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.

This work evaluated the angiotensin-converting-enzyme (ACE)-inhibitory activities of a bovine sodium caseinate fermentate generated using the proteolytic capabilities of the porcine small intestinal isolate Lactobacillus animalis DPC6134 (NCIMB deposit 41355). The crude 10-kDa L. animalis DPC6134 fermentate exhibited ACE-inhibitory activity of 85.51% (؎15%) and had a 50% inhibitory concentration (IC 50 ) of 0.8 mg protein/ml compared to captopril, which had an IC 50 value of 0.005 mg/ml. Fractionation of the crude L. animalis DPC6134 fermentate by membrane filtration and reversed-phase high-performance liquid chromatography (HPLC) generated three bioactive fractions from a total of 72 fractions.

The functional and physicochemical characteristics, and bitterness were evaluated on sodium caseinate hydrolysates generated with a commercial Bacillus proteinase complex. At low degrees of hydrolysis (0.5 and 1.0% DH) the hydrolysates compared to the unhydrolyzed substrate had increased emulsion activity at pH 2 and 4. However, higher DH resulted in lower emulsion activities. At pH 8 and 10, the low DH hydrolysates displayed increased foam expansion and decreased foam drainage compared to the starting substrate. The Bacillus proteinase hydrolysates at higher DH had similar bitterness values to tryptic hydrolysates at equivalent DH. However, the Bacillus hydrolysate at 0.5% DH was less bitter (p<0.05) than the tryptic hydrolysate at the same DH.

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 ...

Immunoglobulin G (IgG) in bovine milk is credited with ensuring efficient passive immunity for newborn calves. Bovine milk IgG glycosylation may also have positive impacts on the health of nonbovine consumers of cow's milk. Milk IgG's glycosylation contributes to effector function and may also protect it from protease digestion, allowing IgG to reach the intestine for absorption. However, relatively little is known about changes in milk IgG oligosaccharide presentation and composition over early lactation. In this work, IgG was isolated from milk pooled from three cows at four time points over the first 10 days of lactation postparturition. Purified IgG was labeled with a fluorescent dye and interrogated with a microarray consisting of 48 carbohydrate‐binding proteins (lectins) from plant, fungal, and bacterial sources. Lectin microarray profiles suggested that only subtle changes in the glycosylation of IgG occurred during days 2 and 3 of lactation, but by day 10, the lectin profile diverged from the other three time points. Monosaccharide analysis carried out after hydrolysis confirmed that the ratios of oligosaccharide components remained relatively stable through day 3 and also that sialylation was substantially reduced by day 10. The differences that were observed for glycosylation suggest that different functionalities associated with IgG glycosylation may be required in the first days of life.

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