Probiotic products on the shelves must label the probiotics in milligrams. This is a regulatory standard, but it is a meaningless measure when it comes to informing the user whether a bacterial strain is alive or dead. In 2016, the International Probiotic Association petitioned the US Food and Drug Administration to allow the voluntary use of CFU on the label. The FDA issued draft guidance in 2018 allowing claims for probiotics in CFUs, in addition to milligrams.
CFUs, or colony-forming units, are measured using a dish-counting technique: This “gold standard” technique involves adding bacterial cells to appropriate media (various types of agar), culturing under specific conditions, and then optically counting the ability of the probiotic to generate a colony of cells.
The technique is not without limitations. “It can take two to three or even five days to count the plates,” explained Andrzej Benkowski, Senior Technical Manager at Eurofins Microbiology Laboratories, Inc. in Madison, Wisconsin. “And because there’s a lot of manual manipulation of plate counts, you can see quite a lot of variability, especially inter-laboratory variation.
“A contract manufacturer can then add more of the probiotic to the formula to cover these variations, so there are overages.”
This has led some to explore alternative cell enumeration methods. Genetic techniques such as whole genome sequencing or 16S rRNA sequencing are primarily applied for identification, although significant progress has been made in the application of PCR techniques for enumeration, called quantitative PCR (qPCR).
Momentum seems to be building for a technique called Flow Cytometry ever since the publication in 2015 of ISO 19344, an international standard for the quantification of lactic acid bacteria by flow cytometry in fermented products, starter cultures and probiotics used in dairy products.
The US Pharmacopoeia devoted almost an entire day to its recent event, “New Technologies in the Analysis of Probiotics, Live Biotherapeutics and Microbiomes”by flow cytometry (October 6-7, 2022).
Flow cytometry itself has been around for decades: it’s used to analyze blood and detect certain types of cancer, for example. It’s a culture-independent technique that works by adding fluorescent dyes called fluorophores to a solution of cells and passing them through a small funnel that sorts them single file. A laser is used as the light source, and fluorophores allow the detector to distinguish between living, dead and/or damaged cells.
At the end, two measures are obtained, TFU or total fluorescent units (all cells regardless of their state) and AFU or active fluorescent units (all viable (living) cells).
Leading brands such as Seed now label their probiotics in AFUs. Dozens of other products in many markets around the world label their products AFU, said Marco Pane, research and development manager for Italy-based Probiotical and a leading proponent of the technique.
“Flow cytometry is optional in the regulations,” said Lord “but by labeling in AFU you become more informationally competent.”
George Paraskevakos, executive director of the IPA, noted that in its first drafts, his association included flow cytometry methodology in its 2016 citizen petition for CFU, “but we decided not to confuse it with what we were trying to achieve at the time, which was to get CFU instead of mg in the supplement facts panel on finished probiotic product labels.
“That being said, we have added wording to leave the door open to the development of new standards and, if necessary, may reopen the conversation about listing through the FC one day.” We’ll see how that plays out.”
Colonies versus active cells
The Postbiotics Consideration
“In gram-positive bacteria like lactobacillus, over 95% of the dry weight is cell wall, and that’s where you find the molecules that are evolutionarily programmed to talk to the host,” explained Marco Pane of Probiotical.
“So if you have a platelet count, CFU, of 10 billion, to get to that number, you probably have a lot more cells (live and dead) than that, and all those cell wall molecules are sensed by the immune system.” You need to know everything that is inside and the total number of cells. Flow cytometry provides this number.
“Flow cytometry is a structural analysis and gives you a more direct picture,” said Pane. In other words, it counts cells – and using the appropriate stains, the technique can tell you very quickly (tens of thousands of cells per minute) whether they are alive or dead and/or damaged. “It doesn’t necessarily answer the question of what’s in my product, just what grows on a plate.”
Given the traditional plating technique, it basically counts a subset of cells that can form a colony, he added.
And for this reason, there is no direct relationship between CFU and AFU. It can be close to 1:1 in fresh produce, but this correlation does not extend over shelf life.
“Flow cytometry is real-time data,” added Benkovski of Eurofins. “You can go from sample preparation to data analysis in an hour.”
This real-time data is a huge advantage, according to Martin Wilkinson of the University of Limerick. “For the guys at the factory level, the growers of these crops, there’s a huge advantage because it provides real-time data on the functionality of the crops,”he told us. Wilkinson is the author of the 2018 review. “Flow cytometry as a potential method for measuring bacterial viability in probiotic products: a review.”“, which was commissioned by the International Probiotic Association.
Wilkinson also noted that for plate counts, the lab footprint is huge: multiple plates, different media, while flow cytometers are benchtop machines that get smaller every year.
“Even from an environmental perspective, flow cytometry makes sense,” he said.
The scientific literature…
However, one huge factor in CFU’s favor is that the majority of clinical trials describe probiotic doses in terms of CFU. This will change as more researchers report data in AFU instead of or in addition to CFU, but this science must grow.
“At Probiotical we measure probiotics by plaque count (CFU) and flow cytometry (AFU)” said Pane. “We prefer flow cytometry, but it’s important to provide both numbers.
“It’s generally a matter of time. I urge companies and scientists to also use flow cytometry and provide AFU. The winds are changing,” he added.
Dr. Wilkinson agreed: “It’s a change in mindset. Stop binding yourself to CFU.
A common criticism of flow cytometry is that it cannot handle multi-strain formulations nor encapsulated products, but both are being addressed and real progress is being made, according to the experts we spoke to.
For example, Ireland-based Anabio developed a method using flow cytometry to enumerate microencapsulated probiotics, which was subsequently validated by Eurofins.
“Flow cytometry uses advanced technology to provide a rapid, accurate, reliable, ISO-accredited method for enumerating encapsulated probiotics,” Dr. Aidan Fitzsimons, CEO of Anabio, told us. “It allows customers, 3rdparty laboratories and regulatory authorities to easily and accurately list encapsulated probiotics in a wide range of applications and is therefore central to enabling AnaBio to deliver the health benefits of encapsulated probiotics to the widest possible audience.
Solutions to the multi-strain formulation problem are also available, although they are not currently cost-effective. BioAster in France, for example, has developed and validated flow cytometry methods to be able to estimate the bacterial load in faecal material, determine the proportions of gram-positive versus gram-negative bacteria, and monitor specific species of interest using polyclonal antibodies.
Directing the conversation
Probiotical’s Pane, Limerick’s Wilkinson, Eurofin’s Benkowski and Anabio’s Fitzsimons are all proponents of flow cytometry, but what is the general industry position?
“With our experts in terms of analytical standards, the IPA’s position on flow cytometry or any other methodology for that matter is that we always have an ear to the ground for new developments and try to be involved in any discussions about new methods.”IPA’s Paraskevakos told us.
“As the voice of our industry, it’s important that we understand and try to steer the conversation in this area. The ultimate goal is to maintain simplicity and understanding for both regulators and consumers when reporting viable probiotic ingredients in finished products.