an-pep Archives - Cooking al Dante

By Dante Moroni

Note: This article is non-peer reviewed and self published. Any statement within are for entertainment purposes and curiosity sake. In no way should any piece from this article be taken as medical advice. This article is also a work in progress and will be updated as necessary by the author.

Current trust in the gluten free community of gluten reduced or gluten removed beer is low. Confusion around FDA labelling rules, lack of in vivo studies verifying their safety, and positive readings on Lateral Flow Device Assays.¹⁰ are some of the main reasons. Aspergillus niger derived Proline Endopeptidase (AN-PEP) is used in production of these beers to degrade intact gluten proteins with a goal of making them safe for individuals with Celiac Disease (CD).¹ Here I present a brief review of the literature surrounding these Gluten Reduced (GR) beers.

The main reason the FDA rescinded its rule allowing GR beers to be labelled gluten-free is that validated quantification of these fragments is currently unavailable.⁵ However, in opposition to the FDA’s ruling, there is evidence that the R5 competitive ELISA binds relevant hydrolyzed gluten fragments and may be a suitable option for testing.⁸

Mass Spectrometry (MS) is one qualitative platform researchers have been using to probe GR beers for celiac reactive epitopes. I’ve reviewed several studies that analyzed AN-PEP barley beers and/or regular barley beers for immune stimulating epitopes using MS and I think the data does support the conclusions of Ackeroyd et al 2018. That is, AN-PEP degrades all immune stimulating epitopes in barley beer.

Mass Spectrometry Studies

Fiedler et al 2018⁷ found reactive epitopes in an AN-PEP treated beer, but there are some serious caveats. First, the beer was gluten-incurred. They used a gluten free beer and added the gluten to the cooled wort. The problem here is that the gluten free beer is made from sorghum syrup. The gluten in barley beer has already been partially hydrolyzed due to endogenous peptidases. So in this study, you are actually testing if AN-PEP by itself breaks down all the CD epitopes. Without the germination stage, and using incurred gluten, their experimental results cannot be applied to that of a barley AN-PEP beer.

Colgrave et al 2017⁴ showed hydrolyzed gluten fragments remaining in an AN-PEP barley beer, but they were not immunogenic epitopes.

Fiedler et al 2019², found hydrolyzed fragments in an AN-PEP barley beer that matched certain portions of immunogenic sequences, but the stimulating epitopes were not present. Figure 5a’s peptide’s C-terminus contains only the Q-Q-P part of the Q-Q-P-F-P epitope. Figure 5b’s peptide has an I-A insertion which interrupts the Q-Q-G-Y-Y-P-T-S epitope of HMW-Glutenin. Figure 5c’s epitopes both have been cut in the middle of their epitope binding region for HMW-Glutenin. Figure 6 references a peptide that was cleaved by trypsin in the middle of its LMW-glutenin sequence (Trypsin is used in these experiments on higher molecular weight peptides for bottom-up proteomics). In vivo chymotryptic digestion is highly likely to also cleave this same sequence rendering it non-immunogenic.

In Vitro Studies

I was able to find two in vitro studies that looked at the immune stimulating potential of hydrolyzed gluten peptides applied to biological samples from patients with active CD.

Allred et al 2017³applied CD serum to ELISA plates coated with GR beer proteins. They found that only 3 of 31 CD serums reacted to the GR beer (9.7%). Considering that all the proteins in the GR beer were coated onto the ELISA plate, there can be no conclusion drawn as to what proteins the serums were binding to. For example, it could have been yeast proteins or hop proteins that the serums were binding to. Further, they removed all data for negative samples, which makes verifying the data impossible. This is a concern since some serums could be reacting to things like rice or GF beer, just below their 3SD threshold, which would lead to a different interpretation of the data.

Real et al 2014⁹ investigated the immune stimulating potential of a regular barley beer that has tested less than 20ppm intact gluten. They did see immunogenicity in the beer due to hydrolyzed peptides. However, the beer was not AN-PEP treated.

Skerritt Antibody Testing

I decided to test a handful of these beers in early 2022 using the EZ lateral flow test kit (Skerritt antibody) and got some uprising results.¹⁰ All 7 gluten reduced beers that I tested has results showing >20ppm gluten. This contradicts lab negative lab results provided by these breweries. It is up to the brewer to send out samples for gluten testing on R5 antibody competitive ELISA format, however they aren’t legally bound to do this. Also the testing results of gluten reduced brewing websites are spotty. Only certain batches of beer are listed… Are >20ppm test result batches still being sold and the breweries just aren’t sharing this information with the public? Otherwise the difference in antibody seems to be the culprit here. I think that my EZ gluten results are actually indicative of the gluten reduced beers still containing a >20ppm concentration of the glutenin subcomponent of gluten, not gliadin. This is partly due to Skerritt antibody’s higher sensitivity to glutenin vs gliadin along with AN-PEPs slow glutenin hydrolysis compared to gliadin.¹⁰ See more about my experiment HERE. Considering several traditional barley beers have negative EZ Gluten results (and R5 ELISA), they may even be safer than gluten reduced beers. Those brewers could also be using enzymes to process their beer, some with even more robust gluten degrading activity, such as Papain in Corona Extra.¹¹

Conclusion

AN-PEP treated GR beers continue to need more scientific studies on their potential for immunogenicity in the celiac population. There is no current in vivo data showing an adverse response to these beers and in vitro data is significantly lacking. Further, researchers have yet to identify immunogenic epitopes in a AN-PEP barley beer. Considering these facts, it continues to be likely that these beers are relatively safe in CD. However, until robust in vivo data is available it is advised that individuals with CD consult with their doctor before consuming these beers.

Examples of Gluten Reduced / Removed AN-PEP Barley Beers

References