Probiotic potential of ß­galactosidase­producing lactic acid bacteria from fermented milk and their molecular characterization.

Probiotics have attained significant interest in recent years as a result of their gut microbiome modulation and gastrointestinal health benefits. Numerous fermented foods contain lactic acid bacteria (LAB) which are considered as GRAS and probiotic bacteria. The present study aimed to investigate indigenous LAB from homemade fermented milk samples collected in remote areas of Karnataka (India), in order to isolate the most potent and well-adapted to local environmental conditions bacteria, which were then evaluated using a step-by-step approach focused on the evaluation of probiotic traits and ß-galactosidase-producing ability. LAB were screened using 5-bromo-4-chloro-3-indole-D-galactopyranoside (X-Gal) and O-nitrophenyl-ß-D-galactopyranoside (ONPG) as substrate, and exhibited ß-galactosidase activity ranging from 728.25 to 1,203.32 Miller units. The most promising isolates were selected for 16S rRNA gene sequence analysis and identified as Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Lactiplantibacillus pentosus and Lactiplantibacillus sp. Furthermore, these isolates were evaluated by in vitro, viz., survival in gastrointestinal tract, antibiotic susceptibility, antimicrobial activity, cell surface characteristics, and haemolytic activity. All eight isolates demonstrated strong adherence and prevented pathogen penetration into HT-29 cells, indicating potential of the bacteria to scale up industrial level production of milk products for lactose intolerants.

Molecular characterization of non-biogenic amines producing Lactobacillus plantarum GP11 isolated from traditional pickles using HRESI-MS analysis.

Fungal spoilage and toxic biogenic amine production is a major risk in fermented products. Therefore, the selection of nontoxic biogenic amines producing probiotic bacteria plays a vital role in the fermentation process. In the present study, a total of 18 bacterial isolates were isolated from eight different homemade pickle samples and 15 lactic acid bacteria (LAB) were identified based on biochemical tests. Out of which only seven isolates (GP1, GP2, GP3, GP4, GP5, GP9, and GP11) exhibited antifungal activity against pickle contaminant Aspergillus sp and Penicillium sp. Among the potential LAB isolates, GP11 showed the highest antifungal activity against Aspergillus sp and Penicillium sp with a zone of inhibition 28.33 ± 0.57and 19.66 ± 0.57 mm respectively. The potent LAB isolates were tested for amino acid decarboxylase activity, in which GP2, GP3, GP4, and GP5 exhibited to produce tyramine, cadaverine, and phenylethylamine while GP1 and GP5 have produced tyramine and phenylethylamine respectively. However, highly potent antifungal active isolate GP11 did not produce biogenic amine. Further, GP1, GP9, and GP11 were subjected to confirmation of biogenic amines production using HRESI-MS. HRESI-MS analysis of the GP1 and GP9 sample confirmed the presence of phenylethylamine and tyramine respectively. Interestingly, GP11 isolate did not show any biogenic amines production and GP11 was further subjected to 16S rRNA typing and identified as Lactobacillus plantarum. On in situ pickle sensory evaluation, GP11 lactopickle was graded as very good quality when compared to traditional one. Therefore L. plantarum GP11 could be developed as an ideal starter culture for the fermented production of a pickle.

High throughput sequencing study of foliose lichen-associated bacterial communities from India.

Lichens comprise highly diverse and complex microbial communities, the majority consisting of mycobiont, photobiont, Basidiomycetes yeast and bacteriobiont (internal bacterial communities). In this study, bacterial diversity of foliose lichen was reported. Next generation sequence (NGS) such as Illumina Sequencing (150*2) of 16S rRNA (V3 and V6 region) was used to delineate the bacterial communities associated with five foliose lichen samples. Bacterial sequences obtained from lichen samples suggested that, they harboured bacterial community with variable relative abundances. Among all bacterial communities, Alphaproteobacteria were dominant in all the tested lichen samples. The principal coordinate analysis, Venn and bar chart showed significant microbial changes between the different useful bacterial lineages across the lichens. The relative abundance of dominant and rare bacterial species found were varied, diverse, distinct and unique in each lichen. The Proteobacteria 48.19%, Actinobacteria 25.70%, Bacteroidetes 8.53%, Acidobacteria 9.36% and Chloroflexi 0.83% were predominant in all tested lichens. The present empirical study enhances the confirmed knowledge of bacterial diversity inevitably associated with lichens and is the first report on lichenized bacterial diversity and perhaps their potential possible role in lichen symbiosis in addition to phycobiont and mycobiont.

In vivo AFB1 detoxification by Lactobacillus fermentum LC5/a with chlorophyll and immunopotentiating activity in albino mice.

The potential Aflatoxin B1 (AFB1) binding Lactobacillus fermentum (LC5/a) was used for in vivo AFB1 binding and detoxification in presence of chlorophyll (CL) in male Swiss albino mice. Mice were randomly divided into seven groups. The control groups (CL, AFB1 and LC5/a) received chlorophyll (250 µg/kg b.w), AFB1 (100 µg/kg b.w) and LC5/a (1 × 108 CFU) for 21 days. The treatment group (AFB1+LC5/a) received 100 µl of lyophilized bacterial suspension (1 × 108 CFU) 2 h before the AFB1 dosage (100µg/kg b.w). The chlorophyll mice group (CL + AFB1) was given single oral dose of CL (250 µg/kg b.w) before AFB1 dosage and last mice group received the combination of CL + LC5/a before the AFB1 dosage over a period of 21 days. Ballooning of cytoplasm and necrosis in liver was evident in histopathological examination of AFB1 mice group, while, marked improvement and nearly normal histology were seen in LC5/a and CL treated mice group. The levels of AST, ALT, GST, and SOD were increased in AFB1 mice group compared to LC5/a and CL treated mice group. Elevated levels of pro-inflammatory cytokines, TNF-α, IL-12, IL-6 (324, 506, 117.25 pg/ml) were observed in AFB1 treated mice serum compared to LC5/a and CL treated mice (249.54, 322.01 and 82.35 pg/ml). Thus, Lactobacillus fermentum LC5/a has certainly sequestered AFB1 from gastrointestinal tract besides regulating the production of pro-inflammatory cytokines.

Gluten hydrolyzing activity of Bacillus spp isolated from sourdough.

BACKGROUND: Celiac disease is an intestinal chronic disorder with multifactorial etiology resulting in small intestinal mucosal injuries and malabsorption. In genetically predisposed individuals with HLA DQ2/DQ8 molecules, the gluten domains rich in glutamine and proline present gluten domains to gluten reactive CD4+ T cells causing injury to the intestine. In the present experimental design, the indigenous bacteria from wheat samples were studied for their gluten hydrolyzing functionality. RESULTS: Proteolytic activity of Bacillus spp. was confirmed spectrophotometrically and studied extensively on gliadin-derived synthetic enzymatic substrates, natural gliadin mixture, and synthetic highly immunogenic 33-mer peptide. The degradation of 33-mer peptide and the cleavage specificities of the selected isolates were analyzed by tandem mass spectrometry. The gluten content of the sourdough fermented by the chosen bacterial isolates was determined by R5 antibody based competitive ELISA. All the tested isolates efficiently hydrolyzed Z-YPQ-pNA, Z-QQP-pNA, Z-PPF-pNA, and Z-PFP-pNA and also cleaved 33-mer immunogenic peptide extensively. The gluten content of wheat sourdough was found to be below 110 mg/kg. CONCLUSION: It has been inferred that four Bacillus spp especially GS 188 could be useful in developing gluten-reduced wheat food product for celiac disease prone individuals.

Characterization of Novel Lactobacillus fermentum from Curd Samples of Indigenous Cows from Malnad Region, Karnataka, for their Aflatoxin B1 Binding and Probiotic Properties.

Thirty-four isolates of Lactobacillus spp. (LAB) from 34 curd samples were evaluated for their aflatoxin B1 (AFB1) binding and probiotic properties. Upon characterization, four LAB isolates (LC3/a, LC4/c, LC/5a, and LM13/b) were found to be effective in removing AFB1 from culture media with a capacity of above 75%. Staining reaction, biochemical tests, pattern of sugar utilization, and 16s rRNA gene sequence analysis revealed the identity of all the four isolates as L. fermentum. All of them could tolerate acidic pH, salt, and bile, which promise the use of these probiotic bacterial isolates for human applications. These isolates showed poor hydrophobicity and higher auto-aggregation properties. All L. fermentum isolates were found susceptible to gentamycin, chloramphenicol, cefoperazone, ampicillin, and resistant to ciprofloxacin and vancomycin. Results of hemolytic and DNase activity indicated their nonpathogenic nature. Though all L. fermentum isolates found inhibiting the growth of Salmonella ebony, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, maximum inhibition was obtained with isolate LC5/a. Kinetic studies revealed that all four bacteria required a minimum of 2 h to reach stationary phase of AFB1 binding. AFB1 binding ability varied from 66 to 85.2% among these four isolates. Bile (0.4%) was significant (P ≤ 0.05) in reducing the AFB1 binding property of isolates LC3/a, LC4/c, and LM13/b, while increased AFB1 binding ability was recorded at acidic pH (2.0). AFB1 binding properties of isolate LC5/a were found least affected by acidic pH and bile. The findings of our study revealed the higher efficiency of L. fermentum isolate LC5/a in reducing the bioavailability of AFB1 in gut, and additionally, it improves the consumers' health by its various probiotic characters. These beneficial characters, L. fermentum isolates, promise them to use as probiotic formulations alone or in combinations with other beneficial probiotic-bacterial isolates.

Draft Genome Sequence of the Gluten-Hydrolyzing Bacterium Bacillus subtilis GS 188, Isolated from Wheat Sourdough.

The draft genome sequence of Bacillus subtilis GS 188, a novel spore-forming probiotic bacterium with gluten-hydrolyzing potential, was isolated from wheat sourdough and provides deep insights into the beneficial features of this strain for its use in the preparation of gluten-reduced wheat foods for humans with celiac disease.

Plant growth promoting potential and phylogenetic characteristics of a lichenized nitrogen fixing bacterium, Enterobacter cloacae.

Lichens are complex symbiotic association of mycobionts, photobionts, and bacteriobionts, including chemolithotropic bacteria. In the present study, 46 lichenized bacteria were isolated by conventional and enrichment culture methods on nitrogen-free bromothymol blue (NFb) medium. Only 11 of the 46 isolates fixed nitrogen on NFb and had reduced acetylene. All these 11 isolates had also produced siderophore and 10 of them the IAA. Further, ammonia production was recorded from nine of these nitrogen fixers (NF). On molecular characterization, 16 S rRNA sequencing recorded that, nine NF belonged to Proteobacteria, within Gammaproteobacteria, and were closely related to Enterobacter sp. with a maximum similarity to Enterobacter cloacae. Each one of our NF isolates was aligned closely to Enterobacter pulveris strain E443, Cronobacter sakazakii strain PNP8 and Providencia rettgeri strain ALK058. Notably, a few strains we examined found to possess plant growth promoting properties. This is the first report of Enterobacter sp. from lichens which may be inhabit lichen thalli extrinsically or intrinsically.

Degradation of 2,4 dichlorobiphenyl via meta-cleavage pathway by Pseudomonas spp. consortium.

Two bacterial isolates (Pseudomonas sp. GSa and Pseudomonas sp. GSb) were in close association able to assimilate 2,4 dichlorobiphenyl (2,4 CB), a PCB congener. GC-MS analysis of spent culture medium of the consortium with 2,4 CB as substrate showed 90 % degradation (according to Electron capture detection values) with catechol as one of the important intermediate compounds through meta-cleavage pathway. Further, ability of the consortium to utilise PCB congeners, Methoxychlor, Aroclor 1016, Chlorobenzoic acids and Monoaromatic compounds indicated that the consortium of GSa and GSb would be an ideal candidate for in situ bioremediation of PCB.

Synergistic impact of Lactobacillus fermentum, Lactobacillus plantarum and vincristine on 1,2-dimethylhydrazine-induced colorectal carcinogenesis in mice.

Lactobacillus sp. is the most dominant probiotic strain of bacteria. Evidence indicates that the consumption of Lactobacillus sp. reduces the risk of colorectal cancer in animal models. The present study was carried out to determine whether administration of Lactobacillus fermentum/ Lactobacillus plantarum alone or in combination with vincristine have a synergistic impact on the control of colorectal cancer in an animal model. Mice with 1,2 dimethylhydrazine (DMH) hydrochloride-induced colon cancer were fed with L. fermentum and L. plantarum isolated along with vincristine. An increase in body weight, a decrease in ammonia concentration, a decrease in ß glucosidase and ß glucuronidase enzyme activity and a reduction in the number of crypts in the mice in the pre-carcinogen-induced group was noted when compared to these variables in the post-carcinogen-induced group. The body weight of the mice fed L. fermentum along with vincristine was increased (6.5 g), and was found to be 3.5 times higher compared to that of the control. A marked decrease in the ammonia concentration (240 mg), and ß glucosidase (0.0023 IU) and ß glucopyranose enzyme activity (0.0027 IU) was observed; 22.59% less ammonia concentration, 73.26% less ß glucosidase activity and 56.46% less ß glucuronidase enzyme activity was noted when compared to the control. A significant reduction in the number of aberrant crypt foci (ACF) (90%) was observed when compared to the control. Maximum protection was observed in the mice fed the probiotics and vincristine prior to cancer induction. Among the different dietary combinations tested in the present study, L. fermentum and vincristine showed a more extensive reduction in ammonia concentration, ß glucosidase, ß glucuronidase activity and the number of ACF.