Corrigendum: Structural and functional changes of bioactive proteins in donor human milk treated by vat-pasteurization, retort sterilization, ultra-high-temperature sterilization, freeze-thawing and homogenization.

[This corrects the article DOI: 10.3389/fnut.2022.926814.].

Investigating Milk Fat Globule Structure, Size, and Functionality after Thermal Processing and Homogenization of Human Milk.

Human milk provides bioactive compounds such as milk fat globules (MFGs), which promote brain development, modulate the immune system, and hold antimicrobial properties. To ensure microbiological safety, donor milk banks apply heat treatments. This study compares the effects of heat treatments and homogenization on MFG's physicochemical properties, bioactivity, and bioavailability. Vat pasteurization (Vat-PT), retort (RTR), and ultra-high temperature (UHT) were performed with or without homogenization. UHT, RTR, and homogenization increased the colloidal dispersion of globules, as indicated by increased zeta potential. The RTR treatment completely inactivated xanthine oxidase activity (a marker of MFG bioactivity), whereas UHT reduced its activity by 93%. Interestingly, Vat-PT resulted in less damage, with 28% activity retention. Sialic acid, an important compound for brain health, was unaffected by processing. Importantly, homogenization increased the in vitro lipolysis of MFG, suggesting that this treatment could increase the digestibility of MFG. In terms of color, homogenization led to higher L* values, indicating increased whiteness due to finer dispersion of the fat and casein micelles (and thus greater light scattering), whereas UHT and RTR increased b* values associated with Maillard reactions. This study highlights the nuanced effects of processing conditions on MFG properties, emphasizing the retention of native characteristics in Vat-PT-treated human milk.

Eat your beets: Conversion of polysaccharides into oligosaccharides for enhanced bioactivity.

Bioactive oligosaccharides with the potential to improve human health, especially in modulating gut microbiota via prebiotic activity, are available from few natural sources. This work uses polysaccharide oxidative cleavage to generate oligosaccharides from beet pulp, an agroindustry by-product. A scalable membrane filtration approach was applied to purify the oligosaccharides for subsequent in vitro functional testing. The combined use of nano-LC/Chip Q-TOF MS and UHPLC/QqQ MS allowed the evaluation of the oligosaccharide profile and their monosaccharide complexity. A final product containing roughly 40 g of oligosaccharide was obtained from 475 g of carbohydrates. Microbiological bioactivity assays indicated that the product obtained herein stimulated desirable commensal gut bacteria. This rapid, reproducible, and scalable method represents a breakthrough in the food industry for generating potential prebiotic ingredients from common plant by-products at scale. INDUSTRIAL RELEVANCE: This work proposes an innovative technology based on polysaccharide oxidative cleavage and multi-stage membrane purification to produce potential prebiotic oligosaccharides from renewable sources. It also provides critical information to evidence the prebiotic potential of the newly generated oligosaccharides on the growth promotion ability of representative probiotic strains of bifidobacteria and lactobacilli.

Plant-based production of diverse human milk oligosaccharides.

Human milk oligosaccharides (HMOs) are a diverse class of carbohydrates that aid in the health and development of infants. The vast health benefits of HMOs have made them a commercial target for microbial production; however, producing the ∼130 structurally diverse HMOs at scale has proven difficult. Here, we produce a vast diversity of HMOs by leveraging the robust carbohydrate anabolism of plants. This diversity includes high value HMOs, such as lacto-N-fucopentaose I, that have not yet been commercially produced using state-of-the-art microbial fermentative processes. HMOs produced in transgenic plants provided strong bifidogenic properties, indicating their ability to serve as a prebiotic supplement. Technoeconomic analyses demonstrate that producing HMOs in plants provides a path to the large-scale production of specific HMOs at lower prices than microbial production platforms. Our work demonstrates the promise in leveraging plants for the cheap and sustainable production of HMOs.

Uncovering the promising role of grape pomace as a modulator of the gut microbiome: An in-depth review.

Grape pomace is the primary wine coproduct consisting primarily of grape seeds and skins. Grape pomace holds immense potential as a functional ingredient to improve human health while its valorization can be beneficial for industrial sustainability. Pomace contains bioactive compounds, including phenols and oligosaccharides, most of which reach the colon intact, enabling interaction with the gut microbiome. Microbial analysis found that grape pomace selectively promotes the growth of many commensal bacteria strains, while other types of bacteria, including various pathogens, are highly sensitive to the pomace and its components and are inactivated. In vitro studies showed that grape pomace and its extracts inhibit the growth of pathogenic bacteria in Enterobacteriaceae family while increasing the growth and survival of some beneficial bacteria, including Bifidobacterium spp. and Lactobacillus spp. Grape pomace supplementation in mice and rats improves their gut microbiome complexity and decreases diet-induced obesity as well as related illnesses, including insulin resistance, indicating grape pomace could improve human health. A human clinical trial found that pomace, regardless of its phenolic content, had cardioprotective effects, suggesting that dietary fiber induced those health benefits. To shed light on the active components, this review explores the potential prebiotic capacity of select bioactive compounds in grape pomace.

Creation of a milk oligosaccharide database, MilkOligoDB, reveals common structural motifs and extensive diversity across mammals.

The carbohydrate fraction of most mammalian milks contains a variety of oligosaccharides that encompass a range of structures and monosaccharide compositions. Human milk oligosaccharides have received considerable attention due to their biological roles in neonatal gut microbiota, immunomodulation, and brain development. However, a major challenge in understanding the biology of milk oligosaccharides across other mammals is that reports span more than 5 decades of publications with varying data reporting methods. In the present study, publications on milk oligosaccharide profiles were identified and harmonized into a standardized format to create a comprehensive, machine-readable database of milk oligosaccharides across mammalian species. The resulting database, MilkOligoDB, includes 3193 entries for 783 unique oligosaccharide structures from the milk of 77 different species harvested from 113 publications. Cross-species and cross-publication comparisons of milk oligosaccharide profiles reveal common structural motifs within mammalian orders. Of the species studied, only chimpanzees, bonobos, and Asian elephants share the specific combination of fucosylation, sialylation, and core structures that are characteristic of human milk oligosaccharides. However, agriculturally important species do produce diverse oligosaccharides that may be valuable for human supplementation. Overall, MilkOligoDB facilitates cross-species and cross-publication comparisons of milk oligosaccharide profiles and the generation of new data-driven hypotheses for future research.

A multi-centre peptidomics investigation of food digesta: current state of the art in mass spectrometry analysis and data visualisation.

Mass spectrometry has become the technique of choice for the assessment of a high variety of molecules in complex food matrices. It is best suited for monitoring the evolution of digestive processes in vivo and in vitro. However, considering the variety of equipment available in different laboratories and the diversity of sample preparation methods, instrumental settings for data acquisition, statistical evaluations, and interpretations of results, it is difficult to predict a priori the ideal parameters for optimal results. The present work addressed this uncertainty by executing an inter-laboratory study with samples collected during in vitro digestion and presenting an overview of the state-of-the-art mass spectrometry applications and analytical capabilities available for studying food digestion. Three representative high-protein foods - skim milk powder (SMP), cooked chicken breast and tofu - were digested according to the static INFOGEST protocol with sample collection at five different time points during gastric and intestinal digestion. Ten laboratories analysed all digesta with their in-house equipment and applying theirconventional workflow. The compiled results demonstrate in general, that soy proteins had a slower gastric digestion and the presence of longer peptide sequences in the intestinal phase compared to SMP or chicken proteins, suggesting a higher resistance to the digestion of soy proteins. Differences in results among the various laboratories were attributed more to the peptide selection criteria than to the individual analytical platforms. Overall, the combination of mass spectrometry techniques with suitable methodological and statistical approaches is adequate for contributing to the characterisation of the recently defined digestome.

Associations among Milk Microbiota, Milk Fatty Acids, Milk Glycans, and Inflammation from Lactating Holstein Cows.

Milk oligosaccharides (MOs) can be prebiotic and antiadhesive, while fatty acids (MFAs) can be antimicrobial. Both have been associated with milk microbes or mammary gland inflammation in humans. Relationships between these milk components and milk microbes or inflammation have not been determined for cows and could help elucidate a novel approach for the dairy industry to promote desired milk microbial composition for improvement of milk quality and reduction of milk waste. We aimed to determine relationships among milk microbiota, MFAs, MOs, lactose, and somatic cell counts (SCC) from Holstein cows, using our previously published data. Raw milk samples were collected at three time points, ranging from early to late lactation. Data were analyzed using linear mixed-effects modeling and repeated-measures correlation. Unsaturated MFA and short-chain MFA had mostly negative relationships with potentially pathogenic genera, including Corynebacterium, Pseudomonas, and an unknown Enterobacteriaceae genus but numerous positive relationships with symbionts Bifidobacterium and Bacteroides. Conversely, many MOs were positively correlated with potentially pathogenic genera (e.g., Corynebacterium, Enterococcus, and Pseudomonas), and numerous MOs were negatively correlated with the symbiont Bifidobacterium. The neutral, nonfucosylated MO composed of eight hexoses had a positive relationship with SCC, while lactose had a negative relationship with SCC. One interpretation of these trends might be that in milk, MFAs disrupt primarily pathogenic bacterial cells, causing a relative increase in abundance of beneficial microbial taxa, while MOs respond to and act on pathogenic taxa primarily through antiadhesive methods. Further research is needed to confirm the potential mechanisms driving these correlations. IMPORTANCE Bovine milk can harbor microbes that cause mastitis, milk spoilage, and foodborne illness. Fatty acids found in milk can be antimicrobial and milk oligosaccharides can have antiadhesive, prebiotic, and immune-modulatory effects. Relationships among milk microbes, fatty acids, oligosaccharides, and inflammation have been reported for humans. To our knowledge, associations among the milk microbial composition, fatty acids, oligosaccharides, and lactose have not been reported for healthy lactating cows. Identifying these potential relationships in bovine milk will inform future efforts to characterize direct and indirect interactions of the milk components with the milk microbiota. Since many milk components are associated with herd management practices, determining if these milk components impact milk microbes may provide valuable information for dairy cow management and breeding practices aimed at minimizing harmful and spoilage-causing microbes in raw milk.

Effects of protease-assisted aqueous extraction on almond protein profile, digestibility, and antigenicity.

Almonds (Prunus dulcis) are one of the most consumed tree nuts worldwide and have been recognized as a healthy and nutritious food. Nevertheless, almonds are also a source of allergenic proteins that can trigger several mild to life-threatening allergic reactions. The effects of selected extraction conditions (aqueous vs. protease-assisted aqueous extraction) on the protein profile determined by proteomics analysis of excised SDS-PAGE gel bands, in vitro protein digestibility, and immunoreactivity of almond protein extracts, were evaluated. Proteolysis altered almond protein sequential and conformational characteristics thus affecting digestibility and antigenicity. Proteomics analysis revealed that enzymatic extraction resulted in the reduction of allergen proteins and epitopes. While complete hydrolysis of Prunin 1 and 2 α-chain was observed, Prunin 1 and 2 ß-chains were more resistant to hydrolysis. Protein in vitro digestibility increased from 79.1 to 88.5% after proteolysis, as determined by a static digestion model. The degree of hydrolysis (DH) and peptide content of enzymatically extracted proteins during gastric and duodenal digestion were significantly higher than the ones from unhydrolyzed proteins. Proteolysis resulted in a 75% reduction in almond protein immunoreactivity as determined by a sandwich enzyme-linked immunosorbent assay and a reduction in IgE and IgG reactivities using human sera. The present study shows that moderated hydrolysis (7% DH) using protease can be used as a strategy to improve almond protein digestibility and reduce antigenicity. This study's findings could further enhance the potential use of almond protein hydrolysates in the formulation of hypoallergenic food products with improved nutritional quality and safety.

Release of bifidogenic N-glycans from native bovine colostrum proteins by an endo-ß-N-acetylglucosaminidase.

Milk glycoproteins play various biological roles including antibacterial, antiviral activities, modulating immune responses in living organisms. Released N-glycans from milk glycoproteins act as growth substrates for infant-associated bifidobacteria, which are key members of the breastfed infant's gut. To date, the mechanisms, and contributions of glycans to the biological activities of glycoproteins remain to be elucidated. Only by testing both the released glycans and the deglycosylated protein in their native (i.e., non-denatured) form, can the individual contribution to the biological activity of glycoproteins be elucidated. However, for conventional enzymatic and chemical deglycosylation strategies to work efficiently, glycoprotein denaturation is required, which alters the protein native shape, hindering further investigations of its biological roles. An endo-ß-N-acetylglucosaminidase (EndoBI-1) from Bifidobacterium longum subsp. infantis ATCC 15697 (B. infantis) was characterized as having the ability to release N-glycans from bovine milk glycoproteins efficiently, without the denaturation. In this study, the activity of EndoBI-1 was compared to a commercial enzyme to release N-glycans, the peptide-N-glycosidase F (PNGase F), using dairy glycoproteins as the substrate. The kinetic evaluation showed that EndoBI-1 displayed higher activity on native glycoproteins than PNGase F, with 0.036 mg/mL×min and 0.012 mg/mL×min glycan release, respectively. EndoBI-1 released a broader array of glycan structures compared to PNGase F from native glycoproteins. Thirty-two and fifteen distinct compositions were released from the native glycoproteins by EndoBI-1 and PNGase F, respectively, as characterized by advanced mass spectrometry. EndoBI-1 can be considered a promising enzyme for the release of N-glycans and their protein backbone in the native form, which will enable effective glycan release and will facilitate subsequent investigations to reveal their contribution to glycoproteins' biological roles.

Coupling an ion chromatography to high resolution mass spectrometry (IC-MS) for the discovery of potentially prebiotic oligosaccharides in Chardonnay grape marc.

Oligosaccharides are carbohydrates made of three to twenty monosaccharide units linked through glycosidic bonds. Emerging research into the potential prebiotic activity of oligosaccharides is creating opportunities to use industrial byproducts as value-added products. Grape marc is a residue left after winemaking and has been shown to provide health benefits to humans. In this study, we analyzed the oligosaccharides in Chardonnay grape marc by utilizing a hyphenated platform in which an ion chromatography (IC) system is coupled to an Orbitrap mass spectrometer (MS). With this platform, we obtained a structural library including 32 oligosaccharides with unique compositions of monosaccharides and 61 oligosaccharide structures. Notably, the ion chromatographic separation provided resolution of charged isomers while maintaining separation capacity for small, neutral oligosaccharides. High-quality tandem MS also facilitated the identification of oligosaccharides with structural modifications including methylation and the presence of sugar alditols and hexuronic acids. The data acquired by the IC-MS system were also compared with previously published LC-MS data. We found that these two platforms are largely complementary and, in combination, provide a more comprehensive characterization of oligosaccharides than either platform achieves alone.

Comprehensive oligosaccharide profiling of commercial almond milk, soy milk, and soy flour.

Oligosaccharides are known for several bioactivities on health, however, in sensitive individuals, can cause intestinal discomfort. This study aimed to investigate the oligosaccharide profiles in selected plant-based food products. A quantification method based on high-performance anion-exchange chromatography-pulsed amperometric detection was developed, validated, and used to measure major oligosaccharides. Additional low-abundant oligosaccharides and glycosides were characterized by liquid chromatography-tandem mass spectrometry and glycosidases. The summed concentration of raffinose, stachyose, and verbascose ranged from 0.12-0.19 mg/g in almond milk, 3.6-6.4 mg/g in soy milk, and 74-77 and 4.8-57 mg/g in defatted and full-fat soy four. Over 80 different oligosaccharides were characterized. Novel compounds, 2,3-butanediol glycosides, were identified in almond milk. Low-abundant oligosaccharides represented 25 %, 6 %, and 10 % of total OS in almond milk, soy milk, and soy flour, respectively. The data here are useful to estimate oligosaccharide consumption from dietary intake and facilitate further studies on their bioactivity.

Chardonnay Marc as a New Model for Upcycled Co-products in the Food Industry: Concentration of Diverse Natural Products Chemistry for Consumer Health and Sensory Benefits.

Research continues to provide compelling insights into potential health benefits associated with diets rich in plant-based natural products (PBNPs). Coupled with evidence from dietary intervention trials, dietary recommendations increasingly include higher intakes of PBNPs. In addition to health benefits, PBNPs can drive flavor and sensory perceptions in foods and beverages. Chardonnay marc (pomace) is a byproduct of winemaking obtained after fruit pressing that has not undergone fermentation. Recent research has revealed that PBNP diversity within Chardonnay marc has potential relevance to human health and desirable sensory attributes in food and beverage products. This review explores the potential of Chardonnay marc as a valuable new PBNP ingredient in the food system by combining health, sensory, and environmental sustainability benefits that serves as a model for development of future ingredients within a sustainable circular bioeconomy. This includes a discussion on the potential role of computational methods, including artificial intelligence (AI), in accelerating research and development required to discover and commercialize this new source of PBNPs.

Structural and functional changes of bioactive proteins in donor human milk treated by vat-pasteurization, retort sterilization, ultra-high-temperature sterilization, freeze-thawing and homogenization.

Background: Donor human milk should be processed to guarantee microbiological safety prior to infant feeding, but this process can influence the structure and quantity of functional proteins. Objective: The aim of this study was to determine the effect of thawing, homogenization, vat-pasteurization (Vat-PT), retort sterilization (RTR) and ultra-high-temperature (UHT) processing on the structure of bioactive proteins in donor milk. Methods: Pooled donor milk was either not treated (Raw) or treated with an additional freeze-thaw cycle with and without homogenization, Vat-PT, RTR with and without homogenization, and UHT processing with and without homogenization. Overall protein retention was assessed via sodium-dodecyl sulfate (SDS-PAGE), and the immunoreactivity of 13 bioactive proteins were assessed via enzyme-linked immunosorbent assay (ELISA). Results: Freeze-thawing, freeze-thawing plus homogenization and Vat-PT preserved all the immunoglobulins (sIgA/IgA, IgG, IgM) in donor milk, whereas RTR and UHT degraded almost all immunoglobulins. UHT did not alter osteopontin immunoreactivity, but Vat-PT and retort decreased it by ~50 and 70%, respectively. Freeze-thawing with homogenization, Vat-PT and UHT reduced lactoferrin's immunoreactivity by 35, 65, and 84%, respectively. Lysozyme survived unaltered throughout all processing conditions. In contrast, elastase immunoreactivity was decreased by all methods except freeze-thawing. Freeze-thawing, freeze-thawing plus homogenization and Vat-PT did not alter polymeric immunoglobulin receptor (PIGR) immunoreactivity, but RTR, RTR plus homogenization and UHT increased detection. All heat processing methods increased α-lactalbumin immunoreactivity. Vat-PT preserved all the growth factors (vascular/endothelial growth factor, and transforming growth factors ß1 and ß2), and UHT treatments preserved the majority of these factors. Conclusion: Different bioactive proteins have different sensitivity to the treatments tested. Overall, Vat-PT preserved more of the bioactive proteins compared with UHT or RTR. Therefore, human milk processors should consider the impact of processing methods on key bioactive proteins in human milk.

Food glycomics: Dealing with unexpected degradation of oligosaccharides during sample preparation and analysis.

This study reveals that unexpected degradation of food oligosaccharides can occur during conventional glycomics workflows, including sample preparation and analysis by liquid chromatography-mass spectrometry (LC-MS). With the present investigation, we aim to alert the scientific community of the susceptibility of specific glycosidic linkages to degradation induced by heat and acid. Key standard oligosaccharides representing the major types found in foods (3'-sialyllactose and 6'-sialyl-N-acetyllactosamine for milk, raffinose and stachyose for legumes) were selected as model systems and underwent each of the following treatments independently: (1) labeled with the derivatizing agent 1-aminopyrene-3,6,8-trisulfonic (APTS) (followed by analysis with a capillary electrophoresis system coupled with a fluorescence detector), (2) dried from an acetonitrile-water mixture containing 0.1% trifluoroacetic acid, and (3) injected into an LC-MS system. We demonstrated that both raffinose and stachyose degraded during APTS-labeling by the acid in the labeling reagents. We also discovered that during centrifugal evaporation at 37 °C, all of the four nonderivatized oligosaccharides tested were partially degraded. Additionally, when the LC-MS eluent contained 0.1% formic acid, 3'-sialyllactose, raffinose, and stachyose underwent extensive in-source fragmentation during analysis. Lastly, we identified a simple strategy that can reduce the probability of incorrect oligosaccharide identification resulting from extensive in-source fragmentation.

Dietary Fiber to Starch Ratio Affects Bovine Milk Oligosaccharide Profiles.

Background: Bovine milk oligosaccharides (BMOs) have several demonstrated and hypothesized benefits including roles in cognitive development and antipathogenic activities, making them promising ingredients for infant formulas and nutraceutical applications. BMO extraction from bovine milk is challenged by low concentrations relative to nonbioactive simple sugars like lactose. BMO abundances are known to vary with a cow's lactation stage, breed, and parity, but these characteristics are difficult to modify in existing dairy herds. In contrast, diet modification is an accessible target, and is already known to influence milk yield, lipid content, protein levels, and monosaccharide compositions. Objectives: To determine the impact of a low starch high fiber versus a high starch low fiber diet on overall BMO profiles and individual BMO abundances in Holstein dairy cattle. Methods: Milk samples were collected from 59 midlactation Holsteins in a crossover study featuring dietary modification with either a low starch high fiber or high starch low fiber feed. BMO profiles were evaluated by nano-LC quadrupole time-of-flight tandem MS, and differences in BMO abundances between diets were evaluated using linear mixed effects modeling. Results: A total of 19 BMOs were identified across the sample set, including 4 large fucosylated compounds. Seven BMOs were found to have significantly more positive percent changes in yield-adjusted abundance from the pre-experiment baseline period for milk samples collected during feeding with the low starch high fiber diet compared with the high starch low fiber diet. Conclusions: Consuming the low starch high fiber diet promoted greater overall BMO production than the high starch low fiber diet in a population of midlactation Holsteins. Additionally, this study afforded the opportunity to investigate the impact of other factors potentially influencing BMO abundances, furthering understanding of how dairy herd management practices can positively impact milk composition and support the potential use of BMOs as functional ingredients.

Solid-Phase Extraction Approaches for Improving Oligosaccharide and Small Peptide Identification with Liquid Chromatography-High-Resolution Mass Spectrometry: A Case Study on Proteolyzed Almond Extract.

Reverse-phase solid-phase extraction (SPE) is regularly used for separating and purifying food-derived oligosaccharides and peptides prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. However, the diversity in physicochemical properties of peptides may prevent the complete separation of the two types of analytes. Peptides present in the oligosaccharide fraction not only interfere with glycomics analysis but also escape peptidomics analysis. This work evaluated different SPE approaches for improving LC-MS/MS analysis of both oligosaccharides and peptides through testing on peptide standards and a food sample of commercial interest (proteolyzed almond extract). Compared with conventional reverse-phase SPE, mixed-mode SPE (reverse-phase/strong cation exchange) was more effective in retaining small/hydrophilic peptides and capturing them in the high-organic fraction and thus allowed the identification of more oligosaccharides and dipeptides in the proteolyzed almond extract, with satisfactory MS/MS confirmation. Overall, mixed-mode SPE emerged as the ideal method for simultaneously improving the identification of food-derived oligosaccharides and small peptides using LC-MS/MS analysis.

A complete workflow for discovering small bioactive peptides in foods by LC-MS/MS: A case study on almonds.

Identification of bioactive peptides is an increasingly important target for food chemists, particularly in consideration of the widespread application of proteolytic enzymes in food processing. Because the characterization of small peptides by LC-MS/MS is challenging, we optimized a dimethyl labeling technique to facilitate small peptide identification, using almond proteins as a model. The method was validated by comparing the MS/MS spectra of standards and almond-derived peptides in their nonderivatized and derivatized forms. Signal enhancement of a1 ions was proved to effectively aid in the full-length sequencing of small peptides. We further validated this method using two industrially-relevant protein-rich extracts from almond flour: 1737 medium-sized peptides (5-39 amino acids) and 843 small peptides (2-4 amino acids) were identified. The use of an online bioactive peptide database, complemented by the existing literature, allowed the discovery of 208 small bioactive peptides, whereas for medium-sized peptides, only one was reported being bioactive.

Naturally Occurring Glycosidases in Milk from Native Cattle Breeds: Activity and Consequences on Free and Protein Bound-Glycans.

Little is known about the extent of variation and activity of naturally occurring milk glycosidases and their potential to degrade milk glycans. A multi-omics approach was used to investigate the relationship between glycosidases and important bioactive compounds such as free oligosaccharides and O-linked glycans in bovine milk. Using 4-methylumbelliferone (4-MU) assays activities of eight indigenous glycosidases were determined, and by mass spectrometry and 1H NMR spectroscopy various substrates and metabolite products were quantified in a subset of milk samples from eight native North European cattle breeds. The results showed a clear variation in glycosidase activities among the native breeds. Interestingly, negative correlations between some glycosidases including ß-galactosidase, N-acetyl-ß-d-glucosaminidase, certain oligosaccharide isomers as well as O-linked glycans of κ-casein were revealed. Further, a positive correlation was found for free fucose content and α-fucosidase activity (r = 0.37, p-value < 0.001) indicating cleavage of fucosylated glycans in milk at room temperature. The results obtained suggest that milk glycosidases might partially degrade valuable glycans, which would result in lower recovery of glycans and thus represent a loss for the dairy ingredients industry if these activities are pronounced.

Human milk oligosaccharide 2'-fucosyllactose supplementation improves gut barrier function and signaling in the vagal afferent pathway in mice.

2'-Fucosyllactose (2'-FL) is one of the predominant oligosaccharides found in human milk and has several well-established beneficial effects in the host. It has previously been shown that 2'-FL can improve the metabolic phenotype in high-fat (HF)-fed mice. Here we investigated whether dietary supplementation with 2'-FL was associated with improved intestinal barrier integrity, signaling in the vagal afferent pathway and cognitive function. Mice were fed either a low-fat (LF, 10% fat per kcal) or HF (45% fat per kcal) diet with or without supplementation of 2'-FL (10% w/w) in the diet for 8 weeks. Body weight, energy intake, fat and lean mass, intestinal permeability (ex vivo in Ussing chambers), lipid profiles, gut microbiome and microbial metabolites, and cognitive functions were measured. Vagal afferent activity was measured via immunohistochemical detection of c-Fos protein in the brainstem in response to peripheral administration of cholecystokinin (CCK). 2'-FL significantly attenuated the HF-induced increase in fat mass and energy intake. 2'-FL significantly reduced intestinal permeability and significantly increased expression of interleukin (IL)-22, a cytokine known for its protective role in the intestine. Additionally, 2'-FL led to changes in the gut microbiota composition and in the associated microbial metabolites. Signaling in the vagal afferent pathway was improved but there was no effect on cognitive function. In conclusion, 2'-FL supplementation improved the metabolic profiles, gut barrier integrity, lipid metabolism and signaling in the vagal afferent pathway. These findings support the utility of 2'-FL in the control of gut barrier function and metabolic homeostasis under a metabolic challenge.