Search Results (81)

This review is dedicated to exploring recent advancements in the study of amaranth grain and presents research primarily on Amaranthus species such as Amaranthus cruentus, Amaranthus hypochondriacus, and Amaranthus caudatus, and to a lesser extent Amaranthus hybridus, Amaranthus mantegazzianus, Amaranthus muricatus, Amaranthus tuberculatus, Amaranthus viridis, Amaranthus spinosus, and Amaranthus tenuifoliu. Amaranth (Amaranthus spp.) is a promising, high-yield pseudocereal crop with significant commercial potential for developing functional food products. It contains a wide range of bioactive compounds, including squalene, tocopherols, phenolic compounds, phytates, and vitamins, which possess important physiological properties. Amaranth grain is characterized by high levels of starch, proteins, minerals, and dietary fiber. Moreover, amaranth proteins are distinguished by a balanced amino acid composition and exhibit greater resistance to external factors compared to animal-derived proteins. Grains of amaranth are free of gliadin, making it a valuable nutritional source for individuals with celiac disease, an immune-mediated disorder. Unlike traditional cereals, where prolamins and glutelins dominate the protein composition, the proteins of pseudocereals like amaranth primarily consist of albumins and globulins. The processing methods of amaranth grain influence their quantitative and qualitative composition, often significantly improving their physicochemical, antioxidant, functional, and rheological properties. This work provides a detailed analysis of amaranth’s chemical composition and bioactive components, along with its evaluation of therapeutic and preventive properties. Amaranth protein fractions (albumin, globulin, and glutelin) and squalene exhibit increased antioxidant activity, contributing to notable resistance to radiation and X-ray exposure. Bioactive compounds such as phytol, α-tocopherol, and a lunasin-like peptide (AhLun) with potential anticancer properties have also been identified in amaranth. Furthermore, six bioactive peptides were isolated and identified from amaranth, which, according to predictive models, demonstrate a high capacity to inhibit angiotensin-converting enzyme (ACE) activity, suggesting potential hypotensive effects. Certain amaranth peptides are considered promising functional food ingredients for the prevention and comprehensive treatment of conditions such as diabetes, inflammatory bowel diseases, hypercholesterolemia, cardiovascular diseases, and obesity. Amaranthus spp. and its processed products hold significant interest for the development of innovative food products, contributing to the expansion of their range and enhancement of nutritional value. Full article

Background: Gluten-related disorders, particularly celiac disease, are triggered in susceptible individuals by the toxic effects of gluten, the major storage protein of wheat grains. This toxicity can be reduced by wheat glutenases. Members of the papain-like cysteine protease family, which can act in the human gastrointestinal tract, are promising candidates for the enzymatic treatment of celiac disease. Methods: Two wheat proteases were selected using AlphaFold2, produced in recombinant forms, and characterized. Their glutenase potentials under acidic or slightly acidic conditions were evaluated and compared with the properties of the previously characterized wheat glutenase Triticain-α. Results: All enzymes tested, Ta-P7, Ta-V6, and Triticain-α, were able to hydrolyze the model substrate (α-gliadin-derived epitope) in the pH range of 3.6–7.5. Nevertheless, Triticain-α performs the most efficient hydrolysis of the peptide substrate under the conditions of the gastrointestinal tract, according to its kinetic characteristics. In the wheat gluten degradation experiment at pH 4.6 and 37 °C, both Ta-P7 and Triticain-α cleaved the mixture almost completely within 5 min. In addition, Triticain-α and Ta-P7 significantly reduced the levels of toxic peptides compared to both intact gluten and gluten treated with pepsin-trypsin digestion as tested by the Ridascreen Gliadin Kit. Conclusions: Novel wheat proteases under investigation possess the expected glutenase activity to varying degrees; however, Triticain-α is a primary candidate for potential use in the enzymatic therapy of gluten-related disorders. Full article

Gliadin is one of the most important fractions of gluten, a glycoprotein closely linked to the development of negative effects on physiological functions and the development of gastrointestinal diseases, such as celiac disease (CD). Research suggests that inadequate stress responses and anxiety states may trigger or at least contribute to the development of these pathological conditions. Peritoneal leukocytes from Prematurely Aging Mice (PAM), which are chronologically adult mice with compromised responses to stress and anxiety, exhibit functional changes when exposed in vitro to gliadin peptides, resembling some immune alterations found also in CD patients. This observation prompted us to investigate the effects of a gliadin-rich diet on immune function and redox state in PAM. In this study, adult female PAM were fed either a gluten-enriched diet (PAMD, 120 g/kg) or a standard diet (PAMC) for four weeks. Immune function parameters in peritoneal, splenic, and thymic leukocytes (phagocytosis, chemotaxis, Natural Killer activity, lymphoproliferation) and redox markers (glutathione reductase, glutathione peroxidase, reduced/oxidized glutathione, xanthine oxidase activity, lipid peroxidation) were evaluated. The results showed that PAMD exhibited more impaired immune function, lower antioxidant enzyme activities, and reduced glutathione concentrations, as well as higher oxidized glutathione and increased xanthine oxidase activity compared to PAMC. These findings suggest that a gliadin-rich diet worsens immune and redox impairments in PAM, resembling some of the alterations previously described in CD, and indicating the potential of this animal for studying gluten-induced immune dysregulation. Full article

Background: Celiac disease (CD) is an autoimmune disease that results from the interaction of genetic, immune, and environmental factors. According to the 2020 European Society for Pediatric Gastroenterology Hepatology and Nutrition (ESPGHAN) guidelines, an elimination diet (i.e., excluding products that may contain gluten) is the basic method of treating celiac disease. Following a gluten-free diet is extremely problematic, and patients often make unconscious deviations from the diet. According to the current Oslo definitions for celiac disease, depending on the clinical picture and adequate tests, several forms of celiac disease have been identified: typical, atypical, asymptomatic, potential, and refractory. Objective: The aim of the study was to assess the frequency of conscious diet mistakes and unconscious deviations from a gluten-free diet in a group of patients with long-standing celiac disease and their impact on the frequency of typical and atypical symptoms. Methods: The study included 57 people diagnosed with celiac disease between 1980 and 2010. After verifying the history of the disease according to the ESPGHAN guidelines from 2020, we excluded 19 patients who had Marsh grade 1 at the time of diagnosis or those without HLA DQ2 or DQ8 haplotypes detected. After verification, the study included 38 patients, 30 women and 8 men, with a verified diagnosis of typical celiac disease. The effectiveness of the gluten-free diet was assessed in all participants. Blood was collected to determine IgA anti-tissue transglutaminase II antibodies (anti-tTG) and IgG antibodies against deamidated gliadin peptides by ELISA. All survey participants provided data concerning current gastrointestinal and systemic symptoms, bowel habits, comorbidities, dietary habits, physical activity, and socioeconomic conditions. Results: A total of 25 patients (65.78%) declared strict adherence to the gluten-free diet. However, in this group, seven (18.4%) patients had significantly increased levels of anti-tTG antibodies (mean 82.3 RU/mL ± 78.9 SD at N < 20 RU/mL). Among the patients who consciously made dietary mistakes, six (46.2%) demonstrated increased levels of anti-tTG antibodies. The analysis did not reveal any difference between the frequency of intestinal and extraintestinal symptoms in patients making dietary mistakes and following the gluten-free diet. Conclusions: More than half of celiac patients unconsciously or consciously make dietary mistakes, which indicates an urgent need to increase their general knowledge of CD and the appropriate diet. Regardless of whether the gluten-free diet is followed, both typical and atypical symptoms of the disease have been observed among celiac patients. Full article

Alzheimer’s disease (AD), a leading neurodegenerative disorder, is closely associated with the accumulation of amyloid-beta (Aβ) peptides in the brain. The enzyme β-secretase (BACE1), pivotal in Aβ production, represents a promising therapeutic target for AD. While bioactive peptides derived from food protein hydrolysates have neuroprotective properties, their inhibitory effects on BACE1 remain largely unexplored. In this study, we evaluated the inhibitory potential of protein hydrolysates from gliadin, whey, and casein proteins prepared using bromelain, papain, and thermolysin. Through in vitro and cellular assays, bromelain-hydrolyzed gliadin (G-Bro) emerged as the most potent BACE1 inhibitor, with an IC₅₀ of 0.408 mg/mL. G-Bro significantly reduced BACE1 expression and amyloid precursor protein (APP) processing in N2a/PS/APP cell cultures, suggesting its potential to attenuate Aβ aggregation. The unique peptide profile of G-Bro likely contributes to its inhibitory effect, with proline residues disrupting β-sheets, lysine residues introducing positive charges that hinder aggregation, hydrophobic residues stabilizing binding interactions, and glutamine residues enhancing solubility and stability. These findings highlight gliadin hydrolysates, particularly G-Bro, as potential natural BACE1 inhibitors with applications in dietary interventions for AD prevention. However, further studies are warranted to elucidate specific peptide interactions and their bioactivity in neural pathways to better understand their therapeutic potential. Full article

Sorghum is a self-pollinating species belonging to the Poaceae family characterized by a resistance to drought higher than that of corn. Sorghum (Sorghum bicolor L. Moench) has been grown for centuries as a food crop in tropical areas where it has an increasing importance, particularly as a cereal option for people with celiac disease. Over the past fifty years, food-grade varieties and hybrid seeds with white pericarp have been developed, particularly in the United States, to maximize sorghum food quality. Nutrient composition, including moisture, protein, carbohydrates, dietary fiber, fat content, fatty acid composition, and mineral content, was determined for nine inbred varieties with a stabilized food-grade sorghum genotype selected in the USA and grown under typical Mediterranean conditions. Differences in these nutritional components were observed among the varieties considered. Notable differences were found for monounsaturated and polyunsaturated fats, while saturated fatty acids were similar in all varieties. Oleic, linoleic, and palmitic acids were the most abundant fatty acids in all nine lines. Differences were also noted in mineral content, particularly for K, Mg, Al, Mn, Fe, Cu, Zn, and Ba. Enzyme-linked immunosorbent assays (ELISAs) demonstrated the absence of gliadin-like peptides in all the sorghum varieties analyzed, confirming, thus, that these analyzed varieties are safe for consumption by celiac patients. Knowledge of the nutritional values of sorghum lines is relevant for breeding programs devoted to sorghum nutritional content and for beneficial properties to human health. Full article

Immunoreactive gluten peptides that are not digested by peptidases produced by humans can trigger celiac disease, allergy and non-celiac gluten hypersensitivity. The aim of this study was to evaluate the ability of selected probiotic strains to hydrolyze immunoreactive gliadin peptides and to identify peptidase-encoding genes in the genomes of the most efficient strains. Residual gliadin immunoreactivity was measured after one- or two-step hydrolysis using commercial enzymes and bacterial peptidase preparations by G12 and R5 immunoenzymatic assays. Peptidase preparations from Lacticaseibacillus casei LC130, Lacticaseibacillus paracasei LPC100 and Streptococcus thermophilus ST250 strains significantly reduced the immunoreactivity of gliadin peptides, including 33-mer, and this effect was markedly higher when a mixture of these strains was used. In silico genome analyses of L. casei LC130 and L. paracasei LPC100 revealed the presence of genes encoding peptidases with the potential to hydrolyze bonds in proline-rich peptides. This suggests that L. casei LC130, L. paracasei LPC100 and S. thermophilus ST250, especially when used as a mixture, have the ability to hydrolyze immunoreactive gliadin peptides and could be administered to patients on a restricted gluten-free diet to help treat gluten-related diseases. Full article

The diagnosis of celiac disease (CD) is complex and requires a multi-step procedure (symptoms, serology, duodenal biopsy, effect of a gluten-free diet, and optional genetic). The aim of the study was to contribute to the improvement of CD diagnosis by preparing a water-soluble gluten peptide fraction (called Solgluten) and by selecting gluten-specific enzyme-linked immunosorbent assays (ELISA) for the detection of gluten immunogenic gluten peptides (GIPs) in urine and blood serum spiked with Solgluten. Food-grade Solgluten was prepared by the extraction of a peptic digest of vital gluten with water, centrifugation, and freeze-drying. The process was relatively easy, repeatable, and cheap. The content of gliadin-derived GIPs was 491 mg/g. Solgluten was used as antigenic material to compare two competitive ELISA kits (R7021 and K3012) and two sandwich ELISA kits (M2114 and R7041) in their quality regarding the quantitation of GIPs in urine and blood serum. The quality parameters were the reactivity, sensitivity, coefficients of variation and determination, and curve shape. The evaluation of the kits showed a number of discrepancies in individual quality parameters measured in urine and serum. Due to the lowest limit of quantitation and the highest coefficient of determination, M2114 may be the first choice, while R7021 appeared to be less suitable because of the high coefficients of variation and unfavorable curve progression. The results set the stage for improving CD diagnosis by supplementing conventional blood tests with oral provocation with Solgluten and subsequent ELISA measurement of GIPs that could support the no-biopsy approach and by better assessing the effect of a gluten-free diet by monitoring adherence to the diet by measuring GIPs in urine and blood. Full article

Serologic measures of tissue transglutaminase (tTG) immunoglobulin A (IgA) and deamidated gliadin peptide (DGP) IgA and immunoglobulin G (IgG) are hallmark tests utilized when diagnosing individuals for celiac disease (CeD) and for monitoring adherence to a gluten-free diet (GFD), currently the only available treatment for CeD. We address two issues in this study: (i) the relapse to seropositivity for CeD patients who resume a gluten containing diet and (ii) the correlation between two different tTG-IgA assays near the upper limit of normal (ULN) designated thresholds. Regarding the first issue, often a suspected CeD individual is put back on a gluten diet to return to their serologic levels. However, we show it requires a substantial amount of gluten for serology to return to a positive level. For example, in one study of 22 patients treated with placebo and taking 84 g of gluten over 6 weeks, only two converted from seronegative to seropositive for tTG-IgA. Regarding the second topic, we compare the relationship for different serologic assays, namely tTG-IgA AB (recombinant, ULN = 4 units/mL) vs. tTG-IgA (non-recombinant, ULN = 20 units). There is a strong correlation between both measurements as evidenced by a Pearson coefficient of R = 0.8584; however, we observed that the cross-correlation in terms of sensitivity and specificity improved substantially by using an ULN value of three instead of four for the tTG-IgA AB (recombinant) assay. This result suggests that assay thresholds used for initial diagnosis in patients who have not yet started a GFD may need to be adjusted for monitoring and in the setting of a diagnostic gluten challenge. Full article

Immunogenic peptides from wheat gluten can be produced during digestion, which are difficult to digest by gastrointestinal proteases and negatively affect immune responses in humans. Gluten intolerance is a problem in countries where wheat is a staple food, and a gluten-free diet is commonly recommended for its treatment and prevention. Enzyme approaches for degradation of the peptides can be considered as a strategy for its prevention. Here, we isolated a gluten-degrading bacterium, Bacillus amyloliquefaciens subsp. plantarum, from wheat grains. The culture conditions for enzyme production or microbial use were considered based on gluten decomposition patterns. Additionally, the pH range for the activity of the crude enzyme was investigated. The bacterium production of gluten-degrading enzymes was temperature-dependent within 25 °C to 45 °C, and the production time decreased with increasing culture temperature. However, it was markedly decreased with increasing biofilm formation. The bacterium decomposed high-molecular-weight glutenin proteins first, followed by gliadin proteins, regardless of the culture temperature. Western blotting with an anti-gliadin antibody revealed that the bacterium decomposed immunogenic proteins related to α/β-gliadins. The crude enzyme was active in the pH ranges of 5 to 8, and enzyme production was increased by adding gliadin into the culture medium. In this study, the potential of the B. amyloliquefaciens subsp. plantarum for gluten-degrading enzyme production was demonstrated. If further studies for purification of the enzyme specific to the immunogenic peptides and its characteristics are conducted, it may contribute as a strategy for prevention of gluten intolerance. Full article

Roughly 1% of the global population is susceptible to celiac disease (CD)—inheritable autoimmune inflammation of the small intestine caused by intolerance to gliadin proteins present in wheat, rye, and barley grains, and called gluten in wheat. Classical treatment is a life-long gluten-free diet, which is constraining and costly. An alternative approach is based upon the development and oral reception of effective peptidases that degrade in the stomach immunogenic proline- and glutamine-rich gliadin peptides, which are the cause of the severe reaction in the intestine. In previous research, we have established that the major digestive peptidase of an insect Tribolium castaneum—cathepsin L—hydrolyzes immunogenic prolamins after Gln residues but is unstable in the extremely acidic environment (pH 2–4) of the human stomach and cannot be used as a digestive aid. In this work, using molecular dynamics simulations, we discover the probable cause of the pH instability of cathepsin L—loss of the catalytically competent rotameric state of one of the active site residues, His 275. To “fix” the correct orientation of this residue, we designed a V277A mutant variant, which extends the range of stability of the peptidase in the acidic environment while retaining most of its activity. We suggest this protein as a lead glutenase for the development of oral medical preparation that fights CD and gluten intolerance in susceptible people. Full article

The enzyme transglutaminase 2 (TG2) plays a key role in celiac disease (CeD) pathogenesis. Active TG2 is located mainly extracellularly in the lamina propria but also in the villous enterocytes of the duodenum. The TG2 inhibitor ZED1227 is a promising drug candidate for treating CeD and is designed to block the TG2-catalyzed deamidation and crosslinking of gliadin peptides. Our aim was to study the accumulation of ZED1227 after oral administration of the drug. We studied duodenal biopsies derived from a phase 2a clinical drug trial using an antibody that detects ZED1227 when bound to the catalytic center of TG2. Human epithelial organoids were studied in vitro for the effect of ZED1227 on the activity of TG2 using the 5-biotin-pentylamine assay. The ZED1227-TG2 complex was found mainly in the villous enterocytes in post-treatment biopsies. The signal of ZED1227-TG2 was strongest in the luminal epithelial brush border, while the intensity of the signal in the lamina propria was only ~20% of that in the villous enterocytes. No signal specific to ZED1227 could be detected in pretreatment biopsies or in biopsies from patients randomized to the placebo treatment arm. ZED1227-TG2 staining co-localized with total TG2 and native and deamidated gliadin peptides on the enterocyte luminal surface. Inhibition of TG2 activity by ZED1227 was demonstrated in epithelial organoids. Our findings suggest that active TG2 is present at the luminal side of the villous epithelium and that inhibition of TG2 activity by ZED1227 occurs already there before gliadin peptides enter the lamina propria. Full article

Background: Non-celiac wheat sensitivity (NCWS) is a poorly understood gluten-related disorder (GRD) and its prominent symptoms can be ameliorated by gluten avoidance. This study aimed to determine the effectiveness of a probiotic mixture in hydrolyzing gliadin peptides (toxic components of gluten) and suppressing gliadin-induced inflammatory responses in Caco-2 cells. Methods: Wheat dough was fermented with a probiotic mix for 0, 2, 4, and 6 h. The effect of the probiotic mix on gliadin degradation was monitored by SDS-PAGE. The expression levels of IL-6, IL-17A, INF-γ, IL-10, and TGF-β were evaluated using ELISA and qRT-PCR methods. Results: According to our findings, fermenting wheat dough with a mix of B. longum, L. acidophilus, and L. plantarum for 6 h was effective in gliadin degradation. This process also reduced levels of IL-6 (p = 0.004), IL-17A (p = 0.004), and IFN-γ (p = 0.01) mRNA, as well as decreased IL-6 (p = 0.006) and IFN-γ (p = 0.0009) protein secretion. 4 h fermentation led to a significant decrease in IL-17A (p = 0.001) and IFN-γ (p = 0.003) mRNA, as well as reduced levels of IL-6 (p = 0.002) and IFN-γ (p < 0.0001) protein secretion. This process was also observed to increase the expression levels of IL-10 (p < 0.0001) and TGF-β (p < 0.0001) mRNA. Conclusions: 4 h fermentation of wheat flour with the proposed probiotic mix might be a good strategy to develop an affordable gluten-free wheat dough for NCWS and probably other GRD patients. Full article

Celiac disease (CD) is a common systemic disorder that results from an abnormal response of human immunity to gluten intake, affecting the small intestine. In individuals who carry a genetic susceptibility, CD is triggered by environmental factors, including viral infections and dysbiosis of the gut microbiota. The gut microbiome is essential in controlling the immune system, and recent findings indicate that changes in the gut microbiome may contribute to various chronic immune disorders, such as CD through mechanisms that still require further exploration. Some bacteria exhibit epitopes that mimic gliadin and may enhance an immune response in the host. Other bacteria, including Pseudomonas aeruginosa, may work in conjunction with gluten to trigger and escalate intestinal inflammation. The microbiota may also directly influence antigen development through the production of immunogenic or tolerogenic gluten peptides or directly influence intestinal permeability through the release of zonulin. Finally, the gut microbiome can impact intestinal inflammation by generating proinflammatory or anti-inflammatory cytokines and metabolites. It is crucial to consider the impact of genetic factors (specifically, HLA-DQ haplotypes), perinatal elements such as birth mode, type of infant feeding, and antibiotic and infection exposure on the composition of the early intestinal microbiome. According to the available studies, the gut microbiome alterations associated with CD tend to exhibit a decreased presence of beneficial bacteria, including some anti-inflammatory Bifidobacterium species. However, some controversy remains as some reports have found no significant differences between the gut microbiomes of individuals with and without CD. A better understanding of the gut microbiome’s role in the development of CD would greatly benefit both prevention and treatment efforts, especially in complicated or treatment-resistant cases. Here, we have attempted to summarize the available evidence on the relationship between the gut microbiota and CD, with a particular focus on potential therapeutic targets. Full article

Enzymatic modification of gliadin peptides by human transglutaminase 2 (TG2) is a key mechanism in the pathogenesis of celiac disease (CD) and represents a potential therapeutic target. Recently, we have identified the small oxidative molecule PX-12 as an effective inhibitor of TG2 in vitro. In this study, we further investigated the effect of PX-12 and the established active-site directed inhibitor ERW1041 on TG2 activity and epithelial transport of gliadin peptides. We analyzed TG2 activity using immobilized TG2, Caco-2 cell lysates, confluent Caco-2 cell monolayers and duodenal biopsies from CD patients. TG2-mediated cross-linking of pepsin-/trypsin-digested gliadin (PTG) and 5BP (5-biotinamidopentylamine) was quantified by colorimetry, fluorometry and confocal microscopy. Cell viability was tested with a resazurin-based fluorometric assay. Epithelial transport of promofluor-conjugated gliadin peptides P31-43 and P56-88 was analyzed by fluorometry and confocal microscopy. PX-12 reduced TG2-mediated cross-linking of PTG and was significantly more effective than ERW1041 (10 µM, 15 ± 3 vs. 48 ± 8%, p < 0.001). In addition, PX-12 inhibited TG2 in cell lysates obtained from Caco-2 cells more than ERW1041 (10 µM; 12 ± 7% vs. 45 ± 19%, p < 0.05). Both substances inhibited TG2 comparably in the intestinal lamina propria of duodenal biopsies (100 µM, 25 ± 13% vs. 22 ± 11%). However, PX-12 did not inhibit TG2 in confluent Caco-2 cells, whereas ERW1041 showed a dose-dependent effect. Similarly, epithelial transport of P56-88 was inhibited by ERW1041, but not by PX-12. Cell viability was not negatively affected by either substance at concentrations up to 100 µM. PX-12 did not reduce TG2 activity or gliadin peptide transport in confluent Caco-2 cells. This could be caused by rapid inactivation or degradation of the substance in the Caco-2 cell culture. Still, our in vitro data underline the potential of the oxidative inhibition of TG2. The fact that the TG2-specific inhibitor ERW1041 reduced the epithelial uptake of P56-88 in Caco-2 cells further strengthens the therapeutic potential of TG2 inhibitors in CD. Full article