Probiotic Enumerations: The Basics for Starting a Third-Party Testing Program
Probiotics have quickly become a household supplement, and the market is expanding rapidly. As consumer interest in overall health and wellness grows, more and more people are introducing probiotic supplements into their supplement routines. Research supports probiotic use for improved digestion, protection against infections, and nutrient absorption.
Market expansion is multiplied by online and international sales, allowing more small brands to enter the probiotics space with great success. Before introducing a new probiotic product to the market, it is important to understand the testing guidelines and options. Proper testing supports consumer health, product reliability, and overall brand reputation.
To simplify your search into probiotic enumeration, we have summarized the main points here.
What is a probiotic?
Probiotics are dietary supplements that have live microorganisms with potential health benefits. Some of the purported benefits of these bacteria or yeasts include supporting digestion, reducing stomach issues, regulating nutrient absorption, and aiding immune cells. Some probiotics introduce bacteria that already live inside humans, and others have similar qualities that support immune function. Each different species and strain of microbe has unique health benefits. In fact, each strain must have an associated clinical trial to support its specific health claims, meaning each product and formulation has a unique niche.
Probiotic formulations?
Formulation is a huge consideration in probiotic supplement development. Considerations include:
- Selection of bacteria: Some of the most popular probiotic genera include Lactobacillus, Bifidobacterium, Bacillus spores, and the yeast Within these microbial genera, there are multiple species and strain options. Additionally, new and novel probiotic genera and strains are in development. Choosing the best bacterial line for your product requires clear goals for the desired health effects and detailed research.
- Microbial count: Each probiotic product can contain a different amount of microorganisms, typically described as Colony Forming Units (CFUs). The amount of organisms in your product must be confirmed with microbial enumeration testing.
- Finished product matrix: Some matrices are easier to test than others. Gummy products require additional preparation steps to promote adequate growth, while powdered products need to be adequately dissolved for even distribution.
- Additional ingredients: Many probiotics are combined with prebiotics (called synbiotics), which include ingredients to support microbial growth. Products can also include botanicals or vitamin to further promote consumer health.
What are the testing requirements and guidelines?
In the United States, the FDA classifies probiotics as dietary supplements, so probiotic supplements must adhere to the guidelines in CFR 21 Part 111 which ensures all manufacturing facilities, personnel, and procedures are compliant with current Good Manufacturing Procedures (cGMP) as outlined by the FDA. This includes proper evaluation of the purity, strength, and composition of your product. Briefly, CFR 21 Part 111 explains that at least one test must be conducted to verify the identity of your ingredients before using in a product and the production process closely monitored to ensure the finished product meets the quality guidelines.
For probiotics, this means ingredients and finished products must be evaluated to confirm the intended microbial species and strain is provided by your raw ingredient supplier and the correct amount of microbes makes it into the final product.
CFR 21 Part 111 includes specifications about manufacturing facilities, personal hygiene, and more details about production, not just ingredient testing. We recommend reading the guidelines and getting in touch with an expert to make sure you fully understand the regulations!
Probiotic enumeration testing: How many bacteria are in your product?
One of the most important sections of CFR 21 Part 111 is requirements for product potency. For probiotics, this means accurately labeling products with the correct amount of live organisms in the finished product. Probiotic labels include the quantity of bacteria by weight and number of Colony Forming Units (CFUs). Including weight and CFUs is important because bacterial weight includes dead and inactive cells, resulting in inaccurate representations of active probiotic quantities.
Typically, enumeration involves testing your raw materials (lyophilized powders) and finished products at the time of manufacture, guaranteeing that you start and end with the correct CFUs. Different testing options exist depending on your product and label claims.
Types of tests:
Plate based enumeration:
Plate and growth media methods are the traditional approach to counting a sample’s microorganisms. Since probiotics have cell counts in the billions, it is impossible to count every cell. Instead, samples are diluted in broth and plated. Scientists count the colonies (visible clumps of microbes) and multiply this number by dilution factor to calculate CFU/g or CFU/serving, which can be reported on a product label. Different bacteria require different growth media and conditions, so it is important to choose the most applicable method for your strain. Plate enumeration is reliable and provides straightforward results; however, it is time and labor intensive.
Flow cytometry:
Flow cytometry is a unique test for probiotic enumeration that quickly provides cell count information. In a flow cytometer, cells in a solution pass one at a time through a detector, which uses lasers and nucleic acid stains to determine each cells type, size, and viability with the use of nucleic acid stains. As opposed to plate enumeration methods, flow cytometry is automated, providing highly accurate and precise cell counts. The benefits of flow cytometry for probiotic enumeration include improved accuracy and precision, rapid turn-around-time, ability to track product stability, and investigations into cell viability.
Eurofins was the first lab to receive ISO 17025 accreditation for probiotic enumeration by flow cytometry following ISO 19344 Protocol B.
Eurofins Testing Partner Program:
Most probiotic strain manufacturers have their own methods optimized for their developed to maximize recovery and minimize variability – meaning these methods provide accurate and reliable results for their specific raw material. Eurofins works with probiotic raw ingredient manufacturers to validate and become fully qualified to run their methods, so when you purchase a strain from a manufacturer, you can rely on us as your third-party testing lab to provide unbiased results using the ideal method for your ingredients. Find a list of our partners here!
How to get started:
Eurofins Microbiology Laboratories, Inc. in Madison, WI is our Probiotic Testing Center of Excellence. Our team of experts is excited to support you at every step of the probiotic formulation, manufacturing, and quality control process.
Fill out this form to get in contact with a Eurofins representative or request more information!
