The Effect of Human Milk Oligosaccharides and Bifidobacterium longum subspecies infantis Bi-26 on Simulated Infant Gut Microbiome and Metabolites.

Human milk oligosaccharides (HMOs) shape the developing infant gut microbiota. In this study, a semi-continuous colon simulator was used to evaluate the effect of 2 HMOs-2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL)-on the composition of infant faecal microbiota and microbial metabolites. The simulations were performed with and without a probiotic Bifidobacterium longum subspecies infantis Bi-26 (Bi-26) and compared with a control that lacked an additional carbon source. The treatments with HMOs decreased α-diversity and increased Bifidobacterium species versus the control, but the Bifidobacterium species differed between simulations. The levels of acetic acid and the sum of all short-chain fatty acids (SCFAs) trended toward an increase with 2'-FL, as did lactic acid with 2'-FL and 3-FL, compared with control. A clear correlation was seen between the consumption of HMOs and the increase in SCFAs (-0.72) and SCFAs + lactic acid (-0.77), whereas the correlation between HMO consumption and higher total bifidobacterial numbers was moderate (-0.46). Bi-26 decreased propionic acid levels with 2'-FL. In conclusion, whereas infant faecal microbiota varied between infant donors, the addition of 2'-FL and 3-FL, alone or in combination, increased the relative abundance and numbers Bifidobacterium species in the semi-continuous colon simulation model, correlating with the production of microbial metabolites. These findings may suggest that HMOs and probiotics benefit the developing infant gut microbiota.

Regulation of hBD-2, hBD-3, hCAP18/LL37, and Proinflammatory Cytokine Secretion by Human Milk Oligosaccharides in an Organotypic Oral Mucosal Model.

Human milk oligosaccharides (HMOs), the third largest solid fraction in human milk, can modulate inflammation through Toll-like receptor signaling, but little is known about their immunomodulatory potential in the oral cavity. In this study, we determined whether the HMOs 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL) regulate human-beta defensin (hBD)-2 and -3, cathelicidin (hCAP18/LL-37), and cytokine responses in human gingival cells using a three-dimensional oral mucosal culture model. The model was incubated with 0.1% or 1% 2'-FL and 3-FL, alone and in combination, for 5 or 24 h, and hBD-2, hBD-3, and hCAP18/LL-37 were analyzed by immunohistochemistry. The expression profiles of interleukin (IL)-1, IL-1RA, IL-8, and monocyte chemoattractant protein (MCP)-1 were determined by LUMINEX immunoassay. The combination of 1% 2'-FL and 1% 3-FL, and 1% 3-FL alone, for 24 h upregulated hBD-2 protein expression significantly (p < 0.001 and p = 0.016, respectively). No changes in the other antimicrobial peptides or proinflammatory cytokines were observed. Thus, 3-FL, alone and in combination with 2'-FL, stimulates oral mucosal secretion of hBD-2, without effecting a proinflammatory response when studied in an oral mucosal culture model.

Selective Utilization of the Human Milk Oligosaccharides 2'-Fucosyllactose, 3-Fucosyllactose, and Difucosyllactose by Various Probiotic and Pathogenic Bacteria.

Prebiotic human milk oligosaccharides (HMOs) are found in human milk, which are not digested by infants but are metabolized by beneficial gut bacteria. We determined the ability of 57 bacterial strains within the Family Lactobacillaceae and genera Bifidobacterium and Bacteroides and potentially pathogenic bacteria to ferment the HMOs 2'-fucosyllactose, 3-fucosyllactose, and difucosyllactose. In addition, prebiotic galacto-oligosaccharides (GOS), lactose, fucose, and glucose were evaluated as carbon sources for these bacterial strains. Bacterial growth was monitored using the automatic Bioscreen C system. Only certain bifidobacteria, such as Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum, as well as Bacteroides fragilis, Bacteroides vulgatus, and Bacteroides thetaiotaomicron utilized the studied HMOs as their sole carbon source, whereas almost all studied bacterial strains were able to utilize GOS, lactose, and glucose. The selectivity in utilization of HMOs by only certain bacteria can be advantageous by promoting beneficial microbes but not supporting the harmful pathogens in contrast to other less selective prebiotics.

The effect of 2'-fucosyllactose on simulated infant gut microbiome and metabolites; a pilot study in comparison to GOS and lactose.

Human milk oligosaccharides (HMOs) shape gut microbiota during infancy by acting as fermentable energy source. Using a semi-continuous colon simulator, effect of an HMO, 2'-fucosyllactose (2'-FL), on composition of the infant microbiota and microbial metabolites was evaluated in comparison to galacto-oligosaccharide (GOS) and lactose and control without additional carbon source. Data was analysed according to faecal sample donor feeding type: breast-fed (BF) or formula-fed (FF), and to rate of 2'-FL fermentation: fast or slow. Variation was found between the simulations in the ability to utilise 2'-FL. The predominant phyla regulated by 2'-FL, GOS and lactose were significant increase in Firmicutes, numerical in Actinobacteria, and numerical decrease in Proteobacteria compared to control. Verrucomicrobia increased in FF accounted for Akkermansia, whereas in fast-fermenting simulations Actinobacteria increased with trend for higher Bifidobacterium, and Proteobacteria decrease accounted for Enterobacteriaceae. Short-chain fatty acids and lactic acid with 2'-FL were produced in intermediate levels being between ones generated by the control and GOS or lactose. In 2'-FL fast-fermenting group, acetic acid specifically increased with 2'-FL, whereas lactose and GOS also increased lactic acid. The results highlight specificity of 2'-FL as energy source for only certain microbes over GOS and lactose in the simulated gut model.

Xylitol's Health Benefits beyond Dental Health: A Comprehensive Review.

Xylitol has been widely documented to have dental health benefits, such as reducing the risk for dental caries. Here we report on other health benefits that have been investigated for xylitol. In skin, xylitol has been reported to improve barrier function and suppress the growth of potential skin pathogens. As a non-digestible carbohydrate, xylitol enters the colon where it is fermented by members of the colonic microbiota; species of the genus Anaerostipes have been reported to ferment xylitol and produce butyrate. The most common Lactobacillus and Bifidobacterium species do not appear to be able to grow on xylitol. The non-digestible but fermentable nature of xylitol also contributes to a constipation relieving effect and improved bone mineral density. Xylitol also modulates the immune system, which, together with its antimicrobial activity contribute to a reduced respiratory tract infection, sinusitis, and otitis media risk. As a low caloric sweetener, xylitol may contribute to weight management. It has been suggested that xylitol also increases satiety, but these results are not convincing yet. The benefit of xylitol on metabolic health, in addition to the benefit of the mere replacement of sucrose, remains to be determined in humans. Additional health benefits of xylitol have thus been reported and indicate further opportunities but need to be confirmed in human studies.

Novel Genes and Metabolite Trends in Bifidobacterium longum subsp. infantis Bi-26 Metabolism of Human Milk Oligosaccharide 2'-fucosyllactose.

Human milk oligosaccharides (HMOs) function as prebiotics for beneficial bacteria in the developing gut, often dominated by Bifidobacterium spp. To understand the relationship between bifidobacteria utilizing HMOs and how the metabolites that are produced could affect the host, we analyzed the metabolism of HMO 2'-fucosyllactose (2'-FL) in Bifidobacterium longum subsp. infantis Bi-26. RNA-seq and metabolite analysis (NMR/GCMS) was performed on samples at early (A600 = 0.25), mid-log (0.5-0.7) and late-log phases (1.0-2.0) of growth. Transcriptomic analysis revealed many gene clusters including three novel ABC-type sugar transport clusters to be upregulated in Bi-26 involved in processing of 2'-FL along with metabolism of its monomers glucose, fucose and galactose. Metabolite data confirmed the production of formate, acetate, 1,2-propanediol, lactate and cleaving of fucose from 2'-FL. The formation of acetate, formate, and lactate showed how the cell uses metabolites during fermentation to produce higher levels of ATP (mid-log compared to other stages) or generate cofactors to balance redox. We concluded that 2'-FL metabolism is a complex process involving multiple gene clusters, that produce a more diverse metabolite profile compared to lactose. These results provide valuable insight on the mode-of-action of 2'-FL utilization by Bifidobacterium longum subsp. infantis Bi-26.

Effects of Xylitol and Sucrose Mint Products on Streptococcus mutans Colonization in a Dental Simulator Model.

Few laboratory methods exist for evaluating the cariogenicity of food ingredients. In this study, a dental simulator was used to determine the effects of commercial sucrose and xylitol mint products on the adherence and planktonic growth of Streptococcus mutans. Solutions (3% w/v) of sucrose, xylitol, sucrose mints, xylitol mints, xylitol with 0.02% peppermint oil (PO), and 0.02% PO alone were used to test the levels of planktonic and adhered S. mutans. A dental simulator with continuous artificial saliva flow, constant temperature, and mixing was used as a test environment and hydroxyapatite (HA) discs were implemented into the model to simulate the tooth surface. Bacterial content was quantified by qPCR. Compared with the artificial saliva alone, sucrose and sucrose mints increased the numbers of HA-attached S. mutans, whereas xylitol decreased them. Similarly, planktonic S. mutans quantities rose with sucrose and declined with xylitol and xylitol mints. Versus sucrose mints, xylitol mints significantly reduced the counts of HA-bound and planktonic S. mutans. Similar results were observed with the main ingredients of both types of mints separately. PO-supplemented artificial saliva did not influence the numbers of S. mutans that attached to HA or planktonic S. mutans compared with artificial saliva control. In our dental simulator model, xylitol reduced the counts of adhering and planktonic S.mutans. The mints behaved similarly as their pure, main ingredients-sucrose or xylitol, respectively. PO, which has been suggested to have antimicrobial properties, did not influence S. mutans colonization.

Novel 6p21.3 Risk Haplotype Predisposes to Acute Coronary Syndrome.

BACKGROUND: The HLA-DRB1*01 allele of the human leukocyte antigen has been associated with acute coronary syndrome. Genome-wide association studies have revealed associations with human leukocyte antigen and non-human leukocyte antigen genes of 3 major histocompatibility complex gene classes but not at allelic level. METHODS AND RESULTS: We conducted a large-scale genetic analysis on a case-control cohort comprising 5376 acute coronary syndrome cases and 4852 unrelated controls from 4 populations of 2 European countries. We analyzed the risk candidate allele of HLA-DRB1*01 by genomic real-time polymerase chain reaction together with high-density single nucleotide polymorphisms of the major histocompatibility complex to precisely identify risk loci for acute coronary syndrome with effective clinical implications. We found a risk haplotype for the disease containing single nucleotide polymorphisms from BTNL2 and HLA-DRA genes and the HLA-DRB1*01 allele. The association of the haplotype appeared in 3 of the 4 populations, and the direction of the effect was consistent in the fourth. Coronary samples from subjects homozygous for the disease-associated haplotype showed higher BTNL2 mRNA levels (r=0.760; P<0.00001).We localized, with immunofluorescence staining, BTNL2 in CD68-positive macrophages of the coronary artery plaques. In homozygous cases, BTNL2 blocking, in T-cell stimulation assays, enhanced CD4(+)FOXP3(+) regulatory T cell proliferation significantly (blocking versus nonblocking; P<0.05). CONCLUSIONS: In cases with the risk haplotype for acute coronary syndrome, these results suggest involvement of enhanced immune reactions. BTNL2 may have an inhibitory effect on FOXP3(+) T cell proliferation, especially in patients homozygous for the risk alleles. CLINICAL TRIAL REGISTRATION: https://www.clinicaltrials.gov; Unique Identifier: NCT00417534.

The use of in vitro model systems to study dental biofilms associated with caries: a short review.

A dental biofilm forms a distinct environment where microorganisms live in a matrix of extracellular polysaccharides. The biofilm favors certain bacteria and creates a habitat that functions differently compared to planktonic bacteria. Reproducible model systems which help to address various questions related to biofilm formation, the process of caries development, and its prevention are needed and are continuously developed. Recent research using both batch culture, continuous culture and flow cells in caries biofilm formation is presented. The development of new techniques and equipment has led to a deeper understanding of how caries biofilms function. Biofilm models have also been used in the development of materials inhibiting secondary caries. This short review summarizes available models to study these questions.

Postprandial effects of polydextrose on satiety hormone responses and subjective feelings of appetite in obese participants.

BACKGROUND: Dietary fibers are associated with enhanced satiety. However, the mechanism of different dietary fibers contributing to satiety-related gastrointestinal (GI) peptide release, especially in an obese population, is still poorly understood. Polydextrose (PDX), a water-soluble glucose polymer, has demonstrated its ability to reduce energy intake at a subsequent meal, but its mechanism of action requires further research. Also, there is limited evidence on its capacity to regulate subjective feelings of appetite. This study examines the effects of PDX on postprandial secretion of satiety-related GI peptides, short chain fatty acids (SCFAs), lactic acid, and subjective appetite ratings in obese participants. METHODS: 18 non-diabetic, obese participants (42.0 y, 33.6 kg/m2) consumed a high-fat meal (4293 kJ, 36% from fat) with or without PDX (15 g) in an acute, multicenter, randomized, double-blind, placebo-controlled and crossover trial. Postprandial plasma concentrations of satiety-related peptides, namely ghrelin, cholecystokinin (CCK), glucagon-like peptide 1 (GLP-1), and peptide YY (PYY), as well as SCFAs and lactic acid were assessed. GI peptide, SCFA and lactate concentrations were then modeled using a linear mixed-effects model.The subjective feelings of hunger, satisfaction, and desire to eat were evaluated using visual analogue scales (VAS), which were analyzed as incremental areas under the curve (iAUC) during the satiation and satiety periods. RESULTS: We found that PDX supplementation increased plasma GLP-1 levels more than the placebo treatment (P = 0.02). In the whole group, GLP-1 concentrations found in participants older than 40 years old were significantly lower (P = 0.01) as compared to those aged 40 years or less. There were no statistically significant differences in postprandial ghrelin, CCK, or PYY responses. The lactic acid concentrations were significantly (P = 0.01) decreased in the PDX group, while no significant changes in SCFAs were found. PDX reduced iAUC for hunger by 40% (P = 0.03) and marginally increased satisfaction by 22.5% (P = 0.08) during the post-meal satiety period. CONCLUSION: Polydextrose increased the postprandial secretion of the satiety hormone GLP-1 and reduced hunger after a high-fat meal. PDX also reduced the elevated postprandial lactic acid levels in plasma. Therefore, PDX may offer an additional means to regulate inter-meal satiety and improve postprandial metabolism in obese participants.

Comparison of bacterial quantities in left and right colon biopsies and faeces.

AIM: To compare quantities of predominant and pathogenic bacteria in mucosal and faecal samples. METHODS: Twenty patients undergoing diagnostic colonoscopy with endoscopically and histologically normal mucosa were recruited to the study, 14 subjects of which also supplied faecal (F) samples between 15 d to 105 d post colonoscopy. Mucosal biopsies were taken from each subject from the midportion of the ascending colon (right side samples, RM) and the sigmoid (left side samples, LM). Predominant intestinal and mucosal bacteria including clostridial 16S rRNA gene clusters IV and XIVab, Bacteroidetes, Enterobacteriaceae, Bifidobacterium spp., Akkermansia muciniphila (A. muciniphila), Veillonella spp., Collinsella spp., Faecalibacterium prausnitzii (F. prausnitzii) and putative pathogens such as Escherichia coli (E. coli), Clostridium difficile (C. difficile), Helicobacter pylori (H. pylori) and Staphylococcus aureus (S. aureus) were analysed by quantitative polymerase chain reaction (qPCR). Host DNA was quantified from the mucosal samples with human glyceraldehyde 3-phosphate dehydrogenase gene targeting qPCR. Paired t tests and the Pearson correlation were applied for statistical analysis. RESULTS: The most prominent bacterial groups were clostridial groups IV and XIVa+b and Bacteroidetes and bacterial species F. prausnitzii in both sample types. H. pylori and S. aureus were not detected and C. difficile was detected in only one mucosal sample and three faecal samples. E. coli was detected in less than half of the mucosal samples at both sites, but was present in all faecal samples. All detected bacteria, except Enterobacteriaceae, were present at higher levels in the faeces than in the mucosa, but the different locations in the colon presented comparable quantities (RM, LM and F followed by P(1) for RM vs F, P(2) for LM vs F and P(3) for RM vs LM: 4.17 ± 0.60 log(10)/g, 4.16 ± 0.56 log(10)/g, 5.88 ± 1.92 log(10)/g, P(1) = 0.011, P(2) = 0.0069, P(3) = 0.9778 for A. muciniphila; 6.25 ± 1.3 log(10)/g, 6.09 ± 0.81 log(10)/g, 8.84 ± 1.38 log(10)/g, P(1) < 0.0001, P(2) = 0.0002, P(3) = 0.6893 for Bacteroidetes; 5.27 ± 1.68 log(10)/g, 5.38 ± 2.06 log(10)/g, 8.20 ± 1.14 log(10)/g, P(1) < 0.0001, P(2) ≤ 0.0001, P(3) = 0.7535 for Bifidobacterium spp.; 6.44 ± 1.15 log(10)/g, 6.07 ±1.45 log(10)/g, 9.74 ±1.13 log(10)/g, P(1) < 0.0001, P(2) ≤ 0.0001, P(3) = 0.637 for Clostridium cluster IV; 6.65 ± 1.23 log(10)/g, 6.57 ± 1.52 log(10)/g, 9.13 ± 0.96 log(10)/g, P(1) < 0.0001, P(2) ≤ 0.0001, P(3) = 0.9317 for Clostridium cluster XIVa; 4.57 ± 1.44 log(10)/g, 4.63 ± 1.34 log(10)/g, 7.05 ± 2.48 log(10)/g, P(1) = 0.012, P(2) = 0.0357, P(3) = 0.7973 for Collinsella spp.; 7.66 ± 1.50 log(10)/g, 7.60 ± 1.05 log(10)/g, 10.02 ± 2.02 log(10)/g, P(1) ≤ 0.0001, P(2) = 0.0013, P(3) = 0.9919 for F. prausnitzsii; 6.17 ± 1.3 log(10)/g, 5.85 ± 0.93 log(10)/g, 7.25 ± 1.01 log(10)/g, P(1) = 0.0243, P(2) = 0.0319, P(3) = 0.6982 for Veillonella spp.; 4.68 ± 1.21 log(10)/g, 4.71 ± 0.83 log(10)/g, 5.70 ± 2.00 log(10)/g, P(1) = 0.1927, P(2) = 0.0605, P(3) = 0.6476 for Enterobacteriaceae). The Bifidobacterium spp. counts correlated significantly between mucosal sites and mucosal and faecal samples (Pearson correlation coefficients 0.62, P = 0.040 and 0.81, P = 0.005 between the right mucosal sample and faeces and the left mucosal sample and faeces, respectively). CONCLUSION: Non-invasive faecal samples do not reflect bacterial counts on the mucosa at the individual level, except for bifidobacteria often analysed in probiotic intervention studies.

Modulation of dimer stability in yeast pyrophosphatase by mutations at the subunit interface and ligand binding to the active site.

Yeast (Saccharomyces cerevisiae) pyrophosphatase (Y-PPase) is a tight homodimer with two active sites separated in space from the subunit interface. The present study addresses the effects of mutation of four amino acid residues at the subunit interface on dimer stability and catalytic activity. The W52S variant of Y-PPase is monomeric up to an enzyme concentration of 300 microm, whereas R51S, H87T, and W279S variants produce monomer only in dilute solutions at pH > or = 8.5, as revealed by sedimentation, gel electrophoresis, and activity measurements. Monomeric Y-PPase is considerably more sensitive to the SH reagents N-ethylmaleimide and p-hydroxymercurobenzosulfonate than the dimeric protein. Additionally, replacement of a single cysteine residue (Cys(83)), which is not part of the subunit interface or active site, with Ser resulted in insensitivity of the monomer to SH reagents and stabilization against spontaneous inactivation during storage. Active site ligands (Mg(2+) cofactor, P(i) product, and the PP(i) analog imidodiphosphate) stabilized the W279S dimer versus monomer predominantly by decreasing the rate of dimer to monomer conversion. The monomeric protein exhibited a markedly increased (5-9-fold) Michaelis constant, whereas k(cat) remained virtually unchanged, compared with dimer. These results indicate that dimerization of Y-PPase improves its substrate binding performance and, conversely, that active site adjustment through cofactor, product, or substrate binding strengthens intersubunit interactions. Both effects appear to be mediated by a conformational change involving the C-terminal segment that generally shields the Cys(83) residue in the dimer.