Liquid Chromatography–Mass Spectrometry Analysis Reveals Hydrolyzed Gluten in Beers Crafted To Remove Gluten

Abstract

During brewing, gluten proteins may be solubilized, modified, complexed, hydrolyzed, and/or precipitate. Gluten fragments that persist in conventional beers render them unsuitable for people with celiac disease (CD) or gluten intolerance. Barley-based beers crafted to remove gluten using proprietary precipitation and/or application of enzymes, e.g. prolyl endopeptidases (PEP) that degrade the proline-rich gluten molecules, are available commercially. Gluten measurement in fermented products remains controversial. The industry standard, a competitive ELISA, may indicate gluten values <20 mg/kg, which is deemed safe for people with CD. However, in this study, liquid chromatography-mass spectrometry analyses revealed gluten peptides derived from hydrolyzed fragments, many >30 kDa in size. Barley gluten (hordeins) were detected in all beers analyzed with peptides representing all hordein classes detected in conventional beers but also, alarmingly, in many gluten-reduced beers. It is evident that PEP digestion was incomplete in several commercial beers, and peptides comprising missed cleavages were identified, warranting further optimization of PEP application in an industrial setting.

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

“…In line with previous findings ( 32 ), the gluten-free beer contained D- and γ-type hordeins, identified with multiple PSMs, as well as B3- and B-hordeins identified with only 2 or 1 PSMs, respectively. The origin of gluten-free beer, which is brewed essentially with barley malt, clearly emerged from the pattern of proteins identified, as they were the typical polypeptides of barley malt beer (e.g., Z4-barley serpin, LTP and ATIs).…”

“…Thus, in agreement with recent findings ( 32 ), the beer labeled as gluten-free, which is crafted with an enzymatic treatment aimed at removing gluten, can contain a multitude of variously sized polypeptides encrypting the immunotoxic epitopes that trigger CD as well as highly homologous sequences. Strikingly, the 13 residues-long N-terminal fragment of α2-gliadin 33-mer has been detected in beer brewed with even high dose of PEP, along with immunotoxic hydrolyzed peptides of HMW glutenins ( 13 ).…”

Characterization and Relative Quantitation of Wheat, Rye, and Barley Gluten Protein Types by Liquid Chromatography–Tandem Mass Spectrometry

“…The LC-MS/MS analysis was performed on an UHPLC system (Shimadzu Nexera, Sydney, Australia) directly coupled to a QTRAP 6500 mass spectrometer (SCIEX). The cycle time was set to 0.3 s, and the MRM transitions were scheduled to be monitored within 60 s of their expected retention time (± 30 s) (Colgrave et al, 2017a).…”

A new microbial gluten-degrading prolyl endopeptidase: Potential application in celiac disease to reduce gluten immunogenic peptides

“…AN-PEP is the only proline-specific PEP that is industrially available and can cope with the acidic conditions of beer fermentation [60]. However, a number of barley-based beers that are labelled “crafted to remove gluten” have recently become commercially available [61].…”