Sialylated Milk Oligosaccharides Research Articles

Structural Characterization and Abundance of Sialylated Milk Oligosaccharides in Holstein Cows during Early Lactation.

Among other bioactive molecules, milk contains high amounts of sialylated milk oligosaccharides (MOs) that influence numerous processes in the offspring. For instance, sialylated MOs inhibit the invasion of pathogens and positively influence the gut microbiome to support the optimal development of the offspring. For these reasons, sialylated MOs are also used in infant formula as well as food supplements and are potential therapeutic substances for humans and animals. Because of the high interest in sialylated bovine MOs (bMOs), we used several analytical approaches, such as gas and liquid chromatography in combination with mass spectrometry, to investigate in detail the profile of sialylated bMOs in the milk of Holstein Friesian cows during early lactation. Most of the 40 MOs identified in this study were sialylated, and a rapid decrease in all detected sialylated bMOs took place during the first day of lactation. Remarkably, we observed a high variance within the sialylation level during the first two days after calving. Therefore, our results suggest that the content of sialylated MOs might be an additional quality marker for the bioactivity of colostrum and transitional milk to ensure its optimized application for the production of milk replacer and food supplements.

Sialic acid-based probiotic intervention in lactating mothers improves the neonatal gut microbiota and immune responses by regulating sialylated milk oligosaccharide synthesis via the gut–breast axis

ABSTRACT Human milk oligosaccharides (HMOs) are vital milk carbohydrates that help promote the microbiota-dependent growth and immunity of infants. Sialic acid (SA) is a crucial component of sialylated milk oligosaccharides (S-MOs); however, the effects of SA supplementation in lactating mothers on S-MO biosynthesis and their breastfed infants are unknown. Probiotic intervention during pregnancy or lactation demonstrates promise for modulating the milk glycobiome. Here, we evaluated whether SA and a probiotic (Pro) mixture could increase S-MO synthesis in lactating mothers and promote the microbiota development of their breastfed neonates. The results showed that SA+Pro intervention modulated the gut microbiota and 6’-SL contents in milk of maternal rats more than the SA intervention, which promoted Lactobacillus reuteri colonization in neonates and immune development. Deficient 6’-SL in the maternal rat milk of St6gal1 knockouts (St6gal1−/−) disturbed intestinal microbial structures in their offspring, thereby impeding immune tolerance development. SA+Pro intervention in lactating St6gal1± rats compromised the allergic responses of neonates by promoting 6′-SL synthesis and the neonatal gut microbiota. Our findings from human mammary epithelial cells (MCF-10A) indicated that the GPR41-PI3K-Akt-PPAR pathway helped regulate 6′-SL synthesis in mammary glands after SA+Pro intervention through the gut – breast axis. We further validated our findings using a human-cohort study, confirming that providing SA+Pro to lactating Chinese mothers increased S-MO contents in their breast milk and promoted gut Bifidobacterium spp. and Lactobacillus spp. colonization in infants, which may help enhance immune responses. Collectively, our findings may help alter the routine supplementation practices of lactating mothers to modulate milk HMOs and promote the development of early-life gut microbiota and immunity.

Review on the Impact of Milk Oligosaccharides on the Brain and Neurocognitive Development in Early Life.

Milk Oligosaccharides (MOS), a group of complex carbohydrates found in human and bovine milk, have emerged as potential modulators of optimal brain development for early life. This review provides a comprehensive investigation of the impact of milk oligosaccharides on brain and neurocognitive development of early life by synthesizing current literature from preclinical models and human observational studies. The literature search was conducted in the PubMed search engine, and the inclusion eligibility was evaluated by three reviewers. Overall, we identified 26 articles for analysis. While the literature supports the crucial roles of fucosylated and sialylated milk oligosaccharides in learning, memory, executive functioning, and brain structural development, limitations were identified. In preclinical models, the supplementation of only the most abundant MOS might overlook the complexity of naturally occurring MOS compositions. Similarly, accurately quantifying MOS intake in human studies is challenging due to potential confounding effects such as formula feeding. Mechanistically, MOS is thought to impact neurodevelopment through modulation of the microbiota and enhancement of neuronal signaling. However, further advancement in our understanding necessitates clinical randomized-controlled trials to elucidate the specific mechanisms and long-term implications of milk oligosaccharides exposure. Understanding the interplay between milk oligosaccharides and cognition may contribute to early nutrition strategies for optimal cognitive outcomes in children.

Short-Term Effects of Human versus Bovine Sialylated Milk Oligosaccharide Microinjection on Zebrafish Larvae Survival, Locomotor Behavior and Gene Expression

Milk oligosaccharides are a complex class of carbohydrates that act as bioactive factors in numerous defensive and physiological functions, including brain development. Early nutrition can modulate nervous system development and can lead to epigenetic imprinting. We attempted to increase the sialylated oligosaccharide content of zebrafish yolk reserves, with the aim of evaluating any short-term effects of the treatment on mortality, locomotor behavior, and gene expression. Wild-type embryos were microinjected with saline solution or solutions containing sialylated milk oligosaccharides extracted from human and bovine milk. The results suggest that burst activity and larval survival rates were unaffected by the treatments. Locomotion parameters were found to be similar during the light phase between control and treated larvae; in the dark, however, milk oligosaccharide-treated larvae showed increased test plate exploration. Thigmotaxis results did not reveal significant differences in either the light or the dark conditions. The RNA-seq analysis indicated that both treatments exert an antioxidant effect in developing fish. Moreover, sialylated human milk oligosaccharides seemed to increase the expression of genes related to cell cycle control and chromosomal replication, while bovine-derived oligosaccharides caused an increase in the expression of genes involved in synaptogenesis and neuronal signaling. These data shed some light on this poorly explored research field, showing that both human and bovine oligosaccharides support brain proliferation and maturation.

Milk Polysialic Acid Levels Rapidly Decrease in Line with the N-Acetylneuraminic Acid Concentrations during Early Lactation in Dairy Cows.

Sialylated milk oligosaccharides and glycoconjugates have several positive effects on the mucosal barrier, the gut microbiome, and an effective immune system. For this reason, they are important biomolecules for mammary gland health and optimal development of offspring. In milk, the major sialic acid, N-acetylneuraminic acid (Neu5Ac), can be attached as monosialyl-residues or as polymers. To investigate the sialylation processes during lactation of German Holstein cows, we analyzed udder tissue in addition to milk at different time points of lactation. The analysis of the milk samples revealed that both the levels of Neu5Ac and its polymer, polysialic acid (polySia), rapidly decreased during the first three days of lactation, and a high interindividual variance was observed. In mature milk, however, the sialylation status remains relatively constant. The results indicate that mammary gland epithelial cells are one source for milk polySia, since immunohistochemistry of udder tissue exhibited strong polySia staining in these cells. Furthermore, both polysialyltransferases, ST8SiaII and ST8SiaIV, are expressed. Based on known functions of monosialyl residues and polySia, we discuss the potential impact of these biomolecules and the consequences of the heterogeneous sialylation status of milk in relation to udder health and offspring health.

Studies and Application of Sialylated Milk Components on Regulating Neonatal Gut Microbiota and Health.

Breast milk is rich in sialic acids (SA), which are commonly combined with milk oligosaccharides and glycoconjugates. As a functional nutrient component, SA-containing milk components have received increasing attention in recent years. Sialylated human milk oligosaccharides (HMOs) have been demonstrated to promote the growth and metabolism of beneficial gut microbiota in infants, bringing positive outcomes to intestinal health and immune function. They also exhibit antiviral and bacteriostatic activities in the intestinal mucosa of new-borns, thereby inhibiting the adhesion of pathogens to host cells. These properties play a pivotal role in regulating the intestinal microbial ecosystem and preventing the occurrence of neonatal inflammatory diseases. In addition, some recent studies also support the promoting effects of sialylated HMOs on neonatal bone and brain development. In addition to HMOs, sialylated glycoproteins and glycolipids are abundant in milk, and are also critical to neonatal health. This article reviews the current research progress in the regulation of sialylated milk oligosaccharides and glycoconjugates on neonatal gut microbiota and health.

Current Perspective of Sialylated Milk Oligosaccharides in Mammalian Milk: Implications for Brain and Gut Health of Newborns.

Human milk oligosaccharides (HMOs) are the third most abundant solid component after lactose and lipids of breast milk. All mammal milk contains soluble oligosaccharides, including neutral milk oligosaccharides (NMOs) without sialic acid (Sia) moieties and acidic oligosaccharides or sialylated milk oligosaccharides (SMOs) with Sia residues at the end of sugar chains. The structural, biological diversity, and concentration of milk oligosaccharides in mammalian milk are significantly different among species. HMOs have multiple health benefits for newborns, including development of immune system, modification of the intestinal microbiota, anti-adhesive effect against pathogens, and brain development. Most infant formulas lack oligosaccharides which resemble HMOs. Formula-fed infants perform poorly across physical and psychological wellbeing measures and suffer health disadvantages compared to breast-fed infants due to the differences in the nutritional composition of breast milk and infant formula. Of these milk oligosaccharides, SMOs are coming to the forefront of research due to the beneficial nature of Sia. This review aims to critically discuss the current state of knowledge of the biology and role of SMOs in human milk, infant formula milks, and milk from several other species on gut and brain health of human and animal offspring.

Sialylated milk oligosaccharides alter neurotransmitters and brain metabolites in piglets: an In vivo magnetic resonance spectroscopic (MRS) study

ABSTRACT Background: Human milk contains high concentrations and diversity of sialylated oligosaccharides that have multifunctional health benefits, however, their potential role in optimizing neurodevelopment remains unknown. Objective: To investigate the effect of sialylated milk oligosaccharides (SMOS) intervention on neurotransmitters and brain metabolites in piglets. Methods: 3-day-old piglets were randomly allocated to one of three groups and fed either standard sow milk replacer (SMR) alone (n = 15), SMR supplemented with sialyllactose 9.5 g/kg (SL, n = 16) or a combination of SL and 6′-sialyllactosamine 9.5 g/kg (SL/SLN, n = 15) for 35 days. Brain spectra were acquired using a 3T Magnetic Resonance Spectroscopic (MRS) system. Results: SMOS fed piglets were observed to have significantly increased the absolute levels of myo-inositol (mIns) and glutamate + glutamine (Glx), in particular, the SL/SLN group. Similar findings were found in the relative amount of these metabolites calculated as ratios to creatine (Cr), choline (Cho) and N-acetylaspartate (NAA) respectively (P < .05). In addition, there were significant positive correlations of brain NAA, total NAA (TNAA), mIns, total Cho (TCho), total Cr (TCr), scyllo-Inositol (SI) and glutathione (Glth) with total white matter volume; Glu and SI with whole brain volume; and SI with whole brain weight respectively (P < .01). SLN and 3′SL intake were closely correlated with the levels of brain Glu, mlns and Glx in the treatment groups only (P < .01–.05). Conclusions: We provide in vivo evidences that milk SMOS can alter many important brain metabolites and neurotransmitters required for optimizing neurodevelopment in piglets, an animal model of human infants.

Profile and content of sialylated oligosaccharides in donkey milk at early lactation

Animal milk oligosaccharides can be used as bioactive ingredients in functional foods and infant milk formulas. Donkey milk is already considered a functional food and a possible substitute for human milk. The aim of this study was to identify the structure and abundance of sialylated milk oligosaccharides from five Amiata donkeys during four different stages of early lactation. Liquid chromatography combined with mass spectrometry analysis led to the identification and quantification of seven sialylated oligosaccharides: 3'-sialyllactose, 6'-sialyllactose, sialyllacto-N-tetraose a, sialyllacto-N-tetraose b, sialyllacto-N-tetraose c, Neu5Ac(α2–3) + Gal(β1–4)GlcNAc(β1–6)(Gal(β1–3))Gal(β1–4)Glc and Neu5Ac(α2–6) + Gal(β1–4)GlcNAc(β1–6)(Gal(β1–3))Gal(β1–4)Glc. The latter five oligosaccharides were identified in donkey milk for the first time. The most abundant sialylated oligosaccharide found was 6-sialyllactose. In addition, variations in the content of four oligosaccharides were highlighted over the experimental period: the highest values were found at 15 days of lactation, and the lowest at 60 days.

Molecular Mechanisms Underlying How Sialyllactose Intervention Promotes Intestinal Maturity by Upregulating GDNF Through a CREB-Dependent Pathway in Neonatal Piglets

Sialylated milk oligosaccharides (SMOs) have a multifunctional health benefit, yet the molecular details underlying their potential role in modulating intestinal maturation remains unknown. To test the hypothesis that sialyllactose (SL) may mediate intestinal maturation and function through controlling neuronal function, studies were carried out where the diet of postnatal piglets was supplemented with a mixture of 3'- and 6'-sialyllactose from postnatal day 3 to 38. Gene transcription pathways regulating enteric nervous system function, polysialic acid (polySia) synthesis, and cell proliferation were quantified. Our new findings show that SL intervention: (1) upregulated the level of gene and protein expression of the glial-derived neurotrophic factor (GDNF) in the ileum; (2) upregulated phosphorylation of the cAMP responsive element-binding protein (CREB), the downstream target of GDNF signaling pathway; (3) promoted cell proliferation based on an increase in the number and density of Ki-67 positive cells in the crypts; (4) increased the crypt width in the ileum by 10%, while gene markers for the functional cells were not affected; (5) upregulated mRNA expression level of ST8Sia IV, a key polysialyltransferase responsible for synthesis of polySia-NCAM; (6) reduced the incidence and severity of diarrhea. These results show that SL promotes intestinal maturation in neonatal piglets by upregulating GDNF, synthesis of polySia and CREB-interactive pathway.

Mechanisms by which sialylated milk oligosaccharides impact bone biology in a gnotobiotic mouse model of infant undernutrition

Undernutrition in children is a pressing global health problem, manifested in part by impaired linear growth (stunting). Current nutritional interventions have been largely ineffective in overcoming stunting, emphasizing the need to obtain better understanding of its underlying causes. Treating Bangladeshi children with severe acute malnutrition with therapeutic foods reduced plasma levels of a biomarker of osteoclastic activity without affecting biomarkers of osteoblastic activity or improving their severe stunting. To characterize interactions among the gut microbiota, human milk oligosaccharides (HMOs), and osteoclast and osteoblast biology, young germ-free mice were colonized with cultured bacterial strains from a 6-mo-old stunted infant and fed a diet mimicking that consumed by the donor population. Adding purified bovine sialylated milk oligosaccharides (S-BMO) with structures similar to those in human milk to this diet increased femoral trabecular bone volume and cortical thickness, reduced osteoclasts and their bone marrow progenitors, and altered regulators of osteoclastogenesis and mediators of Th2 responses. Comparisons of germ-free and colonized mice revealed S-BMO-dependent and microbiota-dependent increases in cecal levels of succinate, increased numbers of small intestinal tuft cells, and evidence for activation of a succinate-induced tuft cell signaling pathway linked to Th2 immune responses. A prominent fucosylated HMO, 2'-fucosyllactose, failed to elicit these changes in bone biology, highlighting the structural specificity of the S-BMO effects. These results underscore the need to further characterize the balance between, and determinants of, osteoclastic and osteoblastic activity in stunted infants/children, and suggest that certain milk oligosaccharides may have therapeutic utility in this setting.

Profiling of Sialylated Oligosaccharides in Mammalian Milk Using Online Solid Phase Extraction-Hydrophilic Interaction Chromatography Coupled with Negative-Ion Electrospray Mass Spectrometry.

Sialylated oligosaccharides are important components in mammalian milk. They play a key role in the health and growth of infants by helping to shape up infant's gastrointestinal microbiota and defense against infection by various pathogenic agents. A detailed knowledge of the structures, compositions, and quantities of the sialylated milk oligosaccharides (SMOs) is a prerequisite for understanding their biological roles. However, because of the presence of very large amounts of lactose and neutral oligosaccharides, accurate analysis of SMOs is difficult. A pretreatment step is required to remove lactose and neutral oligosaccharides but conventional off-line pretreatment methods are time-consuming and of poor reproducibility. In this presentation, we linked solid-phase extraction (SPE) with hydrophilic interaction chromatography (HILIC) followed by mass spectrometry (MS) identification for the analysis of SMOs. A SPE column with electrostatic repulsion function was used for removal of lactose and neutral oligosaccharides, a HILIC analytical column for separation of the SMOs, and negative-ion electrospray ionization tandem MS was used for their identification and sequencing. The success of the established online SPE-HILIC-MS method was demonstrated by profiling of SMOs in human to investigate detailed SMO changes during lactation period and in animals to compare the difference in SMO contents among the different species.

Functional role and mechanisms of sialyllactose and other sialylated milk oligosaccharides.

Human milk is a rich source of oligosaccharides. Acidic oligosaccharides, such as sialyllactose (SL), contain sialic acid (SA) residues. In human milk, approximately 73% of SA is bound to oligosaccharides, whereas only 3% is present in free form. Oligosaccharides are highly resistant to hydrolysis in the gastrointestinal tract. Only a small portion of the available oligosaccharides in breast milk is absorbed in the neonatal small intestine. SL and sialylated oligosaccharides are thought to have significant health benefits for the neonate, because of their roles in supporting resistance to pathogens, gut maturation, immune function, and cognitive development. The need for SA to allow proper development during the neonatal period is thought to exceed the endogenous synthesis. Therefore, these structures are important nutrients for the neonate. Based on the potential benefits, SL and sialylated oligosaccharides may be interesting components for application in infant nutrition. Once the hurdle of limited availability of these oligosaccharides has been overcome, their functionality can be explored in more detail, and supplementation of infant formula may become feasible.

Fucosylated but Not Sialylated Milk Oligosaccharides Diminish Colon Motor Contractions

Human milk oligosaccharides (HMO) are being studied by different groups exploring a broad range of potential beneficial effects to the breastfed infant. Many of these effects have been attributed to a growth promotion effect on certain gut organisms such as bifidobacteria. Additionally, evidence indicates that HMO are able to directly promote positive changes in gut epithelium and immune responses under certain conditions. This study utilizes a standardized ex vivo murine colon preparation to examine the effects of sialylated, fucosylated and other HMO on gut motor contractions. Only the fucosylated molecules, 2’FL and 3’FL, decreased contractility in a concentration dependent fashion. On the basis of IC50 determinations 3’FL was greater than 2 times more effective than 2’FL. The HMO 3’SL and 6’SL, lacto-N-neotetraose (LNnT), and galactooligosaccharides (GOS) elicited no effects. Lactose was used as a negative control. Fucosylation seems to underlie this functional regulation of gut contractility by oligosaccharides, and L-fucose, while it was also capable of reducing contractility, was substantially less effective than 3’FL and 2’FL. These results suggest that specific HMO are unlikely to be having these effects via bifidogenesis, but though direct action on neuronally dependent gut migrating motor complexes is likely and fucosylation is important in providing this function, we cannot conclusively shown that this is not indirectly mediated. Furthermore they support the possibility that fucosylated sugars and fucose might be useful as therapeutic or preventative adjuncts in disorders of gut motility, and possibly also have beneficial central nervous system effects.

Milk 3′ sialyllactose does not increase brain sialic acid concentration in mice

Sialic acid (Sia), a carboxylated 9‐carbon monosaccharide, is a crucial constituent of brain glycoproteins and glycolipids associated with brain development and enhanced cognitive function. Brain Sia concentrations rapidly increase after birth and are significantly higher in breast‐fed than in formula‐fed infants. Compared to formula, human breast milk contains a high amount of sialylated oligosaccharides; yet their direct role in brain Sia accrual is unknown. Our objective was to determine whether sialylated milk oligosaccharides contribute to brain Sia accumulation in mice between birth and weaning. To overcome problems with artificial feeding in newborn pups, we used a cross‐foster approach with pups reared on dams normal or genetically deficient in 3′sialyllactose (3′SL), one of the major sialylated milk oligosaccharides. Urine 3′SL and brain Sia were derivatized and measured by HPLC. While pups reared on dams with normal milk 3′SL had significantly higher urine 3′SL levels than pups fed on 3′SL deficient milk (460 vs. 40 uM at day 7, P&lt;0.001), there was no significant difference in brain Sia one week (6.0±1.1 vs. 6.0±1.2 pmol/ug protein) and three weeks after birth (19.0±9.7 vs. 15.0±4.9 pmol/ug protein). The data indicate that 3′SL, one of the major sialylated milk oligosaccharides, does not contribute to postnatal brain Sia accumulation – at least in mice.Grant Funding Source: NIH

Evolutionary Glycomics: Characterization of Milk Oligosaccharides in Primates

Free oligosaccharides are abundant components of mammalian milk and have primary roles as prebiotic compounds, in immune defense, and in brain development. A mass spectrometry-based technique is applied to profile milk oligosaccharides from apes (chimpanzee, gorilla, and siamang), new world monkeys (golden lion tamarin and common marmoset), and an old world monkey (rhesus). The purpose of this study is to evaluate the patterns of primate milk oligosaccharide composition from a phylogenetic perspective to assess the extent to which the compositions of HMOs derives from ancestral primate patterns as opposed to more recent evolutionary events. Milk oligosaccharides were quantitated by nanoflow liquid chromatography on chip-based devices. The relative abundances of fucosylated and sialylated milk oligosaccharides in primates were also determined. For a systematic and comprehensive study of evolutionary patterns of milk oligosaccharides, cluster analysis of primate milk was performed using the chromatographic profile. In general, the oligosaccharides in primate milk, including humans, are more complex and exhibit greater diversity compared to the ones in nonprimate milk. A detailed comparison of the oligosaccharides across evolution revealed nonsequential developmental pattern, that is, that primate milk oligosaccharides do not necessarily cluster according to the primate phylogeny. This report represents the first comprehensive and quantitative effort to profile and elucidate the structures of free milk oligosaccharides so that they can be related to glycan function in different primates.

Milk sialyllactose influences colitis in mice through selective intestinal bacterial colonization.

Milk oligosaccharides contribute to the development of the intestinal environment by acting as decoy receptors for pathogens and as prebiotics, which promote the colonization of commensal bacteria. Here, using α2,3- and α2,6-sialyltransferase-deficient mice, we investigated the role of the sialylated milk oligosaccharides sialyl(α2,3)lactose and sialyl(α2,6)lactose on mucosal immunity. The exposure of newborn mice to milk containing or deficient in sialyllactose had no impact on the development of mucosal leukocyte populations. However, when challenged by dextran sulfate sodium (DSS) in drinking water, adult mice that had been fostered on sialyl(α2,3)lactose-deficient milk were more resistant to colitis compared with mice fostered on normal milk or sialyl(α2,6)lactose-deficient milk. Analysis of intestinal microbiota showed different colonization patterns depending on the presence or absence of sialyl(α2,3)lactose in the milk. Germ-free mice reconstituted with intestinal microbiota isolated from mice fed on sialyl(α2,3)lactose-deficient milk were more resistant to DSS-induced colitis than germ-free mice reconstituted with standard intestinal microbiota. Thus, exposure to sialyllactose during infancy affects bacterial colonization of the intestine, which influences the susceptibility to DSS-induced colitis in adult mice.