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Flow Cytometry Proponents Discuss Probiotic Enumeration Techniques

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Probiotic products on the shelf must be labeled with milligrams of probiotics. While this is a regulatory standard, it is a pointless vehicle when it comes to letting consumers know if a strain is alive or dead. The International Probiotics Association petitioned the U.S. Food and Drug Administration in 2016 to voluntarily allow her CFUs to be used on labels. The FDA issued his draft guidance in 2018, allowing probiotic declarations in his CFU in addition to milligrams.

CFU (colony forming units) are measured using the plate count method. This ‘gold standard’ method involves adding bacterial cells to a suitable medium (various types of agar), culturing them under specific conditions, and optically counting their ability to produce probiotics. colony of cells.

This technology is not without limitations. “Plate counting can take two to three days, sometimes five days.”Andrzej Benkowski, senior technical leader at Eurofins Microbiology Laboratories, Inc. in Madison, Wisconsin, explains: “Also, plate counting involves a lot of manual manipulation, so we see a lot of variability, especially between

“Contract manufacturers may then add more probiotics to their formulas to cover these variations, so there is an excess.” na

This has led some to consider alternative methods for enumerating cells. Although genetic techniques such as whole-genome sequencing and 16S rRNA sequencing have been mainly applied for identification, notable progress has been made in applying a PCR technique for enumeration called quantitative PCR (qPCR). I was.

flow cytometryna

Since the publication of ISO 19344 in 2015, a technique called flow cytometry seems to be gaining momentum. ISO 19344 is the international standard for the quantification of lactic acid bacteria by flow cytometry in fermented products, starter cultures and probiotics used in dairy products.

The United States Pharmacopoeia has devoted almost a full day to recent events. “Probiotics, live biotherapeutic products, new technologies in microbiome analysis”On Flow Cytometry (October 6-7, 2022).

Flow cytometry itself has been around for decades, with uses such as blood analysis and the detection of certain cancers. This is a culture-independent technique that works by adding a fluorescent stain called a fluorophore to a solution of cells and passing them through a small funnel that sorts them into a single file. A laser is used as the light source, and a fluorophore allows the detector to distinguish between live, dead, and/or damaged cells.

Finally, two measurements are taken: TFU or total fluorescence units (all cells regardless of state) and AFU or active fluorescence units (all viable (living) cells).

Big brands like Seed already label their probiotics with AFU. Marco Pane, research and development manager for probiotics based in Italy and a major proponent of the technology, says dozens of other products in many markets around the world have been added to his AFU. It states that it is labeled with

“Regulations do not mandate flow cytometry.” Payne said, “But labeling them with AFU makes them more competent from an informational point of view.” na

IPA Executive Director George Paraskevakos noted that his association included the flow cytometry methodology in its first draft of a civil petition to the CFU in 2016. “However, we decided not to confuse the issue of what we were trying to accomplish at the time, which was to get CFUs instead of mgs on the Supplement Facts panel on the final product label for probiotics. na

“Having said that, I added wording to keep the door open for new standards in development. We may reopen the conversation for enumeration via FC at some point if needed.” There is. We’ll see how this evolves.”na

Colonies vs activated cellsna

Consideration for postbiotics

nanaIn Gram-positive bacteria, such as lactobacilli, more than 95% of their dry weight is the cell wall, where molecules are evolutionarily programmed to interact with the host. ” Probiotic’s Marco Pane explains:

“So if you have 10 billion plate counts, CFUs, you probably have a lot more cells than that (living cells and dead cells) and their cell walls All the molecules are being sensed by the immune system.You need to know everything there is and the total number of cells.Flow cytometry will give you that number.”na

“Flow cytometry is structural analysis and provides a more direct picture.” Payne said. In other words, count cells. By using appropriate stains, this technique is very rapid (tens of thousands of cells per minute) and can tell whether cells are alive, dead and/or damaged. “It doesn’t necessarily answer the question of what’s in my product, just what grows on the plate.”na

Considering conventional plating techniques, it’s basically counting subgroups of cells that can form colonies, he added.

Therefore, there is no direct correlation between CFU and AFU. Although it may be close to 1:1 for fresh products, this correlation does not extend through shelf life.

“Flow cytometry is real-time data” Added by Benkowski of Eurofins. “We can go from sample preparation to data analysis in an hour.”na

According to Martin Wilkinson of the University of Limerick, this real-time data is a big advantage. “For the factory-level people who are the producers of these cultures, there is a huge advantage because it gives them real-time data on how the cultures work.”he told us Wilkinson is the author of his 2018 review, “Flow Cytometry as a Potential Method to Measure Bacterial Viability of Probiotic Products: A Review.It was commissioned by the International Probiotics Association.

Wilkinson also noted the huge lab footprint when it comes to plate count. Multiple plates, different media, while the flow cytometer is his benchtop machine, which is getting smaller every year.

“Even from an environmental point of view, flow cytometry makes sense.” He said.

Scientific literature…na

However, one of the major factors in favor of CFU is that the majority of clinical studies describe probiotic doses in terms of CFU. This will change as more researchers report data in his AFU instead of or in addition to his CFU, but that scientific body needs to grow.

“At Probiotics, we measure probiotics by plate count (CFU) and flow cytometry (AFU).” Payne said. “We prefer flow cytometry, but it’s important to provide both numbers. na

“Overall, it’s only a matter of time. I would also strongly encourage companies and researchers to use flow cytometry and offer AFU. The wind is turning.” he added.

Dr. Wilkinson agreed: “It’s a mindset change. Let’s not be bound by CFU.”na

Remaining obstacles na

One common criticism of flow cytometry is that it cannot handle multi-strain formulations or encapsulated products, but according to experts we spoke to, both of these things can be addressed and You’ll see real progress.

For example, Ireland-based Anabio developed a method to enumerate microencapsulated probiotics using flow cytometry, which was subsequently validated by Eurofins.

“Flow cytometry uses the latest technology to provide a fast, accurate, reliable and ISO-certified method for enumerating encapsulated probiotics.” Anabio’s CEO, Dr. Aidan Fitzsimons, said: “It allows the client, 3rdParty laboratories and regulatory agencies can easily and accurately enumerate encapsulated probiotics for a wide range of uses. It is therefore fundamental to ensure that AnaBio can bring the health benefits of encapsulated probiotics to the widest possible audience. “na

A solution to the multi-strain prescription problem is also available, although it may not be cost-effective at the moment. For example, BioAster in France has developed and developed a flow cytometry method that can assess the bacterial load in faeces, establish the ratio of Gram-positive and Gram-negative bacteria, and monitor specific species of interest using polyclonal antibodies. verified.

lead the conversationna

Probiotical’s Pane, Limerick’s Wilkinson, Eurofin’s Benkowski, and Anabio’s Fitzsimons are all proponents of flow cytometry, but where is the industry as a whole?

“With our experts on analytical standards, IPA’s position on flow cytometry or any other methodology on the matter is that we are always open to new developments and try to participate in any discussion of new methods. .”IPA’s Paraskevakos told me.

“As the voice of our industry, it is important that we strive to understand and guide the conversation within this area. The ultimate goal is to report viable probiotic ingredients within final products. In doing so, it remains simple and understandable for both regulators and consumers.”na