Probiotic and postbiotic analytical methods: a perspective of available enumeration techniques
***Curated Content*** Source Frontiers in Microbiology: Frontiers | Probiotic and postbiotic analytical methods: a perspective of available enumeration techniques (frontiersin.org)
Front. Microbiol., 06 December 2023
Sec. Systems Microbiology
Volume 14 - 2023
Authors: Marie-Eve Boyte1; Andrzej Benkowski2; Marco Pane3; Hanan R. Shehata4
1 NutraPharma Consulting Services Inc., Sainte-Anne-des-Plaines, QC, Canada
2 Eurofins, Madison, WI, United States
3 Probiotical Research s.r.l., Novara, Italy
4 Purity-IQ Inc., Guelph, ON, Canada
Probiotics are the largest non-herbal/traditional dietary supplements category worldwide. To be effective, a probiotic strain must be delivered viable at an adequate dose proven to deliver a health benefit. The objective of this article is to provide an overview of the various technologies available for probiotic enumeration, including a general description of each technology, their advantages and limitations, and their potential for the future of the probiotics industry. The current “gold standard” for analytical quantification of probiotics in the probiotic industry is the Plate Count method (PC). PC measures the bacterial cell’s ability to proliferate into detectable colonies, thus PC relies on cultivability as a measure of viability. Although viability has widely been measured by cultivability, there has been agreement that the definition of viability is not limited to cultivability. For example, bacterial cells may exist in a state known as viable but not culturable (VBNC) where the cells lose cultivability but can maintain some of the characteristics of viable cells as well as probiotic properties. This led to questioning the association between viability and cultivability and the accuracy of PC in enumerating all the viable cells in probiotic products. PC has always been an estimate of the number of viable cells and not a true cell count. Additionally, newer probiotic categories such as Next Generation Probiotics (NGPs) are difficult to culture in routine laboratories as NGPs are often strict anaerobes with extreme sensitivity to atmospheric oxygen. Thus, accurate quantification using culture-based techniques will be complicated. Another emerging category of biotics is postbiotics, which are inanimate microorganisms, also often referred to as tyndallized or heat-killed bacteria. Obviously, culture dependent methods are not suitable for these products, and alternative methods are needed for their quantification. Different methodologies provide a more complete picture of a heterogeneous bacterial population versus PC focusing exclusively on the eventual multiplication of the cells. Alternative culture-independent techniques including real-time PCR, digital PCR and flow cytometry are discussed. These methods can measure viability beyond cultivability (i.e., by measuring cellular enzymatic activity, membrane integrity or membrane potential), and depending on how they are designed they can achieve strain-specific enumeration.
Introduction
Probiotics, which represent the largest category of non-herbal/traditional dietary supplements worldwide, are experiencing significant growth. The global market size for probiotics was valued at USD 58.17 billion in 2021 and is anticipated to grow at a compound annual growth rate (CAGR) of 7.5% from 2021 to 2030 (Grand-View-Research-Inc, 2022).
The World Health Organization in 2002 initially defined probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host” (FAO/WHO, 2002). The definition was later refined in 2014 to “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (Hill et al., 2014), a statement that has gained broad acceptance within both the scientific community and the industry. According to this definition, a probiotic strain must be viable in an appropriate quantity to confer a health benefit to the consumer. However, this definition does not provide any specific standards to identify or quantify this viability, but the common practice is to measure viability using direct plate count (PC) enumeration which expresses results in Colony Forming Units (CFUs).
Breeuwer and Abee in 2000 proposed a broader definition of bacterial viability as having an “intact cytoplasmic membrane, protein and other cell components synthesis (nucleic acids, polysaccharides, etc.) and energy production necessary to maintain cells metabolism; and, eventually, growth and multiplication” (Breeuwer and Abee, 2000). Building on Breeuwer and Abee’s definition, a variety of methodologies can provide a more comprehensive view of the viability of a heterogeneous bacterial population than the traditional PC method, which focuses solely on growth and multiplication potential of a subset of the bacterial population. Moreover, the emergence of a new generation of probiotics comprising strictly anaerobic bacteria presents significant challenges for enumeration using traditional PC methods, making it necessary to explore alternative techniques that can assess their viability and provide a more accurate cell count.
This paper will delve into the most widely used methods for quantifying and assessing the viability of probiotic strains, discuss their limitations, and explore alternative techniques that overcome these challenges. The paper will also introduce the concepts and applications of culture-dependent and culture-independent enumeration methods. Continue Reading...
