Patterns of Human Milk Oligosaccharides in Mature Milk Are Associated with Certain Gut Microbiota in Infants.

Human milk oligosaccharides (HMOs) are complexes that play a crucial role in shaping the early-life gut microbiota. This study intends to explore whether HMO patterns are associated with the gut microbiota of infants. We included 96 Chinese breastfeeding mother-infant dyads. Breast milk and infant faecal samples were collected and tested. With milk 2'-fucosyllactose, difucosyllactose, and lacto-N-fucopentaose-I as biomarkers, we divided the mothers into secretor and non-secretor groups. HMO patterns were extracted using principal component analysis. The majority (70.7%) of mothers were categorised as secretor and five different HMO patterns were identified. After adjustment, the infants of secretor mothers exhibited a lower relative abundance of Bifidobacterium bifidum (ß = -0.245, 95%CI: -0.465~-0.025). An HMO pattern characterised by high levels of 3-fucosyllactose, lacto-N-fucopentaose-III, and lacto-N-neodifucohexaose-II was positively associated with the relative abundance of Bifidobacterium breve (p = 0.014), while the pattern characterised by lacto-N-neotetraose, 6'-sialyllactose, and sialyllacto-N-tetraose-b was negatively associated with Bifidobacterium breve (p = 0.027). The pattern characterised by high levels of monofucosyl-lacto-N-hexaose-III and monofucosyl-lacto-N-neohexaose was positively associated with Bifidobacterium dentium (p = 0.025) and Bifidobacterium bifidum (p < 0.001), respectively. This study suggests that HMO patterns from mature breast milk were associated with certain gut microbiota of breastfed infants.

Innovative direct introduction-ion mobility-mass spectrometry (DI-IM-MS) approach for fast and robust isomer-specific quantification in a complex matrix: Application to 2'-fucosyllactose (2'-FL) in breast milk.

Identification and specific quantification of isomers in a complex biological matrix by mass spectrometry alone is not an easy task due to their identical chemical formula and therefore their same mass-to-charge ratio (m/z). Here, the potential of direct introduction combined with ion mobility-mass spectrometry (DI-IM-MS) for rapid quantification of isomers as human milk oligosaccharides (HMOs) was investigated. Differences in HMO profiles between various analyzed breast milk samples were highlighted using the single ion mobility monitoring (SIM2) acquisition for high ion mobility resolution detection. Furthermore, the Se+ (secretor) or Se- (non-secretor) phenotype could be assigned to breast milk samples studied based on their HMO contents, especially on the response of 2'-fucosyllactose (2'-FL) and lacto-N-fucopentaose I (LNFP I). The possibility of quantifying a specific isomer in breast milk by DI-IM-MS was also investigated. The standard addition method allowed the determination of the 2'-FL despite the presence of other oligosaccharides, including 3-fucosyllactose (3-FL) isomer in breast milk. This proof-of-concept study demonstrated the high potential of such an approach for the rapid and convenient quantification of isomers in complex mixtures.

Spaceflight impacts xyloglucan oligosaccharide abundance in Arabidopsis thaliana root cell walls.

Over the course of more than a decade, space biology investigations have consistently indicated that cell wall remodeling occurs in a variety of spaceflight-grown plants. Here, we describe a mass spectrometric method to study the fundamental composition of xyloglucan, the most abundant hemicellulose in dicot cell walls, in space-grown plants. Four representative Arabidopsis root samples, from a previously conducted spaceflight experiment - Advanced Plant EXperiment - 04 (APEX-04), were used to investigate changes in xyloglucan oligosaccharides abundances in spaceflight-grown plants compared to ground controls. In situ localized enzymatic digestions and surface sampling mass spectrometry analysis provided spatial resolution of the changes in xyloglucan oligosaccharides abundances. Overall, the results showed that oligosaccharide XXLG/XLXG and XXFG branching patterns were more abundant in the lateral roots of spaceflight-grown plants, while XXXG, XLFG, and XLFG/XLFG were more abundant in the lateral roots of ground control plants. In the primary roots, XXFG had a higher abundance in ground controls than in spaceflight plants. This methodology of analyzing the basic components of the cell wall in this paper highlights two important findings. First, that are differences in the composition of xyloglucan oligosaccharides in spaceflight root cell walls compared to ground controls and, second, most of these differences are observed in the lateral roots. Thus, the methodology described in this paper provides insights into spaceflight cell wall modifications for future investigations.

A universal method for profiling and characterization of oligosaccharides in traditional Chinese medicines.

Oligosaccharides constitute fundamental components in numerous traditional Chinese medicines (TCMs). Conventional chromatographic methods for natural product analysis are not suitable for oligosaccharides due to their large polarity and structural similarity. Herein, an ultra-high performance liquid chromatography with charged aerosol detector (UHPLC-CAD) method was developed for the profiling of oligosaccharides using 9 neutral (DP3-DP11) reference oligosaccharides. Various factors, including columns, mobile phase, elution conditions, flow rate, and column temperature were systematically examined. Optimal separation was achieved using an Amide column with gradient elution within 18 min, at 0.5 mL/min flow rate and 30°C column temperature. Moreover, an ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) method was also optimized to provide structural information. The developed method was applied to detect oligosaccharides in several TCMs, including Morindae Officinalis Radix (MOR), Ziziphi Spinosae Semen (ZSS), Menthae Haplocalycis Herba (MHH) and Chrysanthemi Indici Flos (CIF), revealing 9 and 16 oligosaccharides being uncovered from MHH and CIF respectively for the first time. This study presents a versatile UHPLC-CAD and UHPLC-Q-TOF/MS method with the potential for advancing oligosaccharides discovery and contributing to the quality analysis of TCMs.

Human Milk Oligosaccharides, Growth, and Body Composition in Very Preterm Infants.

Human milk oligosaccharides (HMOs) are bioactive factors that benefit neonatal health, but little is known about effects on growth in very preterm infants (<32 weeks' gestation). We aimed to quantify HMO concentrations in human milk fed to very preterm infants during the neonatal hospitalization and investigate associations of HMOs with infant size and body composition at term-equivalent age. In 82 human-milk-fed very preterm infants, we measured HMO concentrations at two time points. We measured anthropometrics and body composition with air displacement plethysmography at term-equivalent age. We calculated means of individual and total HMOs, constructed tertiles of mean HMO concentrations, and assessed differences in outcomes comparing infants in the highest and intermediate tertiles with the lowest tertile using linear mixed effects models, adjusted for potential confounders. The mean (SD) infant gestational age was 28.2 (2.2) weeks, and birthweight was 1063 (386) grams. Exposure to the highest (vs. lowest) tertile of HMO concentrations was not associated with anthropometric or body composition z-scores at term-corrected age. Exposure to the intermediate (vs. lowest) tertile of 3FL was associated with a greater head circumference z-score (0.61, 95% CI 0.15, 1.07). Overall, the results do not support that higher HMO intakes influence growth outcomes in this very preterm cohort.

High-Performance Thin-Layer Chromatography Coupled with Single Quadrupole: Application the Identification and Differentiation of Rehmanniae Radix and Its Different Processing Products from Raw Materials to Commercial Products.

The authentication of ingredients in formulas is crucial yet challenging, particularly for constituents with comparable compositions but vastly divergent efficacy. Rehmanniae Radix and its derivatives are extensively utilized in food supplements, which contain analogous compositions but very distinct effects. Rehmanniae Radix, also a difficult-to-detect herbal ingredient, was chosen as a case to explore a novel HPTLC-QDa MS technique for the identification of herbal ingredients in commercial products. Through systematic condition optimization, including thin layer and mass spectrometry, a stable and reproducible HPTLC-QDa MS method was established, which can simultaneously detect oligosaccharides and iridoids. Rehmannia Radix and its processed products were then analyzed to screen five markers that could distinguish between raw and prepared Rehmannia Radix. An HPTLC-QDa-SIM method was further established for formula detection by using the five markers and validated using homemade prescriptions and negative controls. Finally, this method was applied to detect raw and prepared Rehmannia Radix in 12 commercial functional products and supplements.

Isolation of Human Milk Difucosyl Nona- and Decasaccharides by Ultrahigh-Temperature Preparative PGC-HPLC and Identification of Novel Difucosylated Heptaose and Octaose Backbones by Negative-Ion ESI-MSn.

Despite their many important physiological functions, past work on the diverse sequences of human milk oligosaccharides (HMOs) has been focused mainly on the highly abundant HMOs with a relatively low degree of polymerization (DP) due to the lack of efficient methods for separation/purification and high-sensitivity sequencing of large-sized HMOs with DP ≥ 10. Here we established an ultrahigh-temperature preparative HPLC based on a porous graphitized carbon column at up to 145 °C to overcome the anomeric α/ß splitting problem and developed further the negative-ion ESI-CID-MS/MS into multistage MSn using a combined product-ion scanning of singly charged molecular ion and doubly charged fragment ion of the branching Gal and adjacent GlcNAc residues. The separation and sequencing method allows efficient separation of a neutral fraction with DP ≥ 10 into 70 components, among which 17 isomeric difucosylated nona- and decasaccharides were further purified and sequenced. As a result, novel branched difucosyl heptaose and octaose backbones were unambiguously identified in addition to the conventional linear and branched octaose backbones. The novel structures of difucosylated DF-novo-heptaose, DF-novo-LNO I, and DF-novo-LNnO I were corroborated by NMR. The various fucose-containing Lewis epitopes identified on different backbones were confirmed by oligosaccharide microarray analysis.

Green manufacturing of a hypoxanthine enzyme sensor for fish freshness based on modified nitrocellulose surface with chito-oligosaccharide.

Hypoxanthine (Hx), produced by adenosine triphosphate (ATP) metabolism, is a valuable indicator that determines the quality and degradation status of meat products and is also an important biochemical marker to certain diseases such as gout. The rapid emergence of paper-based enzyme biosensors has already revolutionized its on-site determination. But it is still limited by the complex patterning and fabrication, unstable enzyme and uneven coloration. This work aims to develop an eco-friendly method to construct engineered paper microfluidic, which seeks to produce reaction and non-reaction zones without any patterning procedure. Chito-oligosaccharide (COS), derived from shrimp shells, was used to modify nitrocellulose membranes and immobilize xanthine oxidase (XOD) and chromogenic agent of nitro blue tetrazolium chloride (NBT). After modification, micro fluids could converge into the modification area and Hx could be detected by XOD-catalyzed conversion. Due to the positively charged cationic basic properties of COS, the enzyme storage stability and the color homogeneity could be greatly strengthened through the electrostatic attraction between COS and XOD and formazan product. The detection limit (LOD) is 2.30 µM; the linear range is 0.05-0.35 mM; the complete test time can be as short as 5 min. The COS-based biosensor shows high specificity and can be used directly for Hx in complex samples such as fish and shrimp samples, and different broths. This biosensor is eco-friendly, nontechnical, economical and therefore a compelling platform for on-site or home-based detection of food freshness.

Air pollution exposure may impact the composition of human milk oligosaccharides.

Human milk oligosaccharides (HMOs) impact neonate immunity and health outcomes. However, the environmental factors influencing HMO composition remain understudied. This study examined the associations between ambient air pollutant (AAP) exposure and HMOs at 1-month postpartum. Human milk samples were collected at 1-month postpartum (n = 185). AAP (PM2.5, PM10, NO2) exposure included the 9-month pregnancy period through 1-month postpartum. Associations between AAP with (1) HMO diversity, (2) the sum of sialylated and fucosylated HMOs, (3) 6 a priori HMOs linked with infant health, and (4) all HMOs were examined using multivariable linear regression and principal component analysis (PCA). Exposure to AAP was associated with lower HMO diversity. PM2.5 and PM10 exposure was positively associated with the HMO 3-fucosyllactose (3FL); PM2.5 exposure was positively associated with the sum of total HMOs, sum of fucosylated HMOs, and the HMO 2'-fucosyllactose (2'FL). PCA indicated the PM2.5, PM10, and NO2 exposures were associated with HMO profiles. Individual models indicated that AAP exposure was associated with five additional HMOs (LNFP I, LNFP II, DFLNT, LNH). This is the first study to demonstrate associations between AAP and breast milk HMOs. Future longitudinal studies will help determine the long-term impact of AAP on human milk composition.

Microbiota from human infants consuming secretors or non-secretors mothers' milk impacts the gut and immune system in mice.

Maternal secretor status is one of the determinants of human milk oligosaccharides (HMOs) composition, which, in turn, influences the gut microbiota composition of infants. To understand if this change in gut microbiota impacts immune cell composition, intestinal morphology, and gene expression, 21-day-old germ-free C57BL/6 mice were transplanted with fecal microbiota from infants whose mothers were either secretors (SMM) or non-secretors (NSM) or from infants consuming dairy-based formula (MFM). For each group, one set of mice was supplemented with HMOs. HMO supplementation did not significantly impact the microbiota diversity; however, SMM mice had a higher abundance of genus Bacteroides, Bifidobacterium, and Blautia, whereas, in the NSM group, there was a higher abundance of Akkermansia, Enterocloster, and Klebsiella. In MFM, gut microbiota was represented mainly by Parabacteroides, Ruminococcaceae_unclassified, and Clostrodium_sensu_stricto. In mesenteric lymph node, Foxp3+ T cells and innate lymphoid cells type 2 were increased in MFM mice supplemented with HMOs, while in the spleen, they were increased in SMM + HMOs mice. Similarly, serum immunoglobulin A was also elevated in MFM + HMOs group. Distinct global gene expression of the gut was observed in each microbiota group, which was enhanced with HMOs supplementation. Overall, our data show that distinct infant gut microbiota due to maternal secretor status or consumption of dairy-based formula and HMO supplementation impacts immune cell composition, antibody response, and intestinal gene expression in a mouse model. IMPORTANCE: Early life factors like neonatal diet modulate gut microbiota, which is important for the optimal gut and immune function. One such factor, human milk oligosaccharides (HMOs), the composition of which is determined by maternal secretor status, has a profound effect on infant gut microbiota. However, how the infant gut microbiota composition determined by maternal secretor status or consumption of infant formula devoid of HMOs impacts infant intestinal ammorphology, gene expression, and immune signature is not well explored. This study provides insights into the differential establishment of infant microbiota derived from infants fed by secretor or non-secretor mothers milk or those consuming infant formula and demonstrates that the secretor status of mothers promotes Bifidobacteria and Bacteroides sps. establishment. This study also shows that supplementation of pooled HMOs in mice changed immune cell composition in the spleen and mesenteric lymph nodes and immunoglobulins in circulation. Hence, this study highlights that maternal secretor status has a role in infant gut microbiota composition, and this, in turn, can impact host gut and immune system.

Consistency and Variability of the Human Milk Oligosaccharide Profile in Repeat Pregnancies.

Human milk oligosaccharides (HMOs) are a set of complex carbohydrates and the third largest solid component of human milk, after lactose and lipids. To date, over 150 HMOs have been identified and the diversity of structures produced by lactating women is influenced by maternal genetics as well as other maternal, infant, and environmental factors. While the concentrations of individual HMOs have been shown to vary between individuals and throughout the course of lactation, the variability of HMO concentration profiles following different pregnancies occurring in the same woman is presently unknown. As such, the objective of this study was to compare HMO concentrations in human milk samples provided by the same women (n = 34) following repeat pregnancies. We leveraged existing human milk samples and metadata from the UC San Diego Human Milk Research Biorepository (HMB) and measured the concentrations of the 19 most abundant HMOs using high-performance liquid chromatography with fluorescence detection (HPLC-FL). By assessing dissimilarities in HMO concentration profiles, as well as concentration trends in individual structures between pregnancies of each participant, we discovered that HMO profiles largely follow a highly personalized and predictable trajectory following different pregnancies irrespective of non-genetic influences. In conclusion, this is the first study to assess the interactions between parity and time following delivery on variations in HMO compositions.

Genome-scale metabolic modeling of the human milk oligosaccharide utilization by Bifidobacterium longum subsp. infantis.

Bifidobacterium longum subsp. infantis is a representative and dominant species in the infant gut and is considered a beneficial microbe. This organism displays multiple adaptations to thrive in the infant gut, regarded as a model for human milk oligosaccharides (HMOs) utilization. These carbohydrates are abundant in breast milk and include different molecules based on lactose. They contain fucose, sialic acid, and N-acetylglucosamine. Bifidobacterium metabolism is complex, and a systems view of relevant metabolic pathways and exchange metabolites during HMO consumption is missing. To address this limitation, a refined genome-scale network reconstruction of this bacterium is presented using a previous reconstruction of B. infantis ATCC 15967 as a template. The latter was expanded based on an extensive revision of genome annotations, current literature, and transcriptomic data integration. The metabolic reconstruction (iLR578) accounted for 578 genes, 1,047 reactions, and 924 metabolites. Starting from this reconstruction, we built context-specific genome-scale metabolic models using RNA-seq data from cultures growing in lactose and three HMOs. The models revealed notable differences in HMO metabolism depending on the functional characteristics of the substrates. Particularly, fucosyl-lactose showed a divergent metabolism due to a fucose moiety. High yields of lactate and acetate were predicted under growth rate maximization in all conditions, whereas formate, ethanol, and 1,2-propanediol were substantially lower. Similar results were also obtained under near-optimal growth on each substrate when varying the empirically observed acetate-to-lactate production ratio. Model predictions displayed reasonable agreement between central carbon metabolism fluxes and expression data across all conditions. Flux coupling analysis revealed additional connections between succinate exchange and arginine and sulfate metabolism and a strong coupling between central carbon reactions and adenine metabolism. More importantly, specific networks of coupled reactions under each carbon source were derived and analyzed. Overall, the presented network reconstruction constitutes a valuable platform for probing the metabolism of this prominent infant gut bifidobacteria.IMPORTANCEThis work presents a detailed reconstruction of the metabolism of Bifidobacterium longum subsp. infantis, a prominent member of the infant gut microbiome, providing a systems view of its metabolism of human milk oligosaccharides.

Extraction of Mucin from Rodent Feces and Determination of O-Linked Oligosaccharide Chain Equivalent Derived from Fecal Mucin.

Since the core protein of mucin in the digesta of the stomach and small intestine, which is less affected by bacteria, remains intact, mucin content can be measured by enzyme-linked immunosorbent assay (ELISA). However, the mucin core protein in bacteria-rich colon digesta and feces is partially hydrolyzed by bacterial enzymes and not fully recognized by mucin antibodies, so mucin cannot be accurately quantified by ELISA. This method quantifies the glycan content linked to the mucin core protein and expresses mucin content in the colon digesta and feces as the equivalent of O-linked oligosaccharide chain. Although mucin glycans are also hydrolyzed by colonic bacteria, this method is a more accurate and simple way to measure mucin content in the digesta of the large intestine and feces than the ELISA method.

Linking human milk oligosaccharide metabolism and early life gut microbiota: bifidobacteria and beyond.

SUMMARYHuman milk oligosaccharides (HMOs) are complex, multi-functional glycans present in human breast milk. They represent an intricate mix of heterogeneous structures which reach the infant intestine in an intact form as they resist gastrointestinal digestion. Therefore, they confer a multitude of benefits, directly and/or indirectly, to the developing neonate. Certain bifidobacterial species, being among the earliest gut colonizers of breast-fed infants, have an adapted functional capacity to metabolize various HMO structures. This ability is typically observed in infant-associated bifidobacteria, as opposed to bifidobacteria associated with a mature microbiota. In recent years, information has been gleaned regarding how these infant-associated bifidobacteria as well as certain other taxa are able to assimilate HMOs, including the mechanistic strategies enabling their acquisition and consumption. Additionally, complex metabolic interactions occur between microbes facilitated by HMOs, including the utilization of breakdown products released from HMO degradation. Interest in HMO-mediated changes in microbial composition and function has been the focal point of numerous studies, in recent times fueled by the availability of individual biosynthetic HMOs, some of which are now commonly included in infant formula. In this review, we outline the main HMO assimilatory and catabolic strategies employed by infant-associated bifidobacteria, discuss other taxa that exhibit breast milk glycan degradation capacity, and cover HMO-supported cross-feeding interactions and related metabolites that have been described thus far.

Effect of fibre fortification of low FODMAP pasta.

Irritable bowel syndrome (IBS) is a condition affecting the digestive system and can be triggered by several different factors, including diet. To ease symptoms of IBS, a diet low in fermentable oligo-, di-, monosaccharides and polyols (FODMAPs) is often recommended. Pasta, as a staple food in the Western World, is naturally high in FODMAPs. This study investigates the impact of insoluble and soluble dietary fibre ingredients in low-FODMAPs pasta. The assessment included physicochemical, sensory, and nutritional quality. Soluble fibre strengthened gluten network, which caused a lower cooking loss and a lower release of sugars during in vitro starch digestion. Insoluble fibre interfered with the gluten network development to a higher extent causing a higher sugar release during digestion. This study reveals the most suitable fibre ingredients for the development of pasta with elevated nutritional value and sensory characteristics compared to commercial products on the market. This type of pasta has a high potential of being suitable for IBS patients.

Sialic acid in human milk and infant formulas in China: concentration, distribution and type.

This study compared the concentrations, types and distributions of sialic acid (SA) in human milk at different stages of the postnatal period with those in a range of infant formulas. Breast milk from mothers of healthy, full-term and exclusively breastfed infants was collected on the 2nd (n 246), 7th (n 135), 30th (n 85) and 90th (n 48) day after birth. The SA profiles of human milk, including their distribution, were analysed and compared with twenty-four different infant formulas. Outcome of this observational study was the result of natural exposure. Only SA of type Neu5Ac was detected in human milk. Total SA concentrations were highest in colostrum and reduced significantly over the next 3 months. Approximately 68·7­76·1 % of all SA in human milk were bound to oligosaccharides. Two types of SA, Neu5Ac and Neu5Gc, have been detected in infant formulas. Most SA was present in infant formulas combined with protein. Breastfed infants could receive more SA than formula-fed infants with the same energy intake. Overall, human milk is a preferable source of SA than infant formulas in terms of total SA content, dynamics, distribution and type. These SA profiles in the natural state are worth to be considered by the production of formulas because they may have a great effect on infant nutrition and development.

Simulated digestions of free oligosaccharides and mucin-type O-glycans reveal a potential role for Clostridium perfringens.

The development of a stable human gut microbiota occurs within the first year of life. Many open questions remain about how microfloral species are influenced by the composition of milk, in particular its content of human milk oligosaccharides (HMOs). The objective is to investigate the effect of the human HMO glycome on bacterial symbiosis and competition, based on the glycoside hydrolase (GH) enzyme activities known to be present in microbial species. We extracted from UniProt a list of all bacterial species catalysing glycoside hydrolase activities (EC 3.2.1.-), cross-referencing with the BRENDA database, and obtained a set of taxonomic lineages and CAZy family data. A set of 13 documented enzyme activities was selected and modelled within an enzyme simulator according to a method described previously in the context of biosynthesis. A diverse population of experimentally observed HMOs was fed to the simulator, and the enzymes matching specific bacterial species were recorded, based on their appearance of individual enzymes in the UniProt dataset. Pairs of bacterial species were identified that possessed complementary enzyme profiles enabling the digestion of the HMO glycome, from which potential symbioses could be inferred. Conversely, bacterial species having similar GH enzyme profiles were considered likely to be in competition for the same set of dietary HMOs within the gut of the newborn. We generated a set of putative biodegradative networks from the simulator output, which provides a visualisation of the ability of organisms to digest HMO and mucin-type O-glycans. B. bifidum, B. longum and C. perfringens species were predicted to have the most diverse GH activity and therefore to excel in their ability to digest these substrates. The expected cooperative role of Bifidobacteriales contrasts with the surprising capacities of the pathogen. These findings indicate that potential pathogens may associate in human gut based on their shared glycoside hydrolase digestive apparatus, and which, in the event of colonisation, might result in dysbiosis. The methods described can readily be adapted to other enzyme categories and species as well as being easily fine-tuneable if new degrading enzymes are identified and require inclusion in the model.

More than nutrition: Therapeutic potential and mechanism of human milk oligosaccharides against necrotizing enterocolitis.

Human milk is the most valuable source of nutrition for infants. The structure and function of human milk oligosaccharides (HMOs), which are key components of human milk, have long been attracting particular research interest. Several recent studies have found HMOs to be efficacious in the prevention and treatment of necrotizing enterocolitis (NEC). Additionally, they could be developed in the future as non-invasive predictive markers for NEC. Based on previous findings and the well-defined functions of HMOs, we summarize potential protective mechanisms of HMOs against neonatal NEC, which include: modulating signal receptor function, promoting intestinal epithelial cell proliferation, reducing apoptosis, restoring intestinal blood perfusion, regulating microbial prosperity, and alleviating intestinal inflammation. HMOs supplementation has been demonstrated to be protective against NEC in both animal studies and clinical observations. This calls for mass production and use of HMOs in infant formula, necessitating more research into the safety of industrially produced HMOs and the appropriate dosage in infant formula.

Evaluation of solid-phase extraction sorbents for purification of oligosaccharides and glycans derivatized by positively charged labels followed by capillary electrophoretic analysis.

The sample preparation including labeling and clean-up represents a key analytical step in the analysis of oligosaccharides and glycans by either chromatographic or electrophoretic separation methods. Although the majority of labeling has been performed by neutral and/or negatively charged tags, the introduction of a positive charge into the saccharide molecule can significantly improve the analysis, especially with mass spectrometry detection. In this work, we present the evaluation of five solid-phase extraction sorbents differing in extraction chemistry for the clean-up and concentration of positively labeled maltooligosaccharides from the reaction mixtures. Maltooligosaccharides containing four to seven glucose units were labeled by cationic tags (2-aminoethyl)trimethylammonium chloride and (carboxymethyl)trimethylammonium chloride hydrazide and the extraction conditions were optimized followed by electrophoretic analysis with conductivity detection. The effects of the solid-phase extraction sorbent chemistry, extraction conditions, and sample composition are discussed. All tested sorbents were capable of cleaning up maltooligosaccharides from the reaction mixtures to some extent after optimization of the solid-phase extraction procedure (51.9%-98.9% recovery). The best-rated amide-based sorbent was used to process the sample of N-linked glycans enzymatically released from ribonuclease B.

Comparative Analysis of Free and Glycoconjugates Oligosaccharide Content in Milk from Different Species.

Human milk is important for infant growth, and oligosaccharides are one of its main functional nutrients. To enable a systematic comparison of free oligosaccharide and glycoconjugate content in milk from different species, the phenol-sulfuric acid and resorcinol assays were combined to determine the content. Using real samples, the method revealed that human milk contained the highest amount of total, neutral (9.84 ± 0.31 g/L), and sialylated (3.21 ± 0.11 g/L) free oligosaccharides, followed by goat milk, with neutral (0.135 ± 0.015 g/L) and sialylated (0.192 ± 0.016 g/L) free oligosaccharides and at a distance by bovine and yak milk. The highest total glycoconjugate content was detected in yak milk (0.798 ± 0.011 g/L), followed by human, bovine, and goat milk. These findings suggest that goat milk is the best source of free oligosaccharides in infant formula and functional dairy products and yak milk is the best source of glycoconjugates.