Search

Synbiotics are products containing both probiotics and prebiotics, in which the prebiotic compound selectively favours the probiotic to provide synergistic activity for gut health promotion. Due to its potential probiotic effect, Lactococcus lactis KA-FF 1-4 showed inhibitory activity against vancomycin-resistant enterococci (VRE). Its synergistic activity with three commercial plant prebiotics namely inulin, fibersol-2, and XOS is presented in the present work. In vitro batch fermentation with each prebiotic supplement had no effect on the growth rate of L. lactis KA-FF 1-4. However, supplementation with either 2% w/v inulin or fibersol-2 dramatically enhanced anti-VRE activity. Further study was performed on the effect of various concentrations (0.5, 1, 2, and 5%) of inulin or fibersol-2 on anti-VRE activity. In co-culture, L. lactis KA-FF 1-4 with 1% inulin supplementation could apparently reduce the VRE concentration from log 4.0 to 2.7 CFU/mL after 12 h, while complete inhibition was observed with 2% fibersol-2 supplementation after 6 h. These results clearly showed the synergistic effect on VRE growth inhibition, which may serve as a guideline for the development of synbiotic formulations in the future. [ABSTRACT FROM AUTHOR]

Exceeding a healthy weight significantly elevates the likelihood of developing type 2 diabetes (T2DM). A commercially available singular constituent, available as either purified vitexin or iso-vitexin, has been associated with a decreased risk of T2DM, but its synergistic effect has not been reported yet. Vitexin and iso-vitexin were extracted using an ethanol-based solvent from mung bean seed coat (MBCE) and subsequently purified using preparative liquid chromatography (Prep-LC). Eleven mixture ratios of vitexin and/or iso-vitexin were determined for their antioxidant and antihyperglycemic activities. The 1:1.5 ratio of vitexin to iso-vitexin from MBCE demonstrated the most synergistic effects for enzyme inhibition and glucose uptake in HepG2 cells within an insulin-resistant system, while these ratios exhibited a significantly lower antioxidant capacity than that of each individual component. In a gut model system, the ratio of 1:1.5 (vitexin and iso-vitexin) regulated the gut microbiota composition in overweight individuals by decreasing the growth of Enterobacteriaceae and Enterococcaceae , while increasing in Ruminococcaceae and Lachnospiraceae . The application of vitexin/iso-vitexin for 24 h fermentation enhanced a high variety of abundances of 21 genera resulting in five genera of Parabacteroides , Ruminococcus , Roseburia , Enterocloster , and Peptacetobacter , which belonged to the phylum Firmicutes , exhibiting high abundant changes of more than 5%. Only two genera of Proteus and Butyricicoccus belonging to Proteobacteria and Firmicutes decreased. The findings suggest that these phytochemicals interactions could have synergistic effects in regulating glycemia, through changes in antihyperglycemic activity and in the gut microbiota in overweight individuals. This optimal ratio can be utilized by industries to formulate more potent functional ingredients for functional foods and to create nutraceutical supplements aimed at reducing the risk of T2DM in overweight individuals.

Introduction: Bacterial urinary tract infections (UTI) are among the most common infectious diseases worldwide. The rise of multidrug-resistant (MDR) uropathogenic Escherichia coli (UPEC) UTI cases is a significant threat to healthcare systems. Several probiotic bacteria have been proposed as an alternative to combat MDR UTI. Lactic acid bacteria in the genus Limosilactobacillus are some of the most studied and used probiotics. However, strain-specific effects play a critical role in probiotic properties. L. reuteri KUB-AC5 (AC5), isolated from the chicken gut, confers antimicrobial and immunobiotic effects against some human pathogens. However, the antibacterial and immune modulatory effects of AC5 on UPEC have never been explored., Methods: Here, we investigated both the direct and indirect effects of AC5 against UPEC isolates (UTI89, CFT073, and clinical MDR UPEC AT31) in vitro . Using a spot-on lawn, agar-well diffusion, and competitive growth assays, we found that viable AC5 cells and cell-free components of this probiotic significantly reduced the UPEC growth of all strains tested. The human bladder epithelial cell line UM-UC-3 was used to assess the adhesion and pathogen-attachment inhibition properties of AC5 on UPEC., Results and Discussion: Our data showed that AC5 can attach to UM-UC-3 and decrease UPEC attachment in a dose-dependent manner. Pretreatment of UPEC-infected murine macrophage RAW264.7 cells with viable AC5 (multiplicity of infection, MOI = 1) for 24 hours enhanced macrophage-killing activity and increased proinflammatory ( Nos2 , Il6 , and Tnfa ) and anti-inflammatory ( Il10 ) gene expression. These findings indicate the gut-derived AC5 probiotic could be a potential urogenital probiotic against MDR UTI., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Tantibhadrasapa, Li, Buddhasiri, Sukjoi, Mongkolkarvin, Boonpan, Wongpalee, Paenkaew, Sutheeworapong, Nakphaichit, Nitisinprasert, Hsieh and Thiennimitr.)

The association between the gut mycobiome and its potential influence on host metabolism in the Thai Cohort was assessed. Two distinct predominant enterotypes, Saccharomyces (Sa) and Aspergillus/Penicillium (Ap/Pe) showed differences in gut mycobiota diversity and composition. Notably, the Sa enterotype exhibited lower evenness and richness, likely due to the prevalence of Saccharomyces, while both enterotypes displayed unique metabolic behaviors related to nutrient metabolism and body composition. Fiber consumption was positively correlated with adverse body composition and fasting glucose levels in individuals with the Sa enterotype, whereas in the Ap/Pe enterotype it was positively correlated with fat and protein intake. The metabolic functional analysis revealed the Sa enterotype associated with carbohydrate metabolism, while the Ap/Pe enterotype involved in lipid metabolism. Very interestingly, the genes involved in the pentose and glucuronate interconversion pathway, such as polygalacturonase and L-arabinose-isomerase, were enriched in the Sa enterotype signifying a metabolic capacity for complex carbohydrate degradation and utilization of less common sugars as energy sources. These findings highlight the interplay between gut mycobiome composition, dietary habits, and metabolic outcomes within the Thai cohort studies., (© 2024. The Author(s).)

A synbiotic is a combination of live microorganisms and specific substrates that are selectively utilized by host microorganisms, resulting in health benefits for the host. Previous studies have demonstrated the protective effects of L. reuteri KUB-AC5 against Salmonella infection in chicken and mouse models. The probiotic activity of L. reuteri KUB-AC5 in these hosts was influenced by nutritional supplements. Water-based plants contain significant amounts of carbohydrates, particularly dietary fiber and proteins, making them potential prebiotic substrates. In this study, four water-based plants (Ulva rigida, Caulerpa lentillifera, Wolffia globosa, and Gracillaria fisheri) were screened for their ability to support the growth of L. reuteri KUB-AC5. Under monoculture testing, U. rigida exhibited the highest capacity to support the growth of L. reuteri KUB-AC5 and the production of organic acids, including acetic acid, lactic acid, and propionic acid (p ≤ 0.05). In co-culture experiments, the synbiotic combination of U. rigida and L. reuteri KUB-AC5 demonstrated the potential to eliminate Salmonella Typhimurium DMST 48437 when inoculated at 10 4  CFU/mL within 9 h. The synbiotic activities of U. rigida and L. reuteri KUB-AC5 were further investigated using an in vitro human gut model. Compared to the probiotic treatment, the synbiotic combination of L. reuteri KUB-AC5 and U. rigida showed significantly higher levels of L. reuteri KUB-AC5 (5.1 log copies/mL) and a reduction of S. Typhimurium by 0.8 log (CFU/ml) after 24 h (p ≤ 0.05). Synbiotic treatment also significantly promoted the production of short-chain fatty acids (SCFAs), including butyric acid, propionic acid, and acetic acid, compared to prebiotic and probiotic treatments alone (p ≤ 0.05). Furthermore, the synbiotic formulation modulated the in vitro simulated gut microbiome, enhancing putatively beneficial gut microbes, including lactobacilli, Faecalibacterium, and Blautia. Our findings demonstrated that L. reuteri KUB-AC5, in combination with U. rigida, exhibited synergistic activity, as indicated by increased viability, higher anti-pathogenicity toward Salmonella, and the ability to modulate the gut microbiome., (© 2024. The Author(s).)

Palm kernel cake (PKC) is a by-product of palm kernel oil extraction with moderate nutritional value, containing 30-35% β-mannan, which is indigestible, slows growth, and reduces feed efficiency. PKC can be improved by mannanase hydrolysis, but the effectiveness of mannanase is dependent on the microbial source. Thus, the effect of steam pretreatment and bacterial mannanases on PKC quality was investigated. PKC was pretreated by steaming and hydrolyzed in the small intestine by various mannanases. The contents of reducing sugar, total sugar, and protein release were measured. Steamed PKC had a significant increase in protein (16.95 ± 0.14 to 20.98 ± 0.13%) and a substantial decrease in hemicellulose (29.52 ± 0.44 to 3.46 ± 0.88%) and lignin (8.94 ± 0.28 to 1.40 ± 0.22%). Mannanases from Escherichia coli-KMAN-3 and E. coli-Man6.7 recorded the highest activities, followed by commercial mannanase, Bacillus circulans NT6.7 and B. amyloliquefaciens NT6.3 mannanases, orderly. B. circulans NT6.7 and B. amyloliquefaciens NT6.3 had multi-activities that include glucanase (3.10 ± 0.04% and 2.47 ± 0.02%) and amylase (1.74 ± 0.03% and 1.38 ± 0.04%), respectively. B. amyloliquefaciens NT6.3 mannanase hydrolyzed steamed PKC to release more reducing sugar, total sugar, and protein than hydrolyzed raw PKC. In raw and steamed PKC, B. amyloliquefaciens NT6.3 mannanase produced the highest reducing sugar release. As a result, steam pretreatment and mannanase hydrolysis, particularly from B. amyloliquefaciens, can be used to increase the functioning of PKC and develop new feed ingredients for monogastric animals at a reasonable cost., (Copyright © 2022 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Limosilactobacillus reuteri KUB-AC5 displays the hallmark features of probiotic properties for food and feed industries. Optimization of cultivation condition for the industrial production is important to reach cell concentration and cost reduction. Considering the strain-specific growth physiology, metabolic capability, and essential nutrients of L. reuteri KUB-AC5, the genome-scale metabolic model (GSMM) of L. reuteri KUB-AC5 was developed. Hereby, the GSMM of i TN656 was successfully constructed which contained 656 genes, 831 metabolites, and 953 metabolic reactions. The i TN656 model could show a metabolic capability under various carbon sources and guide potentially 14 essential single nutrients (e.g., vitamin B complex and amino acids) and 2 essential double nutrients (pairwise glutamine-glutamate and asparagine-aspartate) for L. reuteri KUB-AC5 growth through single and double omission analysis. Promisingly, the i TN656 model was further integrated with transcriptome data suggesting that putative metabolic routes as preferable paths e.g., sucrose uptake, nucleotide biosynthesis, urea cycle, and glutamine transporter for L. reuteri KUB-AC5 growth. The developed GSMM offers a powerful tool for multi-level omics analysis, enabling probiotic strain optimization for biomass overproduction on an industrial scale.

Limosilactobacillus reuteri KUB-AC5 has been widely used as probiotic in chicken for Salmonella reduction. However, a preferable carbon source and growth phase is poorly characterized underlying metabolic responses on growth and inhibition effects of L. reuteri KUB-AC5. This study therefore aimed to investigate transcriptome profiling of L. reuteri KUB-AC5 revealing global metabolic responses when alteration of carbon sources and growth phases. Interestingly, L. reuteri KUB-AC5 grown under sucrose culture showed to be the best for fast growth and inhibition effects against Salmonella Enteritidis S003 growth. Towards the transcriptome profiling and reporter proteins/metabolites analysis, the results showed that amino acid transport via ABC systems as well as sucrose metabolism and transport are key metabolic responses at Logarithmic (L)-phase of L. reuteri KUB-AC5 growth. Considering the Stationary (S)-phase, we found the potential reporter proteins/metabolites involved in carbohydrate metabolism e.g ., levansucrase and levan. Promisingly, levansucrase and levan were revealed to be candidates in relation to inhibition effects of L. reuteri KUB-AC5. Throughout this study, L. reuteri KUB-AC5 had a metabolic control in acclimatization to sucrose and energy pools through transcriptional co-regulation, which supported the cell growth and inhibition potentials. This study offers a perspective in optimizing fermentation condition through either genetic or physiological approaches for enhancing probiotic L. reuteri KUB-AC5 properties., Competing Interests: The authors declare that they have no competing interests., (© 2021 Jatuponwiphat et al.)

The impact of copra meal hydrolysate (CMH) on gut health was assessed by conducting a double-blinded, placebo-controlled study. Sixty healthy adult participants, aged 18-40 years were assigned to daily consume 3 g of CMH, 5 g of CMH or placebo in the form of drink powder for 21 days. Consumption of CMH at 3 g/d improved defecating conditions by reducing stool size and also relieved flatulence and bloating symptoms. Fecal samples were collected serially at the baseline before treatment, after the treatment and after a 2-week washout period. The gut microbiomes were similar among the treatment groups, with microbial community changes observed within the groups. Intake of CMH at 3 g/d led to increase microbial diversity and richness. Reduction of the ratio between Firmicutes to Bacteroidetes was observed, although it was not significantly different between the groups. The 3 g/d CMH treatment increased beneficial microbes in the group of fiber-degrading bacteria, especially human colonic Bacteroidetes , while induction of Bifidobacteriaceae was observed after the washout period. Intake of CMH led to increase lactic acid production, while 3 g/d supplement promoted the present of immunoglobulin A (IgA) in stool samples. The 3 g daily dose of CMH led to the potentially beneficial effects on gut health for healthy individuals., Competing Interests: The authors declare there are no competing interests., (©2021 Sathitkowitchai et al.)

Acute non-typhoidal salmonellosis (NTS) caused by Salmonella enterica Typhimurium (STM) is among the most prevalent of foodborne diseases. A global rising of antibiotic resistance strains of STM raises an urgent need for alternative methods to control this important pathogen. Major human food animals which harbor STM in their gut are cattle, swine, and poultry. Previous studies showed that the probiotic Limosilactobacillus ( Lactobacillus ) reuteri KUB-AC5 (AC5) exhibited anti- Salmonella activities in chicken by modulating gut microbiota and the immune response. However, the immunobiotic effect of AC5 in a mammalian host is still not known. Here, we investigated the anti- Salmonella and anti-inflammatory effects of AC5 on STM infection using a mouse colitis model. Three groups of C57BL/6 mice (prophylactic, therapeutic, and combined) were fed with 10 9 colony-forming units (cfu) AC5 daily for 7, 4, and 11 days, respectively. Then, the mice were challenged with STM compared to the untreated group. By using a specific primer pair, we found that AC5 can transiently colonize mouse gut (colon, cecum, and ileum). Interestingly, AC5 reduced STM gut proliferation and invasion together with attenuated gut inflammation and systemic dissemination in mice. The decreased STM numbers in mouse gut lumen, gut tissues, and spleen possibly came from longer AC5 feeding duration and/or the combinatorial (direct and indirect inhibitory) effect of AC5 on STM. However, AC5 attenuated inflammation (both in the gut and in the spleen) with no difference between these three approaches. This study demonstrated that AC5 confers both direct and indirect inhibitory effects on STM in the inflamed gut., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Buddhasiri, Sukjoi, Kaewsakhorn, Nambunmee, Nakphaichit, Nitisinprasert and Thiennimitr.)

Background: Extracellular metabolites of short chain fatty acids (SCFA) excreted by gut microbiota have been reported to play an important role in the regulation of intestinal homeostasis. Apart from supplying energy, SCFA also elicit immune stimulation in animal and human cells. Therefore, an attempt was conducted to isolate SCFA producing bacteria from healthy human microbiota. The anti-cancer and anti-inflammatory effects of extracellular metabolites and individual SFCA were further investigated by using breast, colon cancer and macrophage cells. Toxin, inflammatory and anti-inflammatory cytokine gene expressions were investigated by RT-qPCR analyses in this study., Results: Escherichia coli KUB-36 was selected in this study since it has the capability to produce seven SCFA extracellularly. It produced acetic acid as the main SCFA. It is a non-exotoxin producer and hence, it is a safe gut microbiota. The IC 50 values indicated that the E. coli KUB-36 metabolites treatment elicited more potent cytotoxicity effect on MCF7 breast cancer cell as compared to colon cancer and leukemia cancer cells but exhibited little cytotoxic effects on normal breast cell. Furthermore, E. coli KUB-36 metabolites and individual SCFA could affect inflammatory responses in lipopolysaccharide-induced THP-1 macrophage cells since they suppressed inflammatory cytokines IL-1β, IL-6, IL-8 and TNF-α well as compared to the control, whilst inducing anti-inflammatory cytokine IL-10 expression., Conclusion: SCFA producing E. coli KUB-36 possessed vast potential as a beneficial gut microbe since it is a non-exotoxin producer that exhibited beneficial cytotoxic effects on cancer cells and elicited anti-inflammatory activity simultaneously. However, the probiotic characteristic of E. coli KUB-36 should be further elucidated using in vivo animal models.

The infant gut microbiome consists of a complex and diverse microbial community. Comprehensive taxonomic and metabolic functional knowledge about microbial communities supports medical and biological applications, such as fecal diagnostics. Among the omics approaches available for the investigation of microbial communities, metaproteomics-based analysis is a very powerful approach; under this method, the activity of microbial communities is explored by investigating protein expression within a sample. Through use of metaproteomics, this study aimed to investigate the microbial community composition of the infant gut to identify different key proteins playing metabolic functional roles in the microbiome of healthy infants and infants with atopic dermatitis in a Thai population-based birth cohort. Here, 18 fecal samples were analyzed by liquid chromatography-tandem mass spectrometry to conduct taxonomic, functional, and pathway-based protein annotation. Accordingly, 49,973 annotated proteins out of 68,232 total proteins were investigated in gut microbiome samples and compared between the healthy and atopic dermatitis groups. Through differentially expressed proteins (DEPs) analysis, 130 significant DEPs were identified between the healthy and atopic dermatitis groups. Among these DEPs, eight significant proteins were uniquely expressed in the atopic dermatitis group. For instance, triosephosphate isomerase (TPI) in Bifidobacteriaceae in the genus Alloscardovia and demethylmenaquinone methyltransferase (DMM) in Bacteroides were shown to potentially play metabolic functional roles related to disease. PPI network analysis revealed seven reporter proteins showing metabolic alterations between the healthy and disease groups associated with the biosynthesis of ubiquinone and other quinones as well as the energy supply. This study serves as a scaffold for microbial community-wide metabolic functional studies of the infant gut microbiome in relation to allergic disease., Competing Interests: The authors declare there are no competing interests., (©2020 Kingkaw et al.)

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., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest in the publication., (© King Abdulaziz City for Science and Technology 2020.)

Probiotic is an alternative method to treat intestinal infection disease caused by antibiotic-resistant bacteria. In this study, Lactococcus lactis KA-FF 1-4 demonstrated to have the potential to inhibit the growth of Vancomycin-resistant enterococci (VRE) by producing anti-microbial substance. In co-culture, L. lactis KA-FF 1-4 (10 8  CFU/mL) inhibited the growth of VRE from 10 3 -10 4  CFU/mL to zero after 6 h of exposure. However, in a gut model contained human gut microbiota, this anti-VRE activity of L. lactis KA-FF 1-4 was reduced to only 3.59-6.12%. The unexpected difference in efficacy between the experimental models could be explained by the fact that the growth of L. lactis KA-FF 1-4 was stable in the gut model. Leaving aside these limitations, we observed that adding L. lactis KA-FF 1-4 into the human gut model containing VRE was able to enhance microbial richness and diversity. Specifically, a higher abundance of beneficial microbes from the group of Bifidobacterium spp. and Bacteroides fragilis . L. lactis KA-FF 1-4 also enhanced the abundance of Parabacteroides , Lactococcus , and Fusobacterium and promoted the production of lactic acid in the gut model. However, these effects were not observed in the gut model without L. lactis KA-FF 1-4. Even though this study could not demonstrate a significant anti-VRE effect of the L. lactis KA-FF 1-4 in a gut model, our results still offer evidence that L. lactis KA-FF 1-4 could positively modulate the gut microbiota by promoting the growth of beneficial microbes and their metabolite. L. lactis KA-FF 1-4 has probiotic properties to fight against VRE infection, therefore further investigation in animal model is needed., Competing Interests: Conflict of interestOn behalf of all authors, the corresponding author states that there is no conflict of interest., (© King Abdulaziz City for Science and Technology 2020.)

The gut microbial diversity of Thai people was investigated between two large cohorts, adult and elderly subjects, from the middle region of Thailand; the cohorts were divided into different age groups of healthy adult ( 73 ) and elderly subjects ( 47 ) . The diversities of the groups were characterized using a pyrosequencing technique with primers targeting the V6-V8 region of the 16S rRNA gene, and a significant decrease in the Firmicutes and Bacteroidetes ratio from 7.3 to 4.5 was observed with increased age . The microbiota of the adult and elderly groups had a significantly higher abundance of the phylum Actinobacteria , including the three species Bifidobacterium adolescentis , Bifidobacterium longum and Bifidobacterium pseudocatenulatum , and the phylum Bacteroidetes containing the four species Bacteroides uniformis , Bacteroides ovatus , Bacteroides caccae and Bacteroides thetaiotaomicron . Firmicutes showed no significant differences between the two groups. Eleven species belonging to Firmicutes , Bacteroidetes and Proteobacteria were shared by at least 90% of all subjects and defined as core gut microbiota of healthy Thai, among which a high abundance of Escherichia coli was particularly characterized in Thai elderly individuals. Multiple linear regression analysis of age, gender, BMI and diet consumption frequency showed the correlation of age with Bacteroides and Bifidobacterium . Rice consumption frequency showed a significant positive correlation with Bacteroides , while no correlation was found for other factors. Taken together, in the gut of Thai adults, Bifidobacterium decreased and Bacteroides increased with age, while rice consumption increased the abundance of Bacteroides . These link of age and food, especially rice carbohydrate, to gut microbiota and health could be ultimately proposed as the Thai feature., Competing Interests: Conflict of interestThe authors declare that have no conflict of interest in the publication., (© King Abdulaziz City for Science and Technology 2020.)

Hydrolysis products of defatted copra meal (DCM) hydrolysis were investigated with either recombinant β-mannanases from Klebsiella oxytoca KUB-CW2-3 (KMAN-3) or Bacillus circulans NT 6.7 (MAN 6.7). Morphological changes and functional groups of solid residues were also determined by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. Results revealed that the Michaelis-Menten constant ( K m ) and maximum velocity ( V max ) values of KMAN-3 on DCM were 2.4 mg/ml and 5.4 U/mg, respectively, while MAN 6.7 recorded K m and V max at 2.0 mg/ml and 4.3 U/mg, respectively. Both enzymes efficiently randomly hydrolysed DCM and produced a range of different manno-oligosaccharides (MOS). The profile of hydrolysis products was different for each enzyme used. Main products from hydrolysis of DCM by KMAN-3 and MAN 6.7 were various MOS including mannobiose (M2), mannotriose (M3), mannotetraose (M4), and mannose, whereas mannopentaose (M5) was only found from KMAN-3. Amount of M3 produced by KMAN-3 was about three times higher than from MAN 6.7. Total MOS yield for KMAN-3 was 1.5-folds higher than for MAN 6.7. SEM analysis showed that enzymatic hydrolysis with KMAN-3 and MAN 6.7 resulted in deconstruction of the DCM structure which generated a variety of MOS products. FTIR spectra revealed that the properties of both hydrolysed solids were not significantly different compared to the original DCM. Results suggested that KMAN-3 was a promising candidate for production of high MOS content from copra meal., Competing Interests: Conflict of interestThe authors declare no conflict of interest., (© King Abdulaziz City for Science and Technology 2020.)

Several studies have reported a complex microbial community in human breast milk. This community impacts the shape of the infant gut microbiota and consequently impacts host health. Lactobacillus is an important probiotic and has many applications in the functional food industry. This study isolated and evaluated the potential probiotic bacteria from human milk. Two Lactobacillus species, L . plantarum and L . pentosus , were isolated from the breast milk of Thai women. L . pentosus HM04-22, L . pentosus HM04-3, L . plantarum HM04-80, L . plantarum HM04-88 and L . plantarum HM01-1 showed good adhesion activity (> 55%) and resistance in gastric (pH 2) and bile (pH 8) conditions. Characterization of the probiotic properties indicated that all selected Lactobacillus isolates had anti-adhesion properties against Escherichia coli and Salmonella Typhimurium. Lactobacillus isolates protected Caco-2 cells from pathogen adhesion at 25-40%. In addition, the five selected strains presented anti-inflammatory properties by reducing interleukin (IL)-8 expression at 0.14 ± 0.16 to 0.52 ± 0.117-fold. However, the strains had no effect on the expression of tight junction genes, including zona occludens (ZO)-1, occludin and claudin-1. In conclusion, five selected Lactobacillus isolates from human milk were candidates for use as probiotics to promote health. However, more tests in animal models and clinical trials need to be performed., Competing Interests: Conflict of interestOn behalf of all authors, the corresponding author states that there is no conflict of interest., (© King Abdulaziz City for Science and Technology 2019.)

Copra meal hydrolysate (CMH) is obtained by hydrolyzing defatted copra meal with β-mannanase from Bacillus circulans 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 Lactobacillus and Bifidobacterium groups ( P  < 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., Competing Interests: Compliance with ethical standardsThe authors declare that there are no conflicts of interest.

Enzymatic hydrolysis of palm kernel cake to improve the quality of substrates with multi-response criteria based on the Taguchi orthogonal array. Nine experimental runs were performed based on an L9 orthogonal array. Percent substrate, incubation time, and enzyme units were optimized considering multiple performance characteristics. Analysis of variance was also applied to identify the most significant factors. Results determined percent substrate as the most important factor for enzymatic hydrolysis followed by incubation time and enzyme units. Enzymatic hydrolysis conditions were optimized as percent substrate, incubation time and enzyme units at 14%, 6 h and 750 units, respectively. Tests were conducted to compare experimental and model results. The experimental result (protein release) at optimal condition were three times higher than the predicted mode., Competing Interests: Compliance with ethical standardsThe authors declare that they have no conflict of interest in the publication.

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.

The aim of this study was to carry out preliminary investigations on the in vitro fermentation selectivity of copra meal hydrolysate (CMH) by chicken gut microbiota. The ileum and cecum contents from three 35-day-old birds were used as inocula. Yeast mannooligosaccharide (yeast-MOS) or α-mannan was selected as a positive control. Batch culture fermentation with fecal bacteria was performed at 42 °C for 24 h in an anaerobic chamber. Samples were collected at 0, 6, 12, 18 and 24 h of fermentation and evaluated using real-time PCR and short-chain fatty acid (SCFA) analysis. Results showed that the medium containing ileum and both CMH and yeast-MOS substrates led to an increase in the growth of the dominant groups as Lactobacillus , Enterobacteriaceae and Enterococcus spp. compared with 0-h fermentation. Campylobacter spp. and Bifidobacterium spp. were not detected in any samples. A significant decrease in Acinetobacter was observed in all substrates tested after 6 h of fermentation ( P  < 0.05). Only the sample from CMH fermentation showed a significantly greater reduction in the population of Pseudomonas after 18-h fermentation with ileum content ( P  < 0.05). Propionate was the main fermentation product found in both ileum and cecum fermentation followed by lactate and acetate. CMH can be utilized by ileum and cecum microbial of chickens, and CMH has a generally desirable effect on the microbiota. CMH has the potential for use as a supplementary diet with similar or improved benefits and lower costs compared to commercial prebiotics. Further experiments in animal trials would seem to be justified., Competing Interests: Compliance with ethical standardsThe authors declare that there is no conflict of interest regarding publication of this paper.

Pyrosequencing analysis of intestinal microflora from healthy Thai vegetarians and non-vegetarians exhibited 893 OTUs covering 189 species. The strong species indicators of vegetarians and non-vegetarians were Prevotella copri and Bacteroides vulgatus as well as bacteria close to Escherichia hermanii with % relative abundance of 16.9 and 4.5-4.7, respectively. Core gut microbiota of the vegetarian and non-vegetarian groups consisted of 11 and 20 different bacterial species, respectively, belonging to Actinobacteria, Firmicutes, and Proteobacteria commonly found in both groups. Two species, Faecalibacterium prausnitzii and Gemmiger formicilis , had a prevalence of 100% in both groups. Three species, Clostridium nexile , Eubacterium eligens , and P. copri , showed up in most vegetarians, whereas more diversity of Collinsella aerofaciens , Ruminococcus torques , various species of Bacteroides , Parabacteroides , Escherichia , and different species of Clostridium and Eubacterium were found in most non-vegetarians. Considering the correlation of personal characters, consumption behavior, and microbial groups, the age of non-vegetarians showed a strong positive correlation coefficient of 0.54 ( p = 0.001) to Bacteroides uniformis but exhibited a moderate one to Alistipes finegoldii and B. vulgatus . Only a positive moderate correlation of body mass index and Parabacteroides distasonis appeared. Based on the significant abundance of potential pathogens, the microbiota of the non-vegetarian group showed an abundance of potential pathogen varieties of Bilophila wadsworthia , Escherichia coli , and E. hermannii , whereas that of the vegetarian group served for only Klebsiella pneumoniae . These results implied that the microbiota of vegetarians with high abundance of P. copri and low potential pathogen variety would be a way to maintain good health in Thais.

Seventy-five bacterial contaminants which still persisted to cleaning system from three puff pastry production lines (dough forming, layer and filling forming, and shock freezing) were identified using 16S rDNA as seven genera of Bacillus , Corynebacterium , Dermacoccus , Enterobacter , Klebsiella, Pseudomonas , and Staphylococcus with detection frequencies of 24.00, 2.66, 1.33, 37.33, 1.33, 2.66, and 30.66, respectively. Seventeen species were discovered while only 11 species Bacillus cereus, B. subtilis, B. pumilus, Corynebacterium striatum , Dermacoccus barathri , Enterobacter asburiae, Staphylococcus kloosii, S. haemolyticus, S. hominis, S. warneri , and S. aureus were detected at the end of production. Based on their abundance, the highest abundance of E. asburiae could be used as a biomarker for product quality. While a low abundance of the mesophile pathogen C. striatum , which causes respiratory and nervous infection and appeared only at the shock freezing step was firstly reported for its detection in bakery product. Six antimicrobial substances (AMSs) from lactic acid bacteria, FF1-4, FF1-7, PFUR-242, PFUR-255, PP-174, and nisin A were tested for their inhibition activities against the contaminants. The three most effective were FF1-7, PP-174, and nisin A exhibiting wide inhibition spectra of 88.00%, 85.33%, and 86.66%, respectively. The potential of a disinfectant solution containing 800 AU/ml of PP-174 and nisin A against the most resistant strains of Enterobacter , Staphylococcus , Bacillus and Klebsiella was determined on artificially contaminated conveyor belt coupons at 0, 4, 8, 12, and 16 hr. The survival levels of the test strains were below 1 log CFU/coupon at 0 hr. The results suggested that a combined solution of PP-174 and nisin A may be beneficial as a sanitizer to inhibit bacterial contaminants in the frozen puff pastry industry.

Lactobacillus salivarius KL-D4 isolated from duck intestine produced bacteriocin which was stable at high temperature and a wide pH range of 3-10. Its cell free supernatant at pH 5.5 exhibited wide inhibitory spectrum against both G+ and G- bacteria. The highest bacteriocin production was obtained in MRS broth supplemented with 0.5 % (w/v) CaCO3 at 6 h by gentle shaking. PCR walking using specific primers at the conserved region of class-II bacteriocin resulted in 4 known genes of kld1, kld2, kld3 and kld4 with 100 % similarity to genes encoding for salivaricin α, β, induction peptide and histidine protein kinase of Lb. salivarius GJ-24 which did not previously report for bacteriocin characterization, while showing 94, 93, 59 and 62 % to other salivaricin gene cluster, respectively. The high activities of 25,600 AU/ml indicated a strong induction peptide expressed by kld3 which has low similarity to previous inducer reported. Based on operon analysis, only kld1, kld3 and kld4 could be expressed and subsequently elucidated that only salivaricin α like bacteriocin was produced and secreted out of the cells. Using protein purification, only a single peptide band obtained showed that this strain produced one bacteriocin which could be salivaricin α namely salivaricin KLD showing about 4.3 kDa on SDS-PAGE. Partial purification by 20 % ammonium sulfate precipitation of the product was tested on the artificial contamination of creamy filling by Bacillus cereus, Enterococcus faecalis, Pseudomonas stutzeri, Staphylococcus sp. and Stenotrophomonas sp. resulting the growth inhibitory efficiency of 4.45-66.9, 11.5-100, 100, 0-28.1 and 5-100 % respectively. Therefore, salivaricin KLD can be a tentative biopreservative for food industry in the future.

Feeding trials were designed to evaluate the nutritive value of feather meal treated by K6 and K82 keratinase. There were five treatments in feather meal preparation: CFM (non-enzymatically treated feather meal), K6FM (K6 keratinase treated feather meal), K82FM (K82 keratinase treated feather meal), K6:K82FM [K6 and K82 keratinase (5:1) treated feather meal] and CMFM (commercial enzyme treated feather meal). The pepsin digestibility of CFM (70 %) and CMFM (68 %) was significantly higher than K6FM (60 %), K82FM (61 %) and K6:K82FM (63 %). Total amino acid content of K82FM (89.65/100 g) was the highest compared with the other treatments. The nutrient digestibility of the feather meals was determined for broiler chicks between 21 and 27 days old. The apparent nitrogen retention of K82FM (85.82 %) and K6FM (77.31 %) was significantly higher than K6:K82FM (55.42 %), CMFM (45.70 %) and CFM (48.16 %). The apparent metabolisable energy (AMEn) was not significantly different between the feather meal treatments, although K82FM, K6FM and K6:K82FM showed AMEn higher than CMFM and CFM. The results indicated that both K6 and K82 keratinase had a positive effect on the protein quality of the feather meal produced by the enzymatic-hydrothermal method.

Gene encoding for β-mannanase (E.C 3.2.1.78) from Klebsiella oxytoca KUB-CW2-3 was cloned and expressed by an E. coli system resulting in 400 times higher mannanase activities than the wild type. A 3314bp DNA fragment obtained revealed an open reading frame of 1164bp, namely kman-2, which encoded for 387 amino acids with an estimated molecular weight of 43.2kDa. It belonged to the glycosyl hydrolase family 26 (GH26) exhibited low similarity of 50-71% to β-mannanase produced by other microbial sources. Interestingly, the enzyme had a broad range of substrate specificity of homopolymer of ivory nut mannan (6%), carboxymethyl cellulose (30.6%) and avicel (5%), and heteropolymer of konjac glucomannan (100%), locust bean gum (92.6%) and copra meal (non-defatted 5.3% and defatted 7%) which would be necessary for in vivo feed digestion. The optimum temperature and pH were 30-50°C and 4-6, respectively. The enzyme was still highly active over a low temperature range of 10-40°C and over a wide pH range of 4-10. The hydrolysates of konjac glucomannan (H-KGM), locust bean gum (H-LBG) and defatted copra meal (H-DCM) composed of compounds which were different in their molecular weight range from mannobiose to mannohexaose and unknown oligosaccharides indicating the endo action of mannanase. Both H-DCM and H-LBG enhanced the growth of lactic acid bacteria and some pathogens except Escherichia coli E010 with a specific growth rate of 0.36-0.83h(-1). H-LBG was more specific to 3 species of Weissella confusa JCM 1093, Lactobacillus reuteri KUB-AC5, Lb salivarius KL-D4 and E. coli E010 while both H-KGM and H-DCM were to Lb. reuteri KUB-AC5 and Lb. johnsonii KUNN19-2. Based on the nucleotide sequence of kman-2 containing two open reading frames of 1 and 2at 5' end of the +1 and +43, respectively, removal of the first open reading frame provided the recombinant clone E. coli KMAN-3 resulting in the mature protein of mannanase composing of 345 amino acid residues confirmed by 3D structure analysis and amino acid sequence at N-terminal namely KMAN (GenBank accession number KM100456). It exhibited 10 times higher extracellular and periplasmic total activities of 17,600 and 14,800 units than E. coli KMAN-2. With its low similarity to mannanases previously proposed, wide range of homo- and hetero-polysaccharide specificity, negative effect to E. coli and most importance of high production, it would be proposed as a novel mannanase source for application in the future., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This study focused on the characterization of mannanase from Bacillus circulans NT 6.7 for mannooligosaccharides (MOS) production. The enzyme from B. circulans NT 6.7 was produced using defatted copra meal as a carbon source. The mannanase was purified by ultrafiltration and column chromatography of Q-Sepharose. The purified protein (M1) was a dimeric protein with a 40 kDa subunit. The purified M1 exhibited optimum pH and temperature at pH 6.0 and 60 °C, respectively. It was activated by Mn(2+,) Mg(2+,) and Cu(2+), and as inhibited by EDTA (45-65 %). The purified enzyme exhibited high specificity to beta-mannan: konjac (glucomannan), locust bean gum (galactomannan), ivory nut (mannan), guar gum (galactomannan) and defatted copra meal (galactomannan). The defatted copra meal could be hydrolyzed by purified M1 into mannooligosaccharides which promoted beneficial bacteria, especially Lactobacillus group, and inhibited pathogenic bacteria; Shigella dysenteria DMST 1511, Staphylococcus aureus TISTR 029, and Salmonella enterica serovar Enteritidis DMST 17368. Therefore, the mannanase from B. circulans NT 6.7 would be a novel source of enzymes for the mannooligosaccharides production as prebiotics.

Asia differs substantially among and within its regions populated by diverse ethnic groups, which maintain their own respective cultures and dietary habits. To address the diversity in their gut microbiota, we characterized the bacterial community in fecal samples obtained from 303 school-age children living in urban or rural regions in five countries spanning temperate and tropical areas of Asia. The microbiota profiled for the 303 subjects were classified into two enterotype-like clusters, each driven by Prevotella (P-type) or Bifidobacterium/Bacteroides (BB-type), respectively. Majority in China, Japan and Taiwan harbored BB-type, whereas those from Indonesia and Khon Kaen in Thailand mainly harbored P-type. The P-type microbiota was characterized by a more conserved bacterial community sharing a greater number of type-specific phylotypes. Predictive metagenomics suggests higher and lower activity of carbohydrate digestion and bile acid biosynthesis, respectively, in P-type subjects, reflecting their high intake of diets rich in resistant starch. Random-forest analysis classified their fecal species community as mirroring location of resident country, suggesting eco-geographical factors shaping gut microbiota. In particular, children living in Japan harbored a less diversified microbiota with high abundance of Bifidobacterium and less number of potentially pathogenic bacteria, which may reflect their living environment and unique diet.

The mannanase gene of B. circulans NT 6.7 was cloned and expressed in an Escherichia coli expression system. The B. circulans NT 6.7 mannanase gene consists of 1,083 nucleotides encoding a 360-amino acid residue long polypeptide, belonging to glycoside hydrolase family 26. The full-length mannanase gene including its native signal sequence was cloned into the vector pET21d and expressed in E. coli BL21 (DE3). β-Mannanase activities in the culture supernatant and crude cell extract were 37.10 and 515 U per ml, respectively, with most of the activity in the cell extract attributed to the periplasmic fraction. In contrast, expression of mannanase was much lower when using the B. circulans NT 6.7 mannanase gene without its signal sequence. The optimum temperature of recombinant β-mannanase activity was 50°C and the optimum pH was 6.0. The enzyme was very specific for β-mannan substrates with a preference for galactomannan. Hydrolysis products of locust bean gum were various mannooligosaccharides including mannohexaose, mannopentaose, mannotetraose, mannotriose and mannobiose, while mannose could not be detected. In conclusion, this expression system is efficient for the secretory production of recombinant β-mannanase from B. circulans NT 6.7, which shows good characteristics for various applications.

The fecal microbiotas were investigated in 13 healthy Thai subjects using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Among the 186 DNA bands detected on the polyacrylamide gel, 37 bands were identified as representing 11 species: Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides uniformis, Bacteroides vulgatus, Clostridium colicanis, Eubacterium eligenes, E. rectale, Faecalibacterium prausnitzii, Megamonas funiformis, Prevotella copri, and Roseburia intestinalis, belonging mainly to the groups of Bacteroides, Prevotella, Clostridium, and F. prausnitzii. A dendrogram of the PCR-DGGE divided the subjects; vegetarians and non-vegetarians. The fecal microbiotas were also analyzed using a quantitative real-time PCR focused on Bacteroides, Bifidobacterium, Enterobacteriaceae, Clostrium coccoides-Eubacterium rectale, C. leptum, Lactobacillus, and Prevotella. The nonvegetarian and vegetarian subjects were found to have significant differences in the high abundance of the Bacteroides and Prevotella genera, respectively. No significant differences were found in the counts of Bifidabacterium, Enterobacteriaceae, C. coccoides-E. rectale group, C. leptum group, and Lactobacillus. Therefore, these findings on the microbiota of healthy Thais consuming different diets could provide helpful data for predicting the health of South East Asians with similar diets.

A bacteriocin-producing strain, N1-33, isolated from fermented bamboo shoot was identified as Enterococcus faecalis. The pH-adjusted culture supernatant of this strain consisted of several peptides with bacteriocin activity, and the supernatant inhibited the growth of pathogenic bacteria such as Listeria monocytogenes. The major peptide with bacteriocin activity was purified, and the first 39 amino acid residues of the bacteriocin were found to be identical to enterocin MR10A produced by E. faecalis MRR10-3. Addition of the pH-adjusted and concentrated culture supernatant of strain N1-33 caused a marked reduction in the growth of Bacillus cereus in custard cream and L. monocytogenes in pickled cucumber. These results suggest the potential use of the bacteriocin produced by strain N1-33 in food biopreservation.

Tempe was prepared using mixtures of natural soybean and genetically modified Roundup Ready (RUR) soybean fermented with natural Rhizopus sp. The amount of RUR soybean was quantified using an ELISA plate test. The RUR signal decreased during fermentation. In the control experiments on fermentation of non-RUR soybean, the tempe gave a false-positive RUR signal. The cross-reacting substance was generated only in non-RUR soybean during fermentation by Rhizopus sp., Rhizopus oligosporus, R. oryzae, Mucor rouxii and Aspergillus awamori.

Site directed mutagenesis has been performed to test hypotheses concerning the putative active sites of Trichoderma reesei cellobiohydrolase I and endoglucanase I. It is shown that mutagenesis of the residue E126, previously proposed to be the proton donor in CBHI, did not totally inactivate the enzyme while mutagenesis of the residue E127 in the homologous enzyme EGI resulted in complete loss of activity. These results are compared with those obtained in similar studies of other glucanases and the effects on enzymatic activity of hyperglycosylation of the yeast produced cellulases are discussed.