Probiotic and postbiotic analytical methods: a perspective of available enumeration techniques

Abstract

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.

Specific cultivation-independent enumeration of viable cells in probiotic products using a combination of fluorescence in situ hybridization and flow cytometry

“…For these reasons, plate count is nowadays considered more of an estimate than an actual quantification of viable cells ( Davey, 2011 ; Boyte et al, 2023 ).…”

“…An alternative method of great interest in the field of probiotics is flow cytometry ( Adan et al, 2017 ; Pane et al, 2018 ; Sielatycka et al, 2021 ; Boyte et al, 2023 ; Pereira et al, 2023 ; Tracey et al, 2023 ) due to its rapid and automatic results. Staining the cells with fluorescent dyes prior to measurement can provide valuable information.…”

Real-time PCR methods for identification and stability monitoring of Bifidobacterium longum subsp. longum UABl-14 during shelf life

“…Because the probiotic products that were used in the stability monitoring experiment were multi-strain products, it was not possible to compare the viable counts to plate counts. Nonetheless, plate count and viability PCR measure viability differently where plate count methods rely on cultivability while viability PCR relies on membrane integrity as a measure of viability (Boyte et al, 2023). Previous studies have reported discrepancies in viable counts determined using culturedependent versus culture-independent methods, especially following storage (Fiore et al, 2020;Wendel, 2022;Shehata et al, 2023).…”

“…PCR based methods are widely used for probiotic identification including species-specific and strain-specific methods (Morovic et al, 2016;Kim et al, 2020;Shehata et al, 2021a,b;Kim et al, 2022). For probiotic enumeration, plate count methods are currently the most commonly used methods for probiotic quantification (Davis, 2014;Weitzel et al, 2021;Boyte et al, 2023), however, other cultureindependent methods such as flow cytometry and PCR based methods are also emerging for probiotic enumeration (Boyte et al, 2023).…”

Proof of concept: real-time viability and metabolic profiling of probiotics with isothermal microcalorimetry

“…The conventional plate count method may not effectively capture the entire spectrum of microbial activity, particularly for those cells termed “viable but non-culturable” (VBNC). This has driven the exploration of alternative methods that can provide a more comprehensive and nuanced picture of the microbial viability ( Boyte et al, 2023 ).…”