Anabio breakthrough opens door to probiotic enumeration advances

Consumers are increasingly aware of CFUs, or colony forming units, on probiotic labels, a number that is obtained using a plate count technique. This “gold standard” technique involves adding bacterial cells to an appropriate medium (different types of agars), culturing under specific conditions, and then optically counting the probiotic’s ability to generate a colony of cells. Agar plating methods dating back to the mid-1800s have been used for many years to count probiotics and bacteria in general, and the vast majority of published clinical studies describe probiotic doses in terms of CFUs. However, the technique is not without its limitations. Notably, it can take two to five days to perform, a large lab footprint is required for multiple plates with different media, the manual manipulation required can lead to variability in the data (especially inter-lab variation), and waste is generated in the process. Flow cytometry is a significantly faster, more accurate technique. It is a culture-independent approach that works by adding fluorescent stains called fluorophores to a solution of cells and passing these through a tiny funnel which sorts them into single file. A laser is used as the light source, and the fluorophores allow the detector to differentiate between live, dead and/or damaged cells. “Flow cytometry is more precise, much quicker, giving results in minutes rather than days, and generates far less environmental waste in the form of discarded plastics and media,” Dr. Aidan Fitzsimons, CEO at Anabio, told NutraIngredients. Two measures are obtained at the end, the TFUs or total fluorescent units (all the cells, regardless of their state) and the AFUs or active fluorescent units (all the viable (alive) cells. Dozens of brands in many markets around the world already label their products in AFUs, but widespread adoption has been hampered by a poor correlation between AFUs and CFUs (and therefore reference to the published clinical data). There is not a direct correlation between CFUs and AFUs. It can be close to 1:1 in a fresh product but does not extend over the course of shelf-life. Why has this proved a challenge? The reason, Dr. Fitzsimons explained, lies in differences in what each method actually measures. Plating techniques detect cells that grow on agar plates, specifically those can actually form colonies (and some cells may still be viable and alive but not form colonies). On the other hand, glow cytometry measures cells with an intact cell structure. “These intact alive cells may have been exposed to stresses in food, beverage and supplement production and storage which prevents them growing under standard plating techniques and therefore they may not all be detected using plating techniques: hence the unpredictable differences frequently encountered when trying to correlate the two results,” he said. But all that may be changing, as Anabio has been able to show a consistent correlation between the old and new technique, which could, over time, facilitate a managed transition to the more accurate, faster flow cytometry method. Dr. Fitzsimons told us that AnaBio has developed a “deep understanding of the factors contributing towards differences in CFU and AFU measurements and leveraged this to develop a novel technique establishing a robust correlation between the two measurements. “By optimising the culture conditions to ensure optimum growth of live cells, AnaBio has successfully achieved a 1:1 ratio between flow cytometry and plate counting results,” he said. “The method has been successfully transferred to and validated by an independent third-party laboratory, specifically Eurofins, thereby satisfying regulatory requirements.” It should be stressed that AnaBio’s advance relates to one microencapsulated probiotic strain and has not yet been validated for other strains/species, but this is still a significant achievement and major step forward in solving the AFU-CFU correlation puzzle the probiotic industry is facing. “AnaBio’s novel technique combined with the supporting validation strategies and acceptance criteria enables consistent correlations to be made between probiotic counts via plating and flow cytometry,” Dr. Fitzsimons said. “This significant breakthrough facilitates the adoption of the faster, more precise and environmentally friendly flow cytometry technique for probiotic enumeration, leading to increased market opportunities for probiotic food, beverage and supplements with the associated follow-on public health benefits.”