UPLC-MS/MS Based Identification of Dietary Steryl Glucosides by Investigation of Corresponding Free Sterols

Dietary plant foods are characterized by a vast molecular diversity of glycosylated sterols (SG) that differ in the structure of the steryl backbone. The identification of these polar steryl conjugates represents a major challenge as they are structurally highly similar, and commercial standards are limited to a few naturally abundant species. Spectral databases do not yet contain MS/MS spectra of these sterol conjugates obtained by electrospray ionization (ESI), which would facilitate their reliable identification. Thus, this study aimed at providing novel information on ESI-MS/MS spectra of both abundant and minor SG found in foods. As a first step, however, free sterols (FS) were investigated for their fragmentation behavior as they share the same intermediate ion as SG. Pure SG were obtained from commercially available standard mixtures and minor SG were extracted from different food sources (oat bran, wheat bran, pumpkin seeds, melon, rapeseeds, and potato peel). ESI-MS/MS spectra of 15 FS were assessed and fragment ions reflective of structural features were identified and rationalized. Subsequently, 14 SG were identified at four different levels, while relative retention times from chromatographic separation and spectral features of FS served to identify five SG. Spectral data from FS were directly transferable to SG when analyzed as aglycone ions as shown by similarity scores while SG were characterized by shorter retention times in reverse phase chromatography and the additional analysis as sodiated adduct confirmed their glycosidic nature. Moreover, we report for the first time the occurrence of 24-methylenecholesterol and a 4-monomethyl sterol as glycosidic conjugates in higher plants. The presented data will serve as a valuable tool for SG profiling of foods by facilitating their identification

Complementary Sample Preparation Strategies for Analysis of Cereal β-Glucan Oxidation Products by UPLC-MS/MS

The oxidation of cereal (1→3,1→4)-β-D-glucan can influence the health promoting and technological properties of this linear, soluble homopolysaccharide by introduction of new functional groups or chain scission. Apart from deliberate oxidative modifications, oxidation of β-glucan can already occur during processing and storage, which is mediated by hydroxyl radicals (HO•) formed by the Fenton reaction. We present four complementary sample preparation strategies to investigate oat and barley β-glucan oxidation products by hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), employing selective enzymatic digestion, graphitized carbon solid phase extraction (SPE), and functional group labeling techniques. The combination of these methods allows for detection of both lytic (C1, C3/4, C5) and non-lytic (C2, C4/3, C6) oxidation products resulting from HO•-attack at different glucose-carbons. By treating oxidized β-glucan with lichenase and β-glucosidase, only oxidized parts of the polymer remained in oligomeric form, which could be separated by SPE from the vast majority of non-oxidized glucose units. This allowed for the detection of oligomers with mid-chain glucuronic acids (C6) and carbonyls, as well as carbonyls at the non-reducing end from lytic C3/C4 oxidation. Neutral reducing ends were detected by reductive amination with anthranilic acid/amide as labeled glucose and cross-ring cleaved units (arabinose, erythrose) after enzyme treatment and SPE. New acidic chain termini were observed by carbodiimide-mediated amidation of carboxylic acids as anilides of gluconic, arabinonic, and erythronic acids. Hence, a full characterization of all types of oxidation products was possible by combining complementary sample preparation strategies. Differences in fine structure depending on source (oat vs. barley) translates to the ratio of observed oxidized oligomers, with in-depth analysis corroborating a random HO•-attack on glucose units irrespective of glycosidic linkage and neighborhood. The method was demonstrated to be (1) sufficiently sensitive to allow for the analysis of oxidation products also from a mild ascorbate-driven Fenton reaction, and (2) to be specific for cereal β-glucan even in the presence of other co-oxidized polysaccharides. This opens doors to applications in food processing to assess potential oxidations and provides the detailed structural basis to understand the effect oxidized functional groups have on β-glucan's health promoting and technological properties

Complementary Sample Preparation Strategies for Analysis of Cereal β-Glucan Oxidation Products by UPLC-MS/MS

The oxidation of cereal (1→3,1→4)-β-D-glucan can influence the health promoting and technological properties of this linear, soluble homopolysaccharide by introduction of new functional groups or chain scission. Apart from deliberate oxidative modifications, oxidation of β-glucan can already occur during processing and storage, which is mediated by hydroxyl radicals (HO•) formed by the Fenton reaction. We present four complementary sample preparation strategies to investigate oat and barley β-glucan oxidation products by hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), employing selective enzymatic digestion, graphitized carbon solid phase extraction (SPE), and functional group labeling techniques. The combination of these methods allows for detection of both lytic (C1, C3/4, C5) and non-lytic (C2, C4/3, C6) oxidation products resulting from HO•-attack at different glucose-carbons. By treating oxidized β-glucan with lichenase and β-glucosidase, only oxidized parts of the polymer remained in oligomeric form, which could be separated by SPE from the vast majority of non-oxidized glucose units. This allowed for the detection of oligomers with mid-chain glucuronic acids (C6) and carbonyls, as well as carbonyls at the non-reducing end from lytic C3/C4 oxidation. Neutral reducing ends were detected by reductive amination with anthranilic acid/amide as labeled glucose and cross-ring cleaved units (arabinose, erythrose) after enzyme treatment and SPE. New acidic chain termini were observed by carbodiimide-mediated amidation of carboxylic acids as anilides of gluconic, arabinonic, and erythronic acids. Hence, a full characterization of all types of oxidation products was possible by combining complementary sample preparation strategies. Differences in fine structure depending on source (oat vs. barley) translates to the ratio of observed oxidized oligomers, with in-depth analysis corroborating a random HO•-attack on glucose units irrespective of glycosidic linkage and neighborhood. The method was demonstrated to be 1) sufficiently sensitive to allow for the analysis of oxidation products also from a mild ascorbate-driven Fenton reaction, and 2) to be specific for cereal β-glucan even in the presence of other co-oxidized polysaccharides. This opens doors to applications in food processing to assess potential oxidations and provides the detailed structural basis to understand the effect oxidized functional groups have on β-glucan’s health promoting and technological properties.ISSN:2296-264

Structural investigation of oxidized arabinoxylan oligosaccharides by negative ionization HILIC-qToF-MS

Owing to the strong structure–function relationship of polysaccharides, the targeted modification of polysaccharides is attracting widespread interest in various fields, such as food industry, nutritional science, and biomedical research. Apart from intended functionalization, polysaccharide degradation mediated by hydroxyl radicals (HO˙) occurs in various industrial processes such as food processing. In particular, the oxidative degradation of feruloylated arabinoxylan (AX), a linearly-branched polysaccharide in cereals, causes chain scissions, and introduces new functional groups in the fiber, which can potentially modify the physicochemical properties and the functionalities of AX. However, the precise characterization of those structural modifications remains challenging due to the diversity of the oxidation products formed, the high molecular weight, and the relatively low quantity of newly formed functional groups. In this paper, selective (TEMPO-mediated) and random (Fenton) oxidations of several commercial xylo- and arabinoxylan oligosaccharides (A)XOS were studied as model systems by hydrophilic interaction UPLC-MS2 in negative ion resolution mode to identify potential oxidation products. An in-depth identification of acidic (A)XOS oxidation products derived from TEMPO-mediated oxidation provided novel insights in the selective functionalization of isomeric oligosaccharides. Furthermore, MS2 enabled the precise localisation of both glycosidic linkages and functional groups in oxidized (A)XOS. An innovative combination of an enzymatic sample preparation combined with a subsequent HILIC-MS2 analysis enabled the unprecedented comprehensive characterization of Fenton-induced oxidation products derived from AX. In future, this holistic analytical approach will enable the characterization of both selective and non-selective AX oxidation procedures in various applications

Nitrogen-to-Protein Conversion Factors for Edible Insects on the Swiss Market: T. molitor, A. domesticus, and L. migratoria

With an increasing worldwide demand for animal protein, insects are becoming a promising sustainable option for meat protein replacement. However, reported protein contents of insects are often overestimated when calculated as “crude protein” = 6.25 × nitrogen content (N), compared to true protein contents quantified from the sum of amino acid (AA) residues. In this study, the main two types of usual nitrogen-to-protein conversion factors kp and kA were determined on the basis of true protein/total nitrogen and true protein/protein nitrogen, respectively, with focus on the three insect species legally sold on the Swiss food market. T. molitor (mealworm larvae), A. domesticus (house crickets), and L. migratoria (locusts) from various breeders were analyzed for total and amide nitrogen, chitin, and AA composition. Careful control experiments of insect samples spiked with a protein standard were conducted to establish the recovery of true protein, which was with >95% excellent. Mealworms, crickets, and locusts exhibited similar AA-profiles and true protein contents of 51, 55, and 47 g/100 g (dry weight basis), respectively. Specific conversion factors kp showed little variability between the three insect species with 5.41, 5.25, and 5.33 for mealworms, crickets, and locusts, respectively, and confirmed an average ~17% overestimation of protein contents when using 6.25 × N. The determined average kp of 5.33 is supported by extracted literature data and is suggested for general use instead of 6.25 × N to calculate more accurate insect protein contents, whereas the average pure protein conversion factor kA of 5.6 is proposed for use in the case of insect protein isolates

Influence of Partial Acid Hydrolysis on Size, Dispersity, Monosaccharide Composition, and Conformation of Linearly-Branched Water-Soluble Polysaccharides

The effect of partial acid hydrolysis on the physical and chemical properties of galactomannan, arabinoxylan, and xyloglucan was investigated. Polysaccharides were treated at 50 °C with hydrochloric acid for 3–48 h. Portions of isopropanol (i-PrOH) were added sequentially to the hydrolyzates, resulting in fractions that were collected by centrifugation. As expected, a significant reduction of weight-average molecular weight (Mw) was observed with increasing hydrolysis time. Fractional precipitation was successfully applied to collect at least one polymer fraction with dispersity (Đ) close to one for each polysaccharide. The monosaccharide composition analysis showed that the partial hydrolysis usually lowered the relative amount of side chains, with the exception of galactomannan, where the composition remained largely unaffected. Estimation of the polymer conformation in solution, through evaluation of the Mark-Houwink parameter coefficient (α), confirmed that acid hydrolysis influenced the polysaccharides’ conformation. It was demonstrated that acid treatment in dilute solution followed by fractional isopropanol precipitation is a method, extendible to a variety of polysaccharides, to obtain materials of decreased molecular weight and low dispersity with slightly altered overall composition and conformation.ISSN:1420-304

UPLC-MS/MS Based Identification of Dietary Steryl Glucosides by Investigation of Corresponding Free Sterols

Dietary plant foods are characterized by a vast molecular diversity of glycosylated sterols (SG) that differ in the structure of the steryl backbone. The identification of these polar steryl conjugates represents a major challenge as they are structurally highly similar, and commercial standards are limited to a few naturally abundant species. Spectral databases do not yet contain MS/MS spectra of these sterol conjugates obtained by electrospray ionization (ESI), which would facilitate their reliable identification. Thus, this study aimed at providing novel information on ESI-MS/MS spectra of both abundant and minor SG found in foods. As a first step, however, free sterols (FS) were investigated for their fragmentation behavior as they share the same intermediate ion as SG. Pure SG were obtained from commercially available standard mixtures and minor SG were extracted from different food sources (oat bran, wheat bran, pumpkin seeds, melon, rapeseeds, and potato peel). ESI-MS/MS spectra of 15 FS were assessed and fragment ions reflective of structural features were identified and rationalized. Subsequently, 14 SG were identified at four different levels, while relative retention times from chromatographic separation and spectral features of FS served to identify five SG. Spectral data from FS were directly transferable to SG when analyzed as aglycone ions as shown by similarity scores while SG were characterized by shorter retention times in reverse phase chromatography and the additional analysis as sodiated adduct confirmed their glycosidic nature. Moreover, we report for the first time the occurrence of 24-methylenecholesterol and a 4-monomethyl sterol as glycosidic conjugates in higher plants. The presented data will serve as a valuable tool for SG profiling of foods by facilitating their identification.ISSN:2296-264

Site-selective and stochastic spin labelling of neutral water-soluble dietary fibers optimized for electron paramagnetic resonance spectroscopy

Use of spin labels to study structures of polymers has been widely spread in polymer science. However, for the studies of neutral water-soluble dietary fibers (DFs), labelling efficiencies in past studies have only been sufficient for application of continuous wave electron paramagnetic resonance spectroscopy (CW-EPR), but still insufficient for some advanced methods such as pulse EPR. Thus, in this paper, two spin labelling strategies, namely, site-selective mono-spin-labelling and stochastic multi-spin-labelling, were examined on linear cereal β-glucan, as well as linearly branched arabinoxylan and galactomannan. The effects of both methods in DF properties were evaluated. For the mono-labelling pathway, labelling efficiency could reach up to 46 %. In the stochastic labelling strategy, a degree of substitution (DS) up to 150 % could be reached, whereas optimized conditions for this strategy were achieved at DS = 3 % to obtain DFs whose bioactivity properties were still preserved while spin labelling efficiency was still sufficient for CW and pulse EPR experiments.ISSN:0144-8617ISSN:1879-134

Estimation of Iron Availability in Modified Cereal beta-Glucan Extracts by an in vitro Digestion Model

For cereal-based foods rich in dietary fibers, iron bioavailability is known to be poor. For native cereal beta-glucan extracts, literature has demonstrated that the main factor impacting the bioavailability is phytic acid, which is often found in association with dietary fibers. During food processing, beta-glucan can undergo modifications which could potentially affect the equilibrium between phytic acid, fiber, and iron. In this study, an in vitro digestion was used to elucidate the iron dialysability, and hence estimate iron availability, in the presence of native, chelating resin (Chelex)-treated, oxidised, or partially hydrolysed oat and barley beta-glucan extracts (at 1% actual beta-glucan concentration), with or without phytase treatment. It was confirmed that pure, phytic acid-free beta-glucan polysaccharide does not impede iron availability in cereal foods, while phytic acid, and to a smaller extent, also proteins, associated to beta-glucan can do so. Neither Chelex-treatment nor partial hydrolysis, 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) or NaIO4 oxidation significantly influenced the phytic acid content of the beta-glucan extracts (ranging 2.0-3.9%; p > 0.05). Consequently, as long as intrinsic phytic acid was still present, the beta-glucan extracts blocked the iron availability regardless of source (oat, barley) or Chelex-treatment, partial hydrolysis or NaIO4-oxidation down to 0-8% (relative to the reference without beta-glucan extract). Remarkably, TEMPO-oxidation released around 50% of the sequestered iron despite unchanged phytic acid levels in the modified extract. We propose an iron-mobilising effect of the TEMPO product beta-polyglucuronan from insoluble Fe(II)/phytate/protein aggregates to soluble Fe(II)/bile salt units that can cross the dialysis membrane. In addition, Chelex-treatment was identified as prerequisite for phytase to dramatically diminish iron retention of the extract for virtually full availability, with implications for optimal iron bioavailability in cereal foods.ISSN:2296-861