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.     

Effects of water content on volatile generation and peptide degradation in the maillard reaction of glycine, diglycine, and triglycine

Peptides abundant in food and protein hydrolysates are known to be important to process flavors. The present study reports the volatile profile of the Maillard reactions of glycine, diglycine, and triglycine. The reaction with glucose was conducted at 0-100% water content in glycerol medium at 160 degrees C for 1 h. Volatile compounds were quantified by stir bar sorptive extraction-gas chromatography-mass spectrometry, and nonvolatile compounds were quantified by high-performance liquid chromatography-tandem mass spectrometry. The major volatiles produced from each of the reaction systems were trimethylpyrazine and 2,5-dimethylpyrazine. Volatile generation increased as water decreased, and the overall reactivity of the glycine and glycine peptides in volatile formation was glycine approximately triglycine > diglycine. Triglycine was very unstable and mainly degraded into cyclic Gly-Gly and glycine, whereas diglycine had a higher stability than triglycine toward hydrolytic cleavage of the peptide bond. The amounts of glycine, diglycine, cyclic (Gly-Gly), and triglycine in the peptide-glucose reaction mixtures at different water content were reported.     

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.     

Identification of fructo- and malto-oligosaccharides in cured tobacco leaves (Nicotiana tabacum)

Fructo-oligosaccharides (FOSs) and malto-oligosaccharides (MOSs) in cured tobacco leaves ( Nicotiana tabacum ) were detected and quantified using high-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). These oligosaccharides were present in several tobacco materials including flue-cured tobacco, sun/air-cured tobacco, and cut filler of commercially available tobacco products, but were not detected in air-cured tobacco. The changes in these oligosaccharides during storage were also investigated. The results revealed that MOSs simply decreased under a warm atmosphere and FOSs increased. In addition, the formation of FOSs in cured tobacco leaves occurred in the presence of sucrose. With regard to FOSs, it has been reported that green tobacco leaves do not contain FOSs such as kestose, nystose, and fructosyl-nystose. The results of a model test suggested that the changes in FOS amount were caused by enzymatic reactions.     

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.     

Analysis of food oligosaccharides using MALDI-MS: quantification of fructooligosaccharides

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful new technique that will have a great impact on food analysis. This study demonstrates the applicability of MALDI-MS performed directly on an aqueous food extract for qualitative and quantitative analysis of food oligosaccharides. 2', 4',6'-Trihydroxyacetophenone was found to be the best matrix for analysis of oligosaccharides in the foods examined. The relationship between laser strength, resolution, and the response factors of individual oligosaccharides using MALDI-MS was investigated. A MALDI-MS method for quantitative analysis of fructooligosaccharides with standard addition of a pure fructooligosaccharide was developed. High performance anion exchange chromatography with pulsed amperometric detection was compared to MALDI-MS for the analysis of fructooligosaccharides. The fructooligosaccharide analyses were performed on red onions, shallots, and elephant garlic.     

Screening for nutritive peptides that modify cholesterol 7alpha-hydroxylase expression

Bioactive peptides with a variety of effects have been described from several nutritive proteins. They exhibit antimicrobial, blood-pressure lowering, antithrombotic, immunomodulatory, and cholesterol-modulating effects. In this study, we have examined whether peptides derived from food proteins might influence bile acid synthesis. A reporter gene cell line that carries a cholesterol 7alpha-hydroxylase promoter fragment fused to firefly luciferase ( cyp7a-luc) was used to screen for nutritive peptides affecting cyp7a expression, the enzyme catalyzing the rate-limiting step in bile acid synthesis. Proteolytic hydrolysates were prepared from soy protein and bovine casein with pepsin, trypsin, chymotrypsin, and elastase and size fractionated using ultrafiltration. Several bioactive hydrolysates could be identified that inhibited luciferase expression. Also, an activation of kinase (AKT, ERK, p38-MAPK) signaling could be observed. Selected hydrolysates were further fractionated by reversed-phase HPLC. Bioactive HPLC-fractions were obtained from casein but not from soy hydrolysates; however, activity could not be recovered in single peak fractions. Peptides in such fractions were identified by mass spectrometry. Five selected peptides from alpha S1-casein present in active fractions were synthesized, but none of these showed activity in the cyp7a-luc screening system. However, two of them activated MAP-kinase signaling similar to the hydrolysates, which suggests, that these peptides are involved in cyp7a regulation by the casein hydrolysates.     

Glycoprofiling of bifidobacterial consumption of human milk oligosaccharides demonstrates strain specific, preferential consumption of small chain glycans secreted in early human lactation

The molecular basis by which human breast milk supports the development of a protective intestinal microbiome in infants is unknown. After lactose and lipids, human milk oligosaccharides (HMOs) are quantitatively the third largest and most diverse component of breast milk. In this work, glycomic profiling of HMO consumption by bifidobacteria using Fourier transform ion cyclotron resonance mass spectrometry reveals that one species, Bifidobacterium longum biovar infantis ATCC 15697, an isolate from the infant gut, preferentially consumes small mass oligosaccharides, representing 63.9% of the total HMOs available. These HMOs were detected in human breast milk at the onset and constantly through the first month of lactation by use of high performance liquid chromatography-chip time-of-flight mass spectrometry. Further characterization revealed that strain ATCC 15697 possesses both fucosidase and sialidase activities not present in the other tested strains. This work provides evidence that these small mass HMOs are selectively metabolized by select bifidobacterial strains and represent a potential new class of bioactive molecules functioning as prebiotics to facilitate a protective gut colonization in breast-fed newborns.     

Radical scavenging and reducing ability of tilapia (Oreochromis niloticus) protein hydrolysates

Enzymatically hydrolyzed fish protein hydrolysates could be used as a source of antioxidative nutraceuticals. In our current work, we have investigated alkali-solubilized tilapia ( Oreochromis niloticus) protein hydrolysates for their ability to scavenge reactive oxygen species (ROS) and for their reducing power. Tilapia protein isolate was prepared by an alkaline solubilization technique and used as a substrate for enzyme hydrolysis. Cryotin, protease A 'Amano' 2, protease N 'Amano', Neutrase and Flavourzyme, were used separately to determine their effectiveness in hydrolyzing tilapia protein isolate. ROS scavenging ability was quantified using an isoluminol enhanced chemiluminescent assay in the presence of a) hydrogen peroxide or b) mononuclear cells isolated from human blood. Ferric reducing antioxidant power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) of the hydrolysates using 2, 2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) or 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), were also investigated. Results showed that, in general, the TEAC, FRAP values and ROS scavenging ability of the hydrolysates increased with an increase in the degree of hydrolysis. Among the different hydrolysates, those prepared using Cryotin were most effective and Amano A2 hydrolysates were least effective in scavenging ABTS*(+) and ROS generated by hydrogen peroxide. However, FRAP assay showed that hydrolysates prepared using Flavourzyme were most effective, and Amano N and Neutrase hydrolysates were least effective in reducing ferric ions. No significant difference was observed among the hydrolysates produced with different enzymes in their ability to scavenge ROS generated by phorbol myristate acetate stimulated mononuclear cells. These results shed light on the in vitro ROS scavenging ability of alkali solubilized tilapia protein hydrolysates, as well as potential nutraceutical use of these hydrolysates.     

Effects of Eucalyptus globulus wood autohydrolysis conditions on the reaction products

Eucalyptus globulus wood samples were reacted in aqueous media (hydrothermal treatments) at 160 degrees C for 30-66 min. Liquors from the several experiments were analyzed by spectrophotometry, high-performance liquid chromatography, or gas chromatography-mass spectrometry for monosaccharides, oligosaccharides, oligosaccharide substituents (arabinose moieties, uronic acids, and acetyl groups), acetic acid, furfural, hydroxymethylfurfural, and dichloromethane-soluble compounds. Individual components of this latter fraction were identified and quantified. The molecular weight distribution of oligosaccharides was studied by high-performance size exclusion chromatography. The kinetics of xylan conversion into high-, medium-, and low-molecular-weight products was assessed in terms of the severity factor and by pseudohomogeneous kinetic models.     

(E)-1-(2,3,6-trimethylphenyl)buta-1,3-diene: a potent grape-derived odorant in wine

(E)-1-(2,3,6-Trimethylphenyl)buta-1,3-diene (TPB) was identified as a potent odorant in acid hydrolysates of crude glycoconjugate fractions isolated from grapes and grape vine leaves. TPB was also identified in a Semillon wine, using gas chromatography/mass spectrometry, by co-injection with an authentic sample. TPB had an aroma detection threshold of 40 ng/L in a neutral white wine and the concentration of TPB in four out of five white wines analyzed ranged from 50 to 210 ng/L.     

MALDI-MS and HPLC quantification of oligosaccharides of lichenase-hydrolyzed water-soluble beta-glucan from ten barley varieties

This study is the first to apply matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to both qualitative and quantitative analyses of oligosaccharides of lichenase-hydrolyzed water-soluble beta-glucan from barley. Compared to high-performance liquid chromatography (HPLC) with an evaporative light-scattering detector, MALDI-MS is a rapid technique with high accuracy and sensitivity and could be used to assess primary structural features of water-soluble beta-glucan from different barley varieties.     

Use of mass spectrometry To rapidly characterize the heterogeneity of bovine alpha-lactalbumin.

From a bovine whey protein fraction the nonglycosylated and glycosylated alpha-lactalbumin fractions were isolated by gel-permeation chromatography followed by reversed-phase high-performance liquid chromatography. Both fractions were studied by electrospray-ionization mass spectrometry (ESI-MS). For the nonglycosylated fraction, a mass of 14 178 Da was found, which was in accordance with the known amino acid sequence of the protein. The glycosylated fraction appeared to be a mixture of components with mass values ranging from ca. 15 840 to 16 690 Da. Given the published carbohydrate composition of the whole glyco-alpha-lactalbumin fraction, these masses could be matched to those of 14 differently glycosylated forms of alpha-lactalbumin. Further support for these forms was obtained from the results of a separate mass spectrometric analysis of the mixture of oligosaccharides released from the protein by treatment with peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase F. ESI-MS was found to be a powerful tool to gain insight into the composition of the complex mixture of glycoforms of alpha-lactalbumin without the need of further purification of these forms or of the oligosaccharides released thereof.     

Analysis of lidocaine and its major metabolite, monoethylglycinexylidide, in elk velvet antler by liquid chromatography with UV detection and confirmation by electrospray ionization tandem mass spectrometry

A sensitive liquid chromatographic (LC) method with UV detection was developed for the determination of residues of lidocaine (LID) and its major metabolite, monoethylglycinexylidide (MEGX), in elk velvet antler. The drugs were extracted from alkaline velvet antler homogenates, cleaned up on a C(18) solid-phase extraction cartridge, and separated on an Inertsil ODS-3 (3.0 x 250 mm, 5 microm) column using an isocratic mobile phase made up of 0.05 M phosphate buffer (pH 4.0)/acetonitrile (88:12, v/v) at a flow rate of 1.0 mL/min. The limits of quantification for LID and its major metabolite, MEGX, were 10 and 20 ng/g, respectively. The method was validated and used to measure the concentration of residues of LID and MEGX in elk velvet antlers harvested after either LID anesthesia or application of a drug-free control method (electro-anesthesia, EA). No LID or MEGX residues were detected in any of the antlers harvested after EA application. No MEGX residues were detected in any of the velvet antlers harvested after LID application, but residues of LID ranging in concentration from 68 to 4300 ng/g were detected in the three sections of the velvet antlers harvested after LID administration. LC-tandem mass spectrometry was used to confirm the presence of lidocaine detected in the velvet antlers.     

Production of galacto-manno-oligosaccharides from guar gum by beta-mannanase from Penicillium oxalicum SO

Beta-mannanase from Penicillium oxalicum SO efficiently hydrolyzed guar galactomannan to galacto-manno-oligosaccharides. Gel filtration estimated the molecular weight of the beta-mannanase as 35 000 and SDS-PAGE as 29 000. The optimum pH was around 5 while a stable pH was reached in the range of 3-6. Optimum temperature was around 60 degrees C at pH 5, while under 60 degrees C activity was stable. HPLC analysis detected oligosaccharides with degrees of polymerization (DP) of 2 to 7 and 2 to 6 released on hydrolysis of guar and locust bean gums, respectively; about 92% of the released sugars were oligosaccharides. In analysis of the sugar distribution on MALDI-TOF-MS, major products of DP 6 and 7 and DP 5 and 6 were confirmed in hydrolysates of guar gum and locust bean gum, respectively. One of the main oligosaccharides released from guar gum, with DP 7, had a high galactose content (Gal/Man = 0.76) and corresponded to a blockwise galactose-substituted mannan type in galactomannan.     

Improved quantitative analysis of oligosaccharides from lichenase-hydrolyzed water-soluble barley beta-glucans by high-performance anion-exchange chromatography

Cereal beta-glucan is a linear biopolymer linked by beta-(1,3)/(1,4)-glycosidic bonds. More specifically, the beta-(1,4)-linked glucose chain is interrupted with beta-(1,3)-linkages in cereal beta-glucan structure. Elucidation of the exact length and distribution of linear beta-(1,4)-linked portion facilitates the understanding of the fine structure of cereal beta-glucan. A HPAEC assisted by lichenase treatment has been used for the structural and quantitative analysis of cereal beta-glucan. The absence of authentic standard oligosaccharides, putatively 3-O-beta-cellobiosyl-D-glucose (DP3) and 3-O-beta-cellotriosyl-D-glucose (DP4), was a potential problem to the characterization of beta-glucan structure. In this study, two major lichenase-hydrolyzed products were generated from the barley beta-glucan, and putative 3-O-beta-cellobiosyl-D-glucose and 3-O-beta-cellotriosyl-D-glucose were separated and highly purified by recycling preparative HPLC technology. Structural analysis of highly purified putative 3-O-beta-cellobiosyl-D-glucose and 3-O-beta-cellotriosyl-D-glucose was performed by TLC and LC-MS analysis. Two putative DP3 and DP4 displayed the nonreducing end/(1,4)/(1,3) linkage ratios of 1:0.96:0.90 and 1:2.18:1.16, respectively; the molecular masses (m/z) of their sodium adducts were 527.0 and 689.0, respectively. Using these structurally confirmed oligosaccharides, the exact amounts of beta-glucan lichenase hydrolysates from domestic barley cultivars were quantified. The amount of two major DP3 and DP4 accounted for only 71.4-73.3% of water-extractable beta-glucan fraction, and the (1,4)/(1,3) linkage ratios of the extracted beta-glucans were almost identical in the range of 2.24-2.25 among the barley cultivars tested.     

Investigations into inhibitor type and mode, simulated gastrointestinal digestion, and cell transport of the angiotensin I-converting enzyme-inhibitory peptides in Pacific hake (Merluccius productus) fillet hydrolysate

Fish protein hydrolysate (FPH) produced by incubation of Pacific hake fillet with 3.00% Protamex at pH 6.5 and 40 degrees C for 125 min demonstrated in vitro ACE-inhibitory activity (IC50 = 165 microg/mL), which was enhanced by ultrafiltration through a 10 kDa molecular weight cutoff membrane (IC50 = 44 microg/mL). However, after simulated gastrointestinal digestion, FPH and ultrafiltrate had similar ACE-inhibitory activity (IC 50 = 90 microg/mL), indicating that FPH peptides act as "pro-drug type" inhibitors and that enrichment by ultrafiltration may be unnecessary. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry confirmed that the molecular weights of major peaks were <1 kDa regardless of ultrafiltration. ACE-inhibitory activities of digested hydrolysates were not significantly affected by preincubation with ACE ( P > 0.05) and exhibited a competitive inhibitory mode. A permeability assay using fully differentiated colorectal adenocarcinoma (Caco-2) cells showed an apical to basolateral transport of peptides that ranged from approximately 2 to 20% after 2 h at 37 degrees C. Pacific hake fillet hydrolysates are a potentially bioavailable source of ACE-inhibitory peptides awaiting further in vivo study.     

Batatinosides II-VI, acylated lipooligosaccharides from the resin glycosides of sweet potato

Sweet potato ( Ipomoea batatas) belongs to the Convolvulaceae (morning glory family) and is native to Mexico and Central America. Its edible tuberous roots have been much appreciated since pre-Hispanic times in Mesoamerica and now play an important role as a basic diet staple and a medicinal plant worldwide. The hexane-soluble extract from roots, through preparative-scale recycling HPLC, yielded five new lipophilic oligosaccharides of jalapinolic acid, batatinosides II-VI ( 1- 5), as well as the known pescapreins I ( 6) and VII ( 7) and murucoidin I ( 8), which are part of the purgative resin glycoside mixture. NMR spectroscopy and FAB mass spectrometry were used to characterize their structures. Compounds 1 and 2 are tetraglycosidic lactones of operculinic acid C. The pentasaccharide structures for compounds 3 and 4 were confirmed to be macrolactones of simonic acid B, and that characterized for 5 was derived from operculinic acid A. The lactonization site of the aglycone was placed at C-3 of the second saccharide unit in all compounds except 4, where it was placed at C-2. All compounds contain an esterifying residue that is composed of a long-chain fatty acid, n-decanoic acid (capric) or n-dodecanoic acid (lauric). In compound 3, an additional short-chain fatty acid, (2 S)-methylbutyric acid, was also identified.     

Antihypertensive effect of angiotensin i converting enzyme-inhibitory peptide from hydrolysates of Bigeye tuna dark muscle, Thunnus obesus

Angiotensin I converting enzyme (ACE) inhibitory peptide was isolated from tuna dark muscle hydrolysate prepared by alcalase, neutrase, pepsin, papain, alpha-chymotrypsin, and trypsin, respectively. Among hydrolysates, the pepsin-derived hydrolysate exhibited the highest ACE I inhibitory activity versus those of other enzyme hydrolysates. The structure of the peptide was identified to be Trp-Pro-Glu-Ala-Ala-Glu-Leu-Met-Met-Glu-Val-Asp-Pro (molecular weight 1581 Da) by time of flight mass spectrometry/mass spectrometry analysis, and the IC 50 value of the peptide was 21.6 microM. The Lineweaver-Burk plots revealed that the peptide acts as a noncompetitive inhibitor, and the inhibitor constant ( K i) was calculated as 26.6 microM using the secondary plots. The peptide had an antihypertensive effect according to the time-course measurement after oral administration to spontaneously hypertensive rats. Maximal reduction was detected 3 h after oral administration at a dose of 10 mg/kg of body weight. These results suggest that the peptide derived from tuna dark muscle would be a beneficial ingredient for functional food or pharmaceuticals against hypertension and its related diseases.     

Galactoglucomannan Oligosaccharides (GGMO) from a molasses byproduct of pine ( Pinus taeda ) fiberboard production

"Temulose" is the trade name for a water-soluble molasses produced on a large scale (300-400 tonnes per year) as a byproduct of the fiberboard industry. The feedstock for Temulose is predominantly a single species of pine ( Pinus taeda ) grown and harvested in stands in southeastern Texas. Because of the method of production, the molasses was predicted to consist of water-soluble hemicelluloses, mainly arabinoxylan-type and galactoglucomannan-type oligosaccharides, plus minor components of lignin, but no detailed structural study had been reported. The structure and composition of the molasses has now been deduced by a combination of MALDI-TOF mass spectrometry, size exclusion chromatography, proton and (13)C NMR techniques, and classic carbohydrate analysis. Limited acid hydrolysis released a series of galactoglucomannan oligosaccharides (GGMO) that were selectively recovered from the acid-labile arabinogalactan by precipitation with ethanol. The precipitate was named "Temulose brown sugar" because of its appearance, and is shown to consist of GGMO with a degree of polymerization (DP) from 4 to 13, with the major component being DP 5-8. The structure of these oligosaccharides is a β-1,4-linked backbone of Man and Glc residues, with occasional α-1,6 branching by single galactosyl units.