Olive-Derived Antioxidant Dietary Fiber Modulates Gut Microbiota Composition and Attenuates Atopic Dermatitis Like Inflammation in Mice.

SCOPE: Epidemiological data suggest that altered gut microbiota contributes to the development of atopic dermatitis (AD). The effect of an olive-derived antioxidant dietary fiber (OADF) in relieving AD symptoms in a murine model of 2,4-dinitrofluorobenzene (DNFB)-induced AD is examined and the effect of OADF in modulating host gut microbiota is explored. METHODS AND RESULTS: Mice are fed with either standard diet or standard diet + OADF for 3 weeks prior to induction of AD and maintained on the same diet throughout the DNFB application period. Dietary OADF causes significant improvement of AD-like symptoms with reduced serum levels of immunoglobulin (Ig)E, interleukin (IL)-1ß, IL-6, C-X-C motif ligand (CXCL)1, and increased serum levels of IL-10. OADF supplementation restore gut microbiota composition that are altered in AD mice. Specifically, OADF increases the proportion of intestinal bacteria (Ruminococcaceae UCG014, GCA900066575, UBA1819) associated with enhanced butyrate production, along with inhibiting Clostridiales vadin BB60 which are more prevalent in AD mice. CONCLUSION: OADF modulates gut microbiota composition, improves cytokine profile and butyrate production influencing AD-associated immune response. Results highlight the importance of the gut-skin axis for the AD dietary therapeutic agents.

Incubation tests mimicking fermentation reveal that phytate breakdown is key to lower the cadmium concentrations in cacao nibs.

Earlier studies revealed that cadmium (Cd) concentrations in cacao nibs can decrease by a factor up to 1.3 during fermentation. Here, fermentation was mimicked by incubating beans at different temperatures, and acetic acid and ethanol concentrations in the incubation media. Nib Cd concentrations decreased during incubation by mobilisation in the nibs and subsequent outward migration to the testa and the incubation solution. This was most pronounced when high concentrations of acetic acid were combined with high temperature, while ethanol had no statistically significant effect. Incubation under typical fermentation conditions (45 °C and 20.0 g acetic acid L-1) reduced the nib Cd concentration by a factor 1.3. This factor increased to 1.6 under more extreme conditions, i.e. 65 °C and 40 g acetic acid L-1. The final nib Cd concentrations correlated well to nib phytate concentrations (R2 = 0.56), suggesting hydrolysis of phytate and mobilisation of the associated Cd2+.

Investigating the Sequence Determinants of the Curling of Amyloid Fibrils Using Ovalbumin as a Case Study.

Highly ordered, straight amyloid fibrils readily lend themselves to structure determination techniques and have therefore been extensively characterized. However, the less ordered curly fibrils remain relatively understudied, and the structural organization underlying their specific characteristics remains poorly understood. We found that the exemplary curly fibril-forming protein ovalbumin contains multiple aggregation prone regions (APRs) that form straight fibrils when isolated as peptides or when excised from the full-length protein through hydrolysis. In the context of the intact full-length protein, however, the regions separating the APRs facilitate curly fibril formation. In fact, a meta-analysis of previously reported curly fibril-forming proteins shows that their inter-APRs are significantly longer and more hydrophobic when compared to straight fibril-forming proteins, suggesting that they may cause strain in the amyloid state. Hence, inter-APRs driving curly fibril formation may not only apply to our model protein but rather constitute a more general mechanism.

Bioavailability and Health Impact of Ingested Amyloid-like Protein Fibrils and their Link with Inflammatory Status: A Need for More Research?

The use of amyloid-like protein fibrils (ALFs) in food formulations looks very promising in terms of improving techno-functional properties, but raises some concerns in terms of food safety, because of their structural resemblance to disease-related endogenous amyloids. This review focuses on the biological fate and potential health implications of ingested ALF structures in both healthy and predisposed individuals. A comprehensive overview of ALF gastrointestinal digestion, intestinal absorption, and systemic dissemination is provided, in addition to a thorough assessment of potential ALF cross-seeding of endogenous precursor proteins linked to (non)neurodegenerative amyloidosis. In general, this study concludes that the health impact of ALF consumption remains widely understudied and merits additional research efforts to determine the exact extent to which ALF ingestion may influence the general health status.

Gas cell stabilization by aqueous-phase constituents during bread production from wheat and rye dough and oat batter: Dough or batter liquor as model system.

Proper gas cell stability during fermentation and baking is essential to obtain high-quality bread. Gas cells in wheat dough are stabilized by the gluten network formed during kneading and, from the moment this network locally ruptures, by liquid films containing nonstarch polysaccharides (NSPs) and surface-active proteins and lipids. Dough liquor (DL), the supernatant after ultracentrifugation of dough, is a model system for these liquid films and has been extensively studied mostly in the context of wheat bread making. Nonwheat breads are often of lower quality (loaf volume and crumb structure) than wheat breads because their doughs/batters lack a viscoelastic wheat gluten network. Therefore, gas cell stabilization by liquid film constituents may be more important in nonwheat than in wheat bread making. This manuscript aims to review the knowledge on DL/batter liquor (BL) and its relevance for studying gas cell stabilization in wheat and nonwheat (rye and oat) bread making. To this end, the unit operations in wheat, rye, and oat bread making are described with emphasis on gas incorporation and gas cell (de)stabilization. A discussion of the knowledge on the recoveries and chemical structures of proteins, lipids, and NSPs in DLs/BLs is provided and key findings of studies dealing with foaming and air-water interfacial properties of DL/BL are discussed. Next, the extent to which DL/BL functionality can be related to bread properties is addressed. Finally, the extent to which DL/BL is a representative model system for the aqueous phase of dough/batter is discussed and related to knowledge gaps and further research opportunities.

Hydrothermal Treatments Cause Wheat Gluten-Derived Peptides to Form Amyloid-like Fibrils.

Formation of amyloid fibrils (i.e., protein structures containing a compact core of ordered ß-sheet structures) from food proteins can improve their techno-functional properties. Wheat gluten is the most consumed cereal protein by humans and extensively present in food and feed systems. Hydrolysis of wheat gluten increases the solubility of its proteins and brings new opportunities for value creation. In this study, the formation of amyloid-like fibrils (ALFs) from wheat gluten peptides (WGPs) under food relevant processing conditions was investigated. Different hydrothermal treatments were tested to maximize the formation of straight ALFs from WGPs. Thioflavin T (ThT) fluorescence measurements and transmission electron microscopy (TEM) were performed to study the extent of fibrillation and the morphology of the fibrils, respectively. First, the formation of fibrils by heating solutions of tryptic WGPs [degrees of hydrolysis 2.0% (DH 2) or 6.0% (DH 6)] was optimized using a response surface design. WGP solutions were incubated at different pH values, times, and temperatures. DH 6 WGPs had a higher propensity for fibrillation than did DH 2 WGPs. Heating DH 6 WGPs at 2.0% (w/v) for 38 h at 85 °C and pH 7.0 resulted in optimal fibrillation. Second, trypsin, chymotrypsin, thermolysin, papain, and proteinase K were used to produce different DH 6 WGPs. After enzyme inactivation and subsequent heating at optimal fibrillation conditions, chymotrypsin and proteinase K DH 6 WGPs produced small worm-like fibrils, whereas fibrils prepared from trypsin DH 6 WGPs were long and straight. The surface hydrophobicity of the peptides was key for fibrillation. Third, peptides from the wheat gluten components gliadin and glutenin fractions formed smaller and worm-like fibrils than did WGPs. Thus, the peptides of both gluten protein fractions jointly contribute to gluten fibrillation.

Processing Induced Changes in Food Proteins: Amyloid Formation during Boiling of Hen Egg White.

Amyloid fibrils (AFs) are highly ordered protein nanofibers composed of cross ß-structure that occur in nature, but that also accumulate in age-related diseases. Amyloid propensity is a generic property of proteins revealed by conditions that destabilize the native state, suggesting that food processing conditions may promote AF formation. This had only been shown for foie gras, but not in common foodstuffs. We here extracted a dense network of fibrillar proteins from commonly consumed boiled hen egg white (EW) using chemical and/or enzymatic treatments. Conversion of EW proteins into AFs during boiling was demonstrated by thioflavin T fluorescence, Congo red staining, and X-ray fiber diffraction measurements. Our data show that cooking converts approximately 1-3% of the protein in EW into AFs, suggesting that they are a common component of the human diet.

The impact of fermentation on the distribution of cadmium in cacao beans.

A large fraction of the South-American cacao production is affected by new cadmium (Cd) regulations in cacao. This work was set up to characterize the distribution and speciation of Cd within the cacao fruit and to monitor potential Cd redistribution during cacao fermentation. In cacao fruits from four locations, Cd concentrations decreased with testa > nib ~ placenta ~ pod husk > mucilage. The distribution of Cd within cacao beans was successfully visualized using laser ablation inductively coupled plasma spectrometry (LA-ICP-MS) and confirmed higher Cd concentrations in the testa than in the nib. Speciation analysis by X-ray absorption spectroscopy (XANES) of unfermented cacao beans revealed that Cd was bound to O/N-ligands in both nib and testa. Fermentation induced an outward Cd migration from the nibs to the testa, i.e. against the total concentration gradient. This migration occurred only if the fermentation was sufficiently extensive to decrease the pH in the nib to <5.0, likely as a result of increased Cd mobility due to organic acid penetration into the nibs. The change in dry weight based nib Cd concentrations during fermentation was, on average, a factor 1.3 decrease. We propose that nib Cd can be reduced if the nib pH is sufficiently acidified during fermentation. However, a balance must be found between flavor development and Cd removal since extreme acidity is detrimental for cacao flavor.

The impact of steeping, germination and hydrothermal processing of wheat (Triticum aestivum L.) grains on phytate hydrolysis and the distribution, speciation and bio-accessibility of iron and zinc elements.

Chelation of iron and zinc in wheat as phytates lowers their bio-accessibility. Steeping and germination (15 °C, 120 h) lowered phytate content from 0.96% to only 0.81% of initial dry matter. A multifactorial experiment in which (steeped/germinated) wheat was subjected to different time (2-24 h), temperature (20-80 °C) and pH (2.0-8.0) conditions showed that hydrothermal processing of germinated (15 °C, 120 h) wheat at 50 °C and pH 3.8 for 24 h reduced phytate content by 95%. X-ray absorption near-edge structure imaging showed that it indeed abolished chelation of iron to phytate. It also proved that iron was oxidized during steeping, germination and hydrothermal processing. It was further shown that zinc and iron bio-accessibility were respectively 3 and 5% in wheat and 27 and 37% in hydrothermally processed wheat. Thus, hydrothermal processing of (germinated) wheat paves the way for increasing elemental bio-accessibility in whole grain-based products.

Element distribution and iron speciation in mature wheat grains (Triticum aestivum L.) using synchrotron X-ray fluorescence microscopy mapping and X-ray absorption near-edge structure (XANES) imaging.

Several studies have suggested that the majority of iron (Fe) and zinc (Zn) in wheat grains are associated with phytate, but a nuanced approach to unravel important tissue-level variation in element speciation within the grain is lacking. Here, we present spatially resolved Fe-speciation data obtained directly from different grain tissues using the newly developed synchrotron-based technique of X-ray absorption near-edge spectroscopy imaging, coupling this with high-definition µ-X-ray fluorescence microscopy to map the co-localization of essential elements. In the aleurone, phosphorus (P) is co-localized with Fe and Zn, and X-ray absorption near-edge structure imaging confirmed that Fe is chelated by phytate in this tissue layer. In the crease tissues, Zn is also positively related to P distribution, albeit less so than in the aleurone. Speciation analysis suggests that Fe is bound to nicotianamine rather than phytate in the nucellar projection, and that more complex Fe structures may also be present. In the embryo, high Zn concentrations are present in the root and shoot primordium, co-occurring with sulfur and presumably bound to thiol groups. Overall, Fe is mainly concentrated in the scutellum and co-localized with P. This high resolution imaging and speciation analysis reveals the complexity of the physiological processes responsible for element accumulation and bioaccessibility.

A Critical Look at Prebiotics Within the Dietary Fiber Concept.

This article reviews the current knowledge of the health effects of dietary fiber and prebiotics and establishes the position of prebiotics within the broader context of dietary fiber. Although the positive health effects of specific fibers on defecation, reduction of postprandial glycemic response, and maintenance of normal blood cholesterol levels are generally accepted, other presumed health benefits of dietary fibers are still debated. There is evidence that specific dietary fibers improve the integrity of the epithelial layer of the intestines, increase the resistance against pathogenic colonization, reduce the risk of developing colorectal cancer, increase mineral absorption, and have a positive impact on the immune system, but these effects are neither generally acknowledged nor completely understood. Many of the latter effects are thought to be particularly elicited by prebiotics. Although the prebiotic concept evolved significantly during the past two decades, the line between prebiotics and nonprebiotic dietary fiber remains vague. Nevertheless, scientific evidence demonstrating the health-promoting potential of prebiotics continues to accumulate and suggests that prebiotic fibers have their rightful place in a healthy diet.

Identification of lanthionine and lysinoalanine in heat-treated wheat gliadin and bovine serum albumin using tandem mass spectrometry with higher-energy collisional dissociation.

The present manuscript reports on the identification of various dehydroamino acid-derived bonds and cross-links resulting from thermal treatment (excess water, 240 min, 130 °C) of two model food proteins, bovine serum albumin, and wheat gliadin. S-Carbamidomethylated tryptic and chymotryptic digests of unheated (control) and heated serum albumin and gliadin, respectively, were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-ESI-MS/MS) with higher-energy collisional dissociation (HCD). Heat-induced ß-elimination of cystine, serine and threonine, and subsequent Michael addition of cysteine and lysine to dehydroalanine and 3-methyl-dehydroalanine were demonstrated. Lanthionine, lysinoalanine, 3-methyl-lanthionine, and 3-methyl-lysinoalanine were identified. The detection of inter-chain lanthionine in both bovine serum albumin and wheat gliadin suggests the significance of these cross-links for food texture.

Molecular Oxygen and Reactive Oxygen Species in Bread-making Processes: Scarce, but Nevertheless Important.

In bread making, O2 is consumed by flour constituents, yeast, and, optionally, some additives optimizing dough processing and/or product quality. It plays a major role especially in the oxidation/reduction phenomena in dough, impacting gluten network structure. The O2 level is about 7.2 mmol/kg dough, of which a significant part stems from wheat flour. We speculate that O2 is quickly lost to the atmosphere during flour hydration. Later, when the gluten network structure develops, some O2 is incorporated in dough through mixing-in of air. O2 is consumed by yeast respiration and in a number of reactions catalyzed by a wide range of enzymes present or added. About 60% of the O2 consumption in yeastless dough is ascribed to oxidation of fatty acids by wheat lipoxygenase activity. In yeasted dough, about 70% of the O2 in dough is consumed by yeast and wheat lipoxygenase. This would leave only about 30% for other reactions. The severe competition between endogenous (and added) O2-consuming systems impacts the gluten network. Moreover, the scarce literature data available suggest that exogenous oxidative enzymes but not those in flour may promote crosslinking of arabinoxylan in yeastless dough. In any case, dough turns anaerobic during the first minutes of fermentation.

Formation and reshuffling of disulfide bonds in bovine serum albumin demonstrated using tandem mass spectrometry with collision-induced and electron-transfer dissociation.

Thermolysin hydrolyzates of freshly isolated, extensively stored (6 years, 6 °C, dry) and heated (60 min, 90 °C, in excess water) bovine serum albumin (BSA) samples were analyzed with liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using alternating electron-transfer dissociation (ETD) and collision-induced dissociation (CID). The positions of disulfide bonds and free thiol groups in the different samples were compared to those deduced from the crystal structure of native BSA. Results revealed non-enzymatic posttranslational modifications of cysteine during isolation, extensive dry storage, and heating. Heat-induced extractability loss of BSA was linked to the impact of protein unfolding on the involvement of specific cysteine residues in intermolecular and intramolecular thiol-disulfide interchange and thiol oxidation reactions. The here developed approach holds promise for exploring disulfide bond formation and reshuffling in various proteins under conditions relevant for chemical, biochemical, pharmaceutical and food processing.

Native and enzymatically modified wheat (Triticum aestivum L.) endogenous lipids in bread making: a focus on gas cell stabilization mechanisms.

Lipopan F and Lecitase Ultra lipases were used in straight dough bread making to study how wheat lipids affect bread loaf volume (LV) and crumb structure setting. Lipase effects on LV were dose and dough piece weight dependent. The bread quality improving mechanisms exerted by endogenous lipids were studied in terms of gluten network strengthening, which indirectly stabilizes gas cells, and in terms of direct interfacial gas cell stabilization. Unlike diacetyl tartaric esters of mono- and diacylglycerols (DATEM, used as control), lipase use did not impact dough extensibility. The effect on dough extensibility was therefore related to its lipid composition at the start of mixing. Both lipases and DATEM strongly increase the levels of polar lipids in dough liquor and their availability for and potential accumulation at gas cell interfaces. Lipases form lysolipids that emulsify other lipids. We speculate that DATEM competes with (endogenous) polar lipids for interacting with gluten proteins.

Wheat bran extract alters colonic fermentation and microbial composition, but does not affect faecal water toxicity: a randomised controlled trial in healthy subjects.

Wheat bran extract (WBE), containing arabinoxylan-oligosaccharides that are potential prebiotic substrates, has been shown to modify bacterial colonic fermentation in human subjects and to beneficially affect the development of colorectal cancer (CRC) in rats. However, it is unclear whether these changes in fermentation are able to reduce the risk of developing CRC in humans. The aim of the present study was to evaluate the effects of WBE on the markers of CRC risk in healthy volunteers, and to correlate these effects with colonic fermentation. A total of twenty healthy subjects were enrolled in a double-blind, cross-over, randomised, controlled trial in which the subjects ingested WBE (10 g/d) or placebo (maltodextrin, 10 g/d) for 3 weeks, separated by a 3-week washout period. At the end of each study period, colonic handling of NH3 was evaluated using the biomarker lactose[15N, 15N']ureide, colonic fermentation was characterised through a metabolomics approach, and the predominant microbial composition was analysed using denaturing gradient gel electrophoresis. As markers of CRC risk, faecal water genotoxicity was determined using the comet assay and faecal water cytotoxicity using a colorimetric cell viability assay. Intake of WBE induced a shift from urinary to faecal 15N excretion, indicating a stimulation of colonic bacterial activity and/or growth. Microbial analysis revealed a selective stimulation of Bifidobacterium adolescentis. In addition, WBE altered the colonic fermentation pattern and significantly reduced colonic protein fermentation compared with the run-in period. However, faecal water cytotoxicity and genotoxicity were not affected. Although intake of WBE clearly affected colonic fermentation and changed the composition of the microbiota, these changes were not associated with the changes in the markers of CRC risk.

Impact of acid and alkaline pretreatments on the molecular network of wheat gluten and on the mechanical properties of compression-molded glassy wheat gluten bioplastics.

Wheat gluten can be converted into rigid biobased materials by high-temperature compression molding at low moisture contents. During molding, a cross-linked protein network is formed. This study investigated the effect of mixing gluten with acid/alkali in 70% ethanol at ambient temperature for 16 h followed by ethanol removal, freeze-drying, and compression molding at 130 and 150 °C on network formation and on types of cross-links formed. Alkaline pretreatment (0-100 mmol/L sodium hydroxide or 25 mmol/L potassium hydroxide) strongly affected gluten cross-linking, whereas acid pretreatment (0-25 mmol/L sulfuric acid or 25 mmol/L hydrochloric acid) had limited effect on the gluten network. Molded alkaline-treated gluten showed enhanced cross-linking but also degradation when treated with high alkali concentrations, whereas acid treatment reduced gluten cross-linking. ß-Elimination of cystine and lanthionine formation occurred more pronouncedly at higher alkali concentrations. In contrast, formation of disulfide and nondisulfide cross-links during molding was hindered in acid-pretreated gluten. Bioplastic strength was higher for alkali than for acid-pretreated samples, whereas the flexural modulus was only slightly affected by either alkaline or acid pretreatment. Apparently, the ratio of disulfide to nondisulfide cross-links did not affect the mechanical properties of rigid gluten materials.

Ferulic Acid content and appearance determine the antioxidant capacity of arabinoxylanoligosaccharides.

To investigate the antioxidant capacity of ferulic acid (FA) in conjunction with prebiotic arabinoxylanoligosaccharides (AXOS), procedures for the production of FA-enriched, -depleted and cross-linked AXOS were developed, and samples were analyzed using the Trolox equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC) assays. Results showed that not only the level of FA but also the condition under which it appears (free, bound, or dimerized) impacts the antioxidant capacity of FA-containing AXOS samples. Although esterification of FA on AXOS and cross-linking of AXOS through dehydrodiferulic acid formation lowered the antioxidant capacity of FA by 30 and 55%, respectively, as determined with the TEAC test, the antioxidant capacity of these components still remained high compared to Trolox, a water-soluble vitamin E analog. Total antioxidant capacity of the AXOS samples determined by the ORAC assay resulted in less prominent differences between the different forms of FA than those seen with the TEAC test. Feruloylated AXOS can hence function as strong, water-soluble antioxidants.

Improved identification of wheat gluten proteins through alkylation of cysteine residues and peptide-based mass spectrometry.

The concentration and composition of wheat gluten proteins and the presence, concentration and location of cysteine residues therein are important for wheat flour quality. However, it is difficult to identify gluten proteins, as they are an extremely polymorphic mixture of prolamins. We here present methods for cysteine labeling of wheat prolamins with 4-vinylpyridine (4-VP) and iodoacetamide (IDAM) which, as compared to label-free analysis, substantially improve identification of cysteine-containing peptides in enzymic prolamin digests by electrospray ionization--tandem mass spectrometry. Both chymotrypsin and thermolysin yielded cysteine-containing peptides from different gluten proteins, but more proteins could be identified after chymotryptic digestion. In addition, to the best of our knowledge, we were the first to label prolamins with isotope coded affinity tags (ICAT), which are commonly used for quantitative proteomics. However, more peptides were detected after labeling gluten proteins with 4-VP and IDAM than with ICAT.

Impact of amylases on biopolymer dynamics during storage of straight-dough wheat bread.

When Bacillus stearothermophilus α-amylase (BStA), Pseudomonas saccharophila α-amylase (PSA), or Bacillus subtilis α-amylase (BSuA) was added to a bread recipe to impact bread firming, amylose crystal formation was facilitated, leading to lower initial crumb resilience. Bread loaves that best retained their quality were those obtained when BStA was used. The enzyme hindered formation of an extended starch network, resulting in less water immobilization and smaller changes in crumb firmness and resilience. BSuA led to extensive degradation of the starch network during bread storage with release of immobilized water, eventually resulting in partial structure collapse and poor crumb resilience. The most important effect of PSA was an increased bread volume, resulting in smaller changes in crumb firmness and resilience. A negative linear relation was found between NMR proton mobilities of water and biopolymers in the crumb and crumb firmness. The slope of that relation gave an indication of the strength of the starch network.