Systematic Characterization of the Oligosaccharide Profile of Human Milk in Rural Areas of Central China: Quantitative Tracking of Human Milk Oligosaccharide Composition during 12 Months of Lactation.

This study investigates changes in human milk oligosaccharide (HMO) composition over a 12 month breastfeeding period in rural central China. The HMO profiles of 97 mothers were analyzed by graphitized carbon liquid chromatography-electrospray ionization-mass spectrometry. This method was simple to prepare samples and can simultaneously and absolutely quantify at least 20 neutral and acidic HMOs. All mothers were classified into four milk groups based on the presence or absence of specific α-1,2 and α-1,4-fucosylated HMOs. The main oligosaccharides in milk groups I and II were 2'-FL, LDFT, LNFP-I, and LNDFH-I, while LNT, 3-FL, LNFP-II, LNFP-V, LNDFH-II, and DFLNH-b were predominant in milk groups III and IV. Additionally, the lactation period was the primary factor affecting the concentration of individual HMOs. The concentrations of most HMOs decreased with lactation and stabilized after 180 days. However, the concentrations of 3-FL, LDFT, and LNDFH II increased gradually over the lactation period, and the concentration of 3'-SL decreased during early lactation (5-180 days) but increased during later lactation (180-365 days). Furthermore, Spearman correlation analysis revealed that maternal factors and infant factors may also affect the concentration of various HMOs. These findings provide fundamental insights for the development of a comprehensive human milk database.

Combining 2'-fucosyllactose and galactooligosaccharides exerts anti-inflammatory effects and promotes gut health.

This study investigated the potential of 2'-Fucosyllactose (2'-FL) and galactooligosaccharides (GOS) combinations as a novel and cost-effective substitute for human milk oligosaccharides (HMOs) in promoting gut health and reducing inflammation. In vitro studies using Caco-2 cells showed that 2'-FL and GOS combinations (H1: GOS:2'-FL ratio of 1.8:1; H2: ratio of 3.6:1) reduced lipopolysaccharide-induced inflammation by decreasing pro-inflammatory markers, while individual treatments had no significant effects. In a mouse model of dextran sulfate sodium (DSS)-induced colitis, combined 2'-FL and GOS supplementation alleviated symptoms, improved gut permeability, and enhanced intestinal structure, with the GH1 group (H1 combo with DSS) being the most effective. 2'-FL and GOS combinations also enhanced short-chain fatty acid production in infant fecal batch fermentation and mouse fecal analysis, with GH1 showing the most promising results. GH1 supplementation altered gut microbiota in mice with DSS-induced colitis, promoting microbial diversity and a more balanced Firmicutes to Bacteroidota ratio. Infant formula products (IFPs) containing 2'-FL and GOS combinations (IFP2: 174 mg GOS and 95 mg 2'-FL per 14 g serving, 1.8:1 ratio; IFP3: 174 mg GOS and 48 mg 2'-FL per 14 g serving, 3.6:1 ratio) demonstrated gastrointestinal protective and anti-inflammatory properties in a coculture model of Caco-2 and THP-1 cells. These findings suggest that 2'-FL and GOS combinations have potential applications in advanced infant formulas and supplements to promote gut health and reduce inflammation.

The Association Between Maternal Psychological Health and Human Milk Oligosaccharide Composition.

Background and Objective: Human milk oligosaccharides (HMOs) are carbohydrates abundant in human breast milk. Their composition varies widely among women, and prior research has identified numerous factors contributing to this variation. However, the relationship between maternal psychological health and HMO levels is currently unknown. Thus, our objective was to identify whether maternal stress, anxiety, or depressive symptoms are associated with HMOs. Methods: Data originated from 926 lactating individuals from the UC San Diego Human Milk Biorepository. Nineteen prevalent HMOs were assayed using high-performance liquid chromatography. Participants self-reported measures of the Edinburgh Postnatal Depression Scale (n = 495), State-Trait Anxiety Inventory S-Scale (n = 486), and/or Perceived Stress Scale (n = 493) within 60 days of their milk collection; their results were categorized using standard screening cutoffs. HMOs were assessed individually and grouped by principal component analysis (PCA), and associations with maternal psychological symptoms were analyzed using multivariable linear regression adjusted for covariates. Results: After Bonferroni correction (p < 0.002), the following HMOs significantly varied with maternal psychological distress in multivariate analysis: lacto-N-fucopentaose III (LNFP III) and lacto-N-hexaose (LNH) among Secretors with depressive symptoms and difucosyllactose (DFLac), LNFP III, and disialyl-LNH (DSLNH) among Secretors with stress. In PCA, depressive symptoms and stress were associated with one principal component among Secretors. No HMOs varied with anxiety symptoms. Conclusions: Several HMOs varied with maternal depressive symptoms and stress, suggesting a relationship between maternal psychological health and breast milk composition. Additional studies are needed to determine the impact of this variation on infant health.

Enzymatic Synthesis of Disialyllacto-N-Tetraose (DSLNT) and Related Human Milk Oligosaccharides Reveals Broad Siglec Recognition to the Atypical Neu5Acα2-6GlcNAc Motif.

Sialic acids (Sias) are ubiquitously expressed on all types of glycans, typically as terminating residues. They usually link to galactose, N-acetylgalactosamine, or other Sia residues, forming ligands of many glycan-binding proteins. An atypical linkage to the C6 of N-acetylglucosamine (GlcNAc) has been identified in human milk oligosaccharides (HMOs, e.g., DSLNT) and tumor-associated glycoconjugates. Herein, we achieved the systematic synthesis of these HMOs in an enzymatic modular manner. The synthetic strategy relies on a novel activity of ST6GalNAc6 for efficient construction of the Neu5Acα2-6GlcNAc linkage, and another 12 specific enzyme modules for sequential HMO assembly. The structures enabled comprehensive exploration into their structure-function relationships using glycan microarray, revealing broad yet distinct recognitions by Siglecs to the atypical Neu5Acα2-6GlcNAc motif. The work provides tools and new insights for functional study and potential applications of Siglecs and HMOs.

[Research progress in the synthesis of 3'- and 6'-sialactose by Escherichia coli].

Human milk oligosaccharides (HMOs) are a structurally complex group of unbound polysaccharides, representing the third-largest solid component in breast milk. They play a crucial role in the intestinal health and immune system development of infants. Sialylated HMOs, including 3'-sialactose (3'-SL) and 6'-sialactose (6'-SL), are major components of HMOs, playing significant roles in immune regulation, anti-inflammatory processes, and promotion of probiotic growth. Currently, the cost-effective production of high-value sialactose by microbial fermentation with readily available raw materials has become a research hotspot due to the high nutritional value and potential applications of sialylated HMOs in infant food. This paper summarizes the functions and biosynthesis of 3'-SL and 6'-SL. Furthermore, it reviews the research progress in the synthesis of sialactose by Escherichia coli, offering valuable insights for future industrial production.

The Role of Milk Oligosaccharides in Enhancing Intestinal Microbiota, Intestinal Integrity, and Immune Function in Pigs: A Comparative Review.

The objective of this review was to identify the characteristics and functional roles of milk coproducts from human, bovine, and porcine sources and their impacts on the intestinal microbiota and intestinal immunity of suckling and nursery pigs. Modern pig production weans piglets at 3 to 4 weeks of age, which is earlier than pigs would naturally be weaned outside of artificial rearing. As a result, the immature intestines of suckling and nursery pigs face many challenges associated with intestinal dysbiosis, which can be caused by weaning stress or the colonization of the intestines by enteric pathogens. Milk oligosaccharides are found in sow milk and function as a prebiotic in the intestines of pigs as they cannot be degraded by mammalian enzymes and are thus utilized by intestinal microbial populations. The consumption of milk oligosaccharides during suckling and through the nursery phase can provide benefits to young pigs by encouraging the proliferation of beneficial microbial populations, preventing pathogen adhesion to enterocytes, and through directly modulating immune responses. Therefore, this review aims to summarize the specific functional components of milk oligosaccharides from human, bovine, and porcine sources, and identify potential strategies to utilize milk oligosaccharides to benefit young pigs through the suckling and nursery periods.

Is it possible to obtain substitutes for human milk oligosaccharides from bovine milk, goat milk, or other mammal milks?

Considering the current level of chemical and biological synthesis technology, it was a sensible selection to obtain milk oligosaccharides (MOs) from other mammals as the potential substitute for human MOs (HMOs) that possessed various structural features in the infant formula. Through a comprehensive analysis of the content, structure, and function of MOs in six distinct varieties of mammal milk, it has been shown that goat milk was the most suitable material for the preparation as a human milk substitute. Goat MOs (GMOs) had a relatively high content and diverse structural features compared to those found in other mammalian milks. The concentration of GMOs in colostrum ranged from 60 to 350 mg/L, whereas in mature milk, it ranged from 200 to 24,00 mg/L. The acidic oligosaccharides in goat milk have attracted considerable attention due to their closeness in acidic content and structural diversity with HMOs. Simultaneously, it was discovered that some structures, like N-glycolylneuraminic acid, were found to have a certain content in GMOs and served essential functional properties. Moreover, studies focused on the extraction of MOs from goat milk indicated that the production of GMOs on an industrial scale was viable. Furthermore, it is imperative to do further study on GMOs to enhance the preparation process, discover of new MOs structures and bioactivity evaluation, which will contribute to the development of both the commercial production of MOs and the goat milk industry.

Substitutive Effects of Milk vs. Vegetable Milk on the Human Gut Microbiota and Implications for Human Health.

Background: In the last two decades, the consumption of plant-based dairy substitutes in place of animal-based milk has increased in different geographic regions of the world. Dairy substitutes of vegetable origin have a quantitative composition of macronutrients such as animal milk, although the composition of carbohydrates, proteins and fats, as well as bioactive components, is completely different from that of animal milk. Many milk components have been shown to have relevant effects on the intestinal microbiota. Methods: Therefore, the aim of this review is to compare the effects obtained by previous works on the composition of the gut microbiota after the ingestion of animal milk and/or vegetable beverages. Results: In general, the results obtained in the included studies were very positive for animal milk intake. Thus, we found an increase in gut microbiota richness and diversity, increase in the production of short-chain fatty acids, and beneficial microbes such as Bifidobacterium, lactobacilli, Akkermansia, Lachnospiraceae or Blautia. In other cases, we found a significant decrease in potential harmful bacteria such as Proteobacteria, Erysipelotrichaceae, Desulfovibrionaceae or Clostridium perfingens after animal-origin milk intake. Vegetable beverages have also generally produced positive results in the gut microbiota such as the increase in the relative presence of lactobacilli, Bifidobacterium or Blautia. However, we also found some potential negative results, such as increases in the presence of potential pathogens such as Enterobacteriaceae, Salmonella and Fusobacterium. Conclusions: From the perspective of their effects on the intestinal microbiota, milks of animal origin appear to be more beneficial for human health than their vegetable substitutes. These different effects on the intestinal microbiota should be considered in those cases where the replacement of animal milks by vegetable substitutes is recommended.

Human Milk Oligosaccharides in Combination with Galacto- and Long-Chain Fructo-Oligosaccharides Enhance Vaccination Efficacy in a Murine Influenza Vaccination Model.

Early-life nutrition significantly impacts vaccination efficacy in infants, whose immune response to vaccines is weaker compared to adults. This study investigated vaccination efficacy in female C57Bl/6JOlaHsd mice (6 weeks old) fed diets with 0.7% galacto-oligosaccharides (GOS)/long-chain fructo-oligosaccharides (lcFOS) (9:1), 0.3% human milk oligosaccharides (HMOS), or a combination (GFH) for 14 days prior to and during vaccination. Delayed-type hypersensitivity (DTH) was measured by assessing ear swelling following an intradermal challenge. Influvac-specific IgG1 and IgG2a levels were assessed using ELISAs, while splenic T and B lymphocytes were analyzed for frequency and activation via flow cytometry. Additionally, cytokine production was evaluated using murine splenocytes co-cultured with influenza-loaded dendritic cells. Mice on the GFH diet showed a significantly enhanced DTH response (p < 0.05), increased serological IgG1 levels, and a significant rise in memory B lymphocytes (CD27+ B220+ CD19+). GFH-fed mice also exhibited more activated splenic Th1 cells (CD69+ CXCR3+ CD4+) and higher IFN-γ production after ex vivo restimulation (p < 0.05). These findings suggest that GOS/lcFOS and HMOS, particularly in combination, enhance vaccine responses by improving memory B cells, IgG production, and Th1 cell activation, supporting the potential use of these prebiotics in infant formula for better early-life immune development.

Modifiable and Non-Modifiable Factors That Affect Human Milk Oligosaccharides Composition.

Human milk, the gold standard in infant nutrition, is a unique fluid that provides essential nutrients such as lactose, lipids, proteins, and free oligosaccharides. While its primary role is nutritional, it also protects against pathogens. This protection mainly comes from immunoglobulins, with human milk oligosaccharides (HMOs) providing additional support by inhibiting pathogen binding to host cell ligands. The prebiotic and immune-modulatory activity of HMOs strongly depends on their structure. Over 200 individual structures have been identified so far, with the composition varying significantly among women. The structure and composition of HMOs are influenced by factors such as the Lewis blood group, secretor status, and the duration of nursing. HMO profiles are heavily influenced by maternal phenotypes, which are defined based on the expression of two specific fucosyltransferases. However, recent data have shown that HMO content can be modified by various factors, both changeable and unchangeable, including diet, maternal age, gestational age, mode of delivery, breastfeeding frequency, and race. The first part of this overview presents the historical background of these sugars and the efforts by scientists to extract them using the latest chromatography methods. The second part is divided into subchapters that examine modifiable and non-modifiable factors, reviewing the most recent articles on HMO composition variations due to specific reasons and summarizing potential future challenges in conducting these types of studies.

Human Milk Oligosaccharides and Their Pivotal Role in Gut-Brain Axis Modulation and Neurologic Development: A Narrative Review to Decipher the Multifaceted Interplay.

BACKGROUND: Human milk oligosaccharides (HMOs), which are unique bioactive components in human milk, are increasingly recognized for their multifaceted roles in infant health. A deeper understanding of the nexus between HMOs and the gut-brain axis can revolutionize neonatal nutrition and neurodevelopmental strategies. METHODS: We performed a narrative review using PubMed, Embase, and Google Scholar to source relevant articles. The focus was on studies detailing the influence of HMOs on the gut and brain systems, especially in neonates. Articles were subsequently synthesized based on their exploration into the effects and mechanisms of HMOs on these interconnected systems. RESULTS: HMOs significantly influence the neonatal gut-brain axis. Specific concentrations of HMO, measured 1 and 6 months after birth, would seem to agree with this hypothesis. HMOs are shown to influence gut microbiota composition and enhance neurotransmitter production, which are crucial for brain development. For instance, 2'-fucosyllactose has been demonstrated to support cognitive development by fostering beneficial gut bacteria that produce essential short-chain fatty acids. CONCLUSIONS: HMOs serve as crucial modulators of the neonatal gut-brain axis, underscoring their importance in infant nutrition and neurodevelopment. Their dual role in shaping the infant gut while influencing brain function presents them as potential game-changers in neonatal health strategies.

Selective human milk oligosaccharide utilization by members of the Bifidobacterium pseudocatenulatum taxon.

Human milk oligosaccharides (HMOs) are essentially unaffected by the digestive enzymes of the nursling and are known for their ability to enrich certain microbial species in the infant gut microbiota, in particular bifidobacteria. HMO metabolism has been studied in various bifidobacterial species such as B. breve, B. bifidum, and B. longum subsp. infantis. In the current study, we describe differential growth abilities elicited by twenty-three newly isolated Bifidobacterium pseudocatenulatum strains on particular HMOs, such as 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT). Through gene-trait matching and comparative genome analysis, we identified genes involved in the degradation of fucosylated HMOs in this strain set, while we employed a transcriptomic approach to facilitate the identification and characterization of genes and associated enzymes involved in LNT metabolism by strain B. pseudocatenulatum MM0196. A total of 252 publicly available genomes of the B. pseudocatenulatum taxon were screened for homologs of the glycosyl hydrolases (GHs) identified here as being required for selected HMO metabolism. From this analysis, it is clear that all members of this species possess homologs of the genes involved in LNT degradation, while genes required for degradation of fucosylated HMOs are variably present.IMPORTANCEOur findings allow a better understanding of the complex interaction between Bifidobacterium and its host and provide a roadmap toward future applications of B. pseudocatenulatum as a probiotic with a focus on infant health. Furthermore, our investigations have generated information on the role of HMOs in shaping the infant gut microbiota, thus also facilitating applications of HMOs in infant nutrition, with potential extension into the mature or adult gut microbiota. Supplementation of HMOs is known to result in the modulation of bacterial communities toward a higher relative abundance of bifidobacteria, which in turn enforces their ability to modulate particular immune functions and strengthen the intestinal barrier. This work may therefore inspire future studies to improve the formulation of neonatal nutritional products, aimed at facilitating the development of a healthy digestive and immune system and reducing the differences in gut microbiota composition observed between breastfed and formula-fed babies or full-term and preterm infants.

Human milk oligosaccharides in preventing food allergy: A review through gut microbiota and immune regulation.

Food allergy (FA) has increasingly attracted global attention in past decades. However, the mechanism and effect of FA are complex and varied, rendering it hard to prevention and management. Most of the allergens identified so far are macromolecular proteins in food and may have potential cross-reactions. Human milk oligosaccharides (HMOs) have been regarded as an ideal nutrient component for infants, as they can enhance the immunomodulatory capacity to inhibit the progress of FA. HMOs may intervene in the development of allergies by modifying gut microbiota and increasing specific short-chain fatty acids levels. Additionally, HMOs could improve the intestinal permeability and directly or indirectly regulate the balance of T helper cells and regulatory T cells by enhancing the inflammatory signaling pathways to combat FA. This review will discuss the influence factors of FA, key species of gut microbiota involved in FA, types of FA, and profiles of HMOs and provide evidence for future research trends to advance HMOs as potential therapeutic aids in preventing the progress of FA.

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.

Dietary human milk oligosaccharides reduce allergic airway inflammation by modulating SCFAs level and ILC2 activity.

Group 2 innate lymphoid cells (ILC2s) play a crucial role in the progression of asthma, yet the regulatory mechanisms modulating ILC2 responses in asthma remain underexplored. Human milk oligosaccharides (HMOs), vital non-nutritive components of breast milk, are known to significantly shape immune system development and influence the incidence of allergic diseases. However, their impact on ILC2-driven asthma is not fully understood. Our research reveals that dietary HMOs act as potent inhibitors of ILC2 responses and allergic airway inflammation. Treatment with 2'-fucosyllactose (2'-FL) and 6'-sialyllactose (6'-SL) significantly reduced ILC2-related airway inflammation induced by papain or Alternaria alternata in mice, evidenced by decreased eosinophil (EOS) infiltration and lower IL-5 and IL-13 levels in BALF. Notably, while ILC2 expresses HMO receptors, HMO did not act directly on ILC2 but potentially modulated their activity through alterations in gut microbiota derived SCFAs. HMO treatments alleviated airway inflammation in SCFA-dependent manners, with SCFA depletion or receptor blocking reversing these beneficial effects. This study reveals the potential of dietary HMOs in managing asthma through modulation of ILC2 activity and the gut-lung axis, proposing a new therapeutic avenue that utilises the immunomodulatory capacities of nutritional components to combat respiratory diseases.

Gut microbiota: A key role for human milk oligosaccharides in regulating host health early in life.

Human milk oligosaccharides (HMOs) are an evolutionarily significant advantage bestowed by mothers for facilitating the development of the infant's gut microbiota. They can avoid absorption in the stomach and small intestine, reaching the colon successfully, where they engage in close interactions with gut microbes. This process also enables HMOs to exert additional prebiotic effects, including regulating the mucus layer, promoting physical growth and brain development, as well as preventing and mitigating conditions such as NEC, allergies, and diarrhea. Here, we comprehensively review the primary ways by which gut microbiota, including Bifidobacteria and other genera, utilize HMOs, and we classify them into five central pathways. Furthermore, we emphasize the metabolic benefits of bacteria consuming HMOs, particularly the recently identified intrinsic link between HMOs and the metabolic conversion of tryptophan to indole and its derivatives. We also examine the extensive probiotic roles of HMOs and their recent research advancements, specifically concentrating on the unsummarized role of HMOs in regulating the mucus layer, where their interaction with the gut microbiota becomes crucial. Additionally, we delve into the principal tools used for functional mining of new HMOs. In conclusion, our study presents a thorough analysis of the interaction mechanism between HMOs and gut microbiota, emphasizing the cooperative utilization of HMOs by gut microbiota, and provides an overview of the subsequent probiotic effects of this interaction. This review provides new insights into the interaction of HMOs with the gut microbiota, which will inform the mechanisms by which HMOs function.

Infant Microbiota Communities and Human Milk Oligosaccharide Supplementation Independently and Synergistically Shape Metabolite Production and Immune Responses in Healthy Mice.

BACKGROUND: Multiple studies have demonstrated associations between the early-life gut microbiome and incidence of inflammatory and autoimmune disease in childhood. Although microbial colonization is necessary for proper immune education, it is not well understood at a mechanistic level how specific communities of bacteria promote immune maturation or drive immune dysfunction in infancy. OBJECTIVES: In this study, we aimed to assess whether infant microbial communities with different overall structures differentially influence immune and gastrointestinal development in healthy mice. METHODS: Germ-free mice were inoculated with fecal slurries from Bifidobacterium longum subspecies infantis positive (BIP) or B. longum subspecies infantis negative (BIN) breastfed infants; half of the mice in each group were also supplemented with a pool of human milk oligosaccharides (HMOs) for 14 d. Cecal microbiome composition and metabolite production, systemic and mucosal immune outcomes, and intestinal morphology were assessed at the end of the study. RESULTS: The results showed that inoculation with a BIP microbiome results in a remarkably distinct microbial community characterized by higher relative abundances of cecal Clostridium senu stricto, Ruminococcus gnavus, Cellulosilyticum sp., and Erysipelatoclostridium sp. The BIP microbiome produced 2-fold higher concentrations of cecal butyrate, promoted branched short-chain fatty acid (SCFA) production, and further modulated serotonin, kynurenine, and indole metabolism relative to BIN mice. Further, the BIP microbiome increased the proportions of innate and adaptive immune cells in spleen, while HMO supplementation increased proliferation of mesenteric lymph node cells to phorbol myristate acetate and lipopolysaccharide and increased serum IgA and IgG concentrations. CONCLUSIONS: Different microbiome compositions and HMO supplementation can modulate SCFA and tryptophan metabolism and innate and adaptive immunity in young, healthy mice, with potentially important implications for early childhood health.

Biochemical characterization of a novel C-terminally truncated ß-galactosidase from Paenibacillus antarcticus with high transglycosylation activity.

The transgalactosylase activity of ß-galactosidases offers a convenient and promising strategy for conversion of lactose into high-value oligosaccharides, such as galacto-oligosaccharides (GOS) and human milk oligosaccharides (HMOs). In this study, we cloned and biochemically characterized a novel C-terminally truncated ß-galactosidase (PaBgal2A-D) from Paenibacillus antarcticus with high transglycosylation activity. PaBgal2A-D is a member of glycoside hydrolase (GH) family 2. The optimal pH and temperature of PaBgal2A-D were determined to be pH 6.5 and 50°C, respectively. It was relatively stable within pH 5.0-8.0 and up to 50°C. PaBgal2A-D showed high transglycosylation activity for GOS synthesis, and the maximum yield of 50.8% (wt/wt) was obtained in 2 h. Moreover, PaBgal2A-D could synthesize lacto-N-neotetraose (LNnT) using lactose and lacto-N-triose II (LNT2), with a conversion rate of 16.4%. This study demonstrated that PaBgal2A-D could be a promising tool to prepare GOS and LNnT.

Effects of multistrain Bifidobacteria and Lactobacillus probiotics on HMO compositions after supplementation to pregnant women at threatening preterm delivery: design of the randomized clinical PROMO trial.

BACKGROUND: As an indigestible component of human breast milk, Human Milk Oligosaccharides (HMOs) play an important role as a substrate for the establishing microbiome of the newborn. They have further been shown to have beneficial effects on the immune system, lung and brain development. For preterm infants HMO composition of human breast milk may be of particular relevance since the establishment of a healthy microbiome is challenged by multiple disruptive factors associated with preterm birth, such as cesarean section, hospital environment and perinatal antibiotic exposure. In a previous study it has been proposed that maternal probiotic supplementation during late stages of pregnancy may change the HMO composition in human milk. However, there is currently no study on pregnancies which are threatened to preterm birth. Furthermore, HMO composition has not been investigated in association with clinically relevant outcomes of vulnerable infants including inflammation-mediated diseases such as sepsis, necrotizing enterocolitis (NEC) or chronic lung disease. MAIN BODY: A randomized controlled intervention study (PROMO = probiotics for human milk oligosaccharides) has been designed to analyze changes in HMO composition of human breast milk after supplementation of probiotics (Lactobacillus acidophilus, Bifidobacterium lactis and Bifidobacterium infantis) in pregnancies at risk for preterm birth. The primary endpoint is HMO composition of 3-fucosyllactose and 3'-sialyllactose in expressed breast milk. We estimate that probiotic intervention will increase these two HMO levels by 50% according to the standardized mean difference between treatment and control groups. As secondary outcomes we will measure preterm infants' clinical outcomes (preterm birth, sepsis, weight gain growth, gastrointestinal complications) and effects on microbiome composition in the rectovaginal tract of mothers at delivery and in the gut of term and preterm infants by sequencing at high genomic resolution. Therefore, we will longitudinally collect bio samples in the first 4 weeks after birth as well as in follow-up investigations at 3 months, one year, and five years of age. CONCLUSIONS: We estimate that probiotic intervention will increase these two HMO levels by 50% according to the standardized mean difference between treatment and control groups. The PROMO study will gain insight into the microbiome-HMO interaction at the fetomaternal interface and its consequences for duration of pregnancy and outcome of infants.

Development of a cyclic ion mobility spectrometry-mass spectrometry-based collision cross-section database of permethylated human milk oligosaccharides.

Human milk oligosaccharides (HMOs) are an important class of biomolecules responsible for the healthy development of the brain-gut axis of infants. Unfortunately, their accurate characterization is largely precluded due to a variety of reasons - there are over 200 possible HMO structures whereas only 10s of these are available as authentic analytical standards. Furthermore, their isomeric heterogeneity stemming from their many possible glycosidic linkage positions and corresponding α/ß anomericities further complicates their analyses. While liquid chromatography coupled to tandem mass spectrometry remains the gold standard for HMO analyses, it often times cannot resolve all possible isomeric species and thus warrants the development of other orthogonal approaches. High-resolution ion mobility spectrometry coupled to mass spectrometry has emerged as a rapid alternative to condensed-phase separations but largely has remained limited to qualitative information related to the resolution of isomers. In this work, we have assessed the use of permethylation to improve both the resolution and sensitivity of HMO analyses with cyclic ion mobility separations coupled with mass spectrometry. In addition to this, we have developed the first-ever high-resolution collision cross-section database for permethylated HMOs using our previously established calibration protocol. We envision that this internal reference database generated from high-resolution cyclic ion mobility spectrometry-mass spectrometry will greatly aid in the accurate characterization of HMOs and provide a valuable, orthogonal, approach to existing liquid chromatography-tandem mass spectrometry-based methods.