In the past decade, technological advances have greatly expanded our ability to analyze the human gut microbiota, identifying and characterizing hundreds of new bacterial species from human gastrointestinal biopsies and stool samples.
Many of these newly characterized organisms have been linked to important health outcomes, creating the hope that some of these species might be delivered as ‘next-gen’ probiotics. Amongst these newly identified organisms associated with health outcomes, Akkermansia muciniphila has emerged as one of the most promising next-gen probiotic candidates.¹⁻²
First characterized in 2004, Akkermansia muciniphila is now recognized as an important keystone organism within the human GI tract associated with numerous health benefits, including insulin-related metabolic functions, satiety-signaling, immunomodulation, gut barrier support, and a host of gut-brain signals.³
However, unlike most first-generation probiotics (for example, Lactobacillus, Bifidobacterium, Streptococcus, and Saccharomyces), Akkermansia and many other next-gen probiotic candidates are difficult to grow at commercial scales – often because they are anaerobic or have unusual growth requirements – and are less stable than a finished dry powder.
Nonetheless, if these products are to be considered ‘true’ probiotics they must fit the definition of being a ‘live organism’ delivered at ‘adequate amounts’.⁴ Therefore, understanding how these organisms are enumerated is critical for determining if they meet the criteria of a live probiotic, as it directly impacts whether the end consumer receives the probiotic viability they assume based on the product label.
Probiotic enumeration: Is it alive?
The most commonly used technique to enumerate probiotics, and the one relied upon for both research and product labeling of probiotics, is by directly counting colonies on plates. This method relies on plating a series of dilutions of the original sample followed by several days of incubation at 37°C.
Single cells that are capable of metabolizing the substrate of the plate will begin to multiply, eventually producing a colony that is large enough to be enumerated. Plates that contain between 30-300 colonies can then be used to calculate the number of ‘live’ organisms in the original samples, expressed as colony forming units (CFU) per gram.
While this plate counting method is slow and may require different media to optimize growth of different organisms, this method is considered the gold standard for enumerating probiotics in both raw material and finished product testing because it only counts live culturable cells.
Recently, other methods have been explored to enumerate probiotics, including flow cytometry, a method mostly employed for counting and differentiating blood cells. By the use of several fluorescent dyes, flow cytometry can differentiate between cells that are damaged/dead from those which have intact cell membranes.
These intact cells are often deemed to be ‘viable’ based on the assumption that intact cells which can exclude various dyes are live organisms. In general, three units of measure are used when using flow cytometry: active fluorescent units (AFU), a measure of cells assumed to be viable; non-active fluorescent units (n-AFU), a measure of dead/damaged cells; and total fluorescent units (TFU), a measure of all cells (viable, dead, damaged).⁵
Importantly, when this method is used to enumerate cells, researchers noted that the AFU measure was always higher than the CFU measure from the same sample. This difference has been referred to as the portion of cells that are ‘viable, but not culturable’ (VBNC).
However, while flow cytometry is often touted as being more advanced and even more accurate, there is limited evidence to confirm that cells deemed viable when measured by AFU alone are as biologically viable as culturable cells.
Thomas G. Guilliams, Ph.D. of The Point Institute, and a recent panelist discussing the future of next-gen probiotics at the IPA World Congress/Probiota 2025, says: “Flow cytometry is a great new advance in bacterial enumeration, but it is incumbent upon each manufacturer who labels a probiotic product using AFUs to explain how this method of enumeration is related to true biological viability over the complete shelf-life of the product; something that is likely to be influenced by strain, growth conditions, drying techniques, excipients and packaging.”
According to Guilliams, “the market is being flooded by Akkermansia products labeled using only AFU or TFU measures, and consumers are unaware of the important differences between these units of measure and the gold standard (CFU) used to define the viability of probiotics”.
Live Akkermansia muciniphila stabilized to deliver CFU
Vidya, a world-renowned manufacturer of herbal extracts and first-generation probiotics, has now developed a patent-pending fermentation and spray-drying process of its proprietary Akkermansia muciniphila strain VHAKM, delivered under the brand NūGensia™.
Notably, the viability and potency of NūGensia is expressed in Colony Forming Units (CFU) to ensure live cells are delivered to support gut health by degrading mucin and producing beneficial metabolites.
“Vidya has invested enormous amounts of time and resources to figure out how to grow and spray-dry our Akkermansia to ensure we can deliver live organisms which, when manufactured correctly, can meet label claims at the time of expiration using CFU enumeration methods,” says Subhendu Nayak, Director R&D, Probiotics at Vidya.
In keeping with the definition of a probiotic, Vidya is also working to determine the adequate dose needed to confer a variety of health benefits by funding a number of studies, including pre-clinical studies showing GLP-1 and insulin modulation, as well as human clinical studies using doses of 5, 7 and 10 billion CFU per day for gut, brain, and metabolic-related outcomes.
“Our current data suggests that most of the current products on the market are not dosed high enough to trigger significant GLP-1 activity, even if all the cells measured as AFUs are viable,” adds Nayak.
Akkermansia muciniphila is one of the first next-gen probiotic organisms available to consumers and is gaining attention. However, while most of these products are derived from nearly identical strains, they deliver products that are likely to differ in their levels of viable organisms.
Some of these products deliver dead organisms (intentionally-killed) which technically makes these products postbiotics; while others deliver Akkermansia enumerated as AFUs, a measure which has an unknown relationship to CFUs over the shelf-life of the product.
Viability is critical for the delivery of all the potential metabolic benefits of keystone microbes, such as Akkermansia, and a significant hurdle to overcome if we are to fully leverage the potential of the next-gen probiotic era.
References
- Jayachandran, M.; et al. A critical review of the relationship between dietary components, the gut microbe Akkermansia muciniphila, and human health. Crit Rev Food Sci Nutr. 2020;60(13):2265-2276.
- Cani, P.D.; et al. Akkermansia muciniphila: paradigm for next-generation beneficial microorganisms. Nat Rev Gastroenterol Hepatol. 2022;19(10):625-637.
- Ioannou, A.; et al. Akkermansia muciniphila: biology, microbial ecology, host interactions and therapeutic potential. Nat Rev Microbiol. 2024.
- McFarland, L.V.; et al. From yaks to yogurt: the history, development, and current use of probiotics. Clin Infect Dis. 2015;60 Suppl 2:S85-S90.
- Wilkinson, M.G.; et al. Flow cytometry as a potential method of measuring bacterial viability in probiotic products: A review. Trends in Food Science & Technology. 2018; 78: 1-10.
