Effects of a Proline Endopeptidase on the Detection and Quantitation of Gluten by Antibody-Based Methods during the Fermentation of a Model Sorghum Beer

Abstract

The effectiveness of a proline endopeptidase (PEP) in hydrolyzing gluten and its putative immunopathogenic sequences was examined using antibody-based methods and mass spectrometry (MS). Based on the results of the antibody-based methods, fermentation of wheat gluten containing sorghum beer resulted in a reduction in the detectable gluten concentration. The addition of PEP further reduced the gluten concentration. Only one sandwich ELISA was able to detect the apparent low levels of gluten present in the beers. A competitive ELISA using a pepsin-trypsin hydrolysate calibrant was unreliable because the peptide profiles of the beers were inconsistent with that of the hydrolysate calibrant. Analysis by MS indicated that PEP enhanced the loss of a fragment of an immunopathogenic 33-mer peptide in the beer. However, Western blot results indicated partial resistance of the high molecular weight (HMW) glutenins to the action of PEP, questioning the ability of PEP in digesting all immunopathogenic sequences present in gluten.

Detection and Quantitation of Gluten in Fermented-Hydrolyzed Foods by Antibody-Based Methods: Challenges, Progress, and a Potential Path Forward

“…With the Skerritt antibody, beers RA (Lane 5) and RF (Lane 15) yielded multiple bands (20–150 kDa) both at higher and lower MW range (Figure 2B). Binding to Skerritt antibody indicates the presence of HMW glutenin (D Hordein) epitopes in gluten-reduced beers and again confirms the results of previous studies (17, 39, 78). The presence of HMW glutenin in gluten-reduced beers may not get accurately detected by gluten detection assays targeting only gliadin proteins.…”

“…Gluten-derived peptides tend to remain in the final beer product and often contain immunopathogenic sequences (33). Studies have detected peptide fragments from the putative immunotoxic 33-mer of α2-gliadin in several wheat and barley beers produced by different manufacturing processes, indicating the resistance of this peptide to proteolytic cleavage during the production of beers (17, 34, 35). The susceptibility of different gluten proteins to proteolysis during fermentation varies, thereby generating a very diverse range of peptides.…”

“…A study by Colgrave et al (36), indicated that B-hordein and D-hordein are more susceptible to hydrolysis compared to γ-3 hordeins. In recent years, several mass spectrometry studies have detected and characterized gluten proteins/peptides in both wheat and barley beers (17, 34–43).…”

“…In contrast, several recent studies utilizing mass spectrometry, ELISA, and western blot analysis indicated that PEP didn't completely degrade all gluten proteins and gluten proteins/peptides remain in the final beer produced by addition of PEP. Specifically, the HMW glutenin were resistant to the action of PEP during beer production (17, 39, 78). In addition, beer treated with PEP has been shown to cause a humoral response toward IgA or IgG antibodies, derived from the sera of 3 celiac disease-active patients, but there was no response from normal control subjects ( n = 31, control group: n = 29), indicating that beers treated with PEP are still immunogenic (79).…”

Immunogenic Potential of Beer Types Brewed With Hordeum and Triticum spp. Malt Disclosed by Proteomics

“…The occurrence of gluten-derived proteins/peptides also including CD-toxic epitopes has been largely demonstrated and confirmed by this study in the five types of beer, also including the one labeled as “gluten-free.” In agreement with previous findings, peptidomic analysis confirmed that PEP enzymes are not able to completely hydrolyze all the immunopathogenic peptides of gluten ( 43 ). The accurate quantification of gluten epitopes remains a main analytical concern, because the current immunochemical methods used to assess gluten in food have limited reliability for beer and other extensively processed products ( 13 , 15 , 33 , 44 ). The unavailability of trustful determinations for the most consumed beer types hampers estimating the contribution of beer consumption to the extent of intestinal tissue damages.…”