Human milk oligosaccharides (HMO) are complex sugars which are found in breast milk at significant concentrations and with unique structural diversity. These sugars are the fourth most abundant component of human milk after water, lipids, and lactose and yet provide no direct nutritional value to the infant. Recent research has highlighted that HMOs have various functional roles to play in infant development. These sugars act as prebiotics by promoting growth of beneficial intestinal bacteria thereby generating short-chain fatty acids which are critical for gut health. HMOs also directly modulate host-epithelial immune responses and can selectively reduce binding of pathogenic bacteria and viruses to the gut epithelium preventing the emergence of a disease. This review covers current knowledge related to the functional biology of HMOs and their associated impact on infant gut health.

In this study, we tested the hypothesis that milk oligosaccharides may contribute not only to selective growth of bifidobacteria, but also to their specific adhesive ability. Human milk oligosaccharides (3′sialyllactose and 6′sialyllactose) and a commercial prebiotic (Beneo Orafti P95; oligofructose) were assayed for their ability to promote adhesion of Bifidobacterium longum subsp. infantis ATCC 15697 to HT-29 and Caco-2 human intestinal cells. Treatment with the commercial prebiotic or 3′sialyllactose did not enhance adhesion. However, treatment with 6′sialyllactose resulted in increased adhesion (4.7 fold), while treatment with a mixture of 3′- and 6′-sialyllactose substantially increased adhesion (9.8 fold) to HT-29 intestinal cells. Microarray analyses were subsequently employed to investigate the transcriptional response of B. longum subsp. infantis to the different oligosaccharide treatments. This data correlated strongly with the observed changes in adhesion to HT-29 cells. The combination of 3′- and 6′-sialyllactose resulted in the greatest response at the genetic level (both in diversity and magnitude) followed by 6′sialyllactose, and 3′sialyllactose alone. The microarray data was further validated by means of real-time PCR. The current findings suggest that the increased adherence phenotype of Bifidobacterium longum subsp. infantis resulting from exposure to milk oligosaccharides is multi-faceted, involving transcription factors, chaperone proteins, adhesion-related proteins, and a glycoside hydrolase. This study gives additional insight into the role of milk oligosaccharides within the human intestine and the molecular mechanisms underpinning host-microbe interactions.

A number of bacterial species are found in high abundance in the faeces of healthy breast-fed infants, an occurrence that is understood to be, at least in part, due to the ability of these bacteria to metabolize human milk oligosaccharides (HMOs). HMOs are the third most abundant component of human milk after lactose and lipids, and represent complex sugars which possess unique structural diversity and are resistant to infant gastrointestinal digestion. Thus, these sugars reach the infant distal intestine intact, thereby serving as a fermentable substrate for specific intestinal microbes, including Firmicutes, Proteobacteria, and especially infant-associated Bifidobacterium spp. which help to shape the infant gut microbiome. Bacteria utilising HMOs are equipped with genes associated with their degradation and a number of carbohydrate-active enzymes known as glycoside hydrolase enzymes have been identified in the infant gut, which supports this hypothesis. The resulting degraded HMOs can also be used as growth substrates for other infant gut bacteria present in a microbe-microbe interaction known as ‘cross-feeding’. This review describes the current knowledge on HMO metabolism by particular infant gut-associated bacteria, many of which are currently used as commercial probiotics, including the distinct strategies employed by individual species for HMO utilisation.

Bifidobacteria are associated with a host of health benefits and are typically dominant in the gut microbiota of healthy, breast-fed infants. A key adaptation, facilitating the establishment of these species, is their ability to consume particular sugars, known as human milk oligosaccharides (HMO), which are abundantly found in breastmilk. In the current study, we aimed to characterise the co-operative metabolism of four commercial infant-derived bifidobacteria (Bifidobacterium bifidum R0071, Bifidobacterium breve M-16V, Bifidobacterium infantis R0033, and Bifidobacterium infantis M-63) when grown on HMO. Three different HMO substrates (2′-fucosyllactose alone and oligosaccharides isolated from human milk representing non-secretor and secretor status) were employed. The four-strain combination resulted in increased bifidobacterial numbers (> 21%) in comparison to single strain cultivation. The relative abundance of B. breve increased by > 30% during co-cultivation with the other strains despite demonstrating limited ability to assimilate HMO in mono-culture. HPLC analysis revealed strain-level variations in HMO consumption. Metabolomics confirmed the production of formate, acetate, 1,2-propanediol, and lactate with an overall increase in such metabolites during co-cultivation. These results support the concept of positive co-operation between multiple bifidobacterial strains during HMO utilisation which may result in higher cell numbers and a potentially healthier balance of metabolites.

Six antimicrobial-producing seaweed-derived Bacillus strains were evaluated in vitro as animal probiotics, in comparison to two Bacillus from an EU-authorized animal probiotic product. Antimicrobial activity was demonstrated on solid media against porcine Salmonella and E. coli. The marine isolates were most active against the latter, had better activity than the commercial probiotics and Bacillus pumilus WIT 588 also reduced E. coli counts in broth. All of the marine Bacillus tolerated physiological concentrations of bile, with some as tolerant as one of the probiotics. Spore counts for all isolates remained almost constant during incubation in simulated gastric and ileum juices. All of the marine Bacillus grew anaerobically and the spores of all except one isolate germinated under anaerobic conditions. All were sensitive to a panel of antibiotics and none harbored Bacillus enterotoxin genes but all, except B. pumilus WIT 588, showed some degree of β-hemolysis. However, trypan blue dye exclusion and xCELLigence assays demonstrated a lack of toxicity in comparison to two pathogens; in fact, the commercial probiotics appeared more cytotoxic than the majority of the marine Bacillus. Overall, some of the marine-derived Bacillus, in particular B. pumilus WIT 588, demonstrate potential for use as livestock probiotics.

Bovine milk glycomacropeptide (GMP) is derived from κ-casein, with exclusively o-linked glycosylation. Glycomacropeptide promoted the growth of Bifidobacterium longum ssp. infantis in a concentration-dependent manner, and this activity was lost following periodate treatment of the GMP (GMP-P), which disables biological recognition of the conjugated oligosaccharides. Transcriptional analysis of B. longum ssp. infantis following exposure to GMP revealed a substantial response to GMP relative to bacteria treated with GMP-P, with a greater number of differentially expressed transcripts and larger fold changes versus the control. Therefore, stimulation of B. longum ssp. infantis growth by GMP is intrinsically linked to the peptide's O-linked glycosylation. The pool of differentially expressed transcripts included 2 glycoside hydrolase (family 25) genes, which were substantially upregulated following exposure to GMP, but not GMP-P. These GH25 genes were present in duplicated genomic islands that also contained genes encoding fibronectin type III binding domain proteins and numerous phage-related proteins, all of which were also upregulated. Homologs of this genomic arrangement were present in other Bifidobacterium species, which suggest it may be a conserved domain for the utilization of glycosylated peptides. This study provides insights into the molecular basis for the prebiotic effect of bovine milk GMP on B. longum ssp. infantis.