Quantification of Distributions of Local Proton Concentrations in Heterogeneous Soft Matter and Non-Anfinsen Biomacromolecules.

A new method to quantitatively analyze heterogeneous distributions of local proton densities around paramagnetic centers in unstructured and weakly structured biomacromolecules and soft matter is introduced, and its feasibility is demonstrated on aqueous solutions of stochastically spin-labeled polysaccharides. This method is based on the pulse EPR experiment ih-RIDME (intermolecular hyperfine relaxation-induced dipolar modulation enhancement). Global analysis of a series of RIDME traces allows for a mathematically stable transformation of the time-domain data to the distribution of local proton concentrations. Two pulse sequences are proposed and tested, which combine the ih-RIDME block and the double-electron-electron resonance (DEER) experiment. Such experiments can be potentially used to correlate the local proton concentration with the macromolecular chain conformation. We anticipate an application of this approach in studies of intrinsically disordered proteins, biomolecular aggregates, and biomolecular condensates.

A novel approach for the protein determination in food-relevant microalgae.

Microalgae are gaining interest as food ingredient. Assessments of functional and nutritional properties are necessary to forward their implementation. In this study, protein content and composition of eight commercially available microalgae biomasses were determined and compared to conventional food proteins. A novel procedure for the determination of the true protein content was proposed: Multiplication of proteinic nitrogen with a sample-specific nitrogen-to-protein conversion factor kA. The proteinic nitrogen was derived from the difference of total nitrogen minus non-protein nitrogen. The average kA for microalgae was 5.3 and considerable variation between different microalgae biomasses were detected. In addition, the content of non-protein nitrogen varied between 3.4% and 15.4%. The amino acid profiles of Chlorella samples were nutritionally superior to the tested plant proteins but indicated lower protein interaction tendency, potentially limiting their structuring functionality. In contrast, Auxenochlorella contained lower amounts of indispensable amino acids while showing comparable interaction potential to plant proteins.

Pulse EPR spectroscopy and molecular modeling reveal the origins of the local heterogeneity of dietary fibers.

Optimizing human diet by including dietary fibers would be more efficient when the fibers' chain interactions with other molecules are understood in depth. Thereby, it is important to develop methods for characterizing the fiber chain to be able to monitor its structural alterations upon intermolecular interactions. Here, we demonstrate the utility of the electron paramagnetic resonance (EPR) spectroscopy, complemented by simulations in probing the atomistic details of the chain conformations for spin-labeled fibers. Barley ß-glucan, a native polysaccharide with linear chain, was utilized as a test fiber system to demonstrate the technique's capabilities. Pulse dipolar EPR data show good agreement with results of the fiber chain modeling, revealing sinuous chain conformations and providing polymer shape descriptors: the gyration tensor, spin-spin distance distribution function, and information about proton density near the spin probe. Results from EPR measurements point to the fiber aggregation in aqueous solution, which agrees with the results of the dynamic light scattering. We propose that the combination of pulse EPR measurements with modeling can be a perfect experimental tool for in-depth structural investigation of dietary fibers and their interaction under such conditions, and that the presented methodology can be extended to other weakly ordered or disordered macromolecules.

Interaction of barley ß-glucan with food dye molecules - An insight from pulse dipolar EPR spectroscopy.

The interactions between dietary fibers (DFs) and small molecules are of great interest to food chemistry and nutrition science. However, the corresponding interaction mechanisms and structural rearrangements of DFs at the molecular level are still opaque due to the usually weak binding and the lack of appropriate techniques to determine details of conformational distributions in such weakly organized systems. By combining our previously established methodology on stochastic spin-labelling of DFs with the appropriately revised set of pulse electron paramagnetic resonance techniques, we present here a toolkit to determine the interactions between DFs and small molecules, using barley ß-glucan as an example for neutral DF and a selection of food dye molecules as examples for small molecules. The proposed here methodology allowed us to observe subtle conformational changes of ß-glucan by detecting multiple details of the local environment of the spin labels. Substantial variations of binding propensities were detected for different food dyes.

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 ß-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.

Estimation of Iron Availability in Modified Cereal ß-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 ß-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, ß-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 ß-glucan extracts (at 1% actual ß-glucan concentration), with or without phytase treatment. It was confirmed that pure, phytic acid-free ß-glucan polysaccharide does not impede iron availability in cereal foods, while phytic acid, and to a smaller extent, also proteins, associated to ß-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 ß-glucan extracts (ranging 2.0-3.9%; p > 0.05). Consequently, as long as intrinsic phytic acid was still present, the ß-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 ß-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 ß-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.

A microcalorimetric and microscopic strategy to assess the interaction between dietary fibers and small molecules.

The interaction between small molecules and neutral soluble dietary fiber is one of the proposed mechanisms determining the bioavailability of these components in the small intestine. However, the weak nature of these interactions makes it difficult to find an analytical method sensitive enough to detect them. Here, we probed the molecular interaction between galactomannan, arabinoxylan, and ß-glucan with gallic acid, cinnamic acid, acetylsalicylic acid, and acetaminophen, using advanced analytical methods, namely isothermal titration calorimetry (ITC) and in the form of gold-nanoparticles, transmission electron microscopy (TEM). The results obtained from ITC analysis were fully consistent with the results obtained from TEM. In short, the interaction of these fibers and small molecules was mainly entropically driven, hence involving hydrophobic type association and possible conformational changes of the polysaccharide. However, the enthalpy contribution (hydrogen interaction) is also significant, especially regarding interactions with the acetylsalicylic acid molecule.

Glucose Decoration on Wall Teichoic Acid Is Required for Phage Adsorption and InlB-Mediated Virulence in Listeria ivanovii.

Listeria ivanovii (Liv) is an intracellular Gram-positive pathogen that primarily infects ruminants but also occasionally causes enteric infections in humans. Albeit rare, this bacterium possesses the capacity to cross the intestinal epithelium of humans, similar to its more frequently pathogenic cousin, Listeria monocytogenes (Lmo). Recent studies in Lmo have shown that specific glycosyl modifications on the cell wall-associated glycopolymers (termed wall teichoic acid [WTA]) of Lmo are responsible for bacteriophage adsorption and retention of the major virulence factor internalin B (InlB). However, the relationship between InlB and WTA in Liv remains unclear. Here, we report the identification of the unique gene liv1070, which encodes a putative glucosyltransferase in the polycistronic WTA gene cluster of the Liv WSLC 3009 genome. We found that in-frame deletion of liv1070 led to loss of the glucose substitution on WTA, as revealed by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) analysis. Interestingly, the glucose-deficient mutant became resistant to phage B025 infection due to an inability of the phage to adsorb to the bacterial surface, a binding process mediated by the receptor-binding protein B025_Gp17. As expected, deletion of liv1070 led to loss of InlB retention on the bacterial cell wall, which corresponded to a drastic decrease in cellular invasion. Genetic complementation of liv1070 restored the characteristic phenotypes, including glucose decoration, phage adsorption, and cellular invasion. Taken together, our data demonstrate that an interplay between phage, bacteria, and host cells also exists in Listeria ivanovii, suggesting that the trade-off between phage resistance and virulence attenuation may be a general feature in the genus Listeria. IMPORTANCE Listeria ivanovii is a Gram-positive bacterial pathogen known to cause enteric infection in rodents and ruminants and occasionally in immunocompromised humans. Recent investigations revealed that in its better-known cousin Listeria monocytogenes, strains develop resistance to bacteriophage attack due to loss of glycosylated surface receptors, which subsequently results in disconnection of one of the bacterium's major virulence factors, InlB. However, the situation in L. ivanovii remains unclear. Here, we show that L. ivanovii acquires phage resistance following deletion of a unique glycosyltransferase. This deletion also leads to dysfunction of InlB, making the resulting strain unable to invade host cells. Overall, this study suggests that the interplay between phage, bacteria, and the host may be a feature common to the genus Listeria.

Effect of Polysaccharide Conformation on Ultrafiltration Separation Performance.

The manifold array of saccharide linkages leads to a great variety of polysaccharide architectures, comprising three conformations in aqueous solution: compact sphere, random coil, and rigid rod. This conformational variation limits the suitability of the commonly applied molecular weight cut-off (MWCO) as selection criteria for polysaccharide ultrafiltration membranes, as it is based on globular marker proteins with narrow Mw and hydrodynamic volume relation. Here we show the effect of conformation on ultrafiltration performance using randomly coiled pullulan and rigid rod-like scleroglucan as model polysaccharides for membrane rejection and molecular weight distribution. Ultrafiltration with a 10 kDa polyethersulfone membrane yielded significant different recoveries for pullulan and scleroglucan showing 1% and 71%, respectively. We found deviations greater than 77-fold between nominal MWCO and apparent Mw of pullulan and scleroglucan, while recovering over 90% polysaccharide with unchanged Mw. We anticipate our work as starting point towards an optimized membrane selection for polysaccharide 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 k p and k A 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 k p 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 k p 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 k A of 5.6 is proposed for use in the case of insect protein isolates.

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.

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 (D) 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.

Galactosylated wall teichoic acid, but not lipoteichoic acid, retains InlB on the surface of serovar 4b Listeria monocytogenes.

Listeria monocytogenes is a Gram-positive, intracellular pathogen harboring the surface-associated virulence factor InlB, which enables entry into certain host cells. Structurally diverse wall teichoic acids (WTAs), which can also be differentially glycosylated, determine the antigenic basis of the various Listeria serovars. WTAs have many physiological functions; they can serve as receptors for bacteriophages, and provide a substrate for binding of surface proteins such as InlB. In contrast, the membrane-anchored lipoteichoic acids (LTAs) do not show significant variation and do not contribute to serovar determination. It was previously demonstrated that surface-associated InlB non-covalently adheres to both WTA and LTA, mediating its retention on the cell wall. Here, we demonstrate that in a highly virulent serovar 4b strain, two genes gtlB and gttB are responsible for galactosylation of LTA and WTA respectively. We evaluated the InlB surface retention in mutants lacking each of these two genes, and found that only galactosylated WTA is required for InlB surface presentation and function, cellular invasiveness and phage adsorption, while galactosylated LTA plays no role thereof. Our findings demonstrate that a simple pathogen-defining serovar antigen, that mediates bacteriophage susceptibility, is necessary and sufficient to sustain the function of an important virulence factor.

Bile acid-retention by native and modified oat and barley ß-glucan.

Foods rich in cereal ß-glucan are efficient dietary tools to help reduce serum cholesterol levels and hence the risk of cardiovascular diseases. However, ß-glucan undergoes various reactions during food processing, which alter its viscous properties and interactions with components of the gastrointestinal tract. It has been proposed in the literature that oxidation and partial hydrolysis increase ß-glucan's bile acid-binding activity, and therefore its effectiveness in lowering cholesterol. Here, the passage kinetics of a bile salt mix across a dialysis membrane was studied with or without oat and barley ß-glucan extracts, native or modified (partial hydrolysis and oxidations by sodium periodate or TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl)). Bile acid-retention turned out to be purely a function of viscosity, with the most viscous native extracts exhibiting the strongest retardation of bile acid permeation. Opposite of what was suggested in the literature, oxidation and molecular weight reduction do not seem to increase the bile acid-binding capability of ß-glucan.

Amyloid Fibrils Aerogel for Sustainable Removal of Organic Contaminants from Water.

Water contamination by organic pollutants is ubiquitous and hence a global concern due to detrimental effects on the environment and human health. Here, it is demonstrated that amyloid fibrils aerogels are ideal adsorbers for removing organic pollutants from water. To this end, amyloid fibrils prepared from ß-lactoglobulin, the major constituent of milk whey protein, are used as building blocks for the fabrication of the aerogels. The adsorption of Bentazone, Bisphenol A, and Ibuprofen, as model pollutants, is evaluated under quasi-static conditions, without use of energy or pressure. Through adsorption by amyloid fibrils aerogel, excellent removal efficiencies of 92%, 78%, and 98% are demonstrated for Bentazone, Bisphenol A, and Ibuprofen, respectively. Furthermore, the maximum adsorption capacity of amyloid fibrils aerogel for Bentazone, Bisphenol A, and Ibuprofen is 54.2, 50.6, and 69.6 mg g-1 , respectively. To shed light on the adsorption equilibrium process, adsorption isotherms, binding constants, saturation limits, and the effect of pH are evaluated. Finally, the regeneration of the aerogel over three consecutive cycles is studied, exhibiting high reusability with no significant changes in its removal performance. These results point at amyloid fibrils aerogels as a sustainable, efficient, and inexpensive technology for alleviating the ubiquitous water contamination by organic pollutants.

Structural basis for recognition of bacterial cell wall teichoic acid by pseudo-symmetric SH3b-like repeats of a viral peptidoglycan hydrolase.

Endolysins are bacteriophage-encoded peptidoglycan hydrolases targeting the cell wall of host bacteria via their cell wall-binding domains (CBDs). The molecular basis for selective recognition of surface carbohydrate ligands by CBDs remains elusive. Here, we describe, in atomic detail, the interaction between the Listeria phage endolysin domain CBD500 and its cell wall teichoic acid (WTA) ligands. We show that 3'O-acetylated GlcNAc residues integrated into the WTA polymer chain are the key epitope recognized by a CBD binding cavity located at the interface of tandem copies of beta-barrel, pseudo-symmetric SH3b-like repeats. This cavity consists of multiple aromatic residues making extensive interactions with two GlcNAc acetyl groups via hydrogen bonds and van der Waals contacts, while permitting the docking of the diastereomorphic ligands. Our multidisciplinary approach tackled an extremely challenging protein-glycopolymer complex and delineated a previously unknown recognition mechanism by which a phage endolysin specifically recognizes and targets WTA, suggesting an adaptable model for regulation of endolysin specificity.

Biowaste treatment with black soldier fly larvae: Increasing performance through the formulation of biowastes based on protein and carbohydrates.

A key challenge for black soldier fly larvae (BSFL) treatment is its variable reliability and efficiency when applied to different biowastes. Similar to other biowaste treatment technologies, co-conversion could compensate for variability in the composition of biowastes. Using detailed nutrient analyses, this study assessed whether mixing biowastes to similar protein and non-fibre carbohydrate (NFC) contents increased the performance and reduced the variability of BSFL treatment in comparison to the treatment of individual wastes. The biowastes examined were mill by-products, human faeces, poultry slaughterhouse waste, cow manure, and canteen waste. Biowaste formulations had a protein-to-NFC ratio of 1:1, a protein content of 14-19%, and a NFC content of 13-15% (dry mass). Performance parameters that were assessed included survival and bioconversion rate, waste reduction, and waste conversion and protein conversion efficiency. In comparison to poultry feed (benchmark), vegetable canteen waste showed the best performance and cow manure performed worst. Formulations showed significantly improved performance and lower variability in comparison to the individual wastes. However, variability in performance was higher than expected for the formulations. One reason for this variability could be different fibre and lipid contents, which correlated with the performance results of the formulations. Overall, this research provides baseline knowledge and guidance on how BSFL treatment facilities may systematically operate using biowastes of varying types and compositions.

Phage resistance at the cost of virulence: Listeria monocytogenes serovar 4b requires galactosylated teichoic acids for InlB-mediated invasion.

The intracellular pathogen Listeria monocytogenes is distinguished by its ability to invade and replicate within mammalian cells. Remarkably, of the 15 serovars within the genus, strains belonging to serovar 4b cause the majority of listeriosis clinical cases and outbreaks. The Listeria O-antigens are defined by subtle structural differences amongst the peptidoglycan-associated wall-teichoic acids (WTAs), and their specific glycosylation patterns. Here, we outline the genetic determinants required for WTA decoration in serovar 4b L. monocytogenes, and demonstrate the exact nature of the 4b-specific antigen. We show that challenge by bacteriophages selects for surviving clones that feature mutations in genes involved in teichoic acid glycosylation, leading to a loss of galactose from both wall teichoic acid and lipoteichoic acid molecules, and a switch from serovar 4b to 4d. Surprisingly, loss of this galactose decoration not only prevents phage adsorption, but leads to a complete loss of surface-associated Internalin B (InlB),the inability to form actin tails, and a virulence attenuation in vivo. We show that InlB specifically recognizes and attaches to galactosylated teichoic acid polymers, and is secreted upon loss of this modification, leading to a drastically reduced cellular invasiveness. Consequently, these phage-insensitive bacteria are unable to interact with cMet and gC1q-R host cell receptors, which normally trigger cellular uptake upon interaction with InlB. Collectively, we provide detailed mechanistic insight into the dual role of a surface antigen crucial for both phage adsorption and cellular invasiveness, demonstrating a trade-off between phage resistance and virulence in this opportunistic pathogen.

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 ß-Glucan Oxidation Products by UPLC-MS/MS.

The oxidation of cereal (1→3,1→4)-ß-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 ß-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 ß-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 ß-glucan with lichenase and ß-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 ß-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 ß-glucan's health promoting and technological properties.