Optimization of Viability Treatment Essential for Accurate Droplet Digital PCR Enumeration of Probiotics

Abstract

Improvements offered by viability droplet digital PCR (v-ddPCR) include increased precision, specificity and decreased time to results making for an attractive alternative method to traditional plate count enumeration of probiotic products. A major hurdle faced in v-ddPCR, however, is distinguishing between live and dead cells. The objective of this study was to evaluate a combination of PMA and EMA (PE51) for viability treatment of freeze-dried probiotic powders. Lactobacillus acidophilus La-14 and Bifidobacterium animalis subsp. lactis Bi-07 were analyzed over a 2-log PE51 concentration gradient to investigate the efficiency across genus and assay targets. Results suggest a need to optimize viability dye concentration based on the genera of the organism, but also the assay target, even when analyzing the same organism. When optimized for PE51 concentration, strain specific v-ddPCR assays for both La-14 and Bi-07 were demonstrated to agree with plate count enumeration results. In conclusion, while these v-ddPCR assays require highly specific optimization, they are better suited for the future of the probiotic industry and are suggested to be implemented in probiotic product testing.

Locked Nucleic Acid Hydrolysis Probes for the Specific Identification of Probiotic Strains Bifidobacterium animalis subsp. lactis DSM 15954 and Bi-07™

“…These assays can be further optimized and validated as quantitative methods for viable count determination in qPCR or droplet digital PCR based methods ( Hansen et al, 2018 , 2020 ; Shehata and Newmaster, 2021 ). This will require the use of viability dyes to distinguish live versus dead cells, and previous work showed that viability dyes must be optimized for each assay ( Kiefer et al, 2020 ). After generating standard curves, colony forming units (CFUs) can be interpolated based on Cq values.…”

Assessing Viability and Stress Tolerance of Probiotics—A Review

“…Culture-dependent methods, where plate count enumeration is the most prevalent, have for long been the standard for viability measurement of probiotic cultures and products (Lahtinen et al, 2006;Davis, 2014;Hansen et al, 2020;Kiefer et al, 2020). They are uncomplicated to perform but laborious and require media and conditions that are well adapted to the strain of interest (Davis, 2014).…”

“…Factors such as oxygen tolerance, antibiotic susceptibility, and nutritional preferences must be considered when choosing and designing suitable media and cultivation conditions. Thus, although sometimes possible with antibiotics, it can be difficult to separate a mixture of two or several strains with highly similar growth requirements (Jackson et al, 2002;Hansen et al, 2018;Vinderola et al, 2019;Kiefer et al, 2020).…”

“…Usage of molecular methods, which are based on nucleic acid detection, enables detection and in combination with identification of bacteria on a strain-level with high specificity and sensitivity in a high-throughput manner (Keer and Birch, 2003). Therefore, molecular methods are of great use in samples or products containing multiple probiotic strains (Keer and Birch, 2003;Postollec et al, 2011;Hansen et al, 2018;Kiefer et al, 2020).…”

“…The awareness of the health benefits of probiotic microorganisms, most often lactic acid bacteria (LAB), has increased rapidly the latest years and so has the global market for probiotic products (Rosenstiel and Stange, 2010;Hill et al, 2014;Hansen et al, 2018;Jackson et al, 2019;Fallico et al, 2020;Kiefer et al, 2020). Consequently, the market supply of probiotic products has become increasingly diverse, with a large number of both fermented food products and supplements, formulations, species, and strains (Kolaček et al, 2017).…”