Flow cytometry: a versatile technology for specific quantification and viability assessment of microorganisms in multi-strain probiotic products

Classical microbiology techniques are the gold standard for probiotic enumeration. However, these techniques are limited by parameters of time, specificity and incapacity to detect viable but non-culturable (VBNC) microorganisms and non-viable cells. The aim of the study was to evaluate flow cytometry as a novel method for the specific quantification of viable and non-viable probiotics in multi-strain products. Custom polyclonal antibodies were produced against five probiotic strains from different species (Bifidobacterium bifidum R0071, Bifidobacterium longum ssp. infantis R0033, Bifidobacterium longum ssp. longum R0175, Lactobacillus helveticus R0052 and Lactobacillus rhamnosus R0011). Evaluation of specificity confirmed that all antibodies were specific at least at the subspecies level. A flow cytometry method combining specific antibodies and viability assessment with SYTO 24 and propidium iodide, was applied to quantify these strains in three commercial products. Analyses were conducted on two flow cytometry instruments by two operators and compared with classical microbiology using selective media. Results indicated that flow cytometry provides higher cell counts than classical microbiology (p < 0.05) in 73% cases highlighting the possible presence of VBNC. Equivalent performances (repeatability & reproducibility) were obtained for both methods. This study showed that flow cytometry methods can be applied to probiotic enumeration and viability assessment. Combination with polyclonal antibodies can achieve sufficient specificity to differentiate closely related strains. Flow cytometry provides absolute and specific quantification of viable and non-viable probiotic strains in a very short time (< 2h) compared to classical techniques (> 48h), bringing efficient tools for research and development and quality control. This article is protected by copyright. All rights reserved.