Bifidobacterial Growth Research Articles

Anti-bacterial effects, and metabolites derived from bifidobacterial fermentation of an exopolysaccharide of Cs-HK1 medicinal fungus

This study was to investigate the antibacterial effects and metabolites derived from bifidobacterial fermentation of an exopolysaccharide EPS-LM produced by a medicinal fungus Cordyceps sinensis, Cs-HK1. EPS-LM was a partially purified polysaccharide fraction which was mainly composed of Man, Glc and Gal at 7.31:12.95:1.00 mol ratio with a maximum molecular weight of 360 kDa. After fermentation of EPS-LM in two bifidobacterial cultures, B. breve and B. longum, the culture digesta showed significant antibacterial activities, inhibiting the proliferation and biofilm formation of Escherichia coli. Based on untargeted metabolomic profiling of the digesta, the levels of short chain fatty acids, carboxylic acids, benzenoids and their derivatives were all increased significantly (p < 0.01), which probably contributed to the enhanced antibacterial activity by EPS-LM. Since EPS-LM was only slightly consumed for the bifidobacterial growth, it mainly stimulated the biosynthesis of bioactive metabolites in the bifidobacterial cells. The results also suggested that EPS-LM polysaccharide may have a regulatory function on the bifidobacterial metabolism leading to production of antibacterial metabolites, which may be of significance for further exploration.

Supplemented Infant Formula and Human Breast Milk Show Similar Patterns in Modulating Infant Microbiota Composition and Function In Vitro.

The gut microbiota of healthy breastfed infants is often dominated by bifidobacteria. In an effort to mimic the microbiota of breastfed infants, modern formulas are fortified with bioactive and bifidogenic ingredients. These ingredients promote the optimal health and development of infants as well as the development of the infant microbiota. Here, we used INFOGEST and an in vitro batch fermentation model to investigate the gut health-promoting effects of a commercial infant formula supplemented with a blend containing docosahexaenoic acid (DHA) (20 mg/100 kcal), polydextrose and galactooligosaccharides (PDX/GOS) (4 g/L, 1:1 ratio), milk fat globule membrane (MFGM) (5 g/L), lactoferrin (0.6 g/L), and Bifidobacterium animalis subsp. lactis, BB-12 (BB-12) (106 CFU/g). Using fecal inoculates from three healthy infants, we assessed microbiota changes, the bifidogenic effect, and the short-chain fatty acid (SCFA) production of the supplemented test formula and compared those with data obtained from an unsupplemented base formula and from the breast milk control. Our results show that even after INFOGEST digestion of the formula, the supplemented formula can still maintain its bioactivity and modulate infants' microbiota composition, promote faster bifidobacterial growth, and stimulate production of SCFAs. Thus, it may be concluded that the test formula containing a bioactive blend promotes infant gut microbiota and SCFA profile to something similar, but not identical to those of breastfed infants.

Trackability of proteins from probiotic Bifidobacterium spp. in the gut using metaproteomics.

Beneficial effects of Bifidobacterium spp. on gut microbiota development and infant health have been reported earlier. Therefore, supplementation of infant formulas with probiotic bifidobacteria, as well as prebiotics stimulating bifidobacterial growth, has been proposed. Here, we studied the faecal microbiome of infants supplemented with specialized nutrition, of which some received a standard amino acid-based formula (AAF) and others an AAF with a specific mixture of prebiotics and a probiotic (synbiotics) using metaproteomics and 16S rRNA gene sequencing. Faecal samples were taken at baseline, as well as after 6 and 12 months fed with the specialized formula. The aim was to compare microbial differences between infants treated with the standard AAF and those who received the additional synbiotics. Our findings show that infants who received AAF with synbiotics have significantly higher levels of Bifidobacteriaceae DNA as well as significantly increased levels of Coriobacteriaceae proteins, over time. Moreover, at visit 12 months higher levels of some bifidobacterial carbohydrate-active enzymes, known to metabolize oligosaccharides, were observed in the synbiotic group compared to the non-synbiotic group. The results indicate that the synbiotics provided in our study are trackable in faecal samples on the DNA and protein level during the intervention period.

Regionalized analysis of gut barrier components in the small intestine of BALB/c mice following chronic immobilization stress.

Microbiota and tight junction proteins (TJPs) are components of the gut barrier, and are considered stress targets that have deleterious effects on intestinal homeostasis. This study aimed to evaluate the effects of chronic immobilization stress on selected small intestine homeostasis parameters. Female BALB/c mice were divided into a stress group that underwent short-term immobilization for 2 h per day for 4 consecutive days, and a non-stressed control group (n = 6 per group). Proximal and distal small intestine samples were excised to assess colony-forming units per gram (CFU/g) of total bifidobacteria in selective agar plates, luminal albumin was assessed using immune-enzymatic assay, pro-inflammatory cytokines were evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and TJPs (pore-forming, claudin (Cld)-2; pore-sealing, Cld-4; ambiguous, Cld-7, -12 and -15) were assessed with RT-qPCR and western blotting. Compared with the control group, the stress group had lower body weight and energy intake. In the distal region, the stressed mice had lower bifidobacteria count and messenger ribonucleic acid (mRNA) expression of Cld-2, Cld-4 and Cld-12, though they had more albumin and higher interleukin (IL)-6 mRNA expression. Within the proximal region, the stressed mice had higher mRNA expression of tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), IL-6, Cld-7, Cld-12, and Cld-15, along with lower levels of IL-10 and Cld-4. However, mRNA and protein expression of TJPs were discordant. These findings indicate divergent stress-induced outcomes in the small intestine, evidenced by the elicitation of a pro-inflammatory response and decreased anti-inflammatory response in the duodenum, and by increased albumin transudation and decreased bifidobacterial growth in the distal region.

Characterization of peptides available to different bifidobacteria

Nitrogen source is a limiting factor for bifidobacterial growth. It is generally believed that the nitrogen metabolism system varies a lot in bifidobacteria, which has a low utilization efficiency of nitrogen sources in the medium. This study aimed to analyze the rules of peptide utilization and to improve the utilization efficiency of nitrogen sources in bifidobacteria. Casein hydrolysates were prepared to assess their growth-promoting effects on different Bifidobacterium. The quantity, molecular weight, and amino acid composition of transportable peptides in each strain were analyzed by ultrafiltration, gel chromatography, and nano-LC-MS/MS. The results revealed that bifidobacteria had a high affinity for hydrophilic peptides containing growth-promoting amino acids and the transportable peptides varied among the strains. B. bifidum could transport peptides containing up to 14 residues and had a high preference for peptides containing 2, 3, or 9 residues. While B. longum could transport peptides containing up to 20 residues and B. brevis mainly transported peptides with 2–4 and 8–20 residues. Significant differences in the amino acid and peptide transport systems were revealed by the genome sequence analysis. This study highlighted the importance of specific peptides as nitrogen sources rather than macromolecules, which can significantly promote the growth of different bifidobacteria.

Bifidobacterial β-Galactosidase-Mediated Production of Galacto-Oligosaccharides: Structural and Preliminary Functional Assessments.

In the current study the ability of four previously characterized bifidobacterial β-galactosidases (designated here as BgaA, BgaC, BgaD, and BgaE) to produce galacto-oligosaccharides (GOS) was optimized. Of these enzymes, BgaA and BgaE were found to be promising candidates for GOS production (and the corresponding GOS mixtures were called GOS-A and GOS-E, respectively) with a GOS concentration of 19.0 and 40.3% (of the initial lactose), respectively. GOS-A and GOS-E were partially purified and structurally characterized. NMR analysis revealed that the predominant (non-lactose) disaccharide was allo-lactose in both purified GOS preparations. The predominant trisaccharide in GOS-A and GOS-E was shown to be 3′-galactosyllactose, with lower levels of 6′-galactosyllactose and 4′-galactosyllactose. These three oligosaccharides have also been reported to occur in human milk. Purified GOS-A and GOS-E were shown to be able to support bifidobacterial growth similar to a commercially available GOS. In addition, GOS-E and the commercially available GOS were shown to be capable of reducing Escherichia coli adhesion to a C2BBe1 cell line. Both in vitro bifidogenic activity and reduced E. coli adhesion support the prebiotic potential of GOS-E and GOS-A.

Synbiotic Effect of Bifidobacterium lactis CNCM I-3446 and Bovine Milk-Derived Oligosaccharides on Infant Gut Microbiota.

Background: This study evaluated the impact of Bifidobacterium animalis ssp. lactis CNCM I-3446, Bovine Milk-derived OligoSaccharides (BMOS) and their combination on infant gut microbiota in vitro. In addition, a novel strategy consisting of preculturing B. lactis with BMOS to further enhance their potential synbiotic effects was assessed. Method: Short-term fecal batch fermentations (48 h) were used to assess the microbial composition and activity modulated by BMOS alone, B. lactis grown on BMOS or dextrose alone, or their combinations on different three-month-old infant microbiota. Results: BMOS alone significantly induced acetate and lactate production (leading to pH decrease) and stimulated bifidobacterial growth in 10 donors. A further in-depth study on two different donors proved B. lactis ability to colonize the infant microbiota, regardless of the competitiveness of the environment. BMOS further enhanced this engraftment, suggesting a strong synbiotic effect. This was also observed at the microbiota activity level, especially in a donor containing low initial levels of bifidobacteria. In this donor, preculturing B. lactis with BMOS strengthened further the early modulation of microbiota activity observed after 6 h. Conclusion: This study demonstrated the strong synbiotic effect of BMOS and B. lactis on the infant gut microbiota, and suggests a strategy to improve its effectiveness in an otherwise low-Bifidobacterium microbiota.

Short-chain fatty acids production by Bifidobacterium species in the presence of salep

Abstract Background Salep is obtained by grinding dried orchid tubers and used as a valuable ingredient in the food industry. Because of the glucomannan content of salep, it is thought to have prebiotic potential. However, there is little information in studies concerning the fermentation characteristics and potential prebiotic properties of salep. The objective of this study was to investigate the effect of salep on bifidobacterial growth by measuring the highest optical density (OD), calculating the specific growth rates, and determining the production of lactic acid and short-chain fatty acids (acetic, propionic, and butyric acid) as a result of bacterial fermentation. Result The OD and pH values obtained in this study showed that salep was utilized as a source of assimilable carbon and energy by the Bifidobacterium species (BS). All Bifidobacterium strains produced lactic, acetic, propionic, and butyric acid, indicating that salep is readily fermented by these bacteria. Salep at 1% (w/v) showed a similar effect on bifidobacterial growth as that promoted by 1% (w/v) glucose used as a traditional carbon source. Conclusions Bifidobacterium species can develop in media containing salep as well as in glucose and exhibit the potential to be used as new sources of prebiotics. How to cite: Usta-Gorgun B, Yilmaz-Ersan L. Short-chain fatty acid production by the Bifidobacterium species in the presence of salep. Electron J Biotechnol 2020;47. https://doi.org/10.1016/j.ejbt.2020.06.004 .

Ketogenic Diets Alter the Gut Microbiome Resulting in Decreased Intestinal Th17 Cells

Very low-carbohydrate, high-fat ketogenic diets (KDs) induce a pronounced shift in metabolic fuel utilization that elevates circulating ketone bodies; however, the consequences of these compounds for host-microbiome interactions remain unknown. Here, we show that KDs alter the human and mouse gut microbiota in a manner distinct from high-fat diets (HFDs). Metagenomic and metabolomic analyses of stool samples from an 8-week inpatient study revealed marked shifts in gut microbial community structure and function during the KD. Gradient diet experiments in mice confirmed the unique impact of KDs relative to HFDs with a reproducible depletion of bifidobacteria. Invitro and invivo experiments showed that ketone bodies selectively inhibited bifidobacterial growth. Finally, mono-colonizations and human microbiome transplantations into germ-free mice revealed that the KD-associated gut microbiota reduces the levels of intestinal pro-inflammatory Th17 cells. Together, these results highlight the importance of trans-kingdom chemical dialogs for mediating the host response to dietary interventions.

Galacto- and Fructo-oligosaccharides Utilized for Growth by Cocultures of Bifidobacterial Species Characteristic of the Infant Gut.

Bifidobacterial species are common inhabitants of the gut of human infants during the period when milk is a major component of the diet. Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum subspecies longum, and B. longum subspecies infantis have been detected frequently in infant feces, but B. longum subsp. infantis may be disadvantaged numerically in the gut of infants in westernized countries. This may be due to the different durations of breast milk feeding in different countries. Supplementation of the infant diet or replacement of breast milk using formula feeds is common in Western countries. Formula milks often contain galacto- and/or fructo-oligosaccharides (GOS and FOS, respectively) as additives to augment the concentration of oligosaccharides in ruminant milks, but the ability of B. longum subsp. infantis to utilize these potential growth substrates when they are in competition with other bifidobacterial species is unknown. We compared the growth and oligosaccharide utilization of GOS and FOS by bifidobacterial species in pure culture and coculture. Short-chain GOS and FOS (degrees of polymerization [DP] 2 and 3) were favored growth substrates for strains of B. bifidum and B. longum subsp. longum, whereas both B. breve and B. longum subsp. infantis had the ability to utilize both short- and longer-chain GOS and FOS (DP 2 to 6). B. breve was nevertheless numerically dominant over B. longum subsp. infantis in cocultures. This was probably related to the slower use of GOS of DP 3 by B. longum subsp. infantis, indicating that the kinetics of substrate utilization is an important ecological factor in the assemblage of gut communities.IMPORTANCE The kinds of bacteria that form the collection of microbes (the microbiota) in the gut of human infants may influence health and well-being. Knowledge of how the composition of the infant diet influences the assemblage of the bacterial collection is therefore important because dietary interventions may offer opportunities to alter the microbiota with the aim of improving health. Bifidobacterium longum subspecies infantis is a well-known bacterial species, but under modern child-rearing conditions it may be disadvantaged in the gut. Modern formula milks often contain particular oligosaccharide additives that are generally considered to support bifidobacterial growth. However, studies of the ability of various bifidobacterial species to grow together in the presence of these oligosaccharides have not been conducted. These kinds of studies are essential for developing concepts of microbial ecology related to the influence of human nutrition on the development of the gut microbiota.

Large scale production of lacto-N-biose I, a building block of type I human milk oligosaccharides, using sugar phosphorylases.

Human milk oligosaccharides (HMOs) have drawn attention for their contribution to the explosive bifidobacterial growth in the intestines of neonates. We found that bifidobacteria can efficiently metabolize lacto-N-biose I (LNB), the major building blocks of HMOs, and we have developed a method to synthesize LNB by applying this system. We produced LNB on a kilogram scale by the method. This proved that, among the enterobacteria, only bifidobacteria can assimilate LNB, and provided the data that supported the explosive growth of bifidobacteria in neonates. Furthermore, we were also able to reveal the structure of LNB crystal and the low stability for heating at neutral pH, which has not been clarified so far. In this paper, using bifidobacteria and LNB as examples, I describe the research on oligosaccharide synthesis that was conducted by utilizing a sugar metabolism.Abbreviations: LNB: lacto-N-biose I; GNB: galacto-N-biose; HMOs: human milk oligosaccharides; GLNBP: GNB/LNB phosphorylase; NahK: N-acetylhexosamine 1-kinase; GalT: UDP-glucose-hexose-1-phosphate uridylyltransferase; GalE: UDP-glucose 4-epimerase; SP: sucrose phosphorylase.

Production of a human milk oligosaccharide 2\u2032-fucosyllactose by metabolically engineered Saccharomyces cerevisiae

Background2′-Fucosyllactose (2-FL), one of the most abundant oligosaccharides in human milk, has potential applications in foods due to its health benefits such as the selective promotion of bifidobacterial growth and the inhibition of pathogenic microbial binding to the human gut. Owing to the limited amounts of 2-FL in human milk, alternative microbial production of 2-FL is considered promising. To date, microbial production of 2-FL has been studied mostly in Escherichia coli. In this study, 2-FL was produced alternatively by using a yeast Saccharomyces cerevisiae, which may have advantages over E. coli.ResultsFucose and lactose were used as the substrates for the salvage pathway which was constructed with fkp coding for a bifunctional enzyme exhibiting l-fucokinase and guanosine 5′-diphosphate-l-fucose phosphorylase activities, fucT2 coding for α-1,2-fucosyltransferase, and LAC12 coding for lactose permease. Production of 2-FL by the resulting engineered yeast was verified by mass spectrometry. 2-FL titers of 92 and 503 mg/L were achieved from 48-h batch fermentation and 120-h fed-batch fermentation fed with ethanol as a carbon source, respectively.ConclusionsThis is the first report on 2-FL production by using yeast S. cerevisiae. These results suggest that S. cerevisiae can be considered as a host engineered for producing 2-FL via the salvage pathway.

Comparative Genomics Reveals the Regulatory Complexity of Bifidobacterial Arabinose and Arabino-Oligosaccharide Utilization.

Members of the genus Bifidobacterium are common inhabitants of the human gastrointestinal tract. Previously it was shown that arabino-oligosaccharides (AOS) might act as prebiotics and stimulate the bifidobacterial growth in the gut. However, despite the rapid accumulation of genomic data, the precise mechanisms by which these sugars are utilized and associated transcription control still remain unclear. In the current study, we used a comparative genomic approach to reconstruct arabinose and AOS utilization pathways in over 40 bacterial species belonging to the Bifidobacteriaceae family. The results indicate that the gene repertoire involved in the catabolism of these sugars is highly diverse, and even phylogenetically close species may differ in their utilization capabilities. Using bioinformatics analysis we identified potential DNA-binding motifs and reconstructed putative regulons for the arabinose and AOS utilization genes in the Bifidobacteriaceae genomes. Six LacI-family transcriptional factors (named AbfR, AauR, AauU1, AauU2, BauR1 and BauR2) and a TetR-family regulator (XsaR) presumably act as local repressors for AOS utilization genes encoding various α- or β-L-arabinofuranosidases and predicted AOS transporters. The ROK-family regulator AraU and the LacI-family regulator AraQ control adjacent operons encoding putative arabinose transporters and catabolic enzymes, respectively. However, the AraQ regulator is universally present in all Bifidobacterium species including those lacking the arabinose catabolic genes araBDA, suggesting its control of other genes. Comparative genomic analyses of prospective AraQ-binding sites allowed the reconstruction of AraQ regulons and a proposed binary repression/activation mechanism. The conserved core of reconstructed AraQ regulons in bifidobacteria includes araBDA, as well as genes from the central glycolytic and fermentation pathways (pyk, eno, gap, tkt, tal, galM, ldh). The current study expands the range of genes involved in bifidobacterial arabinose/AOS utilization and demonstrates considerable variations in associated metabolic pathways and regulons. Detailed comparative and phylogenetic analyses allowed us to hypothesize how the identified reconstructed regulons evolved in bifidobacteria. Our findings may help to improve carbohydrate catabolic phenotype prediction and metabolic modeling, while it may also facilitate rational development of novel prebiotics.

Bifidobacterial growth stimulation by oligosaccharides generated from olive tree pruning biomass

This work aims to evaluate the prebiotic potential of oligosaccharides (OS) obtained from autohydrolysis of olive tree pruning biomass (OTPB). Two selected fractions (F1 and F2) were characterized and used in in vitro fermentations by two Bifidobacterium spp. (B. adolescentis and B. longum) and one fecal inoculum. The fraction F1 presented a lower average degree of polymerization (DP) mainly with OS ranging from 3 to 6 DP, whereas the fraction F2 corresponded to a pool of unsubstituted and acetylated oligomers with DP between 4 and 19. In the fermentation by Bifidobacterium, F1 supported a higher biomass formation, OS consumption and organic acids production than F2. With the fecal inoculum, the accumulation of organic acids, as the sum of acetate, propionate and butyrate, was similar for F1 and F2 (107 and 101mM, respectively). The bifidobacteria counts also increased during the incubation time for both OS fractions.

Molecular Insight into Evolution of Symbiosis between Breast-Fed Infants and a Member of the Human Gut Microbiome Bifidobacterium longum

Breast-fed infants generally have a bifidobacteria-rich microbiota with recent studies indicating that human milk oligosaccharides (HMOs) selectively promote bifidobacterial growth. Bifidobacterium bifidum possesses a glycoside hydrolase family 20lacto-N-biosidase for liberating lacto-N-biose I fromlacto-N-tetraose, an abundant HMO unique to human milk, while Bifidobacterium longum subsp. longum has a non-classified enzyme (LnbX). Here, we determined the crystal structure of the catalytic domain of LnbX and provide evidence for creation of a novel glycoside hydrolase family, GH136. The structure, in combination with inhibition and mutation studies, provides insight into the molecular mechanism and broader substrate specificity of this enzyme. Moreover, through genetic studies, we show that lnbX is indispensable for B.longum growth on lacto-N-tetraose and is a key genetic factor for persistence in the gut of breast-fed infants. Overall, this study reveals possible evolutionary routes for the emergence of symbiosis between humans and bifidobacterial species in the infant gut.

Effect of &lt;em&gt;Asparagus falcatus&lt;/em&gt; and &lt;em&gt;Taraxacum javanicum&lt;/em&gt; Inulins on growth of &lt;em&gt;L. acidophilus&lt;/em&gt; La-5 and &lt;em&gt;B. animalis&lt;/em&gt; subsp lactis Bb-12, co-cultured in skim milk

A study was conducted to evaluate the effect of inulin from Asparagus falcatus and Taraxacum javanicum on growth of Lactobacillus acidophilus (La-5) and Bifidobacterium animalis (Bb-12) in skim milk. Commercially available chicory inulin and chicory fructooligosaccharides (FOS) were used as positive controls. Skim milk (13% (w/v)) samples supplemented with different concentrations (0.5, 1, 2, 3 and 5%) of AF or TJ inulin, 5% Chicory inulin and 5% FOS, inoculated with mix culture of La-5 and Bb-12 (1:1), were incubated at 37 oC for 24 hours. The skim milk samples prepared the same way, but without adding inulin were used as negative controls. Optical density and pH of the milk samples were checked during 0, 6, 12 and 24 hours of incubation. Selective enumeration of La-5 and Bb-12, at 6 hours of incubation, was done using MRS-sorbitol agar and Beerens agar, respectively. The AF and TJ inulin showed enhanced bifidobacterial growth in skim milk, similar to chicory inulin. The growth promotion effect of AF and TJ inulin on bifidobacteria was maximal at 3% inulin concentration. None of the inulin types or FOS significantly influenced the La-5 growth in skim milk. The pH values of fermented milk samples were significantly (P<0.05) lower in inulin or FOS supplemented skim milks compared with the control. AF and TJ inulin showed clear bifidogenic properties in skim milk, indicating their potential use as alternative inulin sources for the Food Industry.

Combinational effects of prebiotic oligosaccharides on bifidobacterial growth and host gene expression in a simplified mixed culture model and neonatal mice.

It is important to provide formula-fed infants with a bifidobacteria-enriched gut microbiota similar to those of breastfed infants to ensure intestinal health. Prebiotics, such as certain oligosaccharides, are a useful solution to this problem, but the combinational benefits of these oligosaccharides have not been evaluated. This study investigated the benefits of oligosaccharide combinations and screened for an optimal combination of oligosaccharides to promote healthy gut microbiota of formula-fed infants. In vitro and in vivo experiments were performed to assess the bifidogenic effects of lactulose (LAC) alone and LAC combined with raffinose (RAF) and/or galacto-oligosaccharide (GOS), using a mixed culture model and neonatal mice orally administered with these oligosaccharides and Bifidobacterium breve. In the in vitro culture model, the combination of the three oligosaccharides (LAC-RAF-GOS) significantly increased cell numbers of B. breve and Bifidobacterium longum (P<0·05) compared with either LAC alone or the combination of two oligosaccharides, and resulted in the production of SCFA under anaerobic conditions. In the in vivo experiment, the LAC-RAF-GOS combination significantly increased cell numbers of B. breve and Bacteroidetes in the large intestinal content (P<0·05) and increased acetate concentrations in the caecal content and serum of neonatal mice. Genes related to metabolism and immune responses were differentially expressed in the liver and large intestine of mice administered with LAC-RAF-GOS. These results indicate a synergistic effect of the LAC-RAF-GOS combination on the growth of bifidobacteria and reveal possible benefits of this combination to the gut microbiota and health of infants.

In Vitro Fermentation of caprine milk oligosaccharides by bifidobacteria isolated from breast-fed infants

This study was conducted to investigate the catabolism and fermentation of caprine milk oligosaccharides (CMO) by selected bifidobacteria isolated from 4 breast-fed infants. Seventeen bifidobacterial isolates consisting of 3 different species (Bifidobacterium breve, Bifidobacterium longum subsp. longum and Bifidobacterium bifidum) were investigated. A CMO-enriched fraction (CMOF) (50% oligosaccharides, 10% galacto-oligosaccharides (GOS), 20% lactose, 10% glucose and 10% galactose) from caprine cheese whey was added to a growth medium as a sole source of fermentable carbohydrate. The inclusion of the CMOF was associated with increased bifidobacterial growth for all strains compared to glucose, lactose, GOS, inulin, oligofructose, 3'-sialyl-lactose and 6'-sialyl-lactose. Only one B. bifidum strain (AGR2166) was able to utilize the sialyl-CMO, 3'-sialyl-lactose and 6'-sialyl-lactose, as carbohydrate sources. The inclusion of CMOF increased the production of acetic and lactic acid (P < 0.001) after 36 h of anaerobic fermentation at 37°C, when compared to other fermentable substrates. Two B. bifidum strains (AGR2166 and AGR2168) utilised CMO, contained in the CMOF, to a greater extent than B. breve or B. longum subsp longum isolates, and this increased CMO utilization was associated with enhanced sialidase activity. CMOF stimulated bifidobacterial growth when compared to other tested fermentable carbohydrates and also increased the consumption of mono- and disaccharides, such as galactose and lactose present in the CMOF. These findings indicate that the dietary consumption of CMO may stimulate the growth and metabolism of intestinal Bifidobacteria spp. including B. bifidum typically found in the large intestine of breast-fed infants.

Selective growth-inhibitory effect of 8-hydroxyquinoline towards Clostridium difficile and Bifidobacterium longum subsp. longum in co-culture analysed by flow cytometry.

The major risk factor for Clostridium difficile infection (CDI) is the use of antibiotics owing to the disruption of the equilibrium of the host gut microbiota. To preserve the beneficial resident probiotic bacteria during infection treatment, the use of molecules with selective antibacterial activity enhances the efficacy by selectively removing C. difficile. One of them is the plant alkaloid 8-hydroxyquinoline (8HQ), which has been shown to selectively inhibit clostridia without repressing bifidobacteria. Selective antimicrobial activity is generally tested by culture techniques of individual bacterial strains. However, the main limitation of these techniques is the inability to describe differential growth dynamics of more bacterial strains in co-culture within the same experiment. In the present study, we combined fluorescent in situ hybridization and flow cytometry to describe the changes in active and non-active cells of a mixed culture formed by the opportunistic pathogen C. difficile CECT 531 and the beneficial Bifidobacterium longum subsp. longum CCMDMND BL1 after exposure to 8HQ. It was observed that without 8HQ, the proportion of both strains was almost equal, oscillating between 22.7 and 77.9 % during a time lapse of 12 h, whereas with 8HQ the proportion of active C. difficile decreased after 4 h, and persisted only between 8.8 and 17.5 %. In contrast, bifidobacterial growth was not disturbed by 8HQ. The results of this study showed the selective inhibitory effect of 8HQ on clostridial and bifidobacterial growth dynamics, and the potential of this compound for the development of selective agents to control CDIs.

Kefir fermented milk and kefiran promote growth of Bifidobacterium bifidum PRL2010 and modulate its gene expression

Bifidobacteria constitute one of the dominant groups of microorganisms colonizing the human gut of infants. Their ability to utilize various host-derived glycans as well as dietary carbohydrates has received considerable scientific attention. However, very little is known about the role of fermented foods, such as kefir, or their constituent glycans, such as kefiran, as substrates for bifidobacterial growth and for the modulation of the expression of bifidobacterial host-effector molecules. Here, we show that Bifidobacterium bifidum PRL2010 exhibits high growth performance among the bifidobacterial strains tested when cultivated on kefir and/or kefiran polymer. Furthermore, a 16S rRNA metagenomic approach revealed that the microbiota of kefir is modified upon the addition of PRL2010 cells to the kefir matrix. Finally, our results show that kefir and kefiran are able to influence the transcriptome of B. bifidum PRL2010 causing increased transcription of genes involved in the metabolism of dietary glycans as well as genes that act as host–microbe effector molecules such as pili. Altogether, these data support the use of kefir as a valuable means for the delivery of effective microbial cells in probiotic therapy.