Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (Hill et al., 2014). Delivering the correct probiotic strains at the correct dose of viable cells is essential to achieve their health benefits (Tripathi and Giri, 2014; Kolaček et al., 2017; Sánchez et al., 2017). However, several studies reported variable rates of non-compliance in probiotic products, more specifically, failure of probiotic products to meet declared strain contents and/or viable counts (Morovic et al., 2016; Shehata and Newmaster, 2020a,b). Thus, reliable, and accurate methods for probiotic strain identification and viable count determination are essential for probiotic authentication and quality assessment.

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 culture-independent methods such as flow cytometry and PCR based methods are also emerging for probiotic enumeration (Boyte et al., 2023).

Plate count methods have several limitations such as the low specificity, i.e., inability to enumerate individual strains in multi-strain blends as these methods enable enumeration at the genus level or species level only if using selective growth media. This is a huge limitation since the health benefits of probiotics are strain specific (Klein et al., 2010; Sánchez et al., 2017; Mcfarland et al., 2018). Furthermore, plate count methods are culture-dependent methods which measure viability as cultivability, and thus these methods do not detect cells that exist in a viable but non culturable (VBNC) state (Wilkinson, 2018; Gorsuch et al., 2019; Wendel, 2022).

Alternative enumeration methods such as flow cytometry and viability PCR based methods are culture-independent methods that measure viability beyond cultivability (ISO, 2015; Hansen et al., 2018, 2020; Foglia et al., 2020; Kim E. et al., 2023; Ma et al., 2023; Shehata et al., 2023). Thus, these methods are able to count VBNC cells, hence, more accurate viable count determination. Additionally, PCR based methods can be designed to achieve strain specific viable count determination (García-Cayuela et al., 2009; Kramer et al., 2009), which is a huge improvement from the traditional plate count methods. PCR methods can be used with viability dyes in what is called viability PCR to quantify viable cells only (Hansen et al., 2020; Shehata et al., 2023). PCR methods are less laborious, high throughput, and offer shorter time to results (~6 h). Given the advantages of PCR methods over the traditional plate count methods, and their wide use for probiotic species and strain identification, PCR methods represent an attractive alternative method for probiotic enumeration, as they enable simultaneous strain-specific qualitative and quantitative detection.

Bifidobacterium longum subsp. longum is a common bacterium in the gut microbiome of both infants and adults (Oki et al., 2018; Díaz et al., 2021), and strains of this sub species were found to have health benefits such as improving chronic constipation in elderly individuals (Takeda et al., 2023), alleviating glucose intolerance in Type 2 diabetic mice (Kim W. J. et al., 2023), improving cognitive functions in healthy elderly adults (Shi et al., 2023), and reducing perceived stress in healthy adults (Boehme et al., 2023).

Strain Bifidobacterium longum subsp. longum UABl-14^™ is a common probiotic strain in probiotic products that was found to support digestive health, modulate bowel functions and increase fibrolytic microbiota in participants with functional constipation when used in combination with other strains (Martoni et al., 2019). Here we present the development and validation of real-time PCR (qPCR) methods for strain specific identification and viable count determination of this important probiotic strain, Bifidobacterium longum subsp. longum UABl-14^™.

Probiotics are sold in food format such as fermented food products as well as in pharmaceutical dosage forms such as capsules and tablets as natural health products or dietary supplements (Health Canada, 2003). The global probiotic market size is growing rapidly, valued at USD 58.17 billion in 2021, and anticipated to reach USD 111.21 billion in 2030 (Grand-View-Research-Inc, 2022). With the expanding market size, multiple reports have shown failure of probiotic products to meet label claims, observed as strain substitution, missing strains, presence of undeclared strains or lower viable counts compared to label claims during shelf life and before expiration dates (Morovic et al., 2016; Patro et al., 2016; Kolaček et al., 2017; Shehata and Newmaster, 2020a,b). This label non-compliance can result in partial or complete loss of efficacy (Tripathi and Giri, 2014; Kolaček et al., 2017; Sánchez et al., 2017; Jackson et al., 2019). Thus, analytical methods that support product authentication via confirming label information about product content is extremely important (Fusco et al., 2023).

B. longum subsp. longum UABl-14 is a common probiotic strain in probiotic products that was proven to support digestive health (Martoni et al., 2019). However, to the best of our knowledge, there are no available methods to achieve strain-specific identification and enumeration of this strain. In this study, real-time PCR based methods for strain-specific identification and enumeration of strain UABl-14 were developed and validated to facilitate the quality assurance of probiotic products that contain this strain.

A strain-specific identification and/or enumeration method requires robust bioinformatic analyses of genome sequences to confirm strain specificity, as well as extensive validation to ensure accurate and precise performance. Bioinformatic analyses identified a unique sequence region in the genome of strain UABl-14. The sequence region showed no similarity to any sequence in the Nucleotide collection database in NCBI GenBank. Primers and a hydrolysis probe were designed to target this unique sequence region. The primers and probe were validated for use in strain-specific identification and enumeration methods. The specificity of the UABl-14 strain-specific assay was evaluated in qPCR where the assay successfully amplified all 22 target samples, which included mono-strain and multi-strain samples. Thirty non-target samples were used in specificity evaluation which included multiple strains of lactobacilli and Bifidobacterium, and included, other strains of B. longum such as Bifidobacterium longum subsp. infantis strains Bi-26, HA-116, and R0033 and Bifidobacterium longum subsp. longum strains Bl-05, HA-135, and R0175 to confirm strain level specificity (Table 1). No amplification was observed from any non-target strains. It is important to note that these non-target strains are commercialized probiotic strains available and common in the market in finished probiotic products. The results confirmed that the assay is strain specific to strain UABl-14 which means the assay will correctly identify strain UABl-14 only. The results also confirmed that the assay works well with both mono-strain and multi-strain samples.

The sensitivity of the UABl-14 strain-specific assay was also evaluated in qPCR. Sensitivity or the LOD is the lowest amount of the target that an assay can detect (Bustin et al., 2009). Standard curves were established and the LOD was determined to be 0.002 ng of DNA (Figure 1). Thus, the assay proved to be highly sensitive, which means the assay is applicable to multi-strain blends and products in which strain UABl-14 is present at low abundance.

The UABl-14 strain-specific assay was further validated for quantitative use for the enumeration of strain UABl-14. To enumerate viable cells only, the assay was used with PMAxx viability dye, a DNA-intercalating dye that inactivates DNA from dead cells. The viability dye treatment is known to vary between strains and thus optimization for each target strain is required (Kiefer et al., 2020). Optimization of PMAxx viability dye treatment with strain UABl-14 showed that 50 μM of PMAxx was effective in inactivating DNA from dead cells from reacting in PCR, achieving 99.97% removal of DNA from heat-killed cells (Figure 2B). Previous studies reported optimal final concentrations of PMA that ranged from 25 μM to 100 μM (Gobert et al., 2018; Hansen et al., 2018; Scariot et al., 2018; Shehata and Newmaster, 2021; Shehata et al., 2023).

A very important parameter to be considered when evaluating a quantitative assay is the reaction efficiency, with the ideal reaction efficiency ranging between 90 and 110% with R² values ≥ 0.98 (Broeders et al., 2014). Reaction efficiency values of the UABl-14 strain-specific assay were 97.2, 95.2, and 95.0% and R² value was 99% in all three replicates (Figure 3). The linear dynamic range covered four dilutions points (Figure 3). An ideal dynamic range covers 5 to 6 dilutions, with a minimum of three dilutions (Bustin et al., 2009). Thus, the UABl-14 strain-specific assay has high efficiency and adequate linear dynamic range.

The repeatability and reproducibility of the UABl-14 strain-specific assay were evaluated. The RSD% for repeatability using three samples tested at five dilutions was below 2.80, and RSD% for reproducibility using three samples tested at five dilutions ranged was below 2.18 (Figure 4). The results indicate that the UABl-14 strain-specific assay is highly precise, since the acceptable value for repeatability and reproducibility is below 25% (Broeders et al., 2014).

The UABl-14 strain-specific assay was evaluated for the ability to monitor strain stability in multi-strain finished products during storage by testing 18 multi-strain finished products at different expiration dates. The methods showed variable viable and total (viable and dead) counts of strain UABl-14 in finished products tested at different expiration dates (Figures 5, 6). Viability of probiotic strains is expected to decline during storage, the decline rate varying with storage conditions such as temperature and moisture levels (Tripathi and Giri, 2014). Improving strain stability during shelf life of probiotic products is a major challenge in the probiotic industry (Morovic et al., 2016). Probiotic products are expected to meet label claims of viable count until expiration dates to maintain efficacy. Thus, methods that enable strain-specific monitoring of stability during shelf life is of great importance.

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 culture-dependent versus culture-independent methods, especially following storage (Fiore et al., 2020; Wendel, 2022; Shehata et al., 2023). This may be attributed to the fact that cell cultivability declines faster than membrane integrity, and to the portion of cells that exist in a VBNC state (Foglia et al., 2020). Since VBNC cells are considered probiotics, viable counts determined using culture-independent methods would be more accurate compared to culture-dependent methods (Foglia et al., 2020).

The real-time PCR methods developed and validated for strain-specific identification and viable count determination of strain UABl-14 are strain-specific, highly sensitive and enable the enumeration of VBNC cells. Thus, the methods offer a significant advancement in viable count determination over the traditional plate count method. The methods allow for simultaneous and cost-effective analyses, serving the dual purpose of identification and enumeration of strain UABl-14 in mono-strain as well as in multi-strain finished products to facilitate quality control measures for efficacious and compliant probiotic products.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

HS: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Methodology, Investigation, Formal analysis, Conceptualization. BH: Writing – review & editing, Methodology, Investigation. SN: Writing – review & editing, Supervision, Resources, Project administration, Conceptualization.