Gluten Content of Gluten Digestive Enzyme Supplements: A Very Brief Summary

Gluten Content of Gluten Digestive Enzyme Supplements: A Very Brief Summary

This is not a commentary about whether individuals with gluten-related disorders should use gluten digestive aids or whether there is any benefit to these supplements. For an excellent article on enzymes and current medical thinking, please see the interview conducted by NFCA available HERE

As many of you know Gluten Free Watchdog has been sending various “glutenase” enzyme supplements to the lab for gluten testing. Initial results were very troublesome. But based on additional testing following a modified testing protocol, the products appear to contain levels of gluten below the lower limit of quantification.

The information provided below may be a bit technical. My apologies.

Background: in May 2015 an enzyme-industry insider working for a manufacturer who supplies enzymes to industry for use in supplements contacted Gluten Free Watchdog. He was concerned because one of the enzyme ingredients widely used in “glutenase” enzyme supplements (supplements marketed to help break down gluten) was testing very high for gluten (> 270 ppm) using the competitive R5 ELISA (the assay used to detect hydrolyzed gluten).

Note: According to the individuals I have communicated with in the enzyme industry, enzymes used in glutenase supplements are generally derived from bacteria grown on wheat or barley. This is necessary to generate the enzymes that are used in these supplements to supposedly hydrolyze gluten.

What GFWD Tested

Three popular glutenase enzyme supplements were purchased for testing. GFWD asked the lab—Bia Diagnostics–to test the enzymes following the same protocol used by the enzyme supplier who contacted GFWD (i.e., product tested using both the sandwich and competitive R5 ELISAs; no modifications made).


Initial testing was alarming, especially for two of the supplements. Extraction values for one supplement ranged from 24 ppm to 138 ppm gluten using the competitive R5 ELISA. However, the lab used by Gluten Free Watchdog–Bia Diagnostics–questioned the findings. They felt that the results were false positives caused by the enzymes reacting with the assay. To determine if this was the case, the enzymes were deactivated via boiling and the product was retested. Results of the retest were below the lower limit of quantification of 10 ppm gluten.

Manufacturer Testing

Gluten Free Watchdog contacted the manufacturer of both enzyme products that tested high for gluten during the first round of testing. Manufacturer testing initially found high levels of gluten for both products using the competitive R5 ELISA (> 270 ppm and 187 ppm). The manufacturer then deactivated the enzymes and the analysis was rerun. Results of follow-up testing were below the limit of detection.

The manufacturer writes, “Our dietary supplements contain a unique enzyme matrix, which includes various proteolytic enzymes. These proteases degrade the protein bonded to the ELISA test wells which creates interference with the assay thus creating a false positive. In order to correctly analyze these products, protease enzymes must be completely deactivated to prevent interfering with this analysis. Because some proteases are stable at higher temperatures, samples are subject to boiling, plus washing with a protease inhibitor cocktail to ensure deactivation.”

LC/MS Testing

Samples were also sent to a noncommercial lab for testing via mass spec. Results indicate no detectible gluten in the enzymes tested. However, testing enzymes via mass spec proved to be difficult and according to the lab more work is needed to determine how best to test enzymes using mass spec.

Bottom Line

The “glutenase” enzyme supplements tested by Gluten Free Watchdog do not appear to be contaminated with residual gluten. It is important to note that when enzyme supplements are tested for gluten, the enzymes must be deactivated to prevent false positives.

© Copyright August, 17 2015 by Tricia Thompson, MS, RD for Gluten Free Watchdog. All Rights Reserved.

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Comments (11)

  • Pam Newbury Reply

    If enzymes must “deactivated” to prevent “false positives”, what effect do un-deactivated enzymes have on celiac guts? Does a celiac gut recognize the un-deactivated enzymes as harmless?

    In other words, if the enzymes have to be deactivated to pass the test, what proof do we have that they would not cause gut damage to a celiac? Has anyone done any research on this?

    Perhaps I just don’t understand the idea of why it is acceptable to change the enzymes before testing them. I understand that the enzymes interfere with the process of the test itself, but not the relationship of the enzymes to any actual gluten in the product.

    Are the enzymes themselves contaminated with gluten from the grain they are grown on? How can we know that the deactivation process does not dilute, wash off, or change the gluten protein?

    August 17, 2015 at 7:21 pm
    • Tricia Thompson Reply

      Pam, the enzyme supplements tested by GFWD are not actually contaminated with gluten. Deactivating the enzymes is done to prevent them from reacting with the assay in an unintended way and causing false positives results.

      August 17, 2015 at 8:27 pm
      • Peter Nowee Reply

        I still think Pam has a point: Even if there is no actual gluten in the supplements and the test is mistaking enzymes for gluten, that does not mean the supplements are safe. CD patients need to take the enzymes without deactivation, so their immune systems might make the same mistake as the test kit, resulting in inflammation etc.

        This calls for further tests of the supplements against CD patient T-cells, without boiling/deactivation of course.

        August 24, 2015 at 1:30 pm
        • Tricia Thompson Reply

          Hi Peter, The assay is not mistaking the enzymes for gluten. The enzymes are actually breaking down the protein that is included in the assay. This protein breakdown is what leads to the false positives. This is why mass spec did not find any gluten protein in the non-deactivated enzyme supplements.

          August 24, 2015 at 1:44 pm
          • Peter Nowee

            Hmm, I see what you mean now, sounds quite exceptional. Still can’t help wondering about the exact process and if somehow something similar might not happen in the celiac gut… Perhaps the test manufacturer could confirm the enzyme producer’s explanation? Anyway, thanks for your great research and good luck!

            August 24, 2015 at 3:37 pm
          • Tricia Thompson

            Hi Peter, The lab that Gluten Free Watchdog uses–Bia Diagnostics–came to this conclusion initially and then the enzyme manufacturer was able to replicate and confirm the findings. I will ask the lab to write something up that is a bit more detailed.

            August 24, 2015 at 5:24 pm
  • Kevin Baker Reply

    The link to the CeliacCentral article in the first paragraph is dead. Anyone else having this trouble?

    August 17, 2015 at 9:55 pm
  • AGK Reply

    Wow…once again…it’s more complicated than it appears. Thanks for the info and the explanation!


    August 18, 2015 at 12:05 am
    • Tricia Thompson Reply

      You are welcome, Al. Accurately testing for gluten is certainly not straightforward or easy!

      August 18, 2015 at 12:20 am

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