A strategy for annotating the human milk glycome

Oligosaccharides in human milk represent a group of bioactive molecules that have evolved to be an abundant and diverse component of human milk, even though they have no direct nutritive value to the infant. A recent hypothesis proposes that they could be substrates for the development of the intestinal microflora and the mucosal immune system. The inability to determine the exact composition of these oligosaccharides limits research and the ability to understand their biological functions. Oligosaccharides isolated from the lipids and proteins of individual human milk samples were analyzed by a combination of techniques including microchip liquid chromatography mass spectrometry (HPLC-Chip/MS) and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT ICR MS). Accurate mass measurements obtained using an orthogonal time-of-flight (o-TOF) mass spectrometry provided oligosaccharide composition for approximately 200 individual molecular species. Comparison of HPLC-Chip/MS profiles from five different women revealed variations in milk oligosaccharide compositions. HPLC-Chip/MS profiling provides a method for routinely identifying milk oligosaccharides. Tandem MS in combination with exoglycosidase digestion provides unambiguous differentiation of structural isomers.     

Natural variability in bovine milk oligosaccharides from Danish Jersey and Holstein-Friesian breeds

Free oligosaccharides are key components of human milk and play multiple roles in the health of the neonate, by stimulating growth of selected beneficial bacteria in the gut, participating in development of the brain, and exerting antipathogenic activity. However, the concentration of oligosaccharides is low in mature bovine milk, normally used for infant formula, compared with both human colostrum and mature human milk. Characterization of bovine milk oligosaccharides in different breeds is crucial for the identification of viable sources for oligosaccharide purification. An improved source of oligosaccharides can lead to infant formula with improved oligosaccharide functionality. In the present study we have analyzed milk oligosaccharides by high-performance liquid chromatography chip quadrupole time-of-flight mass spectrometry and performed a detailed data analysis using both univariate and multivariate methods. Both statistical tools revealed several differences in oligosaccharide profiles between milk samples from the two Danish breeds, Jersey and Holstein-Friesians. Jersey milk contained higher relative amounts of both sialylated and the more complex neutral fucosylated oligosaccharides, while the Holstein-Friesian milk had higher abundance of smaller and simpler neutral oligosaccharides. The statistical analyses revealed that Jersey milk contains levels of fucosylated oligosaccharides significantly higher than that of Holstein-Friesian milk. Jersey milk also possesses oligosaccharides with a higher degree of complexity and functional residues (fucose and sialic acid), suggesting it may therefore offer advantages in term of a wider array of bioactivities.     

Quantification of bovine milk oligosaccharides using liquid chromatography-selected reaction monitoring-mass spectrometry

Oligosaccharides are important components of milk with bioefficacy as prebiotics, anti-infectives, and immune system modulators and as a possible source of sialic acid for neural function. Bovine milk oligosaccharides are lower in concentration and lack the diversity of human milk oligosaccharides but could be a commercial source of milk oligosaccharides for pediatric foods. For this development, an ability to quantify the oligosaccharides is required. This study validated a hydrophilic interaction chromatography high-performance liquid chromatography-high-resolution selected reaction monitoring-mass spectrometry (HILIC HPLC-HRSRM-MS) method for measuring six different oligosaccharides in bovine milk, bovine colostrum, and infant formulas. The extraction resulted in a high recovery (90-103%) with a repeatability coefficient of variation ranging from 2 to 9% for the two dominant oligosaccharides, 3'-sialyllactose and 6'-sialyllactose, and ranging from 1 to 17% for the much lower concentration oligosaccharides, 6'-sialyllactosamine, disialyllactose, and N-acetylgalactosaminyllactose. The sixth oligosaccharide, 3'-sialyllactosamine, was not detected in any of the samples.     

Daily variations in oligosaccharides of human milk determined by microfluidic chips and mass spectrometry

Human milk is a complex biological fluid that provides not only primary nourishment for infants but also protection against pathogens and influences their metabolic, immunologic, and even cognitive development. The presence of oligosaccharides in remarkable abundance in human milk has been associated to provide diverse biological functions including directing the development of an infant's intestinal microflora and immune system. Recent advances in analytical tools offer invaluable insights in understanding the specific functions and health benefits these biomolecules impart to infants. Oligosaccharides in human milk samples obtained from five different individual donors over the course of a 3 month lactation period were isolated and analyzed using HPLC-Chip/TOF-MS technology. The levels and compositions of oligosaccharides in human milk were investigated from five individual donors. Comparison of HPLC-Chip/TOF-MS oligosaccharides profiles revealed heterogeneity among multiple individuals with no significant variations at different stages of lactation within individual donors.     

Structural profiling and quantification of sphingomyelin in human breast milk by HPLC-MS/MS

The sphingolipid composition of food as well as of physiological samples has received considerable interest due to their positive biological activities. This study quantified the total amount of sphingomyelin (SM) in 20 human breast milk samples from healthy volunteers and determined the structures of SM by detailed mass spectrometric studies in combination with enzymatic cleavage. The quantification of SM was performed by hydrophilic interaction liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (HILIC-HPLC-ESI-MS/MS) measuring the characteristic fragment ion of the phosphorylcholine group at m/z 184.2 and by using hexanoylsphingomyelin (C6-SM) and heptadecanoylsphingomyelin (C17-SM) as internal standards. The structures of SM species were identified after enzymatic cleavage with alkaline sphingomyelinase (SMase) to the corresponding ceramides. Structure elucidation of the sphingoid base and fatty acid backbone was performed by reversed-phase HPLC-ESI-MS/MS. The method includes the sphingoid bases dihydrosphingosine (d18:0), sphingosine (d18:1(Δ4)), 4,8-sphingadienine (d18:2(Δ4,8)), 4-hydroxysphinganine (phytosphingosine (t18:0)), and 4-hydroxy-8-sphingenine (t18:1(Δ8)) and fatty acids with even-numbered carbon atoms (C12-C26) as well as their (poly)unsaturated and monohydroxylated analogues. The total amount of SM in human breast milk varied from 3.87 to 9.07 mg/100 g fresh weight. Sphingosine (d18:1) was the predominant sphingoid base, with 83.6 ± 3.5% in human breast milk, followed by 4,8-sphingadienine (d18:2) (7.2 ± 1.9%) and 4-hydroxysphinganine (t18:0) (5.7 ± 0.7%). The main SM species contained sphingosine and palmitic acid (14.9 ± 2.2%), stearic acid (12.7 ± 1.5%), docosanoic acid (16.2 ± 3.6%), and tetracosenoic acid (15.0 ± 3.1%). Interestingly, the fatty acid composition of SM species in this study differs from the total fatty acids in human breast milk, and the fatty acids are not consistently distributed among the different sphingoid bases.     

Bioactivity profiling with parallel mass spectrometry reveals an assemblage of green tea metabolites affording protection against human huntingtin and alpha-synuclein toxicity

Aberrant protein aggregation and misfolding are key pathological features of many neurodegenerative disorders, including Huntington's and Parkinson's diseases. Compounds that offer protection from toxicity associated with aggregation-prone neurodegenerative proteins may have applications for the treatment of a multitude of disorders. A high-throughput bioassay system with parallel electrospray ionization mass spectrometry screening has been designed for critical evaluation of milligram quantities of natural product extracts, including dietary substances, for compounds of pharmacological relevance to the treatment of human neurodegenerative diseases. Using Saccharomyces cerevisiae strains engineered to express mutant human huntingtin and alpha-synuclein, we are able to identify extracts and compounds that protect cells from toxicity associated with these proteins. Applying this screening paradigm, we determined that a bioactive green tea extract contains an assemblage of catechins that were individually characterized for their respective protective effects against huntingtin and alpha-synuclein toxicity.     

Gas chromatographic determination of N-nitrosamines, aromatic amines, and melamine in milk and dairy products using an automatic solid-phase extraction system

A reliable analytical method was presented for the simultaneous determination of six N-nitrosamines, nine aromatic amines, and melamine in milk and dairy products using gas chromatography coupled with mass spectrometry. The sample treatment includes the precipitation of proteins with acetonitrile, centrifugation, solvent changeover by evaporation, and continuous solid-phase extraction for cleanup and preconcentration purposes. Samples (5 g) containing 0.15-500 ng of each amine were analyzed, and low detection limits (15-130 ng/kg) were achieved. Recoveries for milk and dairy products samples spiked with 1, 10, and 50 μg/kg ranged from 92% to 101%, with intraday and interday relative standard deviation values below 7.5%. The method was successfully applied to determine amine residues in several milk types (human breast, cow, and goat) and dairy products.     

Identification and characterization of complex bioactive oligosaccharides in white and red wine by a combination of mass spectrometry and gas chromatography

Over forty-five complex free oligosaccharides (of which several are novel) have been isolated and chemically characterized by gas chromatography and high resolution and high mass accuracy matrix-assisted laser desorption/ionization mass spectrometry (MALDI-FTICR MS) in red and white wines, Grignolino and Chardonnay, respectively. Oligosaccharides with a degree of polymerization between 3 and 14 were separated from simple monosaccharides and disaccharides by solid-phase extraction. The concentrations of free oligosaccharides were over 100 mg/L in both red and white wines. The free oligosaccharides-characterized for the first time in the present study-include hexose-oligosaccharides, xyloglucans, and arabinogalactans and may be the natural byproduct of the degradation of cell wall polysaccharides. The coupled gas chromatography and accurate mass spectrometry approach revealed an effective method to characterize and quantify complex functional oligosaccharides in both red and white wine.     

Simultaneous determination of 20 pharmacologically active substances in cow's milk, goat's milk, and human breast milk by gas chromatography-mass spectrometry

This paper reports a systematic approach to the development of a method that combines continuous solid-phase extraction and gas chromatography-mass spectrometry for the simultaneous determination of 20 pharmacologically active substances including antibacterials (chloramphenicol, florfenicol, pyrimethamine, thiamphenicol), nonsteroideal anti-inflammatories (diclofenac, flunixin, ibuprofen, ketoprofen, naproxen, mefenamic acid, niflumic acid, phenylbutazone), antiseptic (triclosan), antiepileptic (carbamazepine), lipid regulator (clofibric acid), β-blockers (metoprolol, propranolol), and hormones (17α-ethinylestradiol, estrone, 17β-estradiol) in milk samples. The sample preparation procedure involves deproteination of the milk, followed by sample enrichment and cleanup by continuous solid-phase extraction. The proposed method provides a linear response over the range of 0.6-5000 ng/kg and features limits of detection from 0.2 to 1.2 ng/kg depending on the particular analyte. The method was successfully applied to the determination of pharmacologically active substance residues in food samples including whole, raw, half-skim, skim, and powdered milk from different sources (cow, goat, and human breast).     

Concentrations of Nepsilon-carboxymethyllysine in human breast milk, infant formulas, and urine of infants

Maillard products, such as Nepsilon-carboxymethyllysine (CML), are readily formed during the manufacturing of infant formulas. Little has been known, however, about the presence of CML in human breast milk and about the uptake of CML by infants. In this study, CML was measured in the serum and breast milk of 32 healthy mothers by ELISA. CML concentrations in breast milk (137 +/- 82.7 ng/mL) were significantly lower than in the serum (399 +/- 67.8 ng/mL, p < 0.001) and on average 35-fold lower than in infant formulas (4754 +/- 4299.5 ng/mL). CML was also measured in the urine of 21 infants, which were fed with breast milk or formulas. Although there was a tendency toward higher urinary CML excretion in infants fed with hypoallergenic formulas compared to breast-fed ones, the differences were not significant. Neonates that were delivered by vaginal birth had significantly higher concentrations of CML compared to those delivered by caesarean section (1306 +/- 653 vs 601 +/- 220 ng/mL, p = 0.012). It is concluded that CML passes from the serum into the breast milk, but the levels are by far lower than in infant formulas. In very young neonates (< or =3 days), the mode of delivery has a greater influence on urinary CML excretion than the nutrition.     

Influence of glycosidic linkages and molecular weight on the fermentation of maltose-based oligosaccharides by human gut bacteria

A structure-function study was carried out to increase knowledge of how glycosidic linkages and molecular weights of carbohydrates contribute toward the selectivity of fermentation by gut bacteria. Oligosaccharides with maltose as the common carbohydrate source were used. Potentially prebiotic alternansucrase and dextransucrase maltose acceptor products were synthesized and separated into different molecular weights using a Bio-gel P2 column. These fractions were characterized by matrix-assisted laser desorption/ionization time-of-flight. Nonprebiotic maltooligosaccharides with degrees of polymerization (DP) from three to seven were commercially obtained for comparison. Growth selectivity of fecal bacteria on these oligosaccharides was studied using an anaerobic in vitro fermentation method. In general, carbohydrates of DP3 showed the highest selectivity towards bifidobacteria; however, oligosaccharides with a higher molecular weight (DP6-DP7) also resulted in a selective fermentation. Oligosaccharides with DPs above seven did not promote the growth of "beneficial" bacteria. The knowledge of how specific structures modify the gut microflora could help to find new prebiotic oligosaccharides.     

In vitro fermentation of alternansucrase raffinose-derived oligosaccharides by human gut bacteria

In this work, in vitro fermentation of alternansucrase raffinose-derived oligosaccharides, previously fractionated according to their degree of polymerization (DP; from DP4 to DP10), was carried out using small-scale pH-controlled batch cultures at 37 °C under anaerobic conditions with human feces. Bifidogenic activity of oligosaccharides with DP4-6 similar to that of lactulose was observed; however, in general, a significant growth of lactic acid bacteria Bacteroides , Atopobium cluster, and Clostridium histolyticum group was not shown during incubation. Acetic acid was the main short chain fatty acid (SCFA) produced during the fermentation process; the highest levels of this acid were shown by alternansucrase raffinose acceptor pentasaccharides at 10 h (63.11 mM) and heptasaccharides at 24 h (54.71 mM). No significant differences between the gas volume produced by the mixture of raffinose-based oligosaccharides (DP5-DP10) and inulin after 24 h of incubation were detected, whereas lower gas volume was generated by DP4 oligosaccharides. These findings indicate that novel raffinose-derived oligosaccharides (DP4-DP10) could be a new source of prebiotic carbohydrates.     

Enzymatic production and characterization of konjac glucomannan oligosaccharides

Enzymes from a balanced human gut flora are promising tools to design prebiotic oligosaccharides. In this study, we investigated the action of enzymes from fecal bacteria on the complex polysaccharide konjac glucomannan (KGM). The oligosaccharides produced were compared to oligosaccharides from KGM digests with fungal endo-β-(1,4)-glucanase (EG) or endo-β-(1,4)-mannanase (EM). For this purpose, the oligosaccharides from the different digests were first studied for their structural characteristics like monosugar composition and exo-enzymatic degradability, as monitored by capillary electrophoresis with laser-induced fluorescence detection. Whereas the oligosaccharides produced by EG and EM were characteristic for the selectivity of the respective enzyme in cleaving the mannose-/glucose-sugar linkages of KGM, oligosaccharides produced by the fecal enzymes did not point to a sugar-selective degradation. The oligosaccharide fragments from the different digests indicated the KGM polysaccharide to be composed of a backbone composed of short mannose and glucose sequences, to which branches rich in mannose are attached.     

Mass spectral analyses of corn stover prehydrolysates to assess conditioning processes

Flow injection electrospray (FIE) and LC-tandem mass spectrometry techniques were used to characterize corn stover acid hydrolysates before and after overliming and ammonia conditioning steps. Analyses were performed on samples without fractionation (dilution only) in an effort provide an inventory of ionizable substances. Statistical evaluation of the results indicates that the ammonia-treated and crude hydrolysates were more similar to one another than any other pairing, with conditioning leading to a decrease in malate levels. LC-tandem mass spectrometry studies were also developed to characterize the oligosaccharides present in each hydrolysate utilizing a hydrophilic interaction chromatographic separation method. Neutral and acidic pentose-based oligosaccharides (xylodextrins) with degrees of polymerization between 2 and 5 were quantified with 4-O-methyl glucuronic acid-containing dimer and trimers predominating. Conditioning had little effect on the quantified oligosaccharide pool.     

Simple and fast method for recognition of reducing and nonreducing neutral carbohydrates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Negative-ion mode matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) was used for the characterization of storage, neutral oligosaccharides extracted from Jerusalem artichoke, red onion, and wheat. The oligosaccharides from the real samples were analyzed with 2,4,6-trihydroxyacetophenone as the most convenient matrix that was selected in advance with the standard carbohydrate samples (inulin and maltooligosaccharides). The oligosaccharides from Jerusalem artichoke and red onion (similarly as inulin) produced [M - H](-) peaks as the main distribution, which reflects their nonreducing composition. On the contrary, the cross-ring fragmentations [M - H - 120](-) formed the main distribution in the mass spectra of hydrolyzed wheat starch similarly to reducing maltooligosaccharides and dextrans. The negative-ion mode MALDI-TOF MS is capable of recognizing reducing and nonreducing oligosaccharides. Such a simple differentiation of malto or inulin type of oligosaccharides is not possible in the positive-ion mode.     

Characterization of galactomannan derivatives in roasted coffee beverages

In this work, the galactomannans from roasted coffee infusions were purified by 50% ethanol precipitation, anion exchange chromatography, and phenylboronic acid-immobilized Sepharose chromatography. Specific enzymatic hydrolysis of the beta-(1-->4)-D-mannan backbone allowed us to conclude that the galactomannans of roasted coffee infusions are high molecular weight supports of low molecular weight brown compounds. Also, the molecular weight of the brown compounds linked to the galactomannan increases with the increase of the coffee degree of roast. The reaction pathways of galactomannans during the coffee roasting process were inferred from the detection of specific chemical markers by gas chromatography-electron impact mass spectrometry and/or electrospray ionization tandem mass spectrometry. Maillard reaction, caramelization, isomerization, oxidation, and decarboxylation pathways were identified by detection of Amadori compounds, 1,6-beta-anhydromannose, fructose, glucose, mannonic acid, 2-ketogluconic acid, and arabinonic acid in the reducing end of the obtained oligosaccharides. The implication of the several competitive reaction pathways is discussed and related to the structural changes of the galactomannans present in the roasted coffee infusions.     

Oligosaccharide and peptidoglycan of Ganoderma lucidum activate the immune response in human mononuclear cells

The acid-hydrolyzed fragments of Ganoderma lucidum polysaccharides (GLPS) obtained by Smith degradation were separated by size-exclusion chromatography into two major water-soluble fractions: peptidoglycans (GLPS-SF1) and oligosaccharides (GLPS-SF2). Both fractions induced CD69 in human peripheral blood mononuclear cells (hPB-MNCs), and they displayed distinct immunomodulating properties. GLPS-SF1, with a molecular weight of around 20 kDa, were heterogeneous peptidoglycans composed of glucose/mannose (4:1) that exhibited biological activities with Th1 cytokines IL-12, IL-2, TNF-α, and IFN-γ in hPB-MNCs and stimulated macrophage cytokine expression via Toll-like receptor 4 (TLR4) signaling. For GLPS-SF2, with a molecular weight of around several kilodaltons, its sugar sequence was elucidated by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy as [-α-1,4-Glc-(β-1,4-GlcA)(3)-](n). This oligosaccharide displayed specific immune property with low monocyte induction, greatly stimulated cell activation and proliferation of NK and T cells. This oligosaccharide isolated from G. lucidum polysaccharides with internal glucuronic acids/glucose repeat unit in a 3:1 ratio may be responsible for the active stimulation of NK and T cells.     

Whole Grains and Digestive Health

Whole grains contain all parts of the grain: the endosperm, germ, and bran. Whole grains are rich in fermentable carbohydrates that reach the gut: dietary fiber, resistant starch, and oligosaccharides. Most research that supports the importance of grains to gut health was conducted with isolated fiber fractions, rather than whole grains. Whole grains are an important source of dietary fiber and grain fibers such as wheat, oats, barley, and rye increase stool weight, speed intestinal transit, get fermented to short chain fatty acids, and modify the gut microflora. Wheat bran is particularly effective in increasing stool weight; wheat bran increases stool weight by a ratio of 5:1. In contrast, many novel fibers that are easily incorporated into beverages and foods increase stool weight only on a ratio of 1:1. In vitro fermentation studies with whole grains have been published. Carbohydrates of oat bran (rich in β‐glucan) were consumed by bacteria faster than those of rye and wheat brans (rich in arabinoxylan). Grain fibers were fermented more slowly than inulin, causing less gas production. Wheat is particularly high in fructo‐oligosaccharides, while wheat germ is high in raffinose oligosaccharides. Some in vivo studies show the prebiotic potential of whole grains. Whole grain breakfast cereal was more effective than wheat bran breakfast cereal as a prebiotic, increasing fecal bifidobacteria and lactobacilli in human subjects. Wheat bran consumption increased stool frequency. Thus, the gut enhancing effects of cereal fibers are well known. Limited data exist that whole grains alter gut health.     

Shelf-life prediction of processed milk by solid-phase microextraction, mass spectrometry, and multivariate analysis

A technique based on solid-phase microextraction, mass spectrometry, and multivariate analysis (SPME-MS-MVA) was used to predict the shelf life of pasteurized and homogenized reduced-fat milk and whole-fat chocolate milk sampled over a 7 month period. Using SPME-MS-MVA, which is essentially a mass spectrometry-based electronic-nose instrument, volatile bacterial metabolites were extracted from milk with SPME (Carboxen-PDMS) and injected into a GC capillary column at elevated temperature. Mass fragmentation profiles from the unresolved milk volatile components were normalized to the intensity of a chlorobenzene internal standard mass peak (m/z 112) and subjected to MVA. Prediction models based on partial least-squares regression of mass intensity lists were able to predict the shelf life of samples to approximately +/-1 day, with correlation coefficients greater than 0.98 for the two types of milk samples. Using principal component analysis techniques, the procedure was also useful for classifying samples that were rendered unpalatable by nonmicrobial sources (contamination by copper and sanitizer) as well as by bacteria.     

Identification and site-specific relative quantification of beta-lactoglobulin modifications in heated milk and dairy products

During milk processing, proteins can be severely modified by oxidation, condensation, and Maillard reaction, leading to changes in their nutritional and technological properties. In this study, major modifications of beta-lactoglobulin, formed during the heating and processing of milk, were screened by mass spectrometry. For this purpose, beta-lactoglobulin was isolated from the milk samples by gel electrophoresis and analyzed by matrix-assisted laser desorption/ionization mass spectrometry after in-gel digestion with endoproteinase AspN. In heated milk, lactulosyllysine was detected at lysine 47 and 138 or 141 as well as methionine sulfoxide at methionine 7, 24, and 145. All these modifications increased gradually when raw milk was heated for 20, 40, and 60 min at 120 degrees C. The major modifications were also relatively quantified in dairy products, such as raw, high-temperature, ultra-high-temperature, sterilized, and condensed milk as well as infant formulas. The highest contents of lactulosyllysine at Lys47 were detected in powdered infant formulas, whereas lactulosyllysine at Lys138/141 was predominant in condensed milk samples. Methionine sulfoxide at Met7 and Met24 showed a trend toward higher modification rates in more severely processed products.