Transcriptome analysis provides insights into the stress response in cultivated peanut (Arachis hypogaea L.) subjected to drought-stress.

BACKGROUND: Peanut (Arachis hypogaea L.) is one of the valuable oilseed crops grown in drought-prone areas worldwide. Drought severely limits peanut production and productivity significantly. METHOD AND RESULTS: In order to decipher the drought tolerance mechanism in peanut under drought stress, RNA sequencing was performed in TAG - 24 (drought tolerant genotype) and JL-24 (drought susceptible genotype). Approximately 51 million raw reads were generated from four different libraries of two genotypes subjected to drought stress exerted by 20% PEG 6000 stress and control conditions, of which ~ 41 million (80.87%) filtered reads were mapped to the Arachis hypogaea L. reference genome. The transcriptome analysis detected 1,629 differentially expressed genes (DEGs), 186 genes encoding transcription factors (TFs) and 30,199 SSR among the identified DEGs. Among the differentially expressed TF encoding genes, the highest number of genes were WRKY followed by bZIP, C2H2, and MYB during drought stress. The comparative analysis between the two genotypes revealed that TAG-24 exhibits activation of certain key genes and transcriptional factors that are involved in essential biological processes. Specifically, TAG-24 showed activation of genes involved in the plant hormone signaling pathway such as PYL9, Auxin response receptor gene, and ABA. Additionally, genes related to water deprivation such as LEA protein and those involved in combating oxidative damage such as Glutathione reductase were also found to be activated in TAG-24. CONCLUSION: This genome-wide transcription map, therefore, provides a valuable tool for future transcript profiling under drought stress and enriches the genetic resources available for this important oilseed crop.

Antigenotoxic and Antimutagenic Activities of Probiotic Lactobacillus rhamnosus Vc against N-Methyl-N'-Nitro-N-Nitrosoguanidine.

The present study provides experimental evidence of in vivo reduction of genotoxic and mutagenic activities of potent carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by the strain Lactobacillus rhamnosus Vc. In vitro studies revealed that coincubation of MNNG with viable cells of L. rhamnosus Vc resulted in the detoxification of the parent compound accompanied with reduction in genotoxicity (69%) and mutagenicity (61%) as evaluated by SOS-Chromotest and Ames test, respectively. Oral feeding of probiotic bacteria L. rhamnosus Vc (10(9) cfu) to Gallus gallus (chicks) for 30 days provided protection against MNNG-induced damage as evidenced from the significant decrease (P = 0.009) in glutathione S-transferase activity in the L. rhamnosus Vc+MNNG-treated chicks in comparison to the MNNG-treated chicks. Histopathology of colon and liver showed intact cells and mild inflammation in the L. rhamnosus Vc+MNNG-treated chicks, whereas heavy inflammation and degenerative changes were observed in MNNG-treated chicks. The results indicate that the probiotic L. rhamnosus Vc provided in vivo protection against MNNG-induced colon damage by detoxification of MNNG to less toxic metabolites.

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 the following 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, isomalto-oligosaccharides, and resistant starch; Group 2, fed with the same four probiotic strains as Group 1; Group 3, fed with the same prebiotic supplements as Group 1 for 7 days before CD infection; and Group 4 (control group) antibiotic treated and infected with NAP1/027 strain. Feces and cecal contents were collected for microbial cell viability, quantitative PCR (qPCR), toxin analyses and histopathology. Synbiotics- and probiotics-fed mice showed a significant increase in total bifidobacteria (P < 0.05). The total lactobacilli count was increased in Group 1. Tests for cecal toxins were negative in Group 2 mice, whereas one sample each from Group 1 and 3 was positive. qPCR of cecal contents showed significant reduction in NAP1/027 DNA copies in Groups 1 and 2 and significantly higher numbers of B. breve 46, L. plantarum F44, and L. paracasei F8 in Groups 1 and 2 (P < 0.05); these changes were much less pronounced in Groups 3 and 4. Our findings indicate that the newly developed synbiotic or multi-strain probiotic formulation confers protection against NAP1/027 infection in C57BL/6 mice. This holds promise for performing human studies.

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, Bifidobacterium lactis 8:8 and Bifidobacterium longum 6:18 and three reference strains B. breve CCUG 24611, B. lactis JCM 10602, and Bifidobacterium pseudocatenulatum 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.

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.

Metabolomic Profiling of Drought-Tolerant and Susceptible Peanut (Arachis hypogaea L.) Genotypes in Response to Drought Stress.

Peanut is frequently constrained by extreme environmental conditions such as drought. To reveal the involvement of metabolites, TAG 24 (drought-tolerant) and JL 24 (drought-sensitive) peanut genotypes were investigated under control and 20% PEG 6000-mediated water scarcity conditions at the seedling stage. Samples were analyzed by gas chromatography-mass spectrometry (GC-MS) to identify untargeted metabolites and targeted metabolites, i.e., polyamines and polyphenols by high-performance liquid chromatography (HPLC) and ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), respectively. The principal component analysis (PCA), partial least-squares discriminant analysis (PLS-DA), heat map, and cluster analysis were applied to the metabolomics data obtained by the GC-MS technique to determine the important metabolites for drought tolerance. Among 46 resulting metabolites, pentitol, phytol, xylonic acid, d-xylopyranose, stearic acid, and d-ribose were important drought-responsive metabolites. Agmatine and cadaverine were present in TAG 24 leaves and roots, respectively, during water-deficit conditions and believed to be the potential polyamines for drought tolerance. Polyphenols such as syringic acid and vanillic acid were produced more in the leaves of TAG 24, while catechin production was high in JL 24 during stress conditions. Seven metabolic pathways, namely, galactose metabolism, starch and sucrose metabolism, fructose and mannose metabolism, pentose and glucuronate interconversion, propanoate metabolism, amino sugar and nucleotide sugar metabolism, and biosynthesis of unsaturated fatty acids were significantly affected by water-deficit conditions. This study provides valuable information about the metabolic response of peanut to drought stress and metabolites identified, which encourages further study by transcriptome and proteomics to improve drought tolerance in peanut.

Evaluation of Probiotic Properties and Prebiotic Utilization Potential of Weissella paramesenteroides Isolated From Fruits.

Weissella paramesenteroides has gained a considerable attention as bacteriocin and exopolysaccharide producers. However, potential of W. paramesenteroides to utilize different prebiotics is unexplored area of research. Fruits being vectors of various probiotics, five W. paramesenteroides strains, namely, FX1, FX2, FX5, FX9, and FX12, were isolated from different fruits. They were screened and selected based on their ability to survive at pH 2.5 and in 1.0% sodium taurocholate, high cell surface hydrophobicity, mucin adhesion, bile-induced biofilm formation, antimicrobial activity (AMA) against selected enteropathogens, and prebiotic utilization ability, implicating the functional properties of these strains. In vitro safety evaluation showed that strains were susceptible to antibiotics except vancomycin and did not harbor any virulent traits such as biogenic amine production, hemolysis, and DNase production. Based on their functionality, two strains FX5 and FX9 were selected for prebiotic utilization studies by thin layer chromatography (TLC) and short-chain fatty acids (SCFAs) production by high performance liquid chromatography. TLC profile evinced the ability of these two strains to utilize low molecular weight galactooligosaccharides (GOS) and fructooligosaccharides (FOS), as only the upper low molecular weight fractions were disappeared from cell-free-supernatants (CFS). Enhanced ß-galactosidase activity correlated with galactose accumulation in residual CFS of GOS displayed GOS utilization ability. Both the strains exhibited AMA against E. coli and Staph. aureus and high SCFAs production in the presence of prebiotic, suggesting their synbiotic potential. Thus, W. paramesenteroides strains FX5 and FX9 exhibit potential probiotic properties with prebiotic utilization and can be taken forward to evaluate synergistic synbiotic potential in detail.

Probiotic attributes and prevention of LPS-induced pro-inflammatory stress in RAW264.7 macrophages and human intestinal epithelial cell line (Caco-2) by newly isolated Weissella cibaria strains.

Probiotic lactic acid bacteria are known to modulate gut associated immune responses. Not many studies have reported on the role of Weissella species in preventing lipopolysaccharide (LPS) induced proinflammatory stress in murine macrophages as well as in human intestinal epithelial cells (Caco-2). Therefore, the present study was taken up to evaluate the probiotic attributes of four newly isolated Weissella strains (two each from fermented dosa batter and a human infant faecal sample); these attributes are cholesterol reduction, adhesion to Caco-2 cells and mucin and their ability to prevent LPS-induced nitric oxide and proinflammatory cytokine (IL-6, IL-1ß and TNFα) production by the murine macrophages and IL-8 production by the human epithelial cells. Reduction in LPS induced pro-inflammatory stress was compared with a well-studied probiotic bacterium Lactobacillus rhamnosus GG. The results suggested that the strains were tolerant to gastric conditions (pH 3.0) and bile salts. In addition, the strains exhibited moderate cell surface hydrophobicity, cholesterol reduction and adhesion to Caco-2 cells and gastric mucin. All the strains could prevent LPS-induced nitric oxide and IL-6 production in murine macrophages, while strain 28 alone prevented IL-1ß production. All the strains could prevent IL-8 production by the human epithelial cells. The present study led to the first line selection of W. cibaria 28 as a putative strain for future studies as it showed adhesion to Caco-2 cells and gastric mucin and cholesterol reduction besides preventing LPS-induced pro-inflammatory stress in macrophages and in human colonic epithelial cells.

Probiotics, prebiotics and colorectal cancer prevention.

Colorectal cancer (CRC), the third major cause of mortality among various cancer types in United States, has been increasing in developing countries due to varying diet and dietary habits and occupational hazards. Recent evidences showed that composition of gut microbiota could be associated with the development of CRC and other gut dysbiosis. Modulation of gut microbiota by probiotics and prebiotics, either alone or in combination could positively influence the cross-talk between immune system and microbiota, would be beneficial in preventing inflammation and CRC. In this review, role of probiotics and prebiotics in the prevention of CRC has been discussed. Various epidemiological and experimental studies, specifically gut microbiome research has effectively improved the understanding about the role of probiotics and microbial treatment as anticarcinogenic agents. A few human studies support the beneficial effect of probiotics and prebiotics; hence, comprehensive understanding is urgent to realize the clinical applications of probiotics and prebiotics in CRC prevention.

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".

Insight into the Draft Genome Sequence of Human Isolate Lactobacillus rhamnosus LR231, a Bacterium with Probiotic Potential.

Lactobacillus rhamnosus strain LR231 was isolated from the feces of healthy human subjects. It is observed to be a potential probiotic strain, having a broad spectrum of antimicrobial activity against a wide range of human pathogens and food pathogens. Here, we provide the 2.59-Mb draft genome sequence of L. rhamnosus LR231.

Potential of probiotics, prebiotics and synbiotics for management of colorectal cancer.

Colorectal Cancer (CRC) is the second leading cause of cancer-related mortality and is the fourth most common malignant neoplasm in USA. Escaping apoptosis and cell mutation are the prime hallmarks of cancer. It is apparent that balancing the network between DNA damage and DNA repair is critical in preventing carcinogenesis. One-third of cancers might be prevented by nutritious healthy diet, maintaining healthy weight and physical activity. In this review, an attempt is made to abridge the role of carcinogen in colorectal cancer establishment and prognosis, where special attention has been paid to food-borne mutagens and functional role of beneficial human gut microbiome in evading cancer. Further the significance of tailor-made prebiotics, probiotics and synbiotics in cancer management by bio-antimutagenic and desmutagenic activity has been elaborated. Probiotic bacteria are live microorganisms that, when administered in adequate amounts, confer a healthy benefit on the host. Prebiotics are a selectively fermentable non-digestible oligosaccharide or ingredient that brings specific changes, both in the composition and/or activity of the gastrointestinal microflora, conferring health benefits. Synbiotics are a combination of probiotic bacteria and the growth promoting prebiotic ingredients that purport "synergism."

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 dependent on pH and ionic strength and was protease-sensitive. In the presence of 100 µg mL(-1) cholesterol, the CRB was significantly reduced. Autoaggregating (AA) Lactobacillus crispatus strains showed 50% more CRB than the reference strain, the curli-producing Escherichia coli MC4 100. CRB of L. crispatus 12005, L. paracasei F8, L. plantarum F44 and L. paracasei F19 were enhanced when grown in Man Rogosa Sharpe (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.

Protective effect of Lactobacillus rhamnosus 231 against N-Methyl-N'-nitro-N-nitrosoguanidine in animal model.

The protective effect of Lactobacillus rhamnosus 231 (Lr 231) against potent carcinogen N-Methyl-N'-Nitro-N-Nitrosoguanidine (MNNG) in the rat model is studied. Daily feeding with Lr 231 improved the body weight of male Wistar rats compared with control groups. Fecal azoreductase (p < 0.001) and nitroreductase (p < 0.01) enzyme activity decreased significantly in Lr 231 group in comparison with control groups that received only phosphate buffer or MNNG. Oral administration of MNNG led to a significant increase in Glutathione transferase (GST) while Glutathione reductase (GSH) showed decreased activity. Conversely, feeding Lr 231 showed significantly increased GSH and decreased GST activity in comparison to the MNNG group, emphasizing the protection provided by Lr 231 against MNNG. Histopathological analysis of liver, spleen and colon showed decreased signs of inflammation in the Lr 231 group. The present study highlights that inclusion of active Lr 231 in regular diets could be used to prevent MNNG induced colon carcinoma.