α-L-Fucosidases are attractive biocatalysts for the production of bioactive fucosylated oligosaccharides, however, poor regioselectivity and activity for transglycosylation have significantly limited their applications. We have recently derived an α-L-Fucosidase, BF3242, from Bacteroides fragilis NCTC9343, which could efficiently synthesize a mixture of Fuc-α-1,3/1,6-GlcNAc, but its 1,3/1,6-regioselectivity was observably affected by reaction temperature. Here, we integrated loop-targeted random mutagenesis and site-directed mutagenesis to engineer the regioselectivity and transglycosylation activity of BF3242. Loop-targeted random mutagenesis revealed that L266 in the loop-4 (H242-S267) within the model of BF3242 was a key residue for the regioselectivity for transglycosylation, and the saturation mutagenesis at residue L266 uncovered a mutant L266H with a significantly increased 1,3-regioselectivity of 97 % from 69 % of WT BF3242. Subsequently, five designed single-site mutations at the putative aglycone subsites were performed, resulting in a double-site mutant L266H/M285C that increased the overall yield of Fuc-α-1,3/1,6-GlcNAc to 76 % from 68 % of WT BF3242. The saturation mutagenesis at residue M285 finally generated a double-site mutant L266H/M285T with the maximal overall yield of Fuc-α-1,3/1,6-GlcNAc of 85 % and 1,3-regioselectivity of 98 %. The R T/H of L266H/M285T was approximately 2.7-fold higher than that of the WT BF3242. Molecular dynamics simulations revealed that the structural flexibility of the loop-4 was substantially reduced in mutant L266H, and the hydrogen bond formation and binding affinity between mutant L266H/M285T and Fuc-α-1,3-GlcNAc was significantly enhanced. The semi-rationally engineered enzyme L266H/M285T would be a promising biocatalyst for highly 1,3-regioselective synthesis of fucosyl-N-acetylglucosamine disaccharide.

The development and validation of an accurate, selective, and eco-friendly capillary zone electrophoretic detection (CZE) method has been presented for concurrent measurement of inorganic and organic anions including chloride, sulfate, formic acid, citric acid, acetic acid, phosphate, and glutamic acid in Human Milk Oligosaccharides (HMOs) for the first time. An electrolyte composed of an aqueous solution of benzoic acid, 16.38 mM; l-histidine, 24.49 mM; and polyacrylamide 0.0025 % provided a satisfactory separation of all analytes of interest. The electroosmotic flow was reversed using polyacrylamide in the background buffer. UV detection was carried out at a wavelength of 230 nm using benzoic acid in running buffer. Analysis was conducted on a 60 cm (53 cm to the detector) x 0.75 μm i.d. fused-silica capillary at a potential of -30 kV and the temperature of 20 °C. The procedure had a linear response in the concentration range of 2-200 mg/L for all analytes with correlation coefficients ≥0.9997. Validation was performed based on the International Council for Harmonization (ICH). The National Environmental Methods Index and the analytical GREEness metric were the tools applied for greenness assessment. System suitability parameter was determined using the expanded uncertainty of the method estimated from method validation data. The applicability of the proposed procedure was shown in diverse real samples.

Human milk oligosaccharides (HMOs) are essential for babies' growth. The most commonly used method for HMOs analysis combines hydrophilic interaction liquid chromatography (HILIC) with ultra-performance liquid chromatography (UPLC). However, high-pressure operation and the cost of commercial HILIC columns limit progress in the HMOs research. In this study, we synthesized a novel mixed-mode adsorbent, PS-g-PAMPS, by grafting 2-acrylamido-2-methylpropane sulfonic acid (AMPS) onto a Merrifield resin (PS-g-Cl), and characterized it. The adsorbents were packed into a column (Mixed-HILIC column) for efficient high-performance liquid chromatography (HPLC) analysis of four HMOs: 2'-Focusllactose (2'-FL), Lacto-N-Tetraose (LNnT), 3'-Sialyllactose (3'-SL), and 6'-Sialyllactose (6'-SL). Compared to three commercial columns, AMINEX HPX-87H column, ROA-Organic Acid column, Glycan BEH amide column, our column offered several advantages: low-pressure operation, cost-effective adsorbent, and baseline separation of six analytes in the same analysis time. The mixed-mode mechanism of electrostatic repulsion and hydrophilic interactions was verified using zeta potential, and organic phase ratio. Key performance parameters, including spike recovery, limit of detection, and limit of quantification, were evaluated, and the column's reliability was confirmed using a real 2'-FL fermentation sample. These results demonstrated the potential of the Mixed-HILIC column for the development of application chromatography in HMOs.

Human milk oligosaccharides (HMOs) are the third most abundant solid component in breast milk, which is essential for the healthy growth of infants. 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL) are the main components of HMOs. In this study, we developed and validated a quantitative nuclear magnetic resonance (qNMR) method for determining HMOs in different complex food matrices. The spectra of HMOs often overlap with impurities in the samples, which hampers quantification using the conventional integral method. We overcame this obstacle by building the Global Spectrum Deconvolution-area coefficients algorithm. The method proved to be precise, yielding satisfactory results in terms of precision (RSD% 0.5-1.9), trueness (bias% 1.9-8.2), and recovery (90.5-106.6 %). The limit of quantification for 2'-FL was 0.10 mg/mL, and for 3-FL, it was 0.15 mg/mL. A comparison using Deming regression between the existing high-performance liquid chromatography method and the developed qNMR method for determining HMOs demonstrated the high accuracy of the qNMR method. The proposed method allows for high-throughput measurements of large-volume samples due to its simple and fast sample preparation. Therefore, the method is an essential tool for determining HMOs in various complex foods.

Recently, the construction of metabolically engineered strains for the microbial synthesis of human milk oligosaccharides (HMOs) has attracted increasing attention. However, fewer efforts were made in the in vivo biosynthesis of complex HMOs, especially sialylated complex HMOs. In this study, we engineered Escherichia coli BL21(DE3) to efficiently produce sialyllacto-N-tetraose a (LST-a) efficiently. Three sequential glycosylation steps were introduced to construct the LST-a pathway, catalyzed by β1,3-N-acetylglucosaminylation, β1,3-galactosylation, and α2,3-sialylation. Pathway genes for cytidine 5'-monophospho (CMP)-N-acetylneuraminic acid (Neu5Ac) were introduced to support the sialylation donor supply. Production of LST-a was improved by deleting competitive genes of CMP-Neu5Ac synthesis, screening a more efficient α2,3-sialyltransferase, and combinatorial optimization of pathway gene expression. LST-a was finally produced with the titer of 1.235 and 4.85 g/L by shake-flask and fed-batch cultivation, respectively, demonstrating the feasibility of efficient microbial production of complex sialylated HMOs.

Human milk oligosaccharides (HMOs) represent the third most abundant fraction of biomolecules in human milk (HM) and play a crucial role in infant health and development. The unique contributions of HMOs to healthy development of breast-fed infants are assumed to rely on the extraordinary complexity and diversity of HMO isomeric structures, which in turn still cause a huge analytical challenge. Many contemporary analytical methods aiming for more detailed HMO characterization combine ion mobility (IM) with LC-MS for enhanced structural resolution but are typically lacking the robustness necessary for application to HM cohorts with hundreds of samples. To overcome these challenges, we introduce a novel, robust all-ion fragmentation (AIF) LC-ESI-IM-MS method integrating four analytical dimensions: high-resolution LC separation, IM drift time, accurate mass precursor, and fragment ion measurements. This four-dimensional (4D) analytical characterization is sufficient for resolving various HMO structural isomers in an efficient way. Thereby, up to 200 HMO compounds with a maximum degree of polymerization of 13 could be simultaneously identified and relatively quantified. We devised two methods using this 4D analytical approach. One intended for in-depth characterization of multiple known but also novel HMO structures and the second is designed for robust, increased-throughput analyses. With the first approach, five trifucosyl-lacto-N-tetraose isomers (TF-LNTs), four of which were never detected before in HM, as well as additional difucosyl-lacto-N-heaose isomers (DF-LNHs), were revealed and structures fully elucidated by AIF and IM. This exemplifies the potential of our method for in-depth characterization of novel complex HMO structures. Furthermore, the increased-throughput method featuring a shorter LC gradient was applied to real-world HM samples. Here, we could differentiate the HM types I-IV based on a broader range of partly new marker HMOs. We could also derive valuable new insights into variations of multiple and rare HMOs up to DP 11 across lactational stages. Overall, our AIF LC-ESI-IM-MS approach facilitates in-depth monitoring and confident identification of a broad array of distinct and simple to very complex HMOs. We envision this robust AIF LC-ESI-IM-MS approach to advance HMO research by facilitating the characterization of a broad range of HMOs in high numbers of HM samples. This may help to further extend our understanding about HMOs structure-function relationships relevant for infants' healthy development.

The health status of the growing infant is closely related to the development of the gut microbiota during infancy, which is also a major stimulator of the immune system. Goat milk oligosaccharides (gMOs) are a class of bioactive compounds in goat milk, which have attracted extensive research interest in recent years. Recent studies have highlighted that gMOs as prebiotics can regulate the gut microbiota, exhibit multiple health effects, and act as immunomodulators. This article outlines the structure, classification, and functions of gMOs. In addition, we also deeply explored the mechanism of gMO interaction with infant gut microbiota and regulation of infant immunity. Finally, the possibility of gMOs as an effective substitute for natural prebiotics in breast milk is revisited. We concluded that gMOs improve infant immune function by regulating intestinal beneficial bacteria (Bifidobacteria, Lactobacilli, etc.) and their metabolism. Therefore, gMOs are significant to infant immune health and are expected to become a substitute for human milk oligosaccharides (HMOs).

Human milk oligosaccharides (HMOs) are crucial for promoting neonatal health, with sialylated oligosaccharides, a significant subclass, offering a variety of health benefits such as prebiotic effects, anti-inflammatory and antimicrobial properties, antiviral defense, and cognitive development support. Among these, 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) have received "GRAS" status from the U.S. Food and Drug Administration and approval from the European Food Safety Authority for use as novel food additives in infant formula and supplements. This review focuses on the synthesis methods of sialylated human milk oligosaccharides (SHMOs), their functional properties, downstreaming developments and application technologies. Given the challenges associated with achieving sufficient availability for food and medical applications, the review emphasizes the viability and efficiency of various production strategies. The review also highlights recent research advancements and offers insights for optimizing large-scale production to support future applications in the food and pharmaceutical industries.

Biological production of human milk oligosaccharides (HMOs) using metabolically engineered strains is a research hotspot in food biotechnology, but less effort has been made on the biological production of sialylated complex HMOs. Sialyllacto-N-tetraose c is the only monosialylated complex HMO in the top 15 HMOs. In this study, the metabolic pathway of LST c was constructed in Escherichia coli BL21(DE3) by introducing three sequential glycosyltransferases: β1,3-N-acetylglucosaminyltransferase, β1,4-galactosyltransferase, and α2,6-sialyltransferase. The cytidine 5'-monophospho (CMP)-N-acetylneuraminic acid (Neu5Ac) pathway was enhanced to improve LST c production. The β1,4-galactosyltransferase from Helicobacter pylori J99 (HpGalT) and α2,6-sialyltransferase from Vespertiliibacter pulmonis (ED6ST) were screened as a pair of key glycosyltransferases for enhancing LST c production. The final engineered strain could produce 1.718 and 9.745 g/L LST c by shake-flask and fed-batch cultivation, respectively, indicating the feasibility of efficient biosynthesis of complex sialylated HMOs.

2'-Fucosyllactose (2'-FL) is the most abundant human milk oligosaccharide (HMO) and has been approved to be commercially added to infant formula. Microbial synthesis from exogenous lactose via metabolic engineering is currently the major approach to production of 2'-FL. Replacement of lactose with cheaper sugars such as glucose and sucrose has been studied to reduce the production costs. Herein, Escherichia coli BL21(DE3) was engineered to produce 2'-FL by co-culture with glucose and xylose, the main components of lignocellulosic biomass. Firstly, synthetic pathway of lactose from xylose and glucose was constructed by introducing a lactose-forming enzyme, strengthening xylose uptake pathway, and weakening glucose metabolic pathway. Then, a highly-active α1,2-fucosyltransferase BKHT was introduced to produce 2'-FL and GDP-fucose supply was enhanced to increase 2'-FL production. As a result, when cocultured with glucose and xylose, the engineered strain produced 6.53 g/L and 27.53 g/L of 2'-FL by shake-flask and fed-batch cultivation, respectively.

Lacto-N-neotetraose (LNnT) is a primary solid component of human milk oligosaccharides (HMOs) with various promising health effects for infants. LNnT production by GRAS (generally recognized as safe) microorganisms has attracted considerable attention. However, few studies have emphasized Pichia Pastoris as a cell factory for LNnT's production. Here, we have reported the first-ever synthesis of LNnT employing P. pastoris as the host. Initially, LNnT biosynthetic pathway genes β-1,3-N-acetylglucosaminyltransferase (lgtA) and β-1,4-galactostltransferase (lgtB) along with lactose permease (lac12) and galactose epimerase (gal10) were integrated into the genome of P. pastoris, but only 0.139 g/L LNnT was obtained. Second, the titer of LNnT was improved to 0.162 g/L via up-regulating genes to strengthen the supply of precursors, UDP-GlcNAc (Uridine diphosphate N-acetylglucosamine) and UDP-Gal (Uridine diphosphate galactose), for LNnT biosynthesis. Third, by knocking out critical mediator pfk (6-phosphofructokinase) genes in glycolysis, the major glucose metabolic flux was rewired to the LNnT biosynthesis pathway. As a result, the strain accumulated 0.867 g/L LNnT in YPG medium supplemented with glucose and lactose. Finally, LNnT production was increased to 1.24 g/L in a 3 L bioreactor. The work aimed to explore the potential of P. pastoris as a for LNnT production.

Carbohydrates are biologically and medicinally important molecules that are attracting growing attention to their synthesis and applications. Unlike the biosynthetic processes for nucleic acids and proteins, carbohydrate biosynthesis is not template-driven, more challenging, and often leads to product variations. In lieu of templates for carbohydrate biosynthesis, we describe herein a new concept of designing enzyme assembly synthetic maps (EASyMaps) as blueprints to guide glycosyltransferase-dependent stepwise one-pot multienzyme (StOPMe) synthesis to systematically access structurally diverse carbohydrates in a target-oriented manner. The strategy is demonstrated for the construction of a comprehensive library of tetraose-core-containing human milk oligosaccharides (HMOs) presenting diverse functional important glycan epitopes shared by more complex HMOs. The tetraose-core-HMOs are attractive candidates for large-scale production and for the development of HMO-based nutraceuticals. To achieve the preparative-scale synthesis of targets containing a Neu5Acα2-6GlcNAc component, a human α2-6-sialyltransferase hST6GALNAC5 is successfully expressed in E. coli. Neoglycoproteins with controlled glycan valencies are prepared and immobilized on fluorescent magnetic beads. Multiplex bead assays reveal ligands of glycan-binding proteins from plants, influenza viruses, human, and bacteria, identifying promising HMO targets for functional applications. The concept of designing EASyMaps as blueprints to guide StOPMe synthesis in a systematic target-oriented manner is broadly applicable beyond the synthesis of HMOs. The efficient StOPMe process is suitable for the large-scale production of complex carbohydrates and can be potentially adapted for automation.

Covering: up to the end of 2024Breastfeeding is one of the most effective ways to promote child health. However, characterizing the chemistry that fortifies the benefits of breastfeeding remains a grand challenge. Current efforts in the community are focused on characterizing the roles of the different carbohydrates, proteins, and fats in milk. The goal of this review is to highlight and describe current knowledge about the major classes of macromolecules in human milk and their potential role in infant health and wellness.

Background/Objectives: Breastfeeding provides significant health benefits for both infants and mothers, but many women discontinue earlier than recommended. This study investigates the factors contributing to early breastfeeding cessation among Arab women in Israel, focusing on multiple factors, such as socio-demographic, work-related, cultural, and religious, impacting breastfeeding duration and shaping breastfeeding practices. Methods: A cross-sectional survey was conducted among 349 Arab women, 65% of whom were Muslim and 35% Christian. Logistic regression analyses were used to identify key predictors of breastfeeding cessation. Results: Findings showed that Christian Arab women were more likely to stop breastfeeding earlier than their Muslim counterparts. Mothers with four or more children and those balancing work demands were at higher risk of early cessation. Contrary to expectations, higher levels of religiosity were associated with a greater likelihood of stopping breastfeeding. Additionally, mothers who received personal breastfeeding guidance were more likely to discontinue, suggesting potential gaps in the quality of support provided. Conclusions: These findings underscore the importance of tailoring interventions to address the unique cultural and socio-economic challenges faced by Arab women in Israel. Recommendations include improving breastfeeding guidance quality, workplace support for breastfeeding mothers, and culturally sensitive interventions that consider the role of religiosity and family dynamics. This research provides valuable insights for healthcare providers and policymakers aiming to promote sustained breastfeeding practices in diverse populations. The study highlights the complexity of factors affecting breastfeeding cessation among Arab women in Israel, emphasizing the need for targeted interventions that address socio-demographic, cultural, and religious influences to promote sustained breastfeeding.

Lactodifucotetraose (LDFT) is a human milk oligosaccharide (HMO) that might reduce inflammation in infants. In this study, we established a useful production process of LDFT by engineering two key enzymes, α1,2-fucosyltransferase (α1,2-FucT) and α1,3-fucosyltransferase (α1,3-FucT). First, we verified which of 2'-fucosyllactose (2'-FL) or 3-fucosyllactose (3-FL) (mostly unverified) was more useful. We searched for FucTs that functioned efficiently in vivo against the raw material lactose or the two intermediates 2'-FL or 3-FL by external substrate addition to culture medium. We found that α1,2- FucT (HMFT) from Helicobacter mustelae and the N-terminal truncated form of α1,3-FucT from Bacteroides fragilis (BfFucTΔN10) had high potential. 3-FL was not efficiently converted to LDFT, which might be attributed to the low reactivity of HMFT to 3-FL as well as the low uptake efficiency of 3-FL by LacY, as revealed by a growth test with exogenously added FL as the sole carbon source and heterologously expressed intracellular fucosidase. Furthermore, because 3-FL accumulation had a negative impact on cell growth, we avoided the route passing through 3-FL. By adjusting the copy numbers of HMFT and BffucTΔN10, we produced LDFT from lactose predominantly via 2'-FL. Finally, 17.5 g/L of LDFT (with 6.8 g/L 2'-FL and no 3-FL or residual lactose) accumulated in a 3-L fed-batch culture after 77 h. This study reports the detailed analysis of multiple pathways and shows the control of glycosyltransferases can improve the production efficiency of complex HMOs.

Human colostrum and mature milk contain oligosaccharides (Os), designated as human milk oligosaccharides (HMOs). Approximately 200 varieties of HMOs have been characterized. Although HMOs are not utilized as an energy source by infants, they have important protective functions, including pathogenic bacteria and viral infection inhibitors and immune modulators, among other functions, and HMOs stimulate brain-nerve development. The Os concentration is average 11 g/L in human milk but >100 mg/L in mature bovine milk, which is used to manufacture infant formula, suggesting that human-identical milk oligosaccharides (HiMOs) should be incorporated into milk substitutes. Some infant formulas incorporating 2'-fucosyllactose and lacto-N-neotetraose are now commercially available, and intervention trials have been concluded. We review basic HMO information, including their chemical structures and concentrations, attempts to synthesize HMOs at small and plant scale, studies that clarified HMO biological functions, and interventions with milk substitutes incorporating HiMOs in formula-fed infants.

Milk oligosaccharides are high added value compounds that could be obtained by exploiting cheese whey, a byproduct of dairy industry. The objective was to compare the abundance and diversity of oligosaccharides in whey samples from domestic animals and humans. During fresh cheese making, whey samples were collected and analyzed by untargeted and targeted small molecule analysis using high-resolution mass-spectrometry. A great similarity in the metabolite profile between goat and sheep was observed. Up to 11 oligosaccharides were observed in the sheep whey from those typically found in humans. The concentration of 2'-Fucosyllactose (0.136 ± 0.055 g/L) and 3-Fucosyllactose (0.079 ± 0.009 g/L) were significantly higher (p-value <0.01) in sheep whey, while the concentration of 3'-Sialyllactose (0.826 ± 0.638 g/L) was higher in goat whey. No significant differences were observed between goat and sheep whey for the other oligosaccharides (p-value >0.05). Therefore, sheep and goat whey could become an important source of oligosaccharides through their revalorization.

Although approximately 160 human milk oligosaccharides (HMOs) have been identified, current studies on HMO quantitation are limited to the 10-19 most abundant HMOs. We assessed the variations in the relative concentrations of 71 HMO structures over lactation in human milk samples by an advanced liquid chromatography-mass spectrometry approach.

Samples were collected from 64 mothers at 6 weeks, 6 months, and 12 months of lactation in the Ulm SPATZ Health Study, a German birth cohort. In this longitudinal study, we fitted linear mixed-effect models to analyze changes in the log2-transformed and standardized HMO concentration over time. Based on the profile of 71 HMOs, we also fitted a group-based multi-trajectory (GBMT) model to cluster mothers secreting cluster type I milk, who account for the majority of lactating mothers.

We found that 52 HMOs had a decreasing trend (regression coefficients ranging from -1.41 to -0.17) and 9 had an increasing trend (regression coefficients ranging from 0.25 to 0.64) during lactation, and the findings were statistically significant after multiple testing corrections. Using human milk samples of 49 mothers with type I milk, we further identified two novel sub-clusters with distinct longitudinal trajectories of concentrations of 71 HMOs during lactation: Type I-a (N = 20) and I-b (N = 29). These sub-clusters were not associated with maternal non-genetic characteristics.

Our findings extend existing knowledge about the structural diversity of HMOs and their variations over lactation. These may pave the way to investigate the potential nutritional benefits of various HMOs on infant health and early life development in the future.

Difucosyllactose (DFL), an important kind of fucosylated human milk oligosaccharides (HMOs), has garnered considerable attention due to its excellent physiological activities in infants. Previously, we achieved de novo biosynthesis of DFL; however, substantial residual intermediates of fucosyllactoses (FL) were detected. In this study, DFL biosynthesis was optimized, and residual FL were reduced by regulating metabolic pathways. Different plasmid combinations were used to regulate gene expression, achieving an optimal flux balance between 2'-FL and DFL. The expression level of key enzyme α-1,3-fucosyltransferase (α-1,3-FT, FucTa) was then enhanced by increasing plasmid copy number and integrating fucTa gene into the chromosome. Exocytosis of 2'-FL was reduced by deleting the sugar efflux transporter setA gene, thereby minimizing residual FL. Finally, strain BSF41 produced 55.3 g/L of DFL with only 2.59 g/L of residual FL in a 5 L fermentor, representing the highest reported titer to date. This study provides an important foundation for advancing the biosynthesis of fucosylated HMOs.

Breast milk is an essential source of infant nutrition. It is also a vital determinant of the structure and function of the infant intestinal microbial community, and it connects the mother and infant intestinal microbiota. Human milk oligosaccharides (HMOs) are a critical component in breast milk. HMOs can reach the baby's colon entirely from milk and become a fermentable substrate for some intestinal microorganisms. HMOs can enhance intestinal mucosal barrier function and affect the intestinal function of the host through immune function, which has a therapeutic effect on specific infant intestinal diseases, such as necrotizing enterocolitis. In addition, changes in infant intestinal microbiota can reflect the maternal intestinal microbiota. HMOs are a link between the maternal intestinal microbiota and infant intestinal microbiota. HMOs affect the intestinal microbiota of infants and are related to the maternal milk microbiota. Through breastfeeding, maternal microbiota and HMOs jointly affect infant intestinal bacteria. Therefore, HMOs positively influence the establishment and balance of the infant microbial community, which is vital to ensure infant intestinal function. Therefore, HMOs can be used as a supplement and alternative therapy for infant intestinal diseases.

6'-Sialyllactose (6'-SL), one of the most prevalent sialylated human milk oligosaccharides (HMOs), has recently garnered significant attention due to its promising health effects for infants. In this study, the 6'-SL biosynthetic pathway in EZAK (E. coli BL21(DE3) ΔlacZΔnanAΔnanK) was initially constructed by introducing a plasmid expressing the precursor CMP-Neu5Ac synthesis pathway genes neuBCA. A novel α2,6-sialyltransferase Ev6ST (NCBI Reference Sequence: WP_132500470) was selected by introducing plasmids expressing various α2,6-sialyltransferase-encoding genes and subsequent comparisons of the yields of 6'-SL. Subsequently, by integrating neuBCA and ev6st individually or in combination on the chromosome of EZAK, the high-yielding plasmid-free strain EZAKBEP with an extracellular yield of 5.68 g/L. In the 5 L bioreactor, fed-batch fermentation resulted in an extracellular yield of 15.35 g/L of 6'-SL. This work successfully screened a high-efficiency α2,6-sialyltransferase Ev6ST and constructed a high-yielding strain EZAKBEP under plasmid-free conditions, which is the highest yield of shake flask fermentation to date, and provides some reference significance for subsequent research.

Human milk oligosaccharides (HMOs), the third most abundant solid component in human milk, vary significantly among women due to factors such as secretor status, race, geography, season, maternal nutrition and weight, gestational age, and delivery method. In recent studies, HMOs have been shown to have a variety of functional roles in the development of infants. Because HMOs are not digested by infants, they act as metabolic substrates for certain bacteria, helping to establish the infant's gut microbiota. By encouraging the growth of advantageous intestinal bacteria, these sugars function as prebiotics and produce short-chain fatty acids (SCFAs), which are essential for gut health. HMOs can also specifically reduce harmful microbes and viruses binding to the gut epithelium, preventing illness. HMO addition to infant formula is safe and promotes healthy development, infection prevention, and microbiota. Current infant formulas frequently contain oligosaccharides (OSs) that differ structurally from those found in human milk, making it unlikely that they would reproduce the unique effects of HMOs. However, there is a growing trend in producing OSs resembling HMOs, but limited data make it unclear whether HMOs offer additional therapeutic benefits compared to non-human OSs. Better knowledge of how the human mammary gland synthesizes HMOs could direct the development of technologies that yield a broad variety of complex HMOs with OS compositions that closely mimic human milk. This review explores HMOs' complex nature and vital role in infant health, examining maternal variation in HMO composition and its contributing factors. It highlights recent technological advances enabling large-scale studies on HMO composition and its effects on infant health. Furthermore, HMOs' multifunctional roles in biological processes such as infection prevention, brain development, and gut microbiota and immune response regulation are investigated. The structural distinctions between HMOs and other mammalian OSs in infant formulas are discussed, with a focus on the trend toward producing more precise replicas of HMOs found in human milk.

Infants rely on their developing immune system and the protective components of breast milk to defend against bacterial and viral pathogens, as well as immune disorders such as food allergies, prior to the introduction of solid foods. When breastfeeding is not feasible, fortified infant formula will most frequently be offered, usually based on a cow's milk-based substitute. The current study aimed to explore the immunomodulatory effects of combinations of commercially available human milk oligosaccharides (HMOs). An in vitro co-culture model of Caco-2 intestinal epithelial cells and THP-1 macrophages was established to replicate the hallmarks of intestinal inflammation and to evaluate the direct effects of different synthetic HMO combinations. Notably, a blend of the most prevalent fucosylated and sialylated HMOs, 2'-fucosyllactose (2'-FL) and 6'-siallylactose (6'-SL), respectively, resulted in decreased pro-inflammatory cytokine levels. These effects were dependent on the HMO concentration and on the HMO ratio resembling those in breastmilk. Interestingly, adding additional HMO structures did not enhance the anti-inflammatory effects. This research highlights the importance of carefully selecting HMO combinations in nutritional products, particularly for infant milk formulations, to effectively mimic the benefits associated with breastmilk.

Human milk oligosaccharides (HMOs) have attracted considerable interest for their vital role in supporting infant health. Among these, sialyllacto-N-tetraose c (LST c), a pentasaccharide with the structure Neu5Ac(α2,6)Gal(β1,4)GlcNAc(β1,3)Gal(β1,4)Glc, stands out due to its critical importance in the development and application of complex HMOs. In this study, we employed multivariate modular metabolic engineering (MMME) to screen for efficient sialyltransferases and balance metabolic fluxes, successfully constructing strains capable of LST c biosynthesis. Additionally, by blocking competing pathway genes, enhancing the supply of UDP-GlcNAc and UDP-Gal precursors, and establishing a CTP cofactor regeneration system, we developed a high-yielding Escherichia coli strain, W15. This strain achieved an LST c titer of 220.9 mg/L in shake flask cultures. In a 3-L fed-batch fermentation, the LST c concentration reached 922.2 mg/L, with a productivity of 10.25 mg/L/h and a specific yield of 38.70 mg/g DCW. This research provides an effective strategy for producing LST c in microbial cell factories.

This study aimed to evaluate the concentrations of both essential and non-essential elements in the breast milk of lactating mothers from Tehran, Iran, during the colostrum period. Neutron activation analysis (NAA) was used to measure the element concentrations. Additionally, the study assessed how these element concentrations were influenced by maternal factors such as age and economic status. Breast milk samples were collected from 95 lactating mothers, aged 18 to 41, during the early lactation phase, specifically within the colostrum period (2-7 days postpartum). The colostrum milk samples were freeze-dried, powdered, and irradiated at the Tehran Research Reactor for neutron activation analysis (NAA). This method was used to measure the concentrations of essential elements-calcium (Ca), potassium (K), sodium (Na), chlorine (Cl), and iodine (I)-as well as non-essential elements-aluminum (Al), bromine (Br), and rubidium (Rb). Descriptive statistics, including mean, median, maximum, minimum, and standard deviation, were calculated for each element. Statistical analyses, such as Pearson's correlation, were performed to assess relationships between the concentrations of various elements. Additionally, t-tests and p-values were employed to evaluate differences in element levels across maternal age groups (17-34 years vs. 35-45 years) and economic status (high/middle vs. low). The mean concentrations of the elements in dry breast milk powder samples were: Al = 6.9 mg/kg, Br = 11.9 mg/kg, Ca = 2.757 mg/g, Cl = 7.836 mg/g, I = 1.22 mg/kg, K = 5.853 mg/g, Na = 4.932 mg/g, and Rb = 3.69 mg/kg. Significant correlations were found between element pairs, such as Na-Cl, Br-Cl, Na-Br, Rb-K, and I-Cl. Maternal age significantly influenced bromine concentrations, with older mothers showing 22% higher Br levels (p = 0.038), while calcium levels were 15% lower but not statistically significant (p = 0.20). Maternal economic status significantly impacted calcium and potassium concentrations, with higher levels observed in mothers from better economic conditions (p = 0.02 and p = 0.025, respectively). This study highlights the elemental composition of breast milk samples of lactating mothers in Tehran and shows that maternal factors, such as age and economic status, can significantly influence the concentrations of specific elements in breast milk.

Human milk (HM) is a complex biofluid rich in nutrients and bioactive compounds essential for infant health. Recent advances in omics technologies-such as proteomics, metabolomics, and transcriptomics-have shed light on the influence of HM on bone development and health. This review discusses the impact of various HM components, including proteins, lipids, carbohydrates, and hormones, on bone metabolism and skeletal growth. Proteins like casein and whey promote calcium absorption and osteoblast differentiation, supporting bone mineralization. Long-chain polyunsaturated fatty acids like docosahexaenoic acid (DHA) contribute to bone health by modulating inflammatory pathways and regulating osteoclast activity. Additionally, human milk oligosaccharides (HMOs) act as prebiotics, improving gut health and calcium bioavailability while influencing bone mineralization. Hormones present in HM, such as insulin-like growth factor 1 (IGF-1), leptin, and adiponectin, have been linked to infant growth, body composition, and bone density. Research has shown that higher IGF-1 levels in breast milk are associated with increased weight gain, while leptin and adiponectin influence fat mass and bone metabolism. Emerging studies have also highlighted the role of microRNAs (miRNAs) in regulating key processes like adipogenesis and bone homeostasis. Furthermore, microbiome-focused techniques reveal HM's role in establishing a balanced infant gut microbiota, indirectly influencing bone development by enhancing nutrient absorption. Although current findings are promising, comprehensive longitudinal studies integrating omics approaches are needed to fully understand the intricate relationships among maternal diet, HM composition, and infant bone health. Bridging these gaps could offer novel dietary strategies to optimize skeletal health during infancy, advancing early-life nutrition science.

Human milk oligosaccharides (HMOs) are a diverse group of structures and an abundant bioactive component of breastmilk that contribute to infant health and development. Preclinical studies indicate roles for HMOs in shaping the infant gut microbiota, inhibiting pathogens, modulating the immune system, and influencing cognitive development. In the past decade, several industrially produced HMOs have become available to fortify infant formula. Clinical intervention trials with manufactured HMOs have begun to corroborate some of the physiological effects reported in preclinical studies, especially modulation of the gut microbiota in the direction of breastfed infants. As more HMOs become commercially available and as HMOs have some shared mechanisms of action, there is a need to better understand the unique and differential effects of individual HMOs and the benefits of combining multiple HMOs. This review focuses on the differential effects of different HMO structural classes and individual structures and presents a scientific rationale for why combining multiple structurally diverse HMOs is expected to exert greater biological effects.

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.

Lacto-N-neotetraose (LNnT), a representative oligosaccharide found in human milk, has been previously examined for its beneficial traits. However, the LNnT titer is limited by the efficient glycosyltransferase pathway, particularly with respect to the catalysis of rate-limiting steps. As data on the crystal structure of the key enzyme required for synthesizing LNnT are lacking, the synthesis of LNnT remains an uncertainty. Here, for the first time we report the three-dimensional structure of a bacterial β-1,4-galactosyltransferase, Aaβ4GalT, and analyze the critical role played by residues in its catalytic efficacy. Guided by structural insights, we engineered this enzyme to enhance its catalytic efficiency using structure-guided tunnel engineering. The mutant enzyme L5 (K155M/H156D/F157W/K185M/Q216V) so produced, showed a 50-fold enhancement in catalytic activity. Crystal structure analysis revealed that the mechanism underlying the improvement in activity was of the swing door type. The closed conformation formed by dense hydrophobic packing with Q216V-K155M widened and permitted substrate entry. Our results show that altering the tunnel conformation helped appropriately accommodate the substrate for catalysis and provide a structural basis for the modification of other glycosyltransferases.

Human milk Oligosaccharides (HMOs) are a major component in human milk and recognized to play an important role in modulating gut microbiota, intestinal cell response, and the development of the brain and immune system. While HMOs levels from Chinese mothers across different regions of China have been reported, data from Hebei are lacking. Twelve HMOs were measured from a cross-section of Hebei Han mothers over a 15-month lactation period. The average total of the 12 measured HMOs was 4872 ± 1902 mg/L, similar to that reported for Han mothers from other Chinese regions. Hebei Han mothers had much lower LNnT (59.0 ± 53.1 mg/L), LNFP II (257.5 ± 211.0 mg/L) and LNFP III (149.9 ± 121.7 mg/L) levels and higher 3FL levels (1875.2 ± 1065.3 mg/L) compared to other regional Chinese mother cohorts. The distribution of secretor and Lewis status for this Hebei mother cohort was measured at 68.5 %, 21.9 %, 8.2 % and 1.4 % respectively for Se+Le+, Se-Le+, Se+Le- and Se-Le- respectively. The results from this study suggest that location has influence over the HMOs concentration.

Milk oligosaccharides are complex carbohydrates composed of various monosaccharide units linked together by glycosidic bonds. They play an essential role in promoting gut health by fostering beneficial bacteria, supporting the development of the immune system, and protecting against infections and diseases. This work compared the oligosaccharide profiles in widely utilized breeds such as Holstein and Ayrshire (Nordic Red), with the native Northern Finncattle, which is considered an endangered breed. Oligosaccharides were extracted from milk and analyzed by liquid chromatography-mass spectrometry. The composition and relative abundance of the identified oligosaccharides were characterized and compared. The statistical analyses showed that neutral, sialylated, and fucosylated oligosaccharides vary among the breeds. Ayrshire and Northern Finncattle oligosaccharides formed a cluster, while Holstein's profile shared features with both Ayrshire and Northern Finncattle. Holstein had the lowest abundance of fucosylated OS among the three breeds, with Ayrshire having the highest content followed by Northern Finncattle. The relatively higher sialylated over neutral content of Northern Finncattle is an important feature that should be preserved. Ayrshire is a good candidate to recover more diverse oligosaccharides with potential gut health implications for consumers.

Human milk oligosaccharides (HMOs) are important carbohydrates in human milk that infants cannot digest, acting as prebiotics linked to infant health. The risk of childhood obesity increases with maternal obesity, potentially mediated through the gut microbiota affected by the available HMOs. Studies on whether maternal obesity affects HMO abundance, yield conflicting results. This study aimed to investigate the HMO profile and its association with maternal obesity measured by pre-pregnancy body mass index (BMI) and infant anthropometrics. The results were discussed in the context of existing literature. 90 human milk samples were collected at 3 months postpartum from mothers in three BMI-groups: 32 normal weight (BMI: 18.5-24.99 kg/m2), 34 overweight (BMI: 25-30 kg/m2), and 24 obese (BMI > 30 kg/m2). The samples were analyzed using nano liquid chromatography chip quadrupole time-of-flight mass spectrometry yielding 51 HMO structures and isomers. Their peak areas were integrated and normalized to determine relative abundances. Univariate and multivariate analysis showed associations between relative HMO abundance and donors' secretor status and specific infant anthropometric variables, but not with maternal pre-pregnancy BMI. This study does not support the hypothesis that maternal overweight influences the HMO profile and highlights the importance of reporting results despite absence of significant correlations.

The gut microbiota of breast-fed infants is dominated by infant-type human-residential bifidobacteria (HRB) that contribute to infant health; thus, it is crucial to develop infant formulas that promote the establishment of a gut microbiota enriched with infant-type HRB, closely resembling that of breastfed infants.

We compared various non-digestible prebiotic oligosaccharides and their combinations using a fecal culture system to explore which candidates could promote the growth of all infant-type HRB and rarely yield non-responders. The analysis included lactulose (LAC), raffinose (RAF), galactooligosaccharides (GOS), and short- and long-chain fructooligosaccharides. Fecal samples were collected from seven infants aged 1.5-10.2 months and cultured with each oligosaccharide individually or their combinations.

No single oligosaccharide effectively promoted the growth of all infant-type HRB, although GOS promoted the growth of HRB other than Bifidobacterium longum subsp. longum. Only the LAC/RAF/GOS group evenly and effectively promoted the growth of all infant-type HRB. Accordingly, acetate production was higher in fecal cultures supplemented with GOS or LAC/RAF/GOS than in the other cultures, suggesting that it is a superior combination for all infant-type HRB and rarely yields non-responders.

This study can aid in developing infant formulas that help align the gut microbiota of formula-fed infants with that of breastfed infants.

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.

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.

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.

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.

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.

Early nutritional exposures, including during embryogenesis and the immediate postnatal period, affect offspring outcomes in both the short- and long-term. Alterations of these modifiable exposures shape the developing gut microbiome, intestinal development, and even neurodevelopmental outcomes. A gut-brain axis exists, and it is intricately connected to early life feeding and nutritional exposures. Here, we seek to discuss the (1) origins of the gut-brain access and relationship with neurodevelopment, (2) components of human milk (HM) beyond nutrition and their role in the developing newborn, and (3) clinical application of nutritional practices, including fluid management and feeding on the development of the gut-brain axis, and long-term neurodevelopmental outcomes. We conclude with a discussion on future directions and unanswered questions that are critical to provide further understanding and insight into how clinicians and healthcare providers can optimize early nutritional practices to ensure children not only survive, but thrive, free of neurodevelopmental impairment.

As the evidence supporting the beneficial effects of human milk oligosaccharides (HMOs) grows, so does the commercial interest in their inclusion in infant formula products. This also requires analytical methods capable of their quantification from finished infant formula products as well as from premixed ingredients in some cases. The objective of the present study was the development and single-laboratory validation of a method that can be used for this purpose for seven HMOs: 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), difucosyllactose (DFL), 3'-sialyllactose (3'SL), 6'-sialyllactose (6'SL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT). The present method uses labeling by reductive amination, with 4-aminobenzoic acid ethyl ester (benzocaine) as the labeling reagent and picoline borane as the reducing agent, then applies HPLC separation with UV detection. The seven HMOs could be analyzed from infant formula and premix samples with recoveries between 91 and 108 %, relative standard deviations of 4.3 % or lower across all replicates, and limits of quantitation between 0.001 % and 0.004 % of powder sample by weight. The method was found to be rapid and reliable, with a runtime of only 14 min per injection, in contrast to other methods found in literature which typically use nearly or more than an hour. In addition, it uses instrumentation that's readily available in most analytical laboratories.

We aimed to estimate associations between human milk oligosaccharides (HMOs) and infant growth (length-for-age (LAZ) and weight-for-length (WLZ) z-scores) at 12 months postnatal age.

In this secondary analysis of data from a maternal vitamin D trial in Dhaka, Bangladesh (N = 192), absolute concentrations of HMOs were measured in 13 ± 1 week(s) postpartum milk samples, infant anthropometric measurements were obtained soon after birth and at 12 months postpartum, and infant feeding was classified during 6 months postpartum. Associations between individual HMOs or HMO groups and LAZ or WLZ were estimated by multivariable linear regression adjusting for infant feeding pattern, maternal secretor status, and other potential confounders.

The concentrations of 6'sialyllactose, lacto-N-neotetraose, and the non-fucosylated non-sialylated HMOs were inversely associated with LAZ at 12 months of age, whereas the fucosylated non-sialylated HMO concentration was positively associated with LAZ at 12 months. These associations were robust in analyses restricted to infants who were primarily exclusively/predominantly fed human milk during the first 3 (or 6) months.

Since HMOs are both positively and negatively associated with postnatal growth, there is a need for randomized trials to estimate the causal benefits and risks of exogenously administered HMOs on infant growth and other health outcomes.

6'sialyllactose, lacto-N-neotetraose, and the non-fucosylated non-sialylated human milk oligosaccharides (HMOs) were inversely associated with length-for-age z-scores (LAZ) at 12 months, whereas the fucosylated non-sialylated HMO concentration was positively associated with LAZ at 12 months among Bangladeshi infants. Associations between individual and grouped HMOs with infant length growth at 12 months were as strong or stronger in analyses restricted to infants who were exclusively or predominantly fed human milk up to 3 (or 6) months. Randomized trials are needed to characterize the effects of specific HMOs on infant growth, particularly in countries where postnatal linear growth faltering is common.

An accurate method for qualitative and quantitative analysis of lipid-bound (LB), protein-bound (PB), oligosaccharides-bound, and free sialic acids in milk was developed by using high-performance liquid chromatography -triple quadrupole-tandem mass spectrometer. The profile of free and bound sialic acids in milk (human, bovine, goat, and sheep) and infant formula (IF) was examined in the present study. Human milk contains only N-acetylneuraminic acid (Neu5Ac) and was mainly present in the form of oligosaccharide-bound. The content of total Neu5Ac (T-Neu5Ac), free and bound Neu5Ac in human milk decreased with the prolongation of lactation. The most intriguing finding was the increase in the proportion of PB and LB sialic acids. The sialic acids in bovine and sheep milk were mainly PB and oligosaccharides-bound Neu5Ac. T-Neu5Ac in goat milk (GM) was 67.44-89.72 µg/mL and was mainly PB Neu5Ac, but total N-glycolylneuraminic acid (T-Neu5Gc) content of GM can be as high as 100.01 µg/mL. The concentration of T-Neu5Gc in sheep and GM was significantly higher than that of bovine milk (BM). T-Neu5Gc content of GM -based IF was 264.86 µg/g, whereas T-Neu5Gc content of BM -based IF was less (2.26-17.01 µg/g). Additionally, our results found that there were also sialic acids in IF ingredients, which were mainly bound with protein and oligosaccharides, primarily derived from desalted whey powder and whey protein concentrate.

3-Fucosyllactose (3-FL), an important fucosylated human milk oligosaccharide in breast milk, offers numerous health benefits to infants. Previously, we metabolically engineered Escherichia coli BL21(DE3) for the in vivo biosynthesis of 3-FL. In this study, we initially optimized culture conditions to double 3-FL production. Competing pathway genes involved in in vivo guanosine 5'-diphosphate-fucose biosynthesis were subsequently inactivated to redirect fluxes toward 3-FL biosynthesis. Next, three promising transporters were evaluated using plasmid-based or chromosomally integrated expression to maximize extracellular 3-FL production. Additionally, through analysis of α1,3-fucosyltransferase (FutM2) structure, we identified Q126 residues as a highly mutable residue in the active site. After site-saturation mutation, the best-performing mutant, FutM2-Q126A, was obtained. Structural analysis and molecular dynamics simulations revealed that small residue replacement positively influenced helical structure generation. Finally, the best strain BD3-A produced 6.91 and 52.1 g/L of 3-FL in a shake-flask and fed-batch cultivations, respectively, highlighting its potential for large-scale industrial applications.

Adulteration of caprine dairy products raises concerns among consumers. This study aimed to identify the differences in oligosaccharide profiles of caprine dairy products, including raw milk, colostrum powder, and lactose powder, and their corresponding bovine dairy products, and provide new insights for detecting adulteration of bovine dairy products in caprine dairy products. Twenty-seven oligosaccharides were detected in caprine and bovine dairy products. The principal component analysis plot of the oligosaccharide profiles clearly differentiated among the six types of dairy products. Specific oligosaccharides that were most distinctive for caprine and bovine dairy products were identified. Lacto-N-triose (LNTri) could be used as a potential biomarker for distinguishing caprine milk from bovine milk, caprine colostrum powder from bovine colostrum powder, and caprine lactose powder from bovine lactose powder. The results demonstrated that oligosaccharides could be used as biomarkers for detecting bovine dairy products in caprine dairy products, especially caprine lactose powder.

Human milk oligosaccharides (HMOs) are present in high numbers in milk of lactating women. They are beneficial to gut health and the habitant microbiota, but less is known about their effect on cells from the immune system. In this study, we investigated the direct effect of three structurally different HMOs on human derived macrophages before challenge with Staphylococcus aureus (S. aureus). The study demonstrates that individual HMO structures potently affect the activation, differentiation and development of monocyte-derived macrophages in response to S. aureus. 6´-Sialyllactose (6'SL) had the most pronounced effect on the immune response against S. aureus, as illustrated by altered expression of macrophage surface markers, pointing towards an activated M1-like macrophage-phenotype. Similarly, 6'SL increased production of the pro-inflammatory cytokines TNF-α, IL-6, IL-8, IFN-γ and IL-1β, when exposing cells to 6'SL in combination with S. aureus compared with S. aureus alone. Interestingly, macrophages treated with 6'SL exhibited an altered proliferation profile and increased the production of the classic M1 transcription factor NF-κB. The HMOs also enhanced macrophage phagocytosis and uptake of S. aureus. Importantly, the different HMOs did not notably affect macrophage activation and differentiation without S. aureus exposure. Together, these findings show that HMOs can potently augment the immune response against S. aureus, without causing inflammatory activation in the absence of S. aureus, suggesting that HMOs assist the immune system in targeting important pathogens during early infancy.

Human milk oligosaccharides (HMOs) are a diverse class of carbohydrates which support the health and development of infants. The vast health benefits of HMOs have made them a commercial target for microbial production; however, producing the approximately 200 structurally diverse HMOs at scale has proved difficult. Here we produce a diversity of HMOs by leveraging the robust carbohydrate anabolism of plants. This diversity includes high-value and complex HMOs, such as lacto-N-fucopentaose I. HMOs produced in transgenic plants provided strong bifidogenic properties, indicating their ability to serve as a prebiotic supplement with potential applications in adult and infant health. Technoeconomic analyses demonstrate that producing HMOs in plants provides a path to the large-scale production of specific HMOs at lower prices than microbial production platforms. Our work demonstrates the promise in leveraging plants for the low-cost and sustainable production of HMOs.