Utilizing inhibition assays to determine probiotic stability

Creating a successful innovative probiotic dietary supplement can be achieved via many pathways, including wisely choosing the probiotics strains based on clinical substantiation; highlighting the type of packaging chosen; using finished product modalities such as gummies, fast-melt powder, chewing gum and chewables rather than traditional tablets and capsules; and by adding synergistic non-probiotic ingredients. Of the options, the one that proves to be the toughest to execute is combining probiotics with non-probiotic ingredients, particularly botanicals.

Just like the animal kingdom, plants have evolved elaborate defense systems to help prevent invasion by bacteria, molds, yeasts, viruses, etc. And not coincidentally, the phytoconstituents that help make up this aspect of a plant’s defense system are often the same ones that are isolated to address specific health concerns, which makes them potentially harmful to the stability and integrity of probiotics when combined in a condition-specific product. Take turmeric, for example; some of the vast health benefits that have been published on its curcuminoid phytoconstituents are due their potent antimicrobial activities. So how can one determine which ingredients are safe to combine with probiotics in the same product? Two words: inhibition assays.

Inhibition assays modify traditional plating techniques used to measure bacterial colony-forming units (CFUs) in such a way as to determine the manner by which an ingredient affects the growth and integrity of a probiotic. Inhibition assays can quickly help determine if an ingredient truly harms (kills) the probiotic, or if the probiotic colony growth is just delayed in the presence of the ingredient as the growing conditions become less than optimal.

The method is simple: A mixture of bacteria and ingredient are made and rehydrated in standard broth (with a positive control that does not contain the chosen ingredient). A sample is removed from the solution and plated immediately on agar. The remaining solution is kept at room temperature for 2 hours, then remixed and plated. The plates are then incubated in an anaerobic environment for 72 hours. After incubation the plates are counted and the colonies per gram compared between T=0 and T=2 hours.

What sort of information do inhibition assays reveal? If growth does not happen at T=0 or T=2 hours, it is a good bet that the chosen ingredient either kills the probiotic or inhibits it so badly that it will not grow (meaning its presence in the finished product cannot be confirmed). If growth at T=0 is higher than at T=2 hours, it is likely the ingredient can kill off the probiotic over time and, thus, may not be a good match. If growth at T=0 is stymied but growth at T=2 hours compares favorably to the expected CFU count (less than 25 percent difference), it is likely the ingredient of interest inhibits/delays growth of the probiotic but does not kill it. If growth at both T=0 and T= 2 hours compares favorably to the expected CFU count—congratulations on finding a probable match! It is important to note that inhibition assays do not take the place of traditional real-time stability studies for probiotics; rather, they complement stability studies, allowing the team to troubleshoot pilot formulations before they become commercialized product.

Going back to turmeric as a working example, our team found not all turmeric ingredients show negative results with inhibition assays, and not all probiotics are inhibited/killed in the presence of turmeric. Although not all probiotics fared well, a particular strain of Lactobacillus acidophilus thrived, especially in the presence of microencapsulated turmeric ingredients designed to improve bioavailability of the curcuminoids (a “wall” between “bad neighbors” per se). So choose probiotic and non-probiotic ingredients wisely and confirm the choice with inhibition assays to prevent stability trouble down the road.

Jeremy Bartos, Ph.D., is the vice president of research and development (R&D) at MeriCal. MeriCal is presenting a session, “The Art of Manufacturing an Innovative Probiotic Product,” on Thursday, Nov. 8, as part of the IPA Probiotics Resource Center at SupplySide West.