Rita M. Hickey

Method for milk oligosaccharide profiling by 2-aminobenzamide labeling and hydrophilic interaction chromatography

Although the properties of milk oligosaccharides have been of scientific interest for many years, their structural diversity presents a challenging analytical task. In the quest for a simple and robust technology to characterize the different oligosaccharides present in milk, we developed an analytical scheme based on their fluorescent labeling, pre-fractionation by weak anionic exchange chromatography and separation by hydrophilic interaction liquid chromatography (HILIC)-high performance liquid chromatography (HPLC). HILIC relies on the hydrophilic potential of the molecule, which accounts for differences in properties such as molecular volume, lipophilic surface area, charge, composition, structure, linkage and oligosaccharide branching. The robustness of the methodology has been demonstrated using bovine colostrum oligosaccharides as a case study. Structural assignments for 37 free glycans, including 20 sialylated species, were obtained by a combination of HILIC-HPLC, exoglycosidase digestion and offline negative-ion mode mass spectrometry (MS)/MS. Parameters obtained for each glycan, including linkages, enzymatic digestion products and glucose unit values, will be added to GlycoBase, a public access database (http://glycobase.nibrt.ie/glycobase.html). This approach provides a basis for the analysis of free milk oligosaccharides in a fast and sensitive manner and could be adapted for an automated technology platform amenable to diverse environments. Indeed, our approach, in conjunction with bacterial-binding assays, can provide a better understanding of the structural elements required for biological activity of free milk oligosaccharides and could serve as a scientific basis for the selection of such bioactives from various food sources.

Bifidobacteria exhibit social behavior through carbohydrate resource sharing in the gut

Bifidobacteria are common and frequently dominant members of the gut microbiota of many animals, including mammals and insects. Carbohydrates are considered key carbon sources for the gut microbiota, imposing strong selective pressure on the complex microbial consortium of the gut. Despite its importance, the genetic traits that facilitate carbohydrate utilization by gut microbiota members are still poorly characterized. Here, genome analyses of 47 representative Bifidobacterium (sub)species revealed the genes predicted to be required for the degradation and internalization of a wide range of carbohydrates, outnumbering those found in many other gut microbiota members. The glycan-degrading abilities of bifidobacteria are believed to reflect available carbon sources in the mammalian gut. Furthermore, transcriptome profiling of bifidobacterial genomes supported the involvement of various chromosomal loci in glycan metabolism. The widespread occurrence of bifidobacterial saccharolytic features is in line with metagenomic and metatranscriptomic datasets obtained from human adult/infant faecal samples, thereby supporting the notion that bifidobacteria expand the human glycobiome. This study also underscores the hypothesis of saccharidic resource sharing among bifidobacteria through species-specific metabolic specialization and cross feeding, thereby forging trophic relationships between members of the gut microbiota.

A comparative study of free oligosaccharides in the milk of domestic animals

The present study was conducted to obtain a comprehensive overview of oligosaccharides present in the milk of a variety of important domestic animals including cows, goats, sheep, pigs, horses and dromedary camels. Using an analytical workflow that included ultra-performance liquid chromatography-hydrophilic interaction liquid chromatography with fluorescence detection coupled to quadrupole time-of-flight MS, detailed oligosaccharide libraries were established. The partial or full characterisation of the neutral/fucosylated, phosphorylated and sialylated structures was facilitated by sequencing with linkage- and sugar-specific exoglycosidases. Relative peak quantification of the 2-aminobenzamide-labelled oligosaccharides provided additional information. Milk from domestic animals contained a much larger variety of complex oligosaccharides than was previously assumed, and thirteen of these structures have been identified previously in human milk. The direct comparison of the oligosaccharide mixtures reflects their role in the postnatal maturation of different types of gastrointestinal systems, which, in this way, are prepared for certain post-weaning diets. The potential value of animal milk for the commercial extraction of oligosaccharides to be used in human and animal health is highlighted.

The food glycome: A source of protection against pathogen colonization in the gastrointestinal tract

Trillions of microbes inhabit the gastrointestinal tract of humans with significant differences in the composition and distribution of intestinal flora along its length. Normally there is a symbiotic relationship between the intestinal microflora and the host, with mutual advantages for both partners. When this relationship is altered, commensal bacteria can rapidly shift toward pathogenicity resulting in the onset and progression of gastrointestinal infection. Pathogen adhesion and colonization is often a prelude to infection, and intervention at this early stage can help prevent disease. Bacteria have evolved a multitude of adhesion mechanisms commonly targeting surface carbohydrate structures of the host. Here, we review the ability of various dietary carbohydrates to prevent adhesion of pathogens to host cells. Given their significance in disease, and their ability to cause chronic infection, we have focussed on 3 model pathogens, Helicobacter pylori, Campylobacter jejuni and Clostridium difficile, and dietary carbohydrates which can inhibit their adhesion. The discovery of novel anti-adhesive dietary carbohydrates, once developed as nutraceutical ingredients, may serve as a novel method for preventing infectious diseases in the human gastrointestinal tract. Anti-adhesive carbohydrates used in this context are not bactericidal. Therefore, the spread of pathogens with resistance to antibiotics is less likely to occur.

Structural and functional characteristics of bovine milk protein glycosylation

Most secreted and cell membrane proteins in mammals are glycosylated. Many of these glycoproteins are also prevalent in milk and play key roles in the biomodulatory properties of milk and ultimately in determining milks nutritional quality. Although a significant amount of information exists on the types and roles of free oligosaccharides in milk, very little is known about the glycans associated with milk glycoproteins, in particular, the biological properties that are linked to their presence. The main glycoproteins found in bovine milk are lactoferrin, the immunoglobulins, glycomacropeptide, a glycopeptide derived from κ-casein, and the glycoproteins of the milk fat globule membrane. Here, we review the glycoproteins present in bovine milk, the information currently available on their glycosylation and the biological significance of their oligosaccharide chains.

Controlled Autolysis and Enzyme Release in a Recombinant Lactococcal Strain Expressing the Metalloendopeptidase Enterolysin A

ABSTRACT This study concerns the exploitation of the lytic enzyme enterolysin A (EntL), produced by Enterococcus faecalis strain DPC5280, to elicit the controlled autolysis of starter lactococci. EntL, a cell wall metalloendopeptidase secreted by some E. faecalis strains, can kill a wide range of gram-positive bacteria, including lactococci. The controlled expression of entL, which encodes EntL, was achieved using a nisin-inducible expression system in a lactococcal host. Zymographic analysis of EntL activity demonstrated that active enzyme is produced by the recombinant lactococcal host. Indeed, expression of EntL resulted in almost complete autolysis of the host strain 2 h after induction with nisin. Model cheese experiments using a starter strain in addition to the inducible enterolysin-producing strain showed a 27-fold increase in activity with respect to the release of lactate dehydrogenase in the strain overexpressing EntL, demonstrating the potential of EntL production in large-scale cheese production systems. Indeed, the observation that a wide range of lactic bacteria are sensitive to EntL suggests that EntL-induced autolysis has potential applications with a variety of lactic acid bacteria and could be a basis for probiotic delivery systems.

Exposure of Bifidobacterium longum subsp. infantis to Milk Oligosaccharides Increases Adhesion to Epithelial Cells and Induces a Substantial Transcriptional Response

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.

Transcriptional response of HT-29 intestinal epithelial cells to human and bovine milk oligosaccharides.

Human milk oligosaccharides (HMO) have been shown to interact directly with immune cells. However, large quantities of HMO are required for intervention or clinical studies, but these are unavailable in most cases. In this respect, bovine milk is potentially an excellent source of commercially viable analogues of these unique molecules. In the present study, we compared the transcriptional response of colonic epithelial cells (HT-29) to the entire pool of HMO and bovine colostrum oligosaccharides (BCO) to determine whether the oligosaccharides from bovine milk had effects on gene expression that were similar to those of their human counterparts. Gene set enrichment analysis of the transcriptional data revealed that there were a number of similar biological processes that may be influenced by both treatments including a response to stimulus, signalling, locomotion, and multicellular, developmental and immune system processes. For a more detailed insight into the effects of milk oligosaccharides, the effect on the expression of immune system-associated glycogenes was chosen as a case study when performing validation studies. Glycogenes in the current context are genes that are directly or indirectly regulated in the presence of glycans and/or glycoconjugates. RT-PCR analysis revealed that HMO and BCO influenced the expression of cytokines (IL-1β, IL-8, colony-stimulating factor 2 (granulocyte-macrophage) (GM-CSF2), IL-17C and platelet factor 4 (PF4)), chemokines (chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C-X-C motif) ligand 3 (CXCL3), chemokine (C-C motif) ligand 20 (CCL20), chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 6 (CXCL6), chemokine (C-C motif) ligand 5 (CCL5), chemokine (C-X3-C motif) ligand 1 (CX3CL1) and CXCL2) and cell surface receptors (interferon γ receptor 1 (IFNGR1), intercellular adhesion molecule-1 (ICAM-1), intercellular adhesion molecule-2 (ICAM-2) and IL-10 receptor α (IL10RA)). The present study suggests that milk oligosaccharides contribute to the development and maturation of the intestinal immune response and that bovine milk may be an attractive commercially viable source of oligosaccharides for such applications.

Anti-infective bovine colostrum oligosaccharides: Campylobacter jejuni as a case study

Campylobacter jejuni is the leading cause of acute bacterial infectious diarrhea in humans. Unlike in humans, C. jejuni is a commensal within the avian host. Heavily colonized chickens often fail to display intestinal disease, and no cellular attachment or invasion has been demonstrated in-vivo. Recently, researchers have shown that the reason for the attenuation of C. jejuni virulence may be attributed to the presence of chicken intestinal mucus and more specifically chicken mucin. Since mucins are heavily glycosylated molecules this observation would suggest that glycan-based compounds may act as anti-infectives against C. jejuni. Considering this, we have investigated naturally sourced foods for potential anti-infective glycans. Bovine colostrum rich in neutral and acidic oligosaccharides has been identified as a potential source of anti-infective glycans. In this study, we tested oligosaccharides isolated and purified from the colostrum of Holstein Friesian cows for anti-infective activity against a highly invasive strain of C. jejuni. During our initial studies we structurally defined 37 bovine colostrum oligosaccharides (BCO) by HILIC-HPLC coupled with exoglycosidase digests and off-line mass spectroscopy, and demonstrated the ability of C. jejuni to bind to some of these structures, in-vitro. We also examined the effect of BCO on C. jejuni adhesion to, invasion of and translocation of HT-29 cells. BCO dramatically reduced the cellular invasion and translocation of C. jejuni, in a concentration dependent manner. Periodate treatment of the BCO prior to inhibition studies resulted in a loss of the anti-infective activity of the glycans suggesting a direct oligosaccharide-bacterial interaction. This was confirmed when the BCO completely prevented C. jejuni binding to chicken intestinal mucin, in-vitro. This study builds a strong case for the inclusion of oligosaccharides sourced from cows milk in functional foods. However, it is only through further understanding the structure and function of milk oligosaccharides that such compounds can reach their potential as food ingredients.

In vitro assessment of marine Bacillus for use as livestock probiotics.

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.