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.

Prototype Gluten-Free Breads from Processed Durum Wheat: Use of Monovarietal Flours and Implications for Gluten Detoxification Strategies

In this investigation, we reported the production of prototype breads from the processed flours of three specific Triticum turgidum wheat genotypes that were selected in our previous investigation for their potential low toxic/immunogenic activity for celiac disease (CD) patients. The flours were subjected to sourdough fermentation with a mixture of selected Lactobacillus strains, and in presence of fungal endoproteases. The breads were characterized by R5 competitive enzyme linked immunosorbent assay in order to quantify the residual gluten, and the differential efficacy in gluten degradation was assessed. In particular, two of them were classified as gluten-free (<20 ppm) and very low-gluten content (<100 ppm) breads, respectively, whereas the third monovarietal prototype retained a gluten content that was well above the safety threshold prescribed for direct consumption by CD patients. In order to investigate such a genotype-dependent efficiency of the detoxification method applied, an advanced proteomic characterization by high-resolution tandem mass spectrometry was performed. Notably, to the best of our knowledge, this is the first proteomic investigation which benefitted, for protein identification, from the full sequencing of the Triticum turgidum ssp durum genome. The differences of the proteins’ primary structures affecting their susceptibility to hydrolysis were investigated. As a confirmation of the previous immunoassay-based results, two out of the three breads made with the processed flours presented an exhaustive degradation of the epitopic sequences that are relevant for CD immune stimulatory activity. The list of the detected epitopes was analyzed and critically discussed in light of their susceptibility to the detoxification strategy applied. Finally, in-vitro experiments of human gastroduodenal digestion were carried out in order to assess, in-silico, the toxicity risk of the prototype breads under investigation for direct consumption by CD patients. This approach allowed us to confirm the total degradation of the epitopic sequences upon gastro-duodenal digestion.

Commensal Bacterium Rothia aeria Degrades and Detoxifies Gluten via a Highly Effective Subtilisin Enzyme

Celiac disease is characterized by a chronic immune-mediated inflammation of the small intestine, triggered by gluten contained in wheat, barley, and rye. Rothia aeria, a gram-positive natural colonizer of the oral cavity and the upper digestive tract is able to degrade and detoxify gluten in vitro. The objective of this study was to assess gluten-degrading activity of live and dead R. aeria bacteria in vitro, and to isolate the R. aeria gluten-degrading enzyme. Methods: After an overnight fast, Balb/c mouse were fed a 1 g pellet of standard chow containing 50% wheat (and 4% gliadin) with or without 1.6 × 107 live R. aeria bacteria. After 2 h, in vivo gluten degradation was assessed in gastric contents by SDS-PAGE and immunoblotting, and immunogenic epitope neutralization was assessed with the R5 gliadin ELISA assay. R. aeria enzyme isolation and identification was accomplished by separating proteins in the bacterial cell homogenate by C18 chromatography followed by gliadin zymography and mass spectrometric analysis of excised bands. Results: In mice fed with R. aeria, gliadins and immunogenic epitopes were reduced by 20% and 33%, respectively, as compared to gluten digested in control mice. Killing of R. aeria bacteria in ethanol did not abolish enzyme activity associated with the bacteria. The gluten degrading enzyme was identified as BAV86562.1, here identified as a member of the subtilisin family. Conclusion: This study shows the potential of R. aeria to be used as a first probiotic for gluten digestion in vivo, either as live or dead bacteria, or, alternatively, for using the purified R. aeria enzyme, to benefit the gluten-intolerant patient population.