Enzymes are used for many reasons and in different products since they are catalysts designed to do the work, a.k.a., the “heavy lifting." Food, beverage, pharmaceutical and other industries use enzymes as processing aids. In the supplement industry, enzymes are often found in digestive aid products, systemic function products, functional foods or might even be used to smooth out the graininess of a whole food beverage.
Companies utilizing enzymes often use testing to dial in the activity level, so they can determine how potent the enzyme is or how much work it can do (remember, enzymes are heavy lifters). For this reason, enzyme potency is measured in activity instead of weight (mg). To measure the activity in supplements, methods have been developed by the U.S. Pharmacopeia, Food Chemical Codex, British Pharmacopeia, Japanese Pharmacopeia and other compendia sources. Most of these methods are developed for pharmaceutical, single-ingredient formulas or raw materials and are generally designed for concentrated single enzyme samples. When complex products and multi-enzyme blends are manufactured, the methods for testing become more complicated.
It takes years of experience and proficiency testing to prove competency with enzyme testing methods. For this reason, most labs are not able to just pick up one of these complicated wet chemistry methods and run them accurately without an extensive investment in time and equipment. These methods vary in the actual procedures, but they are all pH and temperature controlled reactions that show how an enzyme digests a specific substrate. If any of the conditions are not controlled or an incorrect substrate is used, the enzyme reaction may not be as effective and a low recovery may be found.
When qualifying a material or checking the label claim of a product, verification steps need to be implemented to ensure the reaction performs in the manner it is expected to. Performing enzyme testing methods using reference standards, or “check" standards, is a key factor in obtaining accurate results. These standards have known activity value and can be run with the testing method to verify the assay effectively performed the enzyme reaction. The standards are tracked, and any deviations are calculated. All results from the standards must be within a tight range of accuracy to allow the assay to be reportable with a valid certificate of analysis (CoA). The aggregated historical data points are utilized to validate accuracy is continually monitored within the lab. Additionally, this provides the lab with internal proficiency data among each analyst that run the various methods.
Outside lab proficiency testing is also an option that consists of testing between two or more labs. These results are compared by performing the same method on the same samples in each respective lab. The data can then be used to monitor lab-to-lab proficiency. It is important to ask about what proficiency testing is performed when qualifying a lab for your product testing.
Enzyme interference is when a specific raw material enzyme may have specifications for one activity, but also shows activity for other units, as well. For instance, a protease may have a specification for hemoglobin unit (HUT) activity, but other assays on this protease show neutral protease (PC) activity, acid protease (SAP) or even papain unit (PU) activity. Even though the material only has HUT listed for the specification, it may also have various “side activities" too. When multiple enzymes are blended together with overlapping side activities, a higher input result is often noted. Because the side activities contribute to the combined protease effect, they are the reason the values in the blend may be higher than the raw material claims. Enzyme manufacturers can only guarantee activity for the input units, because the side activities are not always consistent and may vary between each production batch. For this reason, manufacturers often label the product using “by input" claims.
Another interference commonly observed is a reduced lactase activity (ALU) in a high-protease blend. Proteases digest protein, and since enzymes are proteins, lactase tends to be the most affected by a high-protease blend. This is proven by creating a small lab blend, then testing the ALU in the blend without the proteases, and comparing that to the production batch that includes the proteases. The test results will show the activity of the lactase as it differs, with and without the proteases. These small batch studies are commonly performed to help brands prove and account for this type of interference.
The third interference is co-ingredient interference from pH swings in the blend. Co-ingredients, such as betaine hydrogen chloride (HCL), lemon grass, calcium carbonate or other acidic or basic ingredients, may affect the pH of the entire mixture to be tested. This interference with the test method can provide results that are not an accurate representation of the product. Lipase (FIP) is a pH-based USP/FCC titration method, which means the pH for this method is closely monitored and controlled. If the test mixture has a starting pH outside of the normal range for the method, then this could affect the results calculation for the reaction.
Customers are often surprised that brands can effectively test blended enzyme products. Most enzyme blends test well and are more straightforward than expected. A label review will often help evaluate potential interference or explain why testing didn’t work well. And, just because the input claims and output results might not have been what was expected, testing can create a deeper understanding of a product. Proving label claims by testing is always the goal, and using a qualified testing lab partner can go a long way in providing confidence in a product or brand line.
Tammy Blakemore is general manager at SORA Labs (soralabs.com), a third-party dietary supplement testing lab that specializes in enzyme testing.