Diversity of the Microbiota of Traditional Izmir Tulum and Izmir Brined Tulum Cheeses and Selection of Potential Probiotics.

High-throughput DNA sequencing (HTS) was used to study the microbial diversity of commercial traditional Izmir Tulum (IT) and Izmir Brined Tulum (IBT) cheeses from Izmir, Türkiye. Simultaneously, cultivation-dependent methods were used to isolate, identify and characterize bacterial strains displaying probiotic potential. At the phylum level, Firmicutes dominated the microbiota of both cheese types comprising >98% of the population. Thirty genera were observed, with Streptococcus being the most abundant genus and with Streptococcus thermophilus and S. infantarius subsp. infantarius being the most abundant species. Genera, including Bifidobacterium and Chryseobacterium, not previously associated with IT and IBT, were detected. IT cheeses displayed higher operational taxonomic units (OTUs; Richness) and diversity index (Simpson) than IBT cheeses; however, the difference between the diversity of the microbiota of IT and IBT cheese samples was not significant. Three Lacticaseibacillus paracasei strains isolated from IBT cheeses exhibited probiotic characteristics, which included capacity to survive under in vitro simulated gastrointestinal conditions, resistance to bile salts and potential to adhere to HT-29 human intestinal cells. These findings demonstrate that Tulum cheeses harbor bacterial genera not previously reported in this cheese and that some strains display probiotic characteristics.

Immunoglobulin G from bovine milk primes intestinal epithelial cells for increased colonization of bifidobacteria.

A bovine colostrum fraction (BCF) was recently shown to enhance the adherence of several commensal organisms to intestinal epithelial cells through modulating the epithelial cell surface. In this study, the main components of the BCF were examined to investigate the active component/s responsible for driving the changes in the intestinal cells. The adherence of various bifidobacteria to HT-29 cells was increased when the intestinal cells were pre-incubated with immunoglobulin G (IgG). Modulation of the intestinal cells by IgG was concentration dependent with 16 mg/mL IgG resulting in a 43-fold increase in the adhesion of Bifidobacterium longum NCIMB 8809 to HT-29 cells. Periodate treatment of colostral IgG prior to performing the colonization studies resulted in a reduction in the adhesion of the strain to the intestinal cells demonstrating that the glycans of IgG may be important in modulating the intestinal cells for enhanced commensal adhesion. IgG isolated from mature milk also resulted in significant increases in adhesion of the Bifidobacterium strains tested albeit at reduced levels (3.9-fold). The impact of IgG on the HT-29 cells was also visualised via scanning electron microscopy. This study builds a strong case for the inclusion of IgG ingredients sourced from cow's milk in functional foods aimed at increasing numbers of health promoting bacteria in the human gut.

Bovine colostrum-driven modulation of intestinal epithelial cells for increased commensal colonisation.

Nutritional intake may influence the intestinal epithelial glycome and in turn the available attachment sites for bacteria. In this study, we tested the hypothesis that bovine colostrum may influence the intestinal cell surface and in turn the attachment of commensal organisms. Human HT-29 intestinal cells were exposed to a bovine colostrum fraction (BCF) rich in free oligosaccharides. The adherence of several commensal bacteria, comprising mainly bifidobacteria, to the intestinal cells was significantly enhanced (up to 52-fold) for all strains tested which spanned species that are found across the human lifespan. Importantly, the changes to the HT-29 cell surface did not support enhanced adhesion of the enteric pathogens tested. The gene expression profile of the HT-29 cells following treatment with the BCF was evaluated by microarray analysis. Many so called "glyco-genes" (glycosyltransferases and genes involved in the complex biosynthetic pathways of glycans) were found to be differentially regulated suggesting modulation of the enzymatic addition of sugars to glycoconjugate proteins. The microarray data was further validated by means of real-time PCR. The current findings provide an insight into how commensal microorganisms colonise the human gut and highlight the potential of colostrum and milk components as functional ingredients that can potentially increase commensal numbers in individuals with lower counts of health-promoting bacteria.

The Role of Oligosaccharides in Host-Microbial Interactions for Human Health.

Milk oligosaccharides have many associated bioactivities which can contribute to human health and offer protective properties to the host. Such bioactivities include anti-infective properties whereby oligosaccharides interact with bacterial cells and prevent adhesion to the host and subsequent colonization. Milk oligosaccharides have also been shown to alter the glycosylation of intestinal cells, leading to a reduction in pathogenic colonization. In addition, these sugars promote adhesion of commensal bacterial strains to host cells as well as possessing the ability to alter mucin expression in intestinal cells and improve barrier function. The ability of milk oligosaccharides to alter the transcriptome of both commensal bacterial strains and intestinal epithelial cells has also been revealed, indicating the potential of many cell types to detect the presence of milk oligosaccharides and respond accordingly at the genetic level. Interestingly, domestic animal milk may provide a bioactive source of oligosaccharides for formula supplementation with the aim of emulating the gold standard that is human milk. Overall, this review highlights the ability of milk oligosaccharides to promote health in a variety of ways, for example, through direct bacterial interactions, immunomodulatory activities, promotion of gut barrier function, and induction of protective transcriptional responses.

Novel high-molecular weight fucosylated milk oligosaccharides identified in dairy streams.

Oligosaccharides are the third largest component in human milk. This abundance is remarkable because oligosaccharides are not digestible by the newborn, and yet they have been conserved and amplified during evolution. In addition to encouraging the growth of a protective microbiota dominated by bifidobacteria, oligosaccharides have anti-infective activity, preventing pathogens from binding to intestinal cells. Although it would be advantageous adding these valuable molecules to infant milk formula, the technologies to reproduce the variety and complexity of human milk oligosaccharides by enzymatic/organic synthesis are not yet mature. Consequently, there is an enormous interest in alternative sources of these valuable oligosaccharides. Recent research has demonstrated that bovine milk and whey permeate also contain oligosaccharides. Thus, a thorough characterization of oligosaccharides in bovine dairy streams is an important step towards fully assessing their specific functionalities. In this study, bovine milk oligosaccharides (BMOs) were concentrated by membrane filtration from a readily available dairy stream called "mother liquor", and analyzed by high accuracy MALDI FT-ICR mass spectrometry. The combination of HPLC and accurate mass spectrometry allowed the identification of ideal processing conditions leading to the production of Kg amount of BMO enriched powders. Among the BMOs identified, 18 have high-molecular weight and corresponded in size to the most abundant oligosaccharides present in human milk. Notably 6 oligosaccharides contained fucose, a sugar monomer that is highly abundant in human milk, but is rarely observed in bovine milk. This work shows that dairy streams represent a potential source of complex milk oligosaccharides for commercial development of unique dairy ingredients in functional foods that reproduce the benefits of human milk.

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.