Enzymes and the FDA Final Rule on Food Labeling and the Claim of “Gluten-Free”

Let us start with the fact that this is not a legal document, legal advice, or written by a lawyer. Having read the regulation, it is how EDC can best determine how it applies to our enzyme preparations. A Rule by the Food and Drug Administration on 08/13/2020

Our understanding is that the regulation was developed because, at this time, there is no scientifically valid test method to determine the presence of gluten fragments in fermented or hydrolyzed foods.

As such, a food may test negative for gluten but still potentially trigger a reaction by someone with Celiac Disease. The current regulation to make a claim of “gluten-free” is less than 20 parts per million of detectable gluten. The new definition will keep the 20 ppm level but, rather than have a specific gluten test, possible protein contribution from gluten must be taken into account by calculation. As EDC understands the issue, if a gluten source was used in the fermentation media and, if an enzyme has a mix of proteins, then the protein/peptides that are not specifically identified as the enzyme must be considered as gluten. Put another way, if you cannot prove it is not gluten, it must be treated as gluten for purposes of gluten-free claims.

For the enzyme industry, one of the primary types of enzyme fermentation is surface culture. This style of fermentation has been around for centuries. It is the same style used for soy sauce, sake, etc. Wheat bran and rice bran are common media components as well as other grains such as Barley. Many of the original enzymes for commercial uses began as surface culture. While some companies have switched to deep tank fermentation, many enzymes are still produced by surface culture. Regardless of the method, the intact protein sources used for the fermentation media are consumed during the fermentation process. Any residual protein-like material will be peptides and amino acids. The original protein will no longer be present.

After fermentation, enzymes are purified by various means but most often by size exclusion or precipitation. For example, the soluble enzyme broth contains the enzyme protein, other proteins produced by the microorganism, hydrolyzed fragments of the fermentation media and other residuals such as minerals and sugars.

The broth is filtered to remove large proteins and fermentation insolubles. It is then is purified by fine filtration and followed by ultrafiltration. This step removes the unwanted small or larger protein fragments. Most commercial enzymes are in the range of 10,000 to 75,000 Daltons. Size exclusion is done by a two step filtration process removing large fragments greater than 75,000 Daltons. The protein fragments smaller than 75,000 Daltons pass through the filter and remain in the permeate. This is followed by followed by second filtration step where those fragments larger than 10,000 Daltons will remain in the retentate and the smaller fragments and low molecular weight pass through the filter and are discarded. The end result is a liquid retentate with protein fragments in the range of 10,000 to 75,000 Daltons.

The enzyme protein can be identified by various methods, but there will always be other protein materials that are not specific to the enzyme but are of a similar size. Microbes or fungi will produce a variety of proteins during the fermentation. However, in this case, there is no way to state that the protein is produced by the microorganism or if it is a fragment from the original fermentation media. Current test methods will only identify intact gluten. Since there is no way to test for gluten fragments, and, since gluten fragments can trigger a reaction by those with Celiac Disease, the FDA regulation requires that all non-enzyme protein must be calculated as “gluten”. If the level of those protein fragments is less than 20 ppm in the Final Food Product, then the final food product can be labelled as “gluten-free”.

Enzymes and the FDA Final Rule on Food Labeling and the Claim of “Gluten-Free”

As discussed, enzymes are used in the production of a food product that may be used as an ingredient in another product. This leads to significant dilution factors.

Worksheet Example:
Protease QED is produced via surface culture on wheat bran. Wheat bran contains significant amounts of glutenin and gliadin, the proteins that make gluten. After fermentation, the purified concentrated enzyme contains 80% identifiable enzyme protein and 20% non-enzyme protein.

Therefore, 1 gram of the dry “pure” enzyme is 800,000 PPM of identified protein and 200,000 PPM of non-enzyme protein that must be considered as gluten for the calculation. Does Not Qualify for a Gluten-free Claim.

Enzymes, as commercial products, are usually sold by standardizing to a specific activity. Usually this creates a range of protein levels in the commercial product. A review of the production records will indicate the typical range. In this case, the recommendation is to use the upper range of protein content of the blend rather than the average protein content.

The commercial version of Protease QED with a standardized enzyme activity is 80% enzyme and 20% diluent such as starch or maltodextrin. In this case:

1 gram of the QED blend gives a 20% reduction in the non-enzyme protein.

200,000 PPM x 0.80 = 160,000 PPM (non enzyme protein) Does Not Qualify for a Gluten-free Claim

The commercial version of Protease QED enzyme preparation is used to produce a flavor. In this case, it is added to a protein slurry at a level of 0.25% based on the total slurry weight. The reaction proceeds to the desired endpoint and the enzyme is inactivated by heat to stop the reaction. The slurry is cooled and then packaged for sales to customers. The level of non-enzyme protein contribution to the flavor is calculated as:

160,000 x .0025 = 400 PPM (non enzyme protein) Does Not Qualify for a Gluten-free Claim.

This flavor is added to a final food product (e.g. a dairy spread) at a level of 0.5%. The non-enzyme protein contribution from the flavor to the final food is calculated as:

400 PPM x 0.005 = 2 PPM The Final Food May Make a Gluten-free Claim.

© Enzyme Development Corporation, January 2021

These suggestions and data are based on information we believe to be reliable. They are offered in good faith, but without guarantee since conditions and methods of use of our products are beyond our control. Suggestions for use of our products should not be understood as recommendations that they be used in violation of any patents or government regulations.