Bifidobacteria play a vital role in human nutrition and health by shaping and maintaining the gut ecosystem. In order to exert a beneficial effect, a sufficient population of bifidobacteria must colonise the host. In this study, we developed a miniaturised high-throughput in vitro assay for assessing the colonising ability of bacterial strains in human cells. We also investigated a variety of components isolated from different milk sources for their ability to increase the adherence of Bifidobacterium longum subsp. infantis ATCC 15697, a common member of the gastrointestinal microbiota of breastfed infants, to HT-29 cells. Both conventional and miniaturised colonisation assays were employed to examine the effect of 13 different milk-derived powders on bacterial adherence, including positive controls which had previously resulted in increased bifidobacterial adherence (human milk oligosaccharides and a combination of 3′- and 6′-sialylactose) to intestinal cells. Immunoglobulin G enriched from bovine whey and goat milk oligosaccharides resulted in increased adhesion (3.3- and 8.3-fold, respectively) of B. infantis to the intestinal cells and the miniaturised and conventional assays were found to yield comparable and reproducible results. This study highlights the potential of certain milk components to favourably modulate adhesion of bifidobacteria to human intestinal cells.

Bifidobacteria are known to inhibit, compete with and displace the adhesion of pathogens to human intestinal cells. Previously, we demonstrated that goat milk oligosaccharides (GMO) increased the attachment of Bifidobacterium longum subsp. infantis ATCC 15697 to intestinal cells in vitro. In this study, we aimed to exploit this effect as a mechanism for inhibiting pathogen association with intestinal cells. We examined the synergistic effect of GMO-treated B. infantis on preventing the attachment of a highly invasive strain of Campylobacter jejuni to intestinal HT-29 cells. The combination decreased the adherence of C. jejuni to the HT-29 cells by an average of 42% compared to the control (non-GMO treated B. infantis). Increasing the incubation time of the GMO with the Bifidobacterium strain resulted in the strain metabolizing the GMO, correlating with a subsequent 104% increase in growth over a 24 h period when compared to the control. Metabolite analysis in the 24 h period also revealed increased production of acetate, lactate, formate and ethanol by GMO-treated B. infantis. Statistically significant changes in the GMO profile were also demonstrated over the 24 h period, indicating that the strain was digesting certain structures within the pool such as lactose, lacto-N-neotetraose, lacto-N-neohexaose 3 -sialyllactose, 6 -sialyllactose, sialyllacto-N-neotetraose c and disialyllactose. It may be that early exposure to GMO modulates the adhesion of B. infantis while carbohydrate utilisation becomes more important after the bacteria have transiently colonised the host cells in adequate numbers. This study builds a strong case for the use of synbiotics that incorporate oligosaccharides sourced from goat s milk and probiotic bifidobacteria in functional foods, particularly considering the growing popularity of formulas based on goat milk. Foods 2020, 9, 348 2 of 15Listeria monocytogenes, and Clostridium difficile [4][5][6]. Microbe-associated molecular patterns (MAMPs) are recognized by the host s intestinal pattern recognition receptors (PRRs), and these interactions play key roles in the association of pathogens with the intestinal epithelia. Probiotics also express molecular patterns which can recognize the same trans-membrane receptors as the pathogens, thus blocking the sites for pathogenic contact by competitive exclusion and, in some cases, displacing already-attached pathogens [7]. However, the health benefits associated with bifidobacteria are reliant on such strains colonising the host in sufficient numbers [8]. The important step in microbial colonisation of the intestinal epithelium is the attachment of bacterial surface lectins to intestinal sugar structures. Recent studies have suggested that milk oligosaccharides may enhance the specific ability of bifidobacteria to attach to the GI epithelium [9][10][11]. Indeed, our group investigated the ability of goat milk oligosaccharides (GMO) to increase the attachment of Bifidobacterium longum subsp. infantis ATCC 15697 to HT-29 cells. Exposure of the strain ...

Oligosaccharides are the third most abundant component in human milk. It is widely accepted that they play several important protective, physiological, and biological roles, including selective growth stimulation of beneficial gut microbiota, inhibition of pathogen adhesion, and immune modulation. However, until recently, very few commercial products on the market have capitalized on these functions. This is mainly because the quantities of human milk oligosaccharides required for clinical trials have been unavailable. Recently, clinical studies have tested the potential beneficial effects of feeding infants formula containing 2′-fucosyllactose, which is the most abundant oligosaccharide in human milk. These studies have opened this field for further well-designed studies, which are required to fully understand the role of human milk oligosaccharides. However, one of the most striking features of human milk is its diversity of oligosaccharides, with over 200 identified to date. It may be that a mixture of oligosaccharides is even more beneficial to infants than a single structure. For this reason, the milk of domestic animals has become a focal point in recent years as an alternative source of complex oligosaccharides with associated biological activity. This review will focus specifically on free oligosaccharides found in bovine and caprine milk and the biological roles associated with such structures. These dairy streams are ideal sources of oligosaccharides, given their wide availability and use in so many regularly consumed dairy products. The aim of this review was to provide an overview of research into the functional role of bovine and caprine milk oli-gosaccharides in host-microbial interactions in the gut and provide current knowledge related to the isolation of oligosaccharides as ingredients for incorporation in functional or medical foods.

Numerous bioactive components exist in human milk including free oligosaccharides, which represent some of the most important, and provide numerous health benefits to the neonate. Considering the demonstrated value of these compounds, much interest lies in characterising structurally similar oligosaccharides in the dairy industry. In this study, the impacts of days post-parturition and parity of the cows on the oligosaccharide and lactose profiles of their milk were evaluated. Colostrum and milk samples were obtained from 18 cows 1–5 days after parturition. Three distinct phases were identified using multivariate analysis: colostrum (day 0), transitional milk (days 1–2) and mature milk (days 3–5). LS-tetrasaccharide c, lacto-N-neotetraose, disialyllacto-N-tetraose, 3’-sial-N-acetyllactosamine, 3’-sialyllactose, lacto-N-neohexaose and disialyllactose were found to be highly affiliated with colostrum. Notably, levels of lactose were at their lowest concentration in the colostrum and substantially increased 1-day post-parturition. The cow’s parity was also shown to have a significant effect on the oligosaccharide profile, with first lactation cows containing more disialyllacto-N-tetraose, 6’-sialyllactose and LS-tetrasaccharide compared to cows in their second or third parity. Overall, this study identifies key changes in oligosaccharide and lactose content that clearly distinguish colostrum from transitional and mature milk and may facilitate the collection of specific streams with divergent biological functions.

Evidence that whey proteins and peptides have health benefits beyond basic infant nutrition has increased dramatically in recent years. Previously, we demonstrated that a whey-derived immunoglobulin G-enriched powder (IGEP) enhanced adhesion of Bifidobacterium longum subsp. infantis ATCC 15697 (B. infantis) to HT-29 cells. In this study, we investigated the synergistic effect of IGEP-treated B. infantis on preventing the attachment of highly invasive Campylobacter jejuni 81–176 (C. jejuni) to intestinal HT-29 cells. The combination decreased the adherence of C. jejuni to the HT-29 cells by an average of 48% compared to the control (non-IGEP-treated B. infantis). We also confirmed that treatment of IGEP with sodium metaperiodate, which disables the biological recognition of the conjugated oligosaccharides, reduced adhesion of B. infantis to the intestinal cells. Thus, glycosylation of the IGEP components may be important in enhancing B. infantis adhesion. Interestingly, an increased adhesion phenotype was not observed when B. infantis was treated with bovine serum-derived IgG, suggesting that bioactivity was unique to milk-derived immunoglobulin-rich powders. Notably, IGEP did not induce growth of B. infantis within a 24 hours incubation period, as demonstrated by growth curves and metabolite analysis. The current study provides insight into the functionality of bovine whey components and highlights their potential in positively impacting the development of a healthy microbiota.