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Immunochemical and mass spectrometric methods were used to examine the gluten composition of a gluten-reduced beer produced by brewing with barley malt in the presence of prolyl endopeptidase (PEP) and a final filtration treatment with diatomaceous earth and perlite. The competitive ELISA is generally considered appropriate for the analysis of hydrolysed gluten, but it is not considered a scientifically valid method for the quantification of gluten in fermented or hydrolysed foods due to the lack of an appropriate reference standard. As no single analytical method can capture the spectrum of gluten-derived products in beer, a comprehensive approach was employed to analyse the intact and hydrolysed fractions of gluten with complementary methods. The combination of PEP addition and diatomaceous earth/perlite filtration was more effective at reducing the concentration of detectable gluten than each of the treatments alone. However, gluten proteins and/or polypeptides were observed in filtered, PEP-treated beers using sandwich ELISA methods, western blot, and bottom-up mass spectrometry. In addition, mass spectrometry results showed that the number of hydrolysed gluten peptides was almost unaffected by the filtration process. Gluten peptides that contained potentially immunopathogenic sequences were identified in the filtered PEP-containing beers by MS. Variability in gluten composition was observed between three replicate pilot-scale productions, suggesting that the gluten profile in beer could differ from batch to batch. As there is uncertainty in the detection and quantification of gluten in hydrolysed and fermented foods, characterisation of hydrolysed gluten by complementary analytical methodologies is recommended.

Most gluten-reduced beers are produced using an enzyme called proline endopeptidase (PEP), which proteolyzes the gluten by cleaving at proline residues. However, the gluten content of beers brewed in the presence of PEP cannot be verified since current analytical methods are not able to accurately quantitate gluten in fermented foods. In this work, mass spectrometry was used to qualitatively characterize the gluten in a wheat-gluten-incurred sorghum model beer brewed with and without the addition of PEP. Hydrolyzed gluten peptides and chymotryptic gluten peptides produced from intact gluten proteins were detected in beer brewed in the presence of up to 6 times the manufacturer's recommended dosage of PEP. The observation of chymotryptic gluten peptides indicates that some gluten proteins remained, at least partially, intact after fermentation and enzymatic treatment. Less intact gluten was observed in beer brewed in the presence of PEP, but more hydrolyzed gluten peptides were consequently observed in PEP-containing beer. Gluten peptides that contained immunogenic sequences known to be associated with celiac disease were detected in PEP-containing beer.

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.