Optimization of extracellular mannanase production from Penicillium oxalicum KUB-SN2-1 and application for hydrolysis property

Effects of media composition, and physical properties on the production of crude mannanase by Penicillium oxalicumKUB-SN2-1 were investigated. P.oxalicum KUB-SN2-1 was propagated in a shaking incubator at 30°C with rotation speed of200 rpm of 7 days. The specific activity obtained during growth on robusta coffee residues (RCR) of 16.21 U/mg protein wasmuch higher than other carbon sources tested. For nitrogen sources, yeast extract (0.11 U/mg protein) and ammonium nitrate(0.09 U/mg protein) showed maximum specific activity. Hence, guar gum was the best inducer for producing mannanase (14U/mg protein). For evaluating the optimal concentration, the result showed that 1% guar gum, 0.5% yeast extract, 0.25%ammonium nitrate, and 0.25% RCR were the suitable sources of inducer, organic nitrogen, inorganic nitrogen, and carbon,respectively. Modified medium with initial culture pH of 5.0 at 30°C was optimum for mannanase production (53.77 U/ml for3 day). Reducing sugars were analyzed by dinitrosalicylic acid methods. The highest reducing sugar of 7517.82 g/mlwas obtained from copra meal hydrolysate after 30 h

Promising discovery of benefcial Escherichia coli in the human gut

Ingested dietary fibres are hydrolysed by colon microbiota to produce energy-providing short-chain fatty acids (SCFA) that stimulate anti-inflammatory effects. SCFA-producing bacteria were screened from bacteria isolated from human faeces using bromothymol blue as an acid indicator and gas chromatography for SCFA profiling. The beneficial functions (antagonistic activity, haemolytic activities, antibiotic susceptibility, mucus adherent percentage and toxin gene detection) were evaluated for the top five SCFA-producing bacteria isolated from three healthy volunteers that identified as Escherichia coli strains. They produced acetic, propionic, isobutyric, butyric, isovaleric, valeric and caproic acids at average concentrations of 15.9, 1.8, 1.1, 1.9, 1.8, 2.7 and 3.4 mM, respectively. The SCFA production by E. coli strains was rapidly increased during the first 8 h of incubation and gradually decreased after 16 h of incubation. All E. coli strains showed acid and bile tolerance, resulting in a survival rate greater than 70% with no haemolytic activity, mucus adherence greater than 40% and susceptibility to conventional antibiotics. Hence, the selected E. coli strains exhibited promising probiotic properties with neither enterotoxin nor LPS producibility was detected. The present results confirm the existence of friendly and harmless E. coli strains in human microbiota as potential probiotics

Urban Diets Linked to Gut Microbiome and Metabolome Alterations in Children: A Comparative Cross-Sectional Study in Thailand

Loss of traditional diets by food globalization may have adverse impact on the health of human being through the alteration of gut microbial ecosystem. To address this notion, we compared the gut microbiota of urban (n = 17) and rural (n = 28) school-aged children in Thailand in association with their dietary habits. Dietary records indicated that children living in urban Bangkok tended to consume modern high-fat diets, whereas children in rural Buriram tended to consume traditional vegetable-based diets. Sequencing of 16S rRNA genes amplified from stool samples showed that children in Bangkok have less Clostridiales and more Bacteroidales and Selenomonadales compared to children in Buriram and bacterial diversity is significantly less in Bangkok children than in Buriram children. In addition, fecal butyrate and propionate levels decreased in Bangkok children in association with changes in their gut microbial communities. Stool samples of these Thai children were classified into five metabolotypes (MTs) based on their metabolome profiles, each characterized by high concentrations of short and middle chain fatty acids (MT1, n = 17), amino acids (MT2, n = 7), arginine (MT3, n = 6), amino acids, and amines (MT5, n = 8), or an overall low level of metabolites (MT4, n = 4). MT1 and MT4 mainly consisted of samples from Buriram, and MT2 and MT3 mainly consisted of samples from Bangkok, whereas MT5 contained three samples from Bangkok and five from Buriram samples. According to the profiles of microbiota and diets, MT1 and MT2 are characteristic of children in Buriram and Bangkok, respectively. Predicted metagenomics indicated the underrepresentation in MT2 of eight genes involved in pathways of butyrate biosynthesis, notably including paths from glutamate as well as pyruvate. Taken together, this study shows the benefit of high-vegetable Thai traditional diets on gut microbiota and suggests that high-fat and less-vegetable urban dietary habits alter gut microbial communities in Thai children, which resulted in the reduction of colonic short chain fatty acid fermentation

Diversity in gut bacterial community of school-age children in Asia

10.1038/srep08397Scientific Reports

Characterization of Grass Degrading Bacteria Active on β-1,3-1,4-D-glucans from Bacillus subtilis GN156 Potential Use for Grass Silage-Making

ABSTRACT One hundred and sixty-one bacterial isolates were screened for (i) the stability of CM-cellulase at high temperature of 60°C as primary screening, (ii) the stability of pH and temperature of 3-7 and 30-60°C, respectively and (iii) the activities of pH and temperature range following stability study. The isolate GN156 showed high stability of CM-cellulase activity at the pH and temperature of 3.7 -7.2 and 30 -70°C, respectively. Based on physical and biochemical properties, this isolate was identified as Bacillus subtilis. The enzyme system study revealed various hydrolytic enzymes of CM-cellulase, dextrinase, cellobiase, xylanase, polygalacturonase, polymethylgalacturonase, but, β-1,3-1,4-glucanase was the most effective enzyme. Therefore, optimum pH and temperature of β-1,3-1,4-glucanase were further studied. Interestingly, its activities appeared at wide range of pH and temperature of 5.5-9 and 40-60°C, respectively. The profile of growth and enzyme production indicated that β-1,3-1,4-glucanase produced by B. subtilis GN156 was associated with cell growth. Induction of β-1,3-1,4-glucanase production by 1% of CM-cellulose, pectin and xylan revealed an increment of activities of 47, 41 and 11-folds, respectively. When various concentrations of CMC were taken into account, the CMC concentration of 0.8% (w/v) provided the maximum β-1,3-1,4-glucanase production

Purification and Characterization of a Novel Bacteriocin Against Vancomycin Resistant Enterococci Produced by Enterococcus hirae HM02-04

Enterococcus hirae HM02-04 isolated from breast milk exerted growth inhibition against especially vancomycin resistance enterococci (VRE). The aims of this study were to purify and characterize the antimicrobial substance produced by the strain HM02-04. One active peptide was successfully purified by 3 steps of Amberlite XAD-16 adsorption-desorption, cation exchange chromatography, and reverse-phase HPLC. It had 2605.298 Da peptide by MALDI-TOF MS analysis. The peptide sequence determined by LC-MS/MS contained only 23 amino acid residues providing the molecular mass of 2312.67 Da by in silico analysis. However, its amino acid sequence obtained showed no homology to other bacteriocins including enterocins previously reported and proposed as a novel one named Hiracin HM02-04. This is the first report to discover E. hirae isolated from breast milk that was able to produce a bacteriocin against VRE. Hiracin HM02-04 was stable at a high temperature of 121°C for 15 min and at a wide pH range of 3–9. It was sensitive to actinase E, pepsin, proteinase K and trypsin. The Hiracin HM02-04 has the narrow inhibition spectrum and no inhibition against Listeria. This bacteriocin was also found to have a bactericidal mode of action with concomitant cell lysis against the strain VRE 426. The present research addresses Hiracin HM02-04 as a promising alternative to conventional antibiotics in the treatment of enterococcal infections

In vitro adhesion assay of lactic acid bacteria, Escherichia coli and Salmonella sp. by microbiological and PCR methods

In vitro adhesion assay using Lactobacillus reuteri KUB-AC5 as a test strain has been studied by applying simple PCR reaction together with image analysis and plate count techniques. Critical factor affecting the PCR method was quality and quantity of DNA. The cell lysis technique was modified to optimize this method. Thus, lysozyme and proteinase K were added to lyse the cells, followed by SDS solution to obtain a complete cell lysis. Only PCR products from total cells (TC) were obtained, with low consistency, but none from cells bound to mucus (BC) at either 0.1 or 0.5 mg/mL concentration. It was hypothesized that the attached cells might not be extracted into the cell suspension. Therefore, 1% SDS solution and O.1M NaOH were used directly in the extraction. As expected, PCR products were observed when both TC and BC were used as a DNA template. Adhesion appeared at a wide range of 0-45%, with low consistency. Therefore, a simple microbiological method (plate count) was used. The extraction of bound cells into cell suspension was critical in this method. Extraction times of 20, 60, 120 and 150 min were tried. Results showed that maximum cell number was obtained with 120 min extraction. L. reuteri KUB-AC5, L. reuteri KUB-AC16, L. reuteri KUB-AC20, L. salivarius KUB-AC21, L. acidophilus KV-1, Escherichia coli E010, Salmonella sp. S003, E. coli ATCC8739, and S. typhimurium ATCC 13311 exhibited adhesion activity of 21.6%, 0.8%, 5.7%, 1.1%, 23.1%, 10.7%, 103%, 4.4% and 3.2%, respectively. Among the 9 types of microorganisms tested L. acidophilus KV-1 and L. reuteri KUB-AC5 showed higher adhesion activity than the others. (Résumé d'auteur

In vitro fermentation of copra meal hydrolysate by human fecal microbiota

Copra meal hydrolysate (CMH) is obtained by hydrolyzing defatted copra meal with β-mannanase from NT 6.7. In this study, we investigated the resistance of CMH to upper gastrointestinal tract digestion and the fecal fermentation profiles of CMH. Fecal slurries from four healthy human donors were used as inocula, and fructooligosaccharides (FOS) were used as a positive prebiotic control. Fecal batch cultures were performed at 37 °C under anaerobic conditions. Samples were collected at 0, 10, 24 and 34 h for bacterial enumeration via fluorescent in situ hybridization and organic acid (OA) analysis. In vitro gastric stomach and human pancreatic α-amylase simulations demonstrated that CMH was highly resistant to hydrolysis. Acetate was the main fermentation product of all the substrates. The proportions of acetate production of the total OAs from FOS, CMH and yeast mannooligosaccharides (MOS) after 34 h of fermentation did not significantly differ (69.76, 65.24 and 53.93%, respectively). At 24 h of fermentation, CMH promoted the growth of and groups (  < 0.01) and did not significantly differ from the results obtained using FOS. The results of in vitro fecal fermentation of CMH indicate that CMH can promote the growth of beneficial bacteria