Evolution of milk oligosaccharides and lactose: a hypothesis

Mammalian milk or colostrum contains up to 10% of carbohydrate, of which free lactose usually constitutes more than 80%. Lactose is synthesized within lactating mammary glands from uridine diphosphate galactose (UDP-Gal) and glucose by a transgalactosylation catalysed by a complex of β4-galactosyltransferase and α-lactalbumin (α-LA). α-LA is believed to have evolved from C-type lysozyme. Mammalian milk or colostrum usually contains a variety of oligosaccharides in addition to free lactose. Each oligosaccharide has a lactose unit at its reducing end; this unit acts as a precursor that is essential for its biosynthesis. It is generally believed that milk oligosaccharides act as prebiotics and also as receptor analogues that act as anti-infection factors. We propose the following hypothesis. The proto-lacteal secretions of the primitive mammary glands of the common ancestor of mammals contained fat and protein including lysozyme, but no lactose or oligosaccharides because of the absence of α-LA. When α-LA first appeared as a result of its evolution from lysozyme, its content within the lactating mammary glands was low and lactose was therefore synthesized at a slow rate. Because of the presence of glycosyltransferases, almost all of the nascent lactose was utilized for the biosynthesis of oligosaccharides. The predominant saccharides in the proto-lacteal secretions or primitive milk produced by this common ancestor were therefore oligosaccharides rather than free lactose. Subsequent to this initial period, the oligosaccharides began to serve as anti-infection factors. They were then recruited as a significant energy source for the neonate, which was achieved by an increase in the synthesis of α-LA. This produced a concomitant increase in the concentration of lactose in the milk, and lactose therefore became an important energy source for most eutherians, whereas oligosaccharides continued to serve mainly as anti-microbial agents. Lactose, in addition, began to act as an osmoregulatory molecule, controlling the milk volume. Studies on the chemical structures of the milk oligosaccharides of a variety of mammalian species suggest that human milk or colostrum is unique in that oligosaccharides containing lacto-N-biose I (LNB) (Gal(β1 → 3)GlcNAc, type I) predominate over those containing N-acetyllactosamine (Gal(β1 → 4)GlcNAc, type II), whereas in other species only type II oligosaccharides are found or else they predominate over type I oligosaccharides. It can be hypothesized that this feature may have a selective advantage in that it may promote the growth of beneficial colonic bacteria, Bifidobacteria, in the human infant colon.     

人乳寡糖的结构及其分离分析

母乳中存在的人乳寡糖（HMOs）是一类结构高度复杂的低聚糖，对婴儿的肠道菌群、免疫屏障、大脑发育发挥积极作用。由于母乳中基质复杂，寡糖的种类繁多，丰度跨度大，存在众多异构体，这都使得检测面临诸多挑战。现已有多种技术用于HMOs的分析，发现了200多种HMOs，液相色谱和毛细管电泳在分离HMOs方面效果显著，核磁共振、质谱、红外多光子解离光谱推动了对HMOs结构的全面解析。本文回顾了对HMOs实现高灵敏度和高特异性分析的多种技术方法，比较了不同技术的优缺点，还重点介绍了质谱以及不同技术联用在推动HMOs解析和测定方面的突破，为探究寡糖的结构-功能关系、深入理解HMOs的生物学功能提供了全面的技术支持。     

The evolution of milk secretion and its ancient origins

Lactation represents an important element of the life history strategies of all mammals, whether monotreme, marsupial, or eutherian. Milk originated as a glandular skin secretion in synapsids (the lineage ancestral to mammals), perhaps as early as the Pennsylvanian period, that is, approximately 310 million years ago (mya). Early synapsids laid eggs with parchment-like shells intolerant of desiccation and apparently dependent on glandular skin secretions for moisture. Mammary glands probably evolved from apocrine-like glands that combined multiple modes of secretion and developed in association with hair follicles. Comparative analyses of the evolutionary origin of milk constituents support a scenario in which these secretions evolved into a nutrient-rich milk long before mammals arose. A variety of antimicrobial and secretory constituents were co-opted into novel roles related to nutrition of the young. Secretory calcium-binding phosphoproteins may originally have had a role in calcium delivery to eggs; however, by evolving into large, complex casein micelles, they took on an important role in transport of amino acids, calcium and phosphorus. Several proteins involved in immunity, including an ancestral butyrophilin and xanthine oxidoreductase, were incorporated into a novel membrane-bound lipid droplet (the milk fat globule) that became a primary mode of energy transfer. An ancestral c-lysozyme lost its lytic functions in favor of a role as α-lactalbumin, which modifies a galactosyltransferase to recognize glucose as an acceptor, leading to the synthesis of novel milk sugars, of which free oligosaccharides may have predated free lactose. An ancestral lipocalin and an ancestral whey acidic protein four-disulphide core protein apparently lost their original transport and antimicrobial functions when they became the whey proteins β-lactoglobulin and whey acidic protein, which with α-lactalbumin provide limiting sulfur amino acids to the young. By the late Triassic period (ca 210 mya), mammaliaforms (mammalian ancestors) were endothermic (requiring fluid to replace incubatory water losses of eggs), very small in size (making large eggs impossible), and had rapid growth and limited tooth replacement (indicating delayed onset of feeding and reliance on milk). Thus, milk had already supplanted egg yolk as the primary nutrient source, and by the Jurassic period (ca 170 mya) vitellogenin genes were being lost. All primary milk constituents evolved before the appearance of mammals, and some constituents may have origins that predate the split of the synapsids from sauropsids (the lineage leading to 'reptiles' and birds). Thus, the modern dairy industry is built upon a very old foundation, the cornerstones of which were laid even before dinosaurs ruled the earth in the Jurassic and Cretaceous periods.     

研究: 奶牛与水牛初乳中乳寡糖组分比较研究

乳寡糖是由乳汁中含量丰富的固体物质组成．研究结果表明,乳寡糖有提高免疫、益生元及抗感染等作用,已发现与婴儿肠道发育、神经智力发育等多方面关系密切．水牛奶是除牛奶外的第二大奶源,国际上公认其为营养含量高、口感好的优质乳制品,但目前针对水牛乳寡糖的研究多以美洲水牛为研究对象,尚无中国水牛的相关研究．本研究利用固相萃取对已脱脂和除去蛋白质的广西水牛初乳乳汁样品进行纯化,并采用苯胺 (aniline,Bn)衍生化试剂对其进行衍生化处理,通过UPLC-ESI-Q-TOF-MS液相质谱进行优化后,对水牛初乳中的寡糖组分进行测定并与牛乳进行了对比,最终测得奶牛初乳中19种及水牛初乳中的9种乳寡糖组分,并对二者的种类及含量进行比较,发现在两种初乳的乳寡糖中,中性糖二糖m/z 385.15和中性糖三糖m/z 547.21以及酸性糖m/z 635.23均为其主要寡糖成分,与其他乳寡糖相比含量相对较高．总体而言水牛初乳中的中性寡糖占比比奶牛初乳高,二者中性糖占乳寡糖总量的比例分别为88.88%和63.16%．     

Binding of milk oligosaccharides by several enterotoxigenic Escherichia coli strains isolated from calves

Milk oligosaccharides have been proposed to play an important role in newborn defense, blocking bacterial adhesion to the intestinal mucosa and preventing infections. Some studies have been performed on human milk oligosaccharides. Here we checked whether bovine milk oligosaccharides would achieve the same protective action against the most common calf enteric pathogens. Seven enterotoxigenic Escherichia coli strains, isolated from diarrheic calves, were selected. All strains managed to agglutinate horse erythrocytes, and we therefore used the inhibition of hemagglutination in the presence of oligosaccharides as an indicator of the union between oligosaccharide and bacterial adhesins. Oligosaccharides from different stages of bovine lactation and standard oligosaccharides were assayed. Midlactation milk, in particular that corresponding to the transition period, proved to be the most efficient at inhibiting hemagglutination. The standard oligosaccharides used pointed to the preference of several strains (K99-, F41-, and F17-fimbriated) for 2,6-linked sialic acid. By contrast, B23 fimbriae exhibited higher affinity for 2,3-sialylated isomers and B64 seemed to require N-acetylglucosamine for binding.Our results suggest a general trend for milk oligosaccharides. Probably they participate in the protection of newborn mammals from pathogens.     

Host-derived glycans serve as selected nutrients for the gut microbe: human milk oligosaccharides and bifidobacteria

Lactation is a common feeding strategy of eutherian mammals, but its functions go beyond feeding the neonates. Ever since Tissier isolated bifidobacteria from the stool of breast-fed infants, human milk has been postulated to contain compounds that selectively stimulate the growth of bifidobacteria in intestines. However, until relatively recently, there have been no reports to link human milk compound(s) with bifidobacterial physiology. Over the past decade, successive studies have demonstrated that infant-gut-associated bifidobacteria are equipped with genetic and enzymatic toolsets dedicated to assimilation of host-derived glycans, especially human milk oligosaccharides (HMOs). Among gut microbes, the presence of enzymes required for degrading HMOs with type-1 chains is essentially limited to infant-gut-associated bifidobacteria, suggesting HMOs serve as selected nutrients for the bacteria. In this study, I shortly discuss the research on bifidobacteria and HMOs from a historical perspective and summarize the roles of bifidobacterial enzymes in the assimilation of HMOs with type-1 chains. Based on this overview, I suggest the co-evolution between bifidobacteria and human beings mediated by HMOs.     

Biotechnological production of human milk oligosaccharides

Human milk contains a large variety of oligosaccharides (HMOs) that have the potential to modulate the gut flora, affect different gastrointestinal functions, and influence inflammatory processes. This review introduces the recent advances in the microbial and coupled enzymatic methods to produce HMOs with grouping them into trisaccharides (sialyllactose and fucosyllactose) and complex oligosaccharides (lacto-N-biose derivatives). The high purity and low cost of HMOs should make their use possible in new fields such as the food or pharmaceutical industries.     

High-pH anion-exchange chromatography with pulsed amperometric detection and molar response factors of human milk oligosaccharides

A method is described to separate and characterize neutral and acidic lactose-derived oligosaccharides without prior derivatization or reduction by high-pH anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD). This method has been applied to human milk oligosaccharides from donors with different blood group specificity (A, Le^a and A, Le^b). Neutral and acidic components were separated from each other by anion-ecchange chromatography. A distinct separation of individual components was obtained by size-exclusion chromatography on Fractogel TSK HW 50S (acidic oligosaccharides) or Fractogel TSK HW 40S (neutral oligosaccharides containing up to 6 monomers) and Bio-Gel P-4 size exclusion (neutral oligosaccharides containing more than 6 monomers). Furthermore, the molar response factors after HPAEC-PAD have been determined for 8 components.     

Evolution of milk oligosaccharides: Origin and selectivity of the ratio of milk oligosaccharides to lactose among mammals

BackgroundThe carbohydrate fraction of mammalian milk is constituted of lactose and oligosaccharides, most of which contain a lactose unit at their reducing ends. Although lactose is the predominant saccharide in the milk of most eutherians, oligosaccharides significantly predominate over lactose in the milk of monotremes and marsupials.Scope of reviewThis review describes the most likely process by which lactose and milk oligosaccharides were acquired during the evolution of mammals and the mechanisms by which these saccharides are digested and absorbed by the suckling neonates.Major conclusionsDuring the evolution of mammals, c-type lysozyme evolved to α-lactalbumin. This permitted the biosynthesis of lactose by modulating the substrate specificity of β4galactosyltransferase 1, thus enabling the concomitant biosynthesis of milk oligosaccharides through the activities of several glycosyltransferases using lactose as an acceptor. In most eutherian mammals the digestion of lactose to glucose and galactose is achieved through the action of intestinal lactase (β-galactosidase), which is located within the small intestinal brush border. This enzyme, however, is absent in neonatal monotremes and macropod marsupials. It has therefore been proposed that in these species the absorption of milk oligosaccharides is achieved by pinocytosis or endocytosis, after which digestion occurs through the actions of several lysosomal acid glycosidases. This process would enable the milk oligosaccharides of monotremes and marsupials to be utilized as a significant energy source for the suckling neonates.General significanceThe evolution and significance of milk oligosaccharides is discussed in relation to the evolution of mammals.     

Simultaneous quantification of sialyloligosaccharides from human milk by capillary electrophoresis

The acidic oligosaccharides of human milk are predominantly sialyloligosaccharides. Pathogens that bind sialic acid-containing glycans on their host mucosal surfaces may be inhibited by human milk sialyloligosaccharides, but testing this hypothesis requires their reliable quantification in milk. Sialyloligosaccharides have been quantified by anion exchange high-performance liquid chromatography (HPLC), reverse- or normal-phase HPLC, and capillary electrophoresis (CE) of fluorescent derivatives; in milk, these oligosaccharides have been analyzed by high pH anion exchange chromatography with pulsed amperometric detection and, in our laboratory, by CE with detection at 205nm. The novel method described here uses a running buffer of aqueous 200mM NaH2PO4 (pH 7.05) containing 100mM sodium dodecyl sulfate (SDS) mixed with 45% (v/v) methanol to baseline resolve 5 oligosaccharides and separate all 12. This allows automated simultaneous quantification of the 12 major sialyloligosaccharides of human milk in a single 35-min run. This method revealed differences in sialyloligosaccharide concentrations between less and more mature milk from the same donors. Individual donors also varied in expression of sialyloligosaccharides in their milk. Thus, the facile quantification of sialyloligosaccharides by this method is suitable for measuring variation in expression of specific sialyloligosaccharides in milk and their relationship to decreased risk of specific diseases in infants.     

Bio-therapeutics from human milk: prospects and perspectives

Human milk is elixir for neonates and is a rich source of nutrients and beneficial microbiota required for infant growth and development. Its benefits prompted research into probing the milk components and their use as prophylactic or therapeutic agents. Culture-independent estimation of milk microbiome and high-resolution identification of milk components provide information, but a holistic purview of these research domains is lacking. Here, we review the current research on bio-therapeutic components of milk and simplified future directions for its efficient usage. Publicly available databases such as PubMed and Google scholar were searched for keywords such as probiotics and prebiotics related to human milk, microbiome and milk oligosaccharides. This was further manually curated for inclusion and exclusion criteria relevant to human milk and clinical efficacy. The literature was classified into subgroups and then discussed in detail to facilitate understanding. Although milk research is still in infancy, it is clear that human milk has many functions including protection of infants by passive immunization through secreted antibodies, and transfer of immune regulators, cytokines and bioactive peptides. Unbiased estimates show that the human milk carries a complex community of microbiota which serves as the initial inoculum for establishment of infant gut. Our search effectively screened for evidence that shows that milk also harbours many types of prebiotics such as human milk oligosaccharides which encourage growth of beneficial probiotics. The milk also trains the naive immune system of the infant by supplying immune cells and stimulatory factors, thereby strengthening mucosal and systemic immune system. Our systematic review would improve understanding of human milk and the inherent complexity and diversity of human milk. The interrelated functional role of human milk components especially the oligosaccharides and microbiome has been discussed which plays important role in human health.     

Methods for the quantitation of human milk oligosaccharides in bacterial fermentation by mass spectrometry

Oligosaccharides are the third most abundant component in human milk. In the past decades, it became apparent that they would be able to protect against pathogens and participate in the development of the gut microflora for infants. However, their role in infants' nutrition and development remains poorly understood. To better understand this function, it is extremely important to have a quantitative tool for profiling oligosaccharides. In this article, we show the development of a method to quantitatively differentiate the relative amounts of oligosaccharides fermented by different intestinal bacteria. To determine the oligosaccharide consumption, bacteria were grown in a medium using human milk oligosaccharides (HMOs) as the only carbon source purified from breast milk and further analyzed by matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS). A method using an internal deuterium-labeled standard was developed and compared with an external standard method, with the internal standard method giving better precision and unambiguous measurements than the external standard method and providing to be a novel and robust tool for following bacterial fermentation of milk oligosaccharides.     

Growth of bifidobacteria and clostridia on human and cow milk saccharides

For healthy infants, which were born normally and fully breastfed, the dominant component of the intestinal microflora are bifidobacteria. However, infants born by caesarean section possess clostridia as a dominant intestinal bacterial group. The aim of the present study was to determine whether bifidobacteria and clostridia are able to grow on human milk oligosaccharides (HMOs) and other carbon sources - lactose, cow milk (CM) and human milk (HM). Both bifidobacteria and clostridia grew on lactose and in CM. Bifidobacteria grew in HM and on HMOs. In contrast, 3 out of 5 strains of clostridia were not able to grow in HM. No clostridial strain was able to utilise HMOs. While both bifidobacterial strains were resistant to lysozyme, 4 out of 5 strains of clostridia were lysozyme-susceptible. It seems that HMOs together with lysozyme may act as prebiotic-bifidogenic compounds inhibiting intestinal clostridia.     

Chemical characterization of the oligosaccharides in milk of high Arctic harbour seal (Phoca vitulina vitulina)

Carbohydrates were extracted from high Arctic harbour seal milk, Phoca vitulina vitulina (family Phocidae). Free neutral oligosaccharides were separated by gel filtration and preparative thin layer chromatography, while free sialyl oligosaccharides were separated by gel filtration and then purified by ion exchange chromatography, gel filtration and high performance liquid chromatography. Oligosaccharide structures were determined by 1H-NMR spectroscopy. The structures of the neutral oligosaccharides were as follows: lactose, 2'-fucosyllactose, lacto-N-neotetraose, lacto-N-neohexaose, monofucosyl lacto-N-neohexaose and difucosyl lacto-N-neohexaose. Thus, all of the neutral saccharides contained lactose or lacto-N-neotetraose or lacto-N-neohexaose as core units and/or non-reducing alpha(1-2) linked fucose. These oligosaccharides have also been found in hooded seal milk. The structures of the silalyl oligosaccharides were: monosialyl lacto-N-neohexaose, monosialyl monofucosyl lacto-N-neohexaose, monosialyl difucosyl lacto-N-neohexaose and disialyl lacto-N-neohexaose. These oligosaccharides contained lacto-N-neohexaose as core units, and one or two alpha(2-6) linked Neu5Ac, and/or non-reducing alpha(1-2) linked Fuc. The Neu5Ac residues were found to be linked to GlcNAc or penultimate Gal residues. The acidic oligosaccharides are the first to have been characterized in the milk of any species of seal.     

Milk carbohydrates of marsupials. II. Quantitative and qualitative changes in milk carbohydrates during lactation in the tammar wallaby (Macropus eugenii)

Milk was collected at various stages of lactation from a group of tammar wallabies, M. eugenii, in which parturition had been synchronized. The milk carbohydrate was determined by a phenol-sulfuric acid method which had been modified to give equal colour yields for galactose and glucose. The mean carbohydrate content increased gradually during the first 6 months of lactation to a peak of 13 g hexose/100 ml of milk, but then fell rapidly to much lower values, over the following 2 months. Throughouth lactation, galactose was the predominant monosaccharide constituent of acid hydrolysates of the milk carbohydrate. Glucose, glucosamine, galactosamine and sialic acid were the only other monosaccharides present. Qualitative changes were investigated by gel filtration and thin-layer chromatography. During the first 6 months post partum the milk carbohydrate was composed of a variety of oligosaccharides including lactose, but from 8 months onwards it consisted mainly of free monosaccharides. Between 6 and 8 months an intermediate pattern was observed, i.e. a mixture of lower oligosaccharides and free monosaccharides. In two animals which suckled both a new-born pouch young and a young at foot, the mammary gland supplying the new-born secreted milk which was rich in oligosaccharides, whereas that supplying the young at foot produced milk in which the carbohydrates were mainly free monosaccharides, and which had a much lower carbohydrate content.     

Novel High-Molecular Weight Fucosylated Milk Oligosaccharides Identified in Dairy Streams

Oligosaccharides are the third largest component in human milk. This abundance is remarkable because oligosaccharides are not digestible by the newborn, and yet they have been conserved and amplified during evolution. In addition to encouraging the growth of a protective microbiota dominated by bifidobacteria, oligosaccharides have anti-infective activity, preventing pathogens from binding to intestinal cells. Although it would be advantageous adding these valuable molecules to infant milk formula, the technologies to reproduce the variety and complexity of human milk oligosaccharides by enzymatic/organic synthesis are not yet mature. Consequently, there is an enormous interest in alternative sources of these valuable oligosaccharides. Recent research has demonstrated that bovine milk and whey permeate also contain oligosaccharides. Thus, a thorough characterization of oligosaccharides in bovine dairy streams is an important step towards fully assessing their specific functionalities. In this study, bovine milk oligosaccharides (BMOs) were concentrated by membrane filtration from a readily available dairy stream called “mother liquor”, and analyzed by high accuracy MALDI FT-ICR mass spectrometry. The combination of HPLC and accurate mass spectrometry allowed the identification of ideal processing conditions leading to the production of Kg amount of BMO enriched powders. Among the BMOs identified, 18 have high-molecular weight and corresponded in size to the most abundant oligosaccharides present in human milk. Notably 6 oligosaccharides contained fucose, a sugar monomer that is highly abundant in human milk, but is rarely observed in bovine milk. This work shows that dairy streams represent a potential source of complex milk oligosaccharides for commercial development of unique dairy ingredients in functional foods that reproduce the benefits of human milk.     

Structural characterization of neutral and acidic oligosaccharides in the milks of strepsirrhine primates: greater galago, aye-aye, Coquerel’s sifaka and mongoose lemur

The structures of milk oligosaccharides were characterized for four strepsirrhine primates to examine the extent to which they resemble milk oligosaccharides in other primates. Neutral and acidic oligosaccharides were isolated from milk of the greater galago (Galagidae: Otolemur crassicaudatus), aye-aye (Daubentoniidae: Daubentonia madagascariensis), Coquerel's sifaka (Indriidae: Propithecus coquereli) and mongoose lemur (Lemuridae: Eulemur mongoz), and their chemical structures were characterized by (1)H-NMR spectroscopy. The oligosaccharide patterns observed among strepsirrhines did not appear to correlate to phylogeny, sociality or pattern of infant care. Both type I and type II neutral oligosaccharides were found in the milk of the aye-aye, but type II predominate over type I. Only type II oligosaccharides were identified in other strepsirrhine milks. α3'-GL (isoglobotriose, Gal(α1-3)Gal(β1-4)Glc) was found in the milks of Coquerel's sifaka and mongoose lemur, which is the first report of this oligosaccharide in the milk of any primate species. 2'-FL (Fuc(α1-2)Gal(β1-4)Glc) was found in the milk of an aye-aye with an ill infant. Oligosaccharides containing the Lewis x epitope were found in aye-aye and mongoose lemur milk. Among acidic oligosaccharides, 3'-N-acetylneuraminyllactose (3'-SL-NAc, Neu5Ac(α2-3)Gal(β1-4)Glc) was found in all studied species, whereas 6'-N-acetylneuraminyllactose (6'-SL-NAc, Neu5Ac(α2-6)Gal(β1-4)Glc) was found in all species except greater galago. Greater galago milk also contained 3'-N-glycolylneuraminyllactose (3'-SL-NGc, Neu5Gc(α2-3)Gal(β1-4)Glc). The finding of a variety of neutral and acidic oligosaccharides in the milks of strepsirrhines, as previously reported for haplorhines, suggests that such constituents are ancient rather than derived features, and are as characteristic of primate lactation is the classic disaccharide, lactose.     

OMICS-rooted studies of milk proteins,oligosaccharides and lipids

Milk has co-evolved with mammals and mankind to nourish their offspring and is a biological fluid of unique complexity and richness. It contains all necessary nutrients for the growth and development of the newborn. Structure and function of biomolecules in milk such as the macronutrients (glyco-) proteins, lipids, and oligosaccharides are central topics in nutritional research. Omics disciplines such as proteomics, glycomics, glycoproteomics, and lipidomics enable comprehensive analysis of these biomolecule components in food science and industry. Mass spectrometry has largely expanded our knowledge on these milk bioactives as it enables identification, quantification and characterization of milk proteins, carbohydrates, and lipids. In this article, we describe the biological importance of milk macronutrients and review the application of proteomics, glycomics, glycoproteomics, and lipidomics to the analysis of milk. Proteomics is a central platform among the Omics tools that have more recently been adapted and applied to nutrition and health research in order to deliver biomarkers for health and comfort as well as to discover beneficial food bioactives.     

Engineering a colanic acid biosynthesis pathway in E. coli for manufacturing 2’-fucosyllactose

Human milk oligosaccharides (HMOs) are beneficial for infants’ health and growth. As one of the most abundant oligosaccharides in human milk, 2′-fucosyllactose (2′-FL) has been approved to supplement in infant formula. Microbial synthesis of 2′-FL achieved in E. coli tends to use a T7-expression system for the heterologous expression of the fucosyltransferase and/or enzymes involved in fucose metabolism. In this paper, we report a novel bioconversion route of 2′-FL by engineering a low pH triggered colanic acid (CA) synthetic pathway, found in E. coli S17−3, which supplies GDP-l-fucose for in vivo 2′-FL formation catalyzed by the heterologous α-1,2-fucosyltransferases. In medium added with 10 g/L lactose and 20 g/L glycerol, recombinant S17−3 was able to produce 0.617 g/L of 2′-FL. The concentration of 2′-FL came to 1.029 g/L when a heterologous pathway for the synthesis of polyhydroxybutyrate was additionally introduced in the engineered S17−3.     

Offline coupling of low-pressure anion-exchange chromatography with MALDI-MS to determine the elution order of human milk oligosaccharides

Pooled human milk oligosaccharides were separated into neutral and several acidic oligosaccharide fractions by preparative anion-exchange chromatography (AEC) using AG 1-X2. The oligosaccharides were eluted stepwise using deionized water and three different concentrations of ammonium acetate buffer, pH 6.8. The elution order of the compounds was determined directly by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of the AEC effluent without any cleanup or concentration steps. Up to a concentration of 500 mM ammonium acetate, the masses of acidic oligosaccharides could be detected by screening the fractions in an automated mode. The combination of the improved chromatographic procedure, the applied MALDI matrices, and operating parameters is suitable for the detection of neutral oligosaccharides as well as acidic oligosaccharides. The method provides high sensitivity and mass accuracy, including for the high-molecular-weight monosialylated oligosaccharides up to 2751.5 Da. The applied ionic strength of the anion-exchange eluents enables a rapid and an unambiguous composition assignment by MALDI-MS for neutral, monosialylated, and disialylated oligosaccharides from human milk. The acidic fractions have to be desalted by electrodialysis and were finally analyzed by HPAEC-PAD to get a high-resolution "fingerprint" of structures present in each fraction. From these analyses, it can be concluded that the isomeric variety of monosialylated oligosaccharides occurring in human milk is higher than estimated before.