The Italian team has proposed using flow cytometry and strain specific real-time quantitative polymerase chain reactions (RT-qPCRs) as it is “considerably faster” than culture-based quality control methods - that are time-consuming and operator-dependent - and demonstrates lower variability and higher accuracy, they say.
“In pursuit of a rapid, convenient, and cost-effective method for monitoring bacterial viability, costs and time are important aspects to take into consideration,” they write in Nutrients.
Although RT-qPCR analysis is considered expensive, there can be substantial savings in reagent costs, technical resource and increased throughput when combined with flow cytometry, they say.
“Compared to classical microbiological methods, flow cytometry and RT-qPCR provide the optimal balance of cost in quality control screening scenarios.”
Standard culture procedures are not only expensive but time-consuming and have low statistical accuracy. Moreover, tests typically deliver 20-30% variability, compared with a mean 10% standard deviation for flow cytometry, the authors comment.
In addition, plating techniques take two to five days to complete, while the alternative described in the article takes as little as two hours.
“With flow cytometry and molecular biology approaches, the testing time is relatively short - within two hours for results with the first technology and within eight hours with the second one - with a significant gain in ‘worker effort’”, they write.
“Man-hour is the basis for measuring the cost of professional people and their contribution to productivity.”
The team evaluated the efficacy of flow cytometry and RT-qPCR for specific quantification and qualification quality control of six Lactobacillus and three Bifidobacterium strains in two commercial food supplements supplied by Biolife Italiana.
The first powder was formulated with seven species (Bifidobacterium longum, Bifidobacterium animalis spp. lactis, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus plantarum, Bifidobacterium breve, and Lactobacillus helveticus) and was used to set the sensitivity and specificity of the RT-qPCR reactions and define the qualitative composition of the probiotic mix.
The second contained four bacteria strains (Bifidobacterium animalis spp. lactis, Bifidobacterium breve, Lactobacillus rhamnosus, and Lactobacillus paracasei) and served to optimise quantitative RT-qPCR reactions.
The presence and quantity of probiotics were determined through plate count enumeration and with a multiplex qPCR approach based on TaqMan probes.
Researchers report that the technique demonstrated high sensitivity for strain-specific detection, as well as a high degree of specificity, efficiency, and low variance between measurements.
“High-throughput analysis using a flow-cytometric based-count gave the advantages of a lower variation, a reduction in testing time, the differentiation between active and total cells, and quantification of the fraction of active cells per total cells,” the researchers say.
Quantification “over-estimated” the number of probiotics declared by the producer and detected by culture analysis. The authors believe RT-qPCR was able to identify single cells and amplify genetic targets, as well as dead and damaged cells. Furthermore, it detected the presence of several bacterial species simultaneously in food, environmental matrices, cosmetics, faeces, and other complex matrices.
Accurate identification of probiotic strains is important to optimise health benefits “in a human environment”, therefore findings suggest RT-qPCR could contribute significantly to overcome limitations in classical microbiological techniques, they assert.
Results also confirm the long-term, cost-effective benefits of the combined technique in managing routine tests for a high number of samples per day.
Published online, November 29, 2022: http://doi.org/10.3390/nu14235085
‘An Integrated Analytical Approach for the Characterization of Probiotic Strains in Food Supplements’
Authors: Bolzon. V., Pesando .M., Bulfoni. M., Nencioni. A., and Nencioni. E.,