Profiles of human milk oligosaccharides and production of some human milk oligosaccharides in transgenic animals

During the decade of the 1990s and the first years of the current century, our group embarked on a project to study and synthesize human milk oligosaccharides. This report describes 2 unexpected collateral observations from that endeavor. The first observation was the detection and confirmation of 2 rare neutral human milk oligosaccharides profiles that were uncovered while assessing oligosaccharide content in hundreds of samples of human milk. One of these lacked fucosylated structures altogether, and the other lacked the oligosaccharide 3-fucosyllactose [Galβ1-4(Fucα1-3)Glc]. We used glycoconjugate probes to determine whether the unusual profiles were mirrored by fucosylation of milk glycoproteins. The results show that the lack of fucosylated oligosaccharides in these samples corresponds to the absence of equivalent fucosylated motifs in milk glycoproteins. The second finding was a shortened and distinct lactation process in transgenic rabbits expressing the human fucosyltransferase 1. During the first day of lactation, these animals expressed milk that contained both lactose and 2'-fucosylactose, but on the second day, the production of milk was severely diminished, and by the fourth day, no lactose was detected in their milk. Meanwhile, the concentration of fucosylated glycoproteins increased from the onset of lactation through its premature termination. These 2 findings may shed light on the glycobiology of milk and perhaps on mammary gland differentiation.     

Lactose hydrolyzed milk.

Lactose intolerance is being reported in many populations. Yet, milk is highly nutritious and methods are being explored to use milk while limiting the lactose content. Thirty-two blacks 13-19 years of age were studied to determine a blood sugar rise with 8 ounces of the following test milks: 1) untreated whole milk (12 g/lactose); 2) 90% lactose hydrolyzed milk (1.2 g/lactose); and 3) 50% lactose hydrolyzed milk (6 g/lactose). In the 22 lactose malabsorbers, the peak blood sugars were: 1) untreated whole milk--4.4 mg/100 ml, 2) 90% lactose hydrolyzed milk--14.5 mg/100 ml, and 3) 50% lactose hydrolyzed milk--8.8 mg/100 ml. The 10 blacks with normal lactose absorption had a comparably high peak blood sugar on all three test milks. Differences between the blood sugar in the lactose absorbing and malabsorbing subjects when drinking untreated whole milk are significant (P less than 0.001); so are differences in the lactose malabsorbing subjects consuming untreated whole milk and 90% lactose hydrolyzed milk (P less than 0.001) as well as 50 and 90% lactose hydrolyzed milk. Symptoms were reported by three lactose malabsorbing subjects with untreated whole milk with two of the three symptomatic with 90% lactose hydrolyzed milk and none with 50% lactose hydrolyzed milk. No symptoms were reported by the lactose absorbers. Significant improvement in absorption with 90% lactose hydrolyzed milk is seen in low lactase subjects. Lactose hydrolyzed milk may serve as an important alternative for food planners wanting to provide milk to high risk populations with low lactase levels.     

Neutral oligosaccharide content of preterm human milk

Human milk oligosaccharides are known to play a role in protection against certain infectious diseases. Previous reports indicate that the content of human milk oligosaccharides varies widely among individuals at term but such information on preterm milk is lacking. After removal of the fat, protein and most of the lactose from non-pooled human milk samples, a total neutral oligosaccharide fraction was isolated by ion-exchange chromatography followed by gel filtration. A Dionex high-performance anion-exchange chromatography system equipped with a pulsed electrometric detector was then employed to measure the levels of ten neutral oligosaccharides in the individual milk samples. Twenty-three milk samples from thirteen mothers who delivered at a mean gestational age of 29.5 (SD 3.1) weeks were collected between days 0 and 33 of lactation, and compared with three samples of term milk from two mothers. The ranges of the total and individual levels of the ten neutral oligosaccharides in preterm milk were similar to those in term milk. Further, as previously described in term milk, preterm milk exhibited a quantitative individual variation. This variation was independent of the gestational age, day of lactation, and postconceptional age. In conclusion, levels of ten neutral oligosaccharides did not differ between preterm and term human milk.     

Human milk oligosaccharides affect P-selectin binding capacities: in vitro investigation

OBJECTIVE: In the initial phase of cellular immune response, selectins mediate the emigration of leukocytes from the blood stream into inflammatory regions. Human milk oligosaccharides (HMOs) possess binding epitopes of selectin ligands such as sialyl Lewis(x) and sialyl Lewis(a) and therefore might impair the interaction of selectins with cellular ligands. Neutral, acidic, sialylated, or fucosylated HMO fractions with polymerization degrees of 3 to 50 were investigated regarding this interaction in a dynamic flow chamber model that considers physiologic shear stress conditions. METHODS: Human milk oligosaccharides were compared with kappa-carrageenans and pectin oligosaccharides to deduce structure-activity relations. Fucoidan and sialyl Lewis(x) served as positive controls. RESULTS: All HMO fractions affected P-selectin ligand binding capacity but were not comparable to fucoidan. The activity of the acidic HMO fraction resembled sialyl Lewis(x) in decreasing the binding of the ligand to P-selectin. CONCLUSION: Human milk oligosaccharides modulate rather than block the function of P-selectin.     

Oligosaccharides and glycoconjugates in bovine milk and colostrum

Oligosaccharides and glycoconjugates are some of the most important bioactive components in milk. A great deal of information is available on the biological function of the components from human milk. Their primary role seems to be in providing protection against pathogens by acting as competitive inhibitors for the binding sites on the epithelial surfaces of the intestine. Evidence is also available to support the role of some of these components as growth promoters for genera of beneficial microflora in the colon. Compared with human milk, levels of oligosaccharides in bovine milk are very low. Nevertheless, a number of neutral and acidic oligosaccharides have been isolated from bovine milk and characterised. The highest concentration of these molecules is found in early postparturition milk (colostrum). The chemical structure of the oligosaccharides and many of the glycoconjugates from bovine milk are similar to those in human milk. It is likely that bovine oligosaccharides and glycoconjugates can be used in milk products as bioactive components in human nutrition.     

Absorption of fortification iron from milk formulas in infants

The bioavailability of iron added to different types of cows' milk formulas was studied using mono-isotopic and double-isotopic methods in 396 infants aged 5-18 mo. All the milk formulas were fortified with ferrous sulfate in concentrations varying between 10 and 19 mg elemental iron/liter. Iron absorption from low-fat milks and full-fat milks varied from 2.9 to 5.1%. A higher range of mean absorption, 5.9 to 11.3%, was observed in the same formulas with the addition of ascorbic acid at concentrations of 100 mg/l or higher (up to 800 mg/l), demonstrating its enhancing effect on iron absorption in fortified milks. The amount of milk fat, the addition of carbohydrates, or acidification did not seem to influence iron absorption.     

Structure-function relationships of human milk oligosaccharides

Human milk contains more than a hundred structurally distinct oligosaccharides. In this review, we provide examples of how the structural characteristics of these human milk oligosaccharides (HMO) determine functionality. Specific α1-2-fucosylated HMO have been shown to serve as antiadhesive antimicrobials to protect the breast-fed infant against infections with Campylobacter jejuni, one of the most common causes of bacterial diarrhea. In contrast, α1-2-fucosylation may abolish the beneficial effects of HMO against Entamoeba histolytica, a protozoan parasite that causes colitis, acute dysentery, or chronic diarrhea. In a different context, HMO need to be both fucosylated and sialylated to reduce selectin-mediated leukocyte rolling, adhesion, and activation, which may protect breast-fed infants from excessive immune responses. In addition, our most recent data show that a single HMO that carries not 1 but 2 sialic acids protects neonatal rats from necrotizing enterocolitis, one of the most common and often fatal intestinal disorders in preterm infants. Oligosaccharides currently added to infant formula are structurally different from the oligosaccharides naturally occurring in human milk. Thus, it appears unlikely that they can mimic some of the structure-specific effects of HMO. Recent advances in glycan synthesis and isolation have increased the availability of certain HMO tri- and tetrasaccharides for in vitro and in vivo preclinical studies. In the end, intervention studies are needed to confirm that the structure-specific effects observed at the laboratory bench translate into benefits for the human infant. Ultimately, breastfeeding remains the number one choice to nourish and nurture our infants.     

Human milk oligosaccharides are resistant to enzymatic hydrolysis in the upper gastrointestinal tract

BACKGROUND: Human milk oligosaccharides (HMOs) show a complexity and variety not found in milk of any other species. Although progress has been made in the past 3 decades with regard to identification and structural characterization of HMOs, not much is known about the physiologic functions of HMOs. OBJECTIVE: As a prerequisite for biological activity in infant metabolism, HMOs have to resist enzymatic hydrolysis in the gastrointestinal tract. To assess the extent to which selected HMOs are hydrolyzed, we carried out in vitro digestion studies using enzyme preparations of human and porcine pancreas and intestinal brush border membranes (BBMs). DESIGN: Fractions of HMOs, including structurally defined isolated oligosaccharides, were digested for up to 20 h with human pancreatic juice and BBMs prepared from human or porcine intestinal tissue samples. HMOs were incubated by using a porcine pancreatic homogenate and BBMs as enzyme sources. HMOs and digestion products were identified by mass spectrometry and anion-exchange chromatography. Additionally, free D-glucose, L-fucose, and N-acetylneuraminic acid were determined enzymatically. RESULTS: Whereas maltodextrin (control) was rapidly and completely hydrolyzed, neutral and acidic HMOs showed a profound resistance against pancreatic juice and BBM hydrolases. However, cleavage of most of the HMOs was achieved by using a pancreatic homogenate containing intracellular, including lysosomal, enzymes in addition to secreted enzymes. CONCLUSIONS: The results of this study strongly suggest that HMOs are not hydrolyzed by enzymes in the upper small intestine. Although intact HMOs may be absorbed, we postulate that a majority of HMOs reach the large intestine, where they serve as substrates for bacterial metabolism. Therefore, HMOs might be considered the soluble fiber fraction of human milk.     

N-acetylneuraminic acid concentrations in human milk oligosaccharides and glycoproteins during lactation

Human milk contains a variety of N-acetylneuraminic acid (NANA)-containing oligosaccharides, but the expected range of intake of sialic acid in this form by infants fed human milk is unknown. Two quite different amounts have been reported: 120 mg/liter in pooled, mature human milk (1) and 1400 mg/liter in the milk of a single woman on the 1st day of lactation (2). The normal range of NANA intake in human milk glycoproteins likewise does not appear to have been analyzed previously. Data presented here indicate that both human milk oligosaccharide and glycoprotein NANA decline exponentially over the first 2 months of lactation, decreasing little thereafter. During the first 2 months of lactation, milk from women delivering at term cannot be distinguished from that of women delivering significantly before term (less than 32 wks gestation) with regard to oligosaccharide and glycoprotein NANA. The parallel decrease of sialic acid in these fractions suggests a relationship between sialydation of human milk oligosaccharides and glycoproteins. Human milk NANA concentrations are discussed with regard to reports that exogenous administration of NANA can increase cerebral and cerebellar concentrations of NANA in glycoproteins and gangliosides, and produce long term changes in behavior in rats.     

Composition and enrichment of caprine milk oligosaccharides from New Zealand Saanen goat cheese whey

Goat milk contains oligosaccharides that are structurally similar to human milk, which suggests that caprine milk oligosaccharides (CMO) could mimic the beneficial physiological effects described for human milk oligosaccharides for infant health. This study aimed to characterise the nutrient composition of New Zealand Saanen goat colostrum, regular milk and whey samples and to develop an easily scalable approach to produce an enriched CMO product for use in in vivo experimentation. Goat milk whey was processed by a combination of ultrafiltration, enzymatic hydrolysis of the lactose, solid-phase extraction and rotary vacuum evaporation. An 80% recovery of the oligosaccharide fraction with an enrichment of 24-fold was obtained when compared to the starting whey. Lactose was reduced to 2.5% of its initial concentration by enzymatic treatment. From 8 batches (approximately 1200 mL per batch) of whey, 19 g of product were generated of which around 8% were oligosaccharides, 44% monosaccharides, 44% lactose and 4% galacto-oligosaccharides.     

Detection of ligands for selectins in the oligosaccharide fraction of human milk

Summary Background Human milk contains a large variety of oligosaccharides which show structural similarities with ligands for selectins, a family of cell adhesion molecules which are involved in many cell-cell interactions. Aim of the study Due to their structural similarity with selectin ligands, human milk oligosaccharides were labelled with phosphatidyl ethanolamine to be able to investigate specific effects of antibodies against carbohydrate epitopes. Methods Various monoclonal antibodies against physiological selectin ligands were used to determine whether epitopes within human milk oligosaccharides are recognized. Oligosaccharides were isolated from human milk, transferred into neoglycolipids and separated using high performance thin layer chromatography prior to incubation with monoclonal antibodies for the selectin ligands sialyl-Lewis a (sLe a), sialyl-Lewis x (sLe x), Lewis x (Le x) and Lewis y (Le y) after Western blotting. Fast atom bombardment-mass spectrometry was then used to identify antibody-binding compounds. Results In the immunoassays all ligand epitopes except for Le y were detected in the oligosaccharide fraction of human milk. Anti-sLe a showed the most distinct reaction with N-acetylneuraminic acid containing neoglycolipids of which two were identified as neuraminyl-fucosyl-lacto-N-hexaose and neuraminyl-lacto-N-tetraose. Such oligosaccharides as well as similar structures are present in relatively high concentrations in human milk. Conclusions The presence of sialyl-Lewis ligands on milk oligosaccharides together with their abundancy in human milk may suggest that they could be selectin ligands and they may be part of inflammatory processes. Received: 2 September 2001, Accepted: 28 February 2002     

Oligosaccharides from human milk block binding and activity of the Escherichia coli heat-stable enterotoxin (STa) in T84 intestinal cells

Enterotoxin-producing Escherichia coli are major causes of pediatric diarrhea in developing countries. The heat-stable enterotoxin of Escherichia coli (STa) causes diarrhea by virtue of its ability to bind to and stimulate intestinal membrane-bound guanylate cyclase, generating cyclic GMP (cGMP). Previous work showed that a fucosylated oligosaccharide fraction of human milk was able to protect suckling mice from the secretory effects of STa, but the mechanism of the protection could not be determined. Oligosaccharide fractions from human milk were tested for their ability to block the biochemical effects of STa in T84 cells, a human colon carcinoma line responsive to the toxin. Total and fucosylated oligosaccharide fractions were found to inhibit STa-stimulated guanylate cyclase activity in T84 cell membranes and cGMP production in intact T84 cells by 60-80%. In addition, the total oligosaccharide fraction and the fucosylated oligosaccharide fraction inhibited 125I-STa binding significantly (17% and 27% inhibition, respectively). These findings demonstrate the protective activity of human milk oligosaccharides against STa in a human-derived cell line and show that the biochemical step blocked by oligosaccharides is STa-mediated stimulation of guanylate cyclase. This represents a novel mechanism by which human milk oligosaccharides protect against diarrhea.     

Molecular mechanism underlying sialic acid as an essential nutrient for brain development and cognition

The early stages of neurodevelopment in infants are crucial for establishing neural structures and synaptic connections that influence brain biochemistry well into adulthood. This postnatal period of rapid neural growth is of critical importance for cell migration, neurite outgrowth, synaptic plasticity, and axon fasciculation. These processes thus place an unusually high demand on the intracellular pool of nutrients and biochemical precursors. Sialic acid (Sia), a family of 9-carbon sugar acids, occurs in large amounts in human milk oligosaccharides and is an essential component of brain gangliosides and sialylated glycoproteins, particularly as precursors for the synthesis of the polysialic acid (polySia) glycan that post-translationally modify the cell membrane-associated neural cell adhesion molecules (NCAM). Human milk is noteworthy in containing exceptionally high levels of Sia-glycoconjugates. The predominate form of Sia in human milk is N-acetylneuraminic acid (Neu5Ac). Infant formula, however, contains low levels of Sia consisting of both Neu5Ac and N-glycolyneuraminic acid (Neu5Gc). Current studies implicate Neu5Gc in several human inflammatory diseases. Polysialylated NCAM and neural gangliosides both play critical roles in mediating cell-to-cell interactions important for neuronal outgrowth, synaptic connectivity, and memory formation. A diet rich in Sia also increases the level of Sia in the brains of postnatal piglets, the expression level of 2 learning-related genes, and enhances learning and memory.     

Relative effectiveness of milks with reduced amounts of lactose in alleviating milk intolerance

The relative effectiveness of five milk products with various levels of lactose reduction [0%, 50%, 80% (#1), 80% (#2), and 95%] was evaluated in six subjects with lactose malabsorption. Breath hydrogen was measured for 4 h after consumption of 300 mL of each product in a single-blind, randomized design. The mean +/- SEM maximum breath-hydrogen rise (ppm) after the 0%, 50%, 80% (#1), 80% (#2), and 95% lactose-reduced (LR) milks was 31 +/- 6, 7 +/- 3, 5 +/- 3, 5 +/- 2, and 8 +/- 3, respectively. The difference between whole milk and the LR milks was statistically significant (P less than 0.05) but there was no difference between any of the LR milks. Whole milk provoked symptoms in most subjects whereas 95% LR milk produced none. Only one of six subjects reacted to the 50% and 80% LR milks. The results suggest that a 50% level of lactose reduction in milk may be adequate to relieve the signs and symptoms of milk intolerance in the majority of healthy adults with lactose malabsorption.     

The sialylated fraction of milk oligosaccharides is partially responsible for binding to enterotoxigenic and uropathogenic Escherichia coli human strains

Milk oligosaccharides can act as soluble receptors that block bacterial adhesion to the different epithelia. Colonization factor antigens (CFA)/I- and CFA/II-expressing enterotoxigenic Escherichia coli (ETEC) strains constitute one of the main causes of diarrhea in infants. Here, the inhibition of hemagglutination mediated by these strains by milk oligosaccharides was tested. Human milk oligosaccharides showed a strong inhibitory capacity, which decreased when the oligosaccharides were desialylated. Because milk oligosaccharides also are present in the urine of neonates receiving mothers' milk, their ability to bind two uropathogenic Escherichia coli (UPEC) strains was also examined. UPEC strains expressing P (Pap) and P-like (Prs) fimbriae are responsible for infections of the urinary tract such as pyelonephritis and cystitis. The hemagglutination mediated by these strains was inhibited by human milk oligosaccharides. The sialylated fraction was partially responsible for this inhibition in the case of the UPEC expressing the P-like fimbria because differences were found after desialylation. Although bovine milk oligosaccharides were less efficient at inhibiting the hemagglutination of ETEC strains, they were still quite good inhibitors of UPEC strains.     

Human milk oligosaccharides are minimally digested in vitro

In examining the functional aspects of human milk oligosaccharides (HMO), it is not known whether they are digested during the passage through the infant's gastrointestinal tract. HMO were prepared from individual milk samples (n = 6) and separated into neutral and acidic compounds by chromatography. These oligosaccharide fractions were studied for their digestibility by human salivary amylase, porcine pancreatic amylase and brush border membrane vesicles (BBMV) isolated from porcine small intestine; we also examined the effect of low pH on these structures. The characterization of HMO and their digestion products was performed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as well as TLC. It was shown that neither salivary amylase nor pancreatic amylase cleaved HMO. Only after a 2-h incubation with BBMV were slight modifications of the HMO observed. HPAEC-PAD analysis revealed two new components within the neutral oligosaccharide fractions; these were characterized by mass spectrometric analysis as lacto-N:-triose and galactose. Only lacto-N:-triose was present within digestion assays of oligosaccharides, which did not contain fucosyl or N:-acetylneuraminic acid residues. These results suggest that <5% of the HMO are digested in the intestinal tract. Hence, HMO may play a role as prebiotics or as factors influencing the local immune system of the intestine in breast-fed infants.     

The role of fermented milk in complementary feeding of young children: lessons from transition countries

Probiotic bacteria are used for production of fermented dairy products. The use of probiotic bacteria has the potential to replenish the natural intestinal flora of the body. These bacteria competitively inhibit the growth and colonization of pathogenic bacteria. Breastmilk is the best food for babies, also from a probiotic point of view. Human milk, in fact, contains many substances that stimulate the growth of bifidobacteria in vitro and in the small intestine of infants. Improvement of lactose digestion and avoidance of symptoms of intolerance in lactose malabsorbers are the most profoundly studied health-relevant effects of fermented milk. In fact fermented milks are nutritionally similar to unfermented milk, except that some of lactose is broken down to glucose and galactose. The role of fermented milk in complementary feeding and in particular for the prevention of anaemia is an innovative theme, recently focused. Iron deficiency in infants and young children is widespread and has serious consequences for child health. Prevention of iron deficiency should therefore be given high priority. The too-early introduction of unmodified cow's milk and milk products is an important nutritional risk factors for the development of iron-deficiency anaemia. Fermented milks represent an excellent source of nutrients such as calcium, protein, phosphorus and riboflavin. During the fermentation of milk, lactic acid and other organic acids are produced and these increase the absorption of iron. If fermented milk is consumed at mealtimes, these acids are likely to have a positive effect on the absorption of iron from other foods.     

Tolerance of symptomatic lactose malabsorbers to lactose in milk chocolate

OBJECTIVE: To study tolerance to lactose in milk chocolate among symptomatic lactose maldigesters. DESIGN: Randomized cross-over study. SUBJECTS: Twenty-seven adult lactose maldigesters with symptomatic lactose intolerance. METHODS: A 100 g chocolate sample prepared with whole milk (12 g lactose), whole-milk powder (12 g lactose), low-lactose milk powder (2 g lactose) or lactose-free milk powder was eaten after an overnight fast. Gastrointestinal symptoms (flatulence, abdominal bloating, abdominal pain, borgorygmi and nausea) were recorded in a questionnaire during the following 8 h. Bowel movements and stool consistency were also registered during the test day. RESULTS: The numbers of persons reporting different gastrointestinal symptoms or any of the symptoms did not differ significantly after eating the chocolate samples. No statistical differences were found in the estimated strength of the different symptoms or the total strength of all symptoms combined. Differences in the bowel frequency and stool consistency were also non-significant. CONCLUSIONS: Lactose malabsorbers with self-reported lactose intolerence did not differ in their response to milk chocolate samples containing different amounts of lactose.     

Exposure to 3′Sialyllactose-Poor Milk during Lactation Impairs Cognitive Capabilities in Adulthood

Breast milk exerts pivotal regulatory functions early in development whereby it contributes to the maturation of brain and associated cognitive functions. However, the specific components of maternal milk mediating this process have remained elusive. Sialylated human milk oligosaccharides (HMOs) represent likely candidates since they constitute the principal neonatal dietary source of sialic acid, which is crucial for brain development and neuronal patterning. We hypothesize that the selective neonatal lactational deprivation of a specific sialylated HMOs, sialyl(alpha2,3)lactose (3′SL), may impair cognitive capabilities (attention, cognitive flexibility, and memory) in adulthood in a preclinical model. To operationalize this hypothesis, we cross-fostered wild-type (WT) mouse pups to B6.129-St3gal4^tm1.1Jxm/J dams, knock-out (KO) for the gene synthesizing 3′SL, thereby providing milk with approximately 80% 3′SL content reduction. We thus exposed lactating WT pups to a selective reduction of 3′SL and investigated multiple cognitive domains (including memory and attention) in adulthood. Furthermore, to account for the underlying electrophysiological correlates, we investigated hippocampal long-term potentiation (LTP). Neonatal access to 3′SL-poor milk resulted in decreased attention, spatial and working memory, and altered LTP compared to the control group. These results support the hypothesis that early-life dietary sialylated HMOs exert a long-lasting role in the development of cognitive functions.     

Quantification of non-protein nitrogen components of infant formulae and follow-up milks: comparison with cows' and human milk

The composition of fourteen infant formulae and six follow-up milks with regard to their free amino acids (including taurine), free nucleotides, orotic acid, and free and total l-carnitine content was studied. The levels found were compared with the limits established in European legislation and with the composition of human and cows' milk samples. HPLC methodologies, optimized and validated for the matrices under study, were used, except for free and total l-carnitine contents that were quantified using a flow-injection manifold, also optimized and validated for the matrices under study. Global statistical treatment of the results by cluster analysis indicated similarities between the contents of the N compounds under study of infant formulae, follow-up milks and cows' milk and differences with regard to human milk composition. The principal component analysis showed that 60.2 % of the variation in data was due to the first principal component, and the second component represented 23.8 % of the total information. Nucleotide profiles, orotic acid, and free and total l-carnitine contents explain the main differences observed between human milk and the other milks studied (cows' milk, infant formulae and follow-up milks). Cows' milk is distinguished from infant formulae and follow-up milks mainly owing to the different uric acid contents and free amino acids profiles.