Growth of bifidobacteria and clostridia on human and cow milk saccharides

For healthy infants, which were born normally and fully breastfed, the dominant component of the intestinal microflora are bifidobacteria. However, infants born by caesarean section possess clostridia as a dominant intestinal bacterial group. The aim of the present study was to determine whether bifidobacteria and clostridia are able to grow on human milk oligosaccharides (HMOs) and other carbon sources - lactose, cow milk (CM) and human milk (HM). Both bifidobacteria and clostridia grew on lactose and in CM. Bifidobacteria grew in HM and on HMOs. In contrast, 3 out of 5 strains of clostridia were not able to grow in HM. No clostridial strain was able to utilise HMOs. While both bifidobacterial strains were resistant to lysozyme, 4 out of 5 strains of clostridia were lysozyme-susceptible. It seems that HMOs together with lysozyme may act as prebiotic-bifidogenic compounds inhibiting intestinal clostridia.     

An exo-alpha-sialidase from bifidobacteria involved in the degradation of sialyloligosaccharides in human milk and intestinal glycoconjugates

Bifidobacteria are health-promoting enteric commensals that are assumed to proliferate predominantly in the intestines of breast-fed infants by assimilating human milk oligosaccharides (HMOs) that are frequently fucosylated and/or sialylated. We previously identified two different α-l-fucosidases in Bifidobacterium bifidum and showed that the strain furnishes an extracellular degradation pathway for fucosylated HMOs. However, the catabolism of sialylated HMOs by bifidobacteria has remained unresolved. Here we describe the identification and characterization of an exo-α-sialidase in bifidobacteria. By expression cloning, we isolated a novel exo-α-sialidase gene (siabb2) from B. bifidum JCM1254, which encodes a protein (SiaBb2) consisting of 835-amino-acid residues with a predicted molecular mass of 87 kDa. SiaBb2 possesses an N-terminal signal sequence, a sialidase catalytic domain classified into the glycoside hydrolase family 33 (GH33) and a C-terminal transmembrane region, indicating that the mature SiaBb2 is an extracellular membrane-anchored enzyme. The recombinant enzyme expressed in Escherichia coli showed the highest activity in an acidic pH range from 4.0 to 5.0, and at 50 °C. Notably, 80% activity remained after 30 min incubation at 80 °C, indicating that the enzyme is highly thermostable. SiaBb2 liberated sialic acids from sialyloligosaccharides, gangliosides, glycoproteins and colominic acid; however, the linkage preference of the enzyme was remarkably biased toward the α2,3-linkage rather than α2,6- and α2,8-linkages. Expression of siabb2 in B. longum 105-A, which has no endogenous exo-α-sialidase, enabled this strain to degrade sialyloligosaccharides present in human milk. Our results suggest that SiaBb2 plays a crucial role in bifidobacterial catabolism of sialylated HMOs.     

Physiology of consumption of human milk oligosaccharides by infant gut-associated bifidobacteria

The bifidogenic effect of human milk oligosaccharides (HMOs) has long been known, yet the precise mechanism underlying it remains unresolved. Recent studies show that some species/subspecies of Bifidobacterium are equipped with genetic and enzymatic sets dedicated to the utilization of HMOs, and consequently they can grow on HMOs; however, the ability to metabolize HMOs has not been directly linked to the actual metabolic behavior of the bacteria. In this report, we clarify the fate of each HMO during cultivation of infant gut-associated bifidobacteria. Bifidobacterium bifidum JCM1254, Bifidobacterium longum subsp. infantis JCM1222, Bifidobacterium longum subsp. longum JCM1217, and Bifidobacterium breve JCM1192 were selected for this purpose and were grown on HMO media containing a main neutral oligosaccharide fraction. The mono- and oligosaccharides in the spent media were labeled with 2-anthranilic acid, and their concentrations were determined at various incubation times using normal phase high performance liquid chromatography. The results reflect the metabolic abilities of the respective bifidobacteria. B. bifidum used secretory glycosidases to degrade HMOs, whereas B. longum subsp. infantis assimilated all HMOs by incorporating them in their intact forms. B. longum subsp. longum and B. breve consumed lacto-N-tetraose only. Interestingly, B. bifidum left degraded HMO metabolites outside of the cell even when the cells initiate vegetative growth, which indicates that the different species/subspecies can share the produced sugars. The predominance of type 1 chains in HMOs and the preferential use of type 1 HMO by infant gut-associated bifidobacteria suggest the coevolution of the bacteria with humans.     

Bifidobacterium bifidum lacto-N-biosidase, a critical enzyme for the degradation of human milk oligosaccharides with a type 1 structure

Breast-fed infants often have intestinal microbiota dominated by bifidobacteria in contrast to formula-fed infants. We found that several bifidobacterial strains produce a lacto-N-biosidase that liberates lacto-N-biose I (Galbeta1,3GlcNAc; type 1 chain) from lacto-N-tetraose (Galbeta1,3GlcNAcbeta1,3Galbeta1,4Glc), which is a major component of human milk oligosaccharides, and subsequently isolated the gene from Bifidobacterium bifidum JCM1254. The gene, designated lnbB, was predicted to encode a protein of 1,112 amino acid residues containing a signal peptide and a membrane anchor at the N and C termini, respectively, and to possess the domain of glycoside hydrolase family 20, carbohydrate binding module 32, and bacterial immunoglobulin-like domain 2, in that order, from the N terminus. The recombinant enzyme showed substrate preference for the unmodified beta-linked lacto-N-biose I structure. Lacto-N-biosidase activity was found in several bifidobacterial strains, but not in the other enteric bacteria, such as clostridia, bacteroides, and lactobacilli, under the tested conditions. These results, together with our recent finding of a novel metabolic pathway specific for lacto-N-biose I in bifidobacterial cells, suggest that some of the bifidobacterial strains are highly adapted for utilizing human milk oligosaccharides with a type 1 chain.     

Lactoferrin and bifidobacteria

We herein summarized the effects of lactoferrin (LF) on bifidobacteria. Many in vitro studies previously reported the growth-promoting (bifidogenic) effects of LF on bifidobacteria. The involvement of bound iron, sugar chains, and LF peptides has been proposed in this bifidogenic mechanism. Peptides in the LF pepsin hydrolysate (LFH) showed stronger bifidogenic activity than natural LF; therefore, we speculated that peptides may be the bifidogenic active principle of LF. LF or its peptides may be recognized by LF-binding proteins on the surface of bifidobacterial cells, and the cationic nature or disulfide bonds of LF or its peptides may play a crucial role in its recognition by these proteins. Of the bifidobacterial species so far identified, human LF and peptides in human LFH were more likely to show bifidogenic activity especially to Bifidobacterium bifidum, and bovine LF (bLF) and peptides in bovine LFH (bLFH) to B. breve and B. infantis. In animal studies, the administration of LF to mice or piglets increased bifidobacteria levels in the intestine. In human trials, the administration of LF-containing formula to infants increased bifidobacteria levels in the feces; however, human milk achieved better results than LF-containing formula. In the case of breast-fed infants, LF may show bifidogenic activity synergistically with other milk components such as human milk oligosaccharides. As bLFH showed stronger bifidogenic activity than natural bLF, especially to B. breve and B. infantis in vitro, and these species are known to be infant-specific species, bLFH may be a beneficial ingredient in formula.     

Susceptibility of bifidobacteria to lysozyme as a possible selection criterion for probiotic bifidobacterial strains

Resistance or susceptibility of bifidobacteria to lysozyme and growth of bifidobacteria in human milk were tested. Susceptible bifidobacterial strains stopped their growth almost immediately after the addition of lysozyme (400 μg/ml), moderately susceptible strains exhibited reduced growth rate, and growth curves of resistant strains were not affected. Strains of human origin were more resistant to lysozyme than animal strains. While strains of B. bifidum grew well in human milk samples, the growth B. animalis strains was inhibited after inoculation to human milk. The resistance to lysozyme seems to be a promising criterion for the selection of new probiotic bifidobacterial strains.     

Human milk provides peptides highly stimulating the growth of bifidobacteria.

The large intestine of breast-fed infants is colonized predominantly by bifidobacteria, which have a protective effect against acute diarrhea. In this study we report for the first time the identification of human milk peptides that selectively stimulate the growth of bifidobacteria. Several bifidogenic peptides were purified chromatographically from pepsin-treated human milk and identified as proteolytically generated fragments from the secretory component of the soluble polyimmunoglobulin receptor and lactoferrin; both of these proteins exhibit antimicrobial effects. Hydrolysis of the identified peptides with the gastrointestinal proteases pepsin, trypsin and chymotrypsin did not lead to the loss of bifidogenic activity, indicating their potential function in vivo. Sequential comparison revealed a similar structural motif within the identified peptides. A correspondingly designed small peptide (prebiotic lactoferrin-derived peptide-I, PRELP-I) was found to stimulate the growth of bifidobacteria as effectively as the native peptides. The combination of antimicrobial and bifidobacterial growth stimulatory activity in human milk proteins leads to highly specific compounds capable of regulating the microbial composition of infants' large intestine.     

Inter-species differences in the growth of bifidobacteria cultured on human milk oligosaccharides

Human milk (HM) contains as the third most abundant component around 200 different structures of human milk oligosaccharides (HMOs). HMOs are the first and irreplaceable prebiotics for infants, supporting bifidobacteria as the most important bacterial group in an infant intestine. The aim of our study was to test the growth of bifidobacteria in HM and on HMOs. Bifidobacteria were isolated from two groups of infants. The first one (eight strains) were isolated from infants who had bifidobacteria in their feces but, after a short period of time (4 to 24 days), bifidobacteria were no longer detected in their feces (disappeared bifidobacteria [DB]). The second group of bifidobacteria (eight strains) originated from infants with continual presence of bifidobacteria in their feces (persistent bifidobacteria [PB]). There were significant differences (p?Bifidobacterium bifidum and B. breve species were able to utilize HMOs, while B. adolescentis and B. longum subsp. longum species did not. The ability to grow in HM and to utilize HMOs seem to be important properties of bifidobacteria which are able to colonize infant intestinal tract.     

Population dynamics of Bifidobacterium species in human feces during raffinose administration monitored by fluorescence in situ hybridization-flow cytometry

The population dynamics of bifidobacteria in human feces during raffinose administration were investigated at the species level by using fluorescence in situ hybridization (FISH) coupled with flow cytometry (FCM) analysis. Although double-staining FISH-FCM using both fluorescein isothiocyanate (FITC) and indodicarbocyanine (Cy5) as labeling dyes for fecal samples has been reported, the analysis was interfered with by strong autofluorescence at the FITC fluorescence region because of the presence of autofluorescence particles/debris in the fecal samples. We circumvented this problem by using only Cy5 fluorescent dye in the FISH-FCM analysis. Thirteen subjects received 2 g of raffinose twice a day for 4 weeks. Fecal samples were collected, and the bifidobacterial populations were monitored using the established FISH-FCM method. The results showed an increase in bifidobacteria from about 12.5% of total bacteria in the prefeeding period to about 28.7 and 37.2% after the 2-week and 4-week feeding periods, respectively. Bifidobacterium adolescentis, the Bifidobacterium catenulatum group, and Bifidobacterium longum were the major species, in that order, at the prefeeding period, and these bacteria were found to increase nearly in parallel during the raffinose administration. During the feeding periods, indigenous bifidobacterial populations became more diverse, such that minor species in human adults, such as Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium dentium, and Bifidobacterium angulatum, proliferated. Four weeks after raffinose administration was stopped, the proportion of each major bifidobacterial species, as well as that of total bifidobacteria, returned to approximately the original values for the prefeeding period, whereas that of each minor species appeared to differ considerably from its original value. To the best of our knowledge, these results provide the first clear demonstration of the population dynamics of indigenous bifidobacteria at the species level in response to raffinose administration.     

In vitro measurement of the impact of human milk oligosaccharides on the faecal microbiota of weaned formula-fed infants compared to a mixture of prebiotic fructooligosaccharides and galactooligosaccharides

Aims: To investigate the impact of human milk oligosaccharides (HMOs) from a single donor (SO), HMOs from multiple donors (PO), a fructooligosaccharides and galactooligosaccharides mixture (FG) on the composition of a batch culture inoculated with faecal microbiota from formula‐fed infants. Methods and Results: Three substrates were compared using 24‐h pH‐controlled anaerobic batch cultures inoculated with infant faecal slurries. Changes in bacterial populations, short‐chain fatty acids (SCFA) production and bacterial 16S rRNA gene profiles were determined. All three substrates significantly increased numbers of bifidobacteria, bacteroides and those aligning with the clostridial cluster XIVa. Neither the FG nor the HMOs substrates supported the growth of the Clostridium perfringens–histolyticum group. SCFA production corresponded to changes observed in bacterial populations. Denaturing gradient gel electrophoresis fingerprint analysis showed a distinct profile of faecal bacteria present in each infant. Conclusions: HMOs modulated infant faecal culture composition in a similar manner to the prebiotic mixture FG in vitro. Significance and Impact of the Study: This is the first demonstration of the impact of pure HMOs on the mixed culture of infant faecal bacteria. HMOs induced the growth of several saccharolytic bacterial groups and may thus play a role in the health‐promoting attributes of human breast milk and have an extended significance in infant diet during/after weaning.     

Effect of a synbiotic yogurt on levels of fecal bifidobacteria, clostridia, and enterobacteria

While ingestion of synbiotic yogurts containing Bifidobacterium animalis subsp. lactis and inulin is increasing, their effect on certain microbial groups in the human intestine is unclear. To further investigate this, a large-scale, crossover-design, placebo-controlled study was utilized to evaluate the effect of a synbiotic yogurt containing B. animalis subsp. lactis Bb-12 and inulin on the human intestinal bifidobacteria, clostridia, and enterobacteria. Fecal samples were collected at 14 time points from 46 volunteers who completed the study, and changes in the intestinal bacterial levels were monitored using real-time PCR. Strain Bb-12 could not be detected in feces after 2 weeks of washout. A live/dead PCR procedure indicated that the Bb-12 strain detected in the fecal samples was alive. A significant increase (P < 0.001) in the total bifidobacterial numbers was seen in both groups of subjects during the final washout period compared to the prefeeding period. This increase in total bifidobacteria corresponded with a significant decrease (P < 0.05) in numbers of clostridia but not enterobacteria. No significant differences in numbers of bifidobacteria, clostridia, or enterobacteria were observed between the probiotic and placebo groups during any of the feeding periods. However, subgrouping subjects based on lower initial bifidobacterial numbers or higher initial clostridial numbers did show corresponding significant differences between the synbiotic yogurt and placebo groups. This was not observed for a subgroup with higher initial enterobacterial numbers. While this synbiotic yogurt can increase bifidobacterial numbers and decrease clostridial numbers (but not enterobacterial numbers) in some individuals, it cannot modulate these microbial groups in the majority of individuals.     

Population Dynamics of Bifidobacterium Species in Human Feces during Raffinose Administration Monitored by Fluorescence In Situ Hybridization-Flow Cytometry

The population dynamics of bifidobacteria in human feces during raffinose administration were investigated at the species level by using fluorescence in situ hybridization (FISH) coupled with flow cytometry (FCM) analysis. Although double-staining FISH-FCM using both fluorescein isothiocyanate (FITC) and indodicarbocyanine (Cy5) as labeling dyes for fecal samples has been reported, the analysis was interfered with by strong autofluorescence at the FITC fluorescence region because of the presence of autofluorescence particles/debris in the fecal samples. We circumvented this problem by using only Cy5 fluorescent dye in the FISH-FCM analysis. Thirteen subjects received 2 g of raffinose twice a day for 4 weeks. Fecal samples were collected, and the bifidobacterial populations were monitored using the established FISH-FCM method. The results showed an increase in bifidobacteria from about 12.5% of total bacteria in the prefeeding period to about 28.7 and 37.2% after the 2-week and 4-week feeding periods, respectively. Bifidobacterium adolescentis, the Bifidobacterium catenulatum group, and Bifidobacterium longum were the major species, in that order, at the prefeeding period, and these bacteria were found to increase nearly in parallel during the raffinose administration. During the feeding periods, indigenous bifidobacterial populations became more diverse, such that minor species in human adults, such as Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium dentium, and Bifidobacterium angulatum, proliferated. Four weeks after raffinose administration was stopped, the proportion of each major bifidobacterial species, as well as that of total bifidobacteria, returned to approximately the original values for the prefeeding period, whereas that of each minor species appeared to differ considerably from its original value. To the best of our knowledge, these results provide the first clear demonstration of the population dynamics of indigenous bifidobacteria at the species level in response to raffinose administration.     

Effect of production conditions on the stability of a human bifidobacterial species Bifidobacterium longum in yogurt

Aims:  Human bifidobacteria are more sensitive to external environmental factors than animal bifidobacteria, and it is difficult to ensure their stable survival in yogurt. The purpose of this investigation was to observe the survival of human bifidobacteria in yogurts produced under various production conditions.
Methods:  Frozen or lyophilized bifidobacteria starters containing Bifidobacterium longum BB536 originally isolated from an infant, and commercial lyophilized yogurt starters were used for yogurt preparation. After producing yogurts under various conditions, the survival of bifidobacteria in these yogurts over various storage periods was observed.
Results:  Although there were some differences in bifidobacterial survival in yogurt between various production conditions, more than 1·0 × 10⁷ CFU g⁻¹ of Bif. longum survived in yogurt after 35 days' storage at 5°C. Lower fermentation temperature (37°C) and inclusion of Lactococcus lactis in the starter significantly ( P  < 0·05) improved survival of Bif. longum in the yogurt.
Conclusion:  In this investigation, the human bifidobacterial strain Bif. longum survived adequately in yogurt, although the fermentation temperature and starter composition affect bifidobacterial survival.
Significance and Impact of the Study:  This investigation indicates that stable probiotic yogurt using human bifidobacteria can be produced by choosing optimal production conditions.     

Evolutionary development and co-phylogeny of primate-associated bifidobacteria

In recent years, bifidobacterial populations in the gut of various monkey species have been assessed in several ecological surveys, unveiling a diverse, yet unexplored ecosystem harbouring novel species. In the current study, we investigated the species distribution of bifidobacteria present in 23 different species of primates, including human samples, by means of 16S rRNA microbial profiling and internal transcribed spacer bifidobacterial profiling. Based on the observed bifidobacterial-host co-phylogeny, we found a statistically significant correlation between the Hominidae family and particular bifidobacterial species isolated from humans, indicating phylosymbiosis between these lineages. Furthermore, phylogenetic and glycobiome analyses, based on 40 bifidobacterial species isolated from primates, revealed that members of the Bifidobacterium tissieri phylogenetic group, which are typical gut inhabitants of members of the Cebidae family, descend from an ancient ancestor with respect to other bifidobacterial taxa isolated from primates.     

Molecular analysis of the composition of the bifidobacterial and lactobacillus microflora of humans.

The bifidobacterial and lactobacillus populations of fecal samples collected from two human subjects during a 12-month period were studied. The total numbers of bifidobacteria were stable throughout the study period in both subjects, but lactobacillus numbers were less constant. Analysis of the composition of the bifidobacterial populations by using ribotyping or pulsed-field gel electrophoresis to differentiate between bacterial strains demonstrated major differences between the subjects. Subject 1 harbored five strains of bifidobacteria throughout the 12-month period, and one strain was numerically predominant. In contrast, subject 2 harbored a more complex bifidobacterial population (five to six strains per sample) whose composition fluctuated throughout the 12 months. One lactobacillus strain was numerically predominant throughout the study in both subjects. Strains of bifidobacteria and lactobacilli common to both subjects were not detected.     

Carbohydrate metabolism in Bifidobacteria

Members of the genus Bifidobacterium can be found as components of the gastrointestinal microbiota, and are believed to play an important role in maintaining and promoting human health by eliciting a number of beneficial properties. Bifidobacteria can utilize a diverse range of dietary carbohydrates that escape degradation in the upper parts of the intestine, many of which are plant-derived oligo- and polysaccharides. The gene content of a bifidobacterial genome reflects this apparent metabolic adaptation to a complex carbohydrate-rich gastrointestinal tract environment as it encodes a large number of predicted carbohydrate-modifying enzymes. Different bifidobacterial strains may possess different carbohydrate utilizing abilities, as established by a number of studies reviewed here. Carbohydrate-degrading activities described for bifidobacteria and their relevance to the deliberate enhancement of number and/or activity of bifidobacteria in the gut are also discussed in this review.     

Isolation of Bifidobacteria from Breast Milk and Assessment of the Bifidobacterial Population by PCR-Denaturing Gradient Gel Electrophoresis and Quantitative Real-Time PCR

The objective of this work was to elucidate if breast milk contains bifidobacteria and whether they can be transmitted to the infant gut through breastfeeding. Twenty-three women and their respective infants provided samples of breast milk and feces, respectively, at days 4 to 7 after birth. Gram-positive and catalase-negative isolates from specific media with typical bifidobacterial shapes were identified to the genus level by F6PPK (fructose-6-phosphate phosphoketolase) assays and to the species level by 16S rRNA gene sequencing. Bifidobacterial communities in breast milk were assessed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE), and their levels were estimated by quantitative real-time PCR (qRTi-PCR). Bifidobacteria were present in 8 milk samples and 21 fecal samples. Bifidobacterium breve, B. adolescentis, and B. bifidum were isolated from milk samples, while infant feces also contained B. longum and B. pseudocatenulatum. PCR-DGGE revealed the presence of one to four dominant bifidobacterial bands in 22 milk samples. Sequences with similarities above 98% were identified as Bifidobacterium breve, B. adolescentis, B. longum, B. bifidum, and B. dentium. Bifidobacterial DNA was detected by qRTi-PCR in the same 22 milk samples at a range between 40 and 10,000 16S rRNA gene copies per ml. In conclusion, human milk seems to be a source of living bifidobacteria for the infant gut.     

α-N-acetylgalactosaminidase from infant-associated bifidobacteria belonging to novel glycoside hydrolase family 129 is implicated in alternative mucin degradation pathway

Bifidobacteria inhabit the lower intestine of mammals including humans where the mucin gel layer forms a space for commensal bacteria. We previously identified that infant-associated bifidobacteria possess an extracellular membrane-bound endo-α-N-acetylgalactosaminidase (EngBF) that may be involved in degradation and assimilation of mucin-type oligosaccharides. However, EngBF is highly specific for core-1-type O-glycan (Galβ1-3GalNAcα1-Ser/Thr), also called T antigen, which is mainly attached onto gastroduodenal mucins. By contrast, core-3-type O-glycans (GlcNAcβ1-3GalNAcα1-Ser/Thr) are predominantly found on the mucins in the intestines. Here, we identified a novel α-N-acetylgalactosaminidase (NagBb) from Bifidobacterium bifidum JCM 1254 that hydrolyzes the Tn antigen (GalNAcα1-Ser/Thr). Sialyl and galactosyl core-3 (Galβ1-3/4GlcNAcβ1-3(Neu5Acα2-6)GalNAcα1-Ser/Thr), a major tetrasaccharide structure on MUC2 mucin primarily secreted from goblet cells in human sigmoid colon, can be serially hydrolyzed into Tn antigen by previously identified bifidobacterial extracellular glycosidases such as α-sialidase (SiaBb2), lacto-N-biosidase (LnbB), β-galactosidase (BbgIII), and β-N-acetylhexosaminidases (BbhI and BbhII). Because NagBb is an intracellular enzyme without an N-terminal secretion signal sequence, it is likely involved in intracellular degradation and assimilation of Tn antigen-containing polypeptides, which might be incorporated through unknown transporters. Thus, bifidobacteria possess two distinct pathways for assimilation of O-glycans on gastroduodenal and intestinal mucins. NagBb homologs are conserved in infant-associated bifidobacteria, suggesting a significant role for their adaptation within the infant gut, and they were found to form a new glycoside hydrolase family 129.