resistance to digestion, crystallinity, molecular weight, polydispersity and molecular order was observed in the optimal SDS product. Notably, the FT-IR peak at 947cm-1 and XRD peaks at 2θ≈13° and 20° in the optimal SDS product indicated the formation of V-type complexes even without the presence of co-polymers. Birefringence studies showed a loss of typical Maltese cross in the SDS product and revealed a reorientation of crystalline structures within starch granules, suggestive of imperfect crystallite development.

In silico approaches and proportional odds model towards identifying selective ADAM17 inhibitors from anti-inflammatory natural molecules

ADAM metallopeptidase domain 17 (ADAM17) is an attractive target for the development of new anti-inflammatory drugs. We aimed to identify selective inhibitors of ADAM17 against matrix metalloproteinase enzymes (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13, and MMP-16) which have substantial structural similarity. Target proteins were docked with 29 anti-inflammatory natural molecule ligands and a known selective inhibitor IK682. The ligands were screened based on Lipinski rules, interaction with the ADAM17 active site cavity, and then ranked using the proportional odds model multinomial logistic regression. Silymarin was the most selective inhibitor of ADAM17 exhibiting H-bonding with Glu 406, Gly 349, Glu 398, Asn 447, Tyr 433, and Lys 432. Molecular dynamics simulations were carried out for 10ns. The root mean square deviation (RMSD), root mean squared fluctuations (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), and H-bonding indicated the induced metastability. A comparison of the principal component analysis revealed that the silymarin complex also explored lesser region compared to IK682 complex. A control study on ADAM17 protein (2OI0) is included. These observations present silymarin (widely present in plants such as milk thistle (Silybum maianum), wild artichokes (Cynara cardunculus), turmeric (Curcuma longa) roots, coriander (Coriandrum sativum) seeds, etc.) as a promising natural template for development of ADAM17 selective drugs.

Characterizing and evaluating putative indigenous probiotic lactobacilli strain from human feces against inflammatory disorders

M syndrome is a growing epidemic, not only in western cultures but in developing nations as well. The economic toll of the various components that make up metabolic syndrome is staggering. The role of the microbiota is being demonstrated in many applications that can result in phenotype transfer, and improvement in physiological function. This talk will discuss metabolic syndrome, and the latest developments in the roles of active ingredients, specifically Probiotics, on metabolic syndrome and specifically the impact on Type 2 diabetes. Gregory Leyer, J Prob Health 2013, 1:4 http://dx.doi.org/10.4172/2329-8901.S1.013S baicalensis Georgi has been known to have various biological activities such as strong antioxidant activities and whitening effects, etc.; however, its applications have not been much increased due to the existence of small amounts of active compounds of Baicalein, and Baicalin. Most common extraction processes have been operated at 100 oC or 80 OC even though high operation temperature could generate large amounts of carbon dioxide into air as well as possibly degrade biological activities. In this work, high temperature lactic acid bacteria, Bacillus coagulans were cultivated to simultaneously ferment and extract S. baicalensis Georgi. The bacteria were grown with 15% (w/v) of powdered S. baicalensis Georgi at 6 0C for 10 days until pH of the medium was down to 4.5. After that, the culture broth contained 176 ppm of baicalein without further extracting the broth, which was higher than those from hot water extraction such as 17.6 ppm. It was also found that the broth showed very high antioxidant activities such as 23.7% of DPPH scavenging activity and the secretion of TNF-α, IFN-γ and IL-1β from human T cells was measured as 0.76 ng/ml, 0.17 ng/ml and 2.35 ng/ml, respectively. It proves that simultaneous fermentation and extraction can greatly improve the elution yield of active components due to easily breaking down the hard outer shells of the plants and secondary bioconversion by lactic acid at relatively high environmental temperature. This simple system can be applied to most fermentation processes with high efficiency as well as low carbon dioxide emission that is most serious concern in cosmetic and food industries. Hyeon-Yong Lee, J Prob Health 2013, 1:4 http://dx.doi.org/10.4172/2329-8901.S1.013