A wide variety of testing can be conducted on probiotic and enzyme products. These products can be single strains of raw materials or multi-strain, enzyme blends that make up complex finished products. Testing starts with individual raw materials, and can be completed at each stage of the manufacturing process, on the finished product or even to test shelf-life expiration accuracy. Each company must set its specifications and testing requirements to fit its operation and comply with the Code of Federal Regulations—CFR title 21 part 111 for the dietary supplement industry.
The CFR lists many requirements that need to be met in the manufacturing process, but for testing, it is required that “appropriate scientifically valid methods" should be used. When looking for valid testing methods for products, following the methods from the United States Pharmacopeia (USP), Food Chemicals Codex (FCC), British Pharmacopeia (BP), European Pharmacopeia (Ph. Eur.) and other resources is a great place to start.
The CFR clearly identifies the importance of raw material and component testing to determine the identity, purity and strength, and to also ensure these materials are free from contaminants. Looking at the FCC, Ninth Edition, Third Supplement, several different probiotic strains and testing recommendations are listed. Testing methods for probiotic strains include genetic and microbiology tests designed for specific live microorganism or bacteria. For Lactobacillus acidophilus La-14, identification using nucleic acid, such as polymerase chain reaction (PCR) testing, is recommended for positive strain identification. Enumeration assay methods for potency are listed to give colony forming units (CFU)/g viable counts. The CFU/g measurements are usually tested for label claims in the millions or billions of CFU/g. There are also specific tests for microbial analysis that are listed to screen for contamination of unwanted coliforms, E.coli, Salmonella, Staph aureus and other microbiologic contaminants. The FCC even lists additional information such as labeling requirements for these probiotic strains.
Each probiotic strain has a variation in the assay method to determine the CFU/g viable count for each particular strain. Many of the strains are incubated under anaerobic conditions, but there are a few strains, including Bacillus coagulans GBI-30, 6086, that are incubated under aerobic conditions. Some assay methods may add cysteine hydrochloride solutions to the agar for specificity of growth of that particular strain. Other assay methods do not have the addition of cysteine. When looking at the variations of these assay methods for strain-specific testing, one can understand why it might be difficult to test a blended-strain probiotic finished product. There is not one overall test method that can provide an accurate total viable count for the finished product. Testing an enumeration or assay for these products would provide more general information.
Probiotic strains are living organisms, enzymes are not. Instead, they are ingredients used to catalyze reactions. As a dietary supplement, enzymes are used to aid in digestion and the breakdown of food. The FCC gives testing methods for measuring enzyme activity, and these activity assays are listed in Appendix V. Activity units are used instead of milligram or weight to measure the “work" the enzyme is doing. These activity assays are designed to measure the outcome of a chemical reaction to determine potency of the enzyme. Proteases, carbohydrases and lipases are enzymes that catalyze the hydrolysis of bonds in proteins, carbohydrates and fats, respectively. These activity assays have acronyms for the particular assay such as: Protease HUT (hemoglobin unit tyrosine), Amylase DU (alpha-amylase Dextrinizing Units) and Maltase DP (degrees Diastatic power). All of these are examples of how enzyme activity is listed on the label. These activities are often labeled “by input" due to the enzyme interactions in the finished product matrix. Each enzyme blend is unique and the interactions between the enzymes can vary with each unique blend. Finished product and blend testing is possible, but results may vary from the “input" because of the synergistic or diminishing nature of the enzyme interactions. Testing an enzyme blend will give information about the enzyme interactions of a specific product, and will provide the necessary information to determine product specifications.
Storage and handling for probiotics and enzymes can also be a challenge. Some probiotic strains and enzyme ingredients are not tolerant of heat or humidity. Probiotics often need to be stored in refrigerated or even freezer conditions. Some probiotic strains can lose viability with over-blending or mixing, and because of this sensitive nature, many manufacturers over-formulate probiotic blends to account for some loss in the manufacturing process. There is also a notable decrease in activity, as enzymes are sensitive to heat and humidity. Most digestive enzymes will deactivate with elevated heat conditions or exposure to moisture. The most heat-sensitive enzymes tested are papain, bromelain and catalase. These enzymes can show activity declines over time, even at ambient temperature exposure.
Even standard warehouse conditions can affect these enzymes over time. When tested with activity below the label claim, the manufacturer should be questioned about storage conditions. If the product has been stored in standard warehouse conditions, manufacturers are often surprised that these enzymes have lost activity. A decrease in activity is also possible during handling or exposure to heat during shipping. The sample to be tested should represent the same conditions it is stored in, to ensure the most accurate test results. Shipping these sensitive samples in an expedited fashion, with ice packs, will help ensure the testing sample represents the product accurately. Monitoring storage conditions, baseline testing and stability studies can help track potency and determine an appropriate shelf life for the finished product.
Probiotics and enzyme products have challenges, and blending them together with multiple probiotic strains and a variety of enzymes creates complex products. The more complex the matrix, the more difficult it will be to produce, store and test. Starting with raw material testing and ensuring the initial ingredients are of the highest quality can help build a great foundation for the complicated road ahead. Using scientifically valid testing methods and labs with the expertise in performing these specific tests is the key to manufacturing quality products.
Tammy Blakemore guides SORA Labs (soralabs.com) with her proven track record for business management. With her passion to take SORA Labs to a new level of business success, she has become the driving force behind the company’s continued growth. Blakemore’s unique combination of science and sales makes her an invaluable asset to SORA Labs.