Gluten Degradation Research Articles

Changes of gluten protein composition during sourdough fermentation in rye flour

AbstractBackground and ObjectivesCeliac disease (CD) is an immune‐mediated condition triggered by gluten consumption. Although more studies stated that gluten proteins are partially degrading during sourdough fermentation, it is still unclear whether the CD toxic epitopes are affected. Our aim was to examine the effect of controlled sourdough fermentation on the gluten content and composition with different sourdough starters in rye flour.FindingsDespite significant changes in protein size distribution and a decrease in secalin types confirmed by high‐performance liquid chromatography methods, the enzyme‐linked immunosorbent assay results showed increased gluten content after fermentation. The incomplete degradation of gluten and the potential resistance of celiac toxic epitopes may explain our results. As part of the R5 antibody binding, epitopes are supposed to be unavailable in the native form of secalins; they might be released and become accessible in the smaller protein fragments.ConclusionsIn comparison of the effect of different starter cultures, the results show that the different microbiological composition of the sourdough samples might be the reason for the differences in the protein degradation.Significance and NoveltyResults highlight the importance of a deeper investigation of the effects of gluten protein degradation on CD toxicity and the standardization of the fermentation process.

Effect of NaCl on heat-denatured wheat gluten in freeze-thaw cycles: Physicochemical properties and molecular structure

Heat-denatured wheat gluten (HDWG) degradation during freeze-thaw (FT) cycling is a key factor in the quality deterioration of pre-cooked noodles (PCNs). This study investigated the effects and underlying mechanisms of sodium chloride (NaCl) on HDWG. Adding NaCl enhanced the luminance of PCNs and reduced chromatic aberration. Scanning electron microscopy revealed that NaCl facilitated a more continuous microstructure, resulting in a reduction the average hole size of HDWG from 84.44 μm to 56.59 μm after 10 FT cycles. With the FT cycling, gluten macropolymer (GMP) depolymerization occurs, causing the collapse of HDWG network structure. However, NaCl contributed to the formation of β-sheet and gauche-gauche-gauche (g-g-g) conformations, resulting in a stronger three-dimensional structure of HDWG, thus alleviating GMP depolymerization. The analysis of intermolecular interactions shows that NaCl primarily affects hydrogen bonds, disulfide bonds, and hydrophobic interactions, and stabilizes the structural integrity of HDWG by balancing these forces. NaCl altered the three-dimensional structural composition of HDWG by influencing the intermolecular interactions, thereby fortifying the PCNs against FT cycling. This study provides a robust theoretical basis for using NaCl to regulate the quality of thermally precooked flour products.

Assessment of Acrylamide Levels and Evaluation of Physical Attributes in Bread Made with Sourdough and Prolonged Fermentation

Over the past decades, there has been an increasing awareness regarding acrylamide content in cooked foods. The use of natural, homemade, organic and ecological ingredients has been promoted in our society as a strategy to combat the increase in cancer ratios. In this work, the use of sourdough and prolonged fermentation as a mitigation of acrylamide formation in bread were studied. In addition, to accomplish the required standards of industrial production, the dimension and acidity were also assessed. For this purpose, samples were elaborated using diverse fermentation times (0, 60, 120, 180, 240, 300 and 360 minutes) of the dough before baking and different sourdough percentages (0, 20 and 50%). High-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method for acrylamide determination in bread crust was applied. We could corroborate that a decrease in acrylamide levels was observed with fermentation time for samples containing 0 and 20% sourdough, being sharper the decrease for the latter. However, the samples containing 50% sourdough showed a quite different behaviour depending on the fermentation time interval. The physical study revealed that dimension of the pieces increased with fermentation time and sourdough content, until the excessive gluten degradation and acidification of the medium led to a flattening and decrease in elevation of the samples. This study revealed that the samples containing a percentage of 20% sourdough maintained the proper quality of gluten for longer fermentation times than the 50% sourdough samples, delaying the flattening of the samples with fermentation time. Statistical calculations were accomplished in order to elucidate the correlations between the different variables, observing that the titratable index was associated with the fermentation time, the sourdough content, the bulk density and the acrylamide content.

Structural and time-resolved mechanistic investigations of protein hydrolysis by the acidic proline-specific endoprotease from Aspergillus niger.

Proline-specific endoproteases have been successfully used in, for example, the in-situ degradation of gluten, the hydrolysis of bitter peptides, the reduction of haze during beer production, and the generation of peptides for mass spectroscopy and proteomics applications. Here we present the crystal structure of the extracellular proline-specific endoprotease from Aspergillus niger (AnPEP), a member of the S28 peptidase family with rarely observed true proline-specific endoprotease activity. Family S28 proteases have a conventional Ser-Asp-His catalytic triad, but their oxyanion-stabilizing hole shows a glutamic acid, an amino acid not previously observed in this role. Since these enzymes have an acidic pH optimum, the presence of a glutamic acid in the oxyanion hole may confine their activity to an acidic pH. Yet, considering the presence of the conventional catalytic triad, it is remarkable that the A. niger enzyme remains active down to pH 1.5. The determination of the primary cleavage site of cytochrome c along with molecular dynamics-assisted docking studies indicate that the active site pocket of AnPEP can accommodate a reverse turn of approximately 12 amino acids with proline at the S1 specificity pocket. Comparison with the structures of two S28-proline-specific exopeptidases reveals not only a more spacious active site cavity but also the absence of any putative binding sites for amino- and carboxyl-terminal residues as observed in the exopeptidases, explaining AnPEP's observed endoprotease activity.

A gluten degrading probiotic Bacillus subtilis LZU-GM relieve adverse effect of gluten additive food and balances gut microbiota in mice

Gluten accumulation damages the proximal small intestine and causes celiac disease (CeD) which has not been effectively treated except by using a gluten-free diet. In this study, strain Bacillus subtilis LZU-GM was isolated from Pakistani traditional fermented sourdough and could degrade 73.7% of gluten in 24 h in vitro. Strain LZU-GM was employed for practical application to investigate gluten degradation in mice models. The results showed that strain LZU-GM was colonized in mice and the survival rate was around 0.95 % (P < 0.0001). The gluten degradation was 3-fold higher in the small intestine of the strain LZU-GM treated mice group remaining 1511.96 ng/mL of gluten peptides than the untreated mice group (6500.38 ng/mL). Immunochemical analysis showed that gluten-treated mice established positive antigliadin antibodies (AGA) in serum (IgA, IgG, and anti-TG2 antibodies) as compared to the strain LZU-GM treatment group. Furthermore, the number of IFN-γ, TNF-α, IL-10, and COX-2 cells decrease in the lamina propria of the strain LZU-GM treatment group (P < 0.0001). Microbial community bar plot analysis showed that Lactobacillus, Dubosiella, and Enterococcus genera were restored and stabilized in the LZU-GM treatment group while Blautia and Ruminococcus were found lower. The oral gavage of probiotic strain LZU-GM might be useful for gluten metabolism in the intestine during digestion and would be a long-term dietary treatment for CeD management.

Gluten Degradation by the Gut Microbiota of Ulcerative Colitis Patients.

Several studies have reported improved disease symptomatology in ulcerative colitis (UC) patients consuming a gluten free diet. This observation coupled with diversity depletion in the gut microbiota of UC patients led us to hypothesize that UC-associated enteric microbes differentially metabolize dietary gluten to produce immunogenic products that promote inflammation. Gluten concentration in stool was determined using gluten-specific ELISA, and gluten intake was assessed by food frequency questionnaire (FFQ) in UC (n = 12) and healthy controls (HC; n = 13). Gluten-metabolizing bacteria were isolated on minimal media supplemented with 1% gluten from UC and HC and identified by 16S rRNA profiling. Cell-free culture media from gluten metabolizing gut bacterial isolates was assessed for immunogenicity in vitro using HT29 colonocytes. Compared to HC, UC patients did not consume gluten differently (Mann-Whitney; p &gt; 0.10) and exhibited equivalent levels of gluten in their feces (Mann-Whitney; p = 0.163). The profile of gluten-degrading bacteria isolated from UC stool was distinct (Chi-square; p ≤ 0.0001). Compared with Enterococcus isolates, products of gluten degradation by Bacillus strains induced higher IL8 and lower occludin (Mann-Whitney; p = 0.002 and p = 0.059, respectively) gene expression in colonocytes irrespective of whether they originated from UC or healthy gut. Members of HC and UC microbiota exhibit gluten-degrading ability, metabolites of which influence genes involved in inflammation and barrier function in enteric colonocyte cultures. Preliminary findings of this study warrant further investigations into the mechanisms by which gut microbiota contribute to UC pathogenesis through gluten degradation.

A short update on new approaches to celiac disease.

Celiac disease is an autoimmune enteropathy of the small intestine, related to gluten intolerance occurring in genetically predisposed patients. Currently, the only available treatment is a lifelong gluten-free diet. However, the total avoidance of gluten is difficult and poses a challenge to patients, nutritionists and treating physicians. For this reason, scientists have developed in recent years new therapeutic approaches complementary to dietary treatment, such as modification of gluten to make gliadin non-toxic, reduction of the inflammatory response with elafin and Lactococcus Lactis, degradation of gluten by endoproteolytic enzymes, and correction of nutritional deficiencies by adding pseudo-cereals to the diet of celiac patients. This literature review focuses on the different treatment strategies for celiac disease previously studied and summarizes the latest advances in this field.

Wheat Glu-A1a encoded 1Ax1 subunit enhances gluten physicochemical properties and molecular structures that confer superior breadmaking quality

Wheat gluten proteins serve as the largest protein molecules in nature and play key roles in breadmaking quality formation. In this study, we used a pair of Glu-A1 allelic variation lines to perform a comprehensive investigation on the effects of Glu-A1a encoded 1Ax1 subunit on gluten physicochemical properties, molecular structures and breadmaking quality. The results showed that the presence of the 1Ax1 subunit significantly increased gluten content, leading to marked improvement of dough rheological properties. Meanwhile, gluten physicochemical properties such as foaming ability and foaming stability, oil/water-holding capacity, emulsifying activity, disulfide bond content, and gluten degradation temperature were significantly improved. A confocal laser scanning microscope analysis revealed that the 1Ax1 subunit drastically enhanced gluten microstructure. Gluten secondary structure analysis by Fourier transform infrared spectroscopy and laser scanning microscope-Raman spectroscopy indicated that 1Ax1 subunit significantly promoted β-turn and β-sheet content and reduced α-helix content. Three-dimensional structure analysis by AlphaFold2 revealed a similar structural feature of 1Ax1 with the superior quality subunit 1Ax2*. Correlation and principal component analyses demonstrated that α-helix and β-sheet content had a significant correlation with dough rheological properties, gluten physicochemical properties and breadmaking quality. Our results showed that 1Ax1 subunit positively affected gluten molecular structure and quality formation.

Celiac Disease and Possible Dietary Interventions: From Enzymes and Probiotics to Postbiotics and Viruses

Celiac Disease (CeD) is a chronic small intestinal immune-mediated enteropathy caused by the ingestion of dietary gluten proteins in genetically susceptible individuals. CeD is one of the most common autoimmune diseases, affecting around 1.4% of the population globally. To date, the only acceptable treatment for CeD is strict, lifelong adherence to a gluten-free diet (GFD). However, in some cases, GFD does not alter gluten-induced symptoms. In addition, strict adherence to a GFD reduces patients’ quality of life and is often a socio-economic burden. This narrative review offers an interdisciplinary overview of CeD pathomechanism and the limitations of GFD, focusing on current research on possible dietary interventions. It concentrates on the recent research on the degradation of gluten through enzymes, the modulation of the microbiome, and the different types of “biotics” strategies, from probiotics to the less explored “viromebiotics” as possible beneficial complementary interventions for CeD management. The final aim is to set the context for future research that may consider the role of gluten proteins and the microbiome in nutritional and non-pharmacological interventions for CeD beyond the sole use of the GFD.

Mouse feeding study and microbiome analysis of sourdough bread for evaluation of its health effects.

Sourdough bread fermented with yeast and lactic acid bacteria (LAB) is thought to have various beneficial health effects. However, its beneficial effects were not fully evaluated with in vivo mouse model. To evaluate these effects in vivo, a mouse feeding study and microbiome analysis of white bread containing 40% sourdough (WBS) and yeast-leavened white bread (WB) were performed. Although feed consumption and body weight increased with WBS, the glycemic index was reduced, suggesting a diabetes-lowering effect, probably due to the presence of dietary fiber and short-chain fatty acids (SCFA). In addition, a mineral absorption test showed that WBS increased magnesium absorption owing to phytate degradation during fermentation. Interestingly, WBS decreased total cholesterol and triglycerides, probably due to the dietary fiber and SCFA in LAB. In addition, the ratio of low- and high-density lipoprotein was decreased in WBS, implying potential risk reduction for cardiovascular disease. An immunomodulatory assay of WBS revealed that pro-inflammatory cytokines TNF-α and IL-6 were decreased, suggesting anti-inflammatory activity. Gluten degradation by fermentation and antioxidation activity of menaquinol/ubiquinol by gut microbiota also supported the anti-inflammatory activity of sourdough bread. Furthermore, some beneficial gut bacteria, including Akkermansia, Bifidobacterium, and Lactobacillus, were increased in WBS. In particular, Akkermansia has been associated with anti-inflammatory properties. Consequently, WBS has beneficial effects on health, including decreased glycemic index and cholesterol, increased mineral availability and absorption, anti-inflammatory properties, and establishment of healthy gut microbiota.

Digestion of protein and toxic gluten peptides in wheat bread, pasta and cereal and the effect of a supplemental enzyme mix.

There has been an increasing interest in the relationship between wheat digestibility and potential toxicity to the host. However, there is a lack of understanding about temporal profile of digestion of wheat proteins from different food matrices under physiologically relevant conditions. In this study, digestion of three wheat-based foods (bread, pasta and cereal) was conducted based on the INFOGEST semi-dynamic protocol in the absence and presence of a commercial supplemental enzyme preparation (a Glutalytic® based supplement, which will be marketed as Elevase®). Protein hydrolysis (OPA- ortho-phthalaldehyde - assay), molecular weight distribution (SEC-HPLC) and potential toxicity (R5 antibody-based competitive ELISA), were assessed. Our results demonstrated that under normal conditions, the complexity of the food influenced the temporal profile of protein hydrolysis and gluten breakdown throughout simulated gastric and intestinal digestion. However, treatment with the enzyme supplement significantly and acutely increased protein hydrolysis and gluten degradation in the gastric stage, and this enhanced digestion was maintained into the intestinal environment. These findings highlight the limitations of temporal gastric proteolysis and gluten degradation under normal conditions to different food types. They also show that supplemental enzyme mixes can effectively accelerate the breakdown of protein and hydrolysis of toxic gliadin fractions from the early stages of gastric digestion, thereby reducing intestinal exposure and potentially limiting the sensitization of the host.

Biogeographic Variation and Functional Pathways of the Gut Microbiota in Celiac Disease

Genes and gluten are necessary but insufficient to cause celiac disease (CeD). Altered gut microbiota has been implicated as an additional risk factor. Variability in sampling site may confound interpretation and mechanistic insight, as CeD primarily affects the small intestine. Thus, we characterized CeD microbiota along the duodenum and in feces and verified functional impact in gnotobiotic mice. We used 16S rRNA gene sequencing (Illumina) and predicted gene function (PICRUSt2) in duodenal biopsies (D1, D2 and D3), aspirates, and stool from patients with active CeD and controls. CeD alleles were determined in consented participants. A subset of duodenal samples stratified according to similar CeD risk genotypes (controls DQ2-/- or DQ2+/- and CeD DQ2+/-) were used for further analysis and to colonize germ-free mice for gluten metabolism studies. Microbiota composition and predicted function in CeD was largely determined by intestinal location. In the duodenum, but not stool, there was higher abundance of Escherichia coli (D1), Prevotella salivae (D2), and Neisseria (D3) in CeD vs controls. Predicted bacterial protease and peptidase genes were altered in CeD and impaired gluten degradation was detected only in mice colonized with CeD microbiota. Our results showed luminal and mucosal microbial niches along the gut in CeD. We identified novel microbial proteolytic pathways involved in gluten detoxification that are impaired in CeD but not in controls carrying DQ2, suggesting an association with active duodenal inflammation. Sampling site should be considered a confounding factor in microbiome studies in CeD.

Rising stars in the bakery: novel yeasts for modern bread

Bread is a widely consumed fermented food whose taste, aroma, and texture are partly determined by the choice of microbe(s) employed in dough fermentation. Consumer preferences and dietary considerations are currently changing; in addition to a desire for novel, complex flavour profiles, foods low in gluten and fermentable oligo-, di-, monosaccharides and polyols (FODMAPs) are becoming increasingly important. The potential of non-conventional yeasts to improve and diversify key aspects of breadmaking is highlighted in this mini-review. Researchers have investigated species from the genera Kazachstania, Kluyveromyces, Lachancea, Pichia, Torulaspora, and Wickerhamomyces to this end. Some species have demonstrated comparable leavening capacity to baker’s yeast, as well as improved tolerance of baking-related stresses such as high salt and low pH conditions. Others have demonstrated valuable functional properties permitting the degradation of gluten and FODMAPs. Future research directions include the establishment of safe use status and the improvement of novel yeasts’ baking traits through techniques such as evolutionary engineering.

Investigating the Effect of Cloned AN-PEP Enzyme in Yeast on Gluten Degradation and Rheological Features of Dough

Investigating the Effect of Cloned AN-PEP Enzyme in Yeast on Gluten Degradation and Rheological Features of Dough

Comparison of the Effects of Native and Commercial Probiotic on Gluten Degradation

Background and Objectives: The main treatment for celiac disease is a gluten-free diet. Gluten infection occurs in 55%-32% of these patients, which can cause symptoms in these patients. Many patients with celiac disease are dissatisfied with a gluten-free diet and are interested in a non-dietary alternative treatment. Methods: In this study, probiotic samples of local dairy (milk, yogurt, and cheese) from Iran were collected in sterile conditions and after enzymatic extraction using a lysis buffer and a sonication method for enzyme activity of prolyl endopeptidase and aminopeptidase N were evaluated. Results: The highest absorption rate for Z-Gly-Pro-4-nitroanilide substrate that performs the evaluation of prolyl endopeptidase enzyme, respectively, for strains C10-1 (L. pentosus), B6-1 (L. fermentum), C5-2 (L.paracasei), Y5 (L.paracasei) and C11-1 (L. pentosus) and in relation to Leu-p-NA which assesses aminopeptidase N The highest uptake was observed for strains K1 (L. helveticus), B8-2 (L. plantarum), Y5 (L.paracasei), P2-3 and P35 (L. plantarum), respectively. All of the above strains showed significantly different uptake than the negative control containing buffer and substrate (P&lt;0.05). Finally, the 16s rRNA sequence of the isolated strains was registered in NCBI database. Conclusion: The results of the present study showed that number of native Iranian probiotics have effective activity of prolyl endopeptidase and aminopeptidase N which can be used as a selective combination for gluten degradation and to help celiac patients.

Effective Degradation of Gluten and Its Fragments by Gluten-Specific Peptidases: A Review on Application for the Treatment of Patients with Gluten Sensitivity.

To date, there is no effective treatment for celiac disease (CD, gluten enteropathy), an autoimmune disease caused by gluten-containing food. Celiac patients are supported by a strict gluten-free diet (GFD). However, in some cases GFD does not negate gluten-induced symptoms. Many patients with CD, despite following such a diet, retain symptoms of active disease due to high sensitivity even to traces of gluten. In addition, strict adherence to GFD reduces the quality of life of patients, as often it is difficult to maintain in a professional or social environment. Various pharmacological treatments are being developed to complement GFD. One promising treatment is enzyme therapy, involving the intake of peptidases with food to digest immunogenic gluten peptides that are resistant to hydrolysis due to a high prevalence of proline and glutamine amino acids. This narrative review considers the features of the main proline/glutamine-rich proteins of cereals and the conditions that cause the symptoms of CD. In addition, we evaluate information about peptidases from various sources that can effectively break down these proteins and their immunogenic peptides, and analyze data on their activity and preliminary clinical trials. Thus far, the data suggest that enzyme therapy alone is not sufficient for the treatment of CD but can be used as a pharmacological supplement to GFD.

Ekşi Maya Fermantasyonu ile Üretilen Ekmeklerdeki Biyoaktif Bileşenlerin In Vitro Biyoerişilebilirliği ve Sağlık Üzerine Etkileri

Sourdough bread is a traditional product, which is produced by mixing wheat, rye or other grain flours with water and lactic acid fermentation. The supposed mechanisms for the effect of foods produced by sourdough fermentation on health were probiotic effect of microorganisms, production of bioactive peptides and organic acids (acetic acid, butyrate, propionic acid), decreased amount of anti-nutrients (phytic acid, etc.), digestibility of starch and protein, increased bioaccessibility of phenolic compounds and bioavailability of minerals, degradation of gluten, new product to celiac patients. It was reported to have effects on nutrition, such as product development. In this review, the effect of sourdough fermentation on the ingredients in bread, in vitro bioaccessibility and health benefits are examined.

Review of Recent Advances in Treatment of Celiac Disease Using Enzymatic Gluten Degradation

Review of Recent Advances in Treatment of Celiac Disease Using Enzymatic Gluten Degradation

Characterization of Bacillus cereus AFA01 Capable of Degrading Gluten and Celiac-Immunotoxic Peptides.

Wheat gluten elicits a pro-inflammatory immune response in patients with celiac disease. The only effective therapy for this disease is a life-long gluten-free diet. Gluten detoxification using glutenases is an alternative approach. A key step is to identify useful glutenases or glutenase-producing organisms. This study investigated the gluten-degrading activity of three Bacillus cereus strains using gluten, gliadin, and highly immunotoxic 33- and 13-mer gliadin peptides. The strain AFA01 was grown on four culture media for obtaining the optimum gluten degradation. Complete genome sequencing was performed to predict genes of enzymes with potential glutenase activity. The results showed that the three B. cereus strains can hydrolyze gluten, immunotoxic peptides, and gliadin even at pH 2.0. AFA01 was the most effective strain in degrading the 33-mer peptide into fractions containing less than nine amino acid residues, the minimum peptide to induce celiac responses. Moreover, growth on starch casein broth promoted AFA01 to degrade immunotoxic peptides. PepP, PepX, and PepI may be responsible for the hydrolysis of immunotoxic peptides. On the basis of the potential of gluten degradation, AFA01 or its derived enzymes may be the best option for further research regarding the elimination of gluten toxicity.

New Insights into Non-Dietary Treatment in Celiac Disease: Emerging Therapeutic Options.

To date, the only treatment for celiac disease (CD) consists of a strict lifelong gluten-free diet (GFD), which has numerous limitations in patients with CD. For this reason, dietary transgressions are frequent, implying intestinal damage and possible long-term complications. There is an unquestionable need for non-dietary alternatives to avoid damage by involuntary contamination or voluntary dietary transgressions. In recent years, different therapies and treatments for CD have been developed and studied based on the degradation of gluten in the intestinal lumen, regulation of the immune response, modulation of intestinal permeability, and induction of immunological tolerance. In this review, therapeutic lines for CD are evaluated with special emphasis on phase III and II clinical trials, some of which have promising results.