Postbiotics: Considerations for Safety and Quality Management
Andrzej A. Benkowski 1 , Emily Schmitt , Eric Williams , Clinton Copple , and J. David Legan 4
Eurofins Microbiology Laboratories, Inc. Madison, WI
ABSTRACT
Introduction
Postbiotics are preparations of inanimate microorganisms and/or their components that confer a health benefit on the host. They are a rapidly growing (11.5 % CAGR, 2022 32), emerging category of functional ingredients in part because of their safety profiles, but pose challenges related to defining efficacy and measuring potency compared to their traditional probiotic counterparts.
Purpose
To demonstrate a proof of concept for a reliable methodology evaluating postbiotic materials as characterized by inanimate cells by flow cytometry.
Methods
Commercial postbiotic materials were evaluated by flow cytometry following ISO 19344 Protocol B, a viability assay based on cell membrane integrity using dual nucleic acid stains SYTO24 and Propidium Iodide (PI). PI cannot penetrate intact cell membranes but penetrates damaged membranes and binds to the cellular DNA, indicating cell death or injury. The test therefore separates the respective populations (live, injured, dead) based on the different emission spectra of bound and unbound PI. Direct microscopic counting using a hemacytometer was used to determine baseline concentration of inanimate microorganisms present in the samples. Five test samples at different concentrations were each tested five times by both flow cytometry and direct cell counting.
Results
Each cellular concentration tested by flow cytometry and count via microscopy was spaced at logarithmic intervals with the most concentrated cell population falling at ~3.0E11 cells per gram and the least concentrated falling at ~3.0E7 cells per gram spanning a range of four logs. A linear regression analysis was performed on the data set using log transformed results to establish a correlation coefficient between the flow cytometry data and the direct cell counts. The correlation coefficient was >95%, which indicates that flow cytometry can accurately determine postbiotic concentration in a pure material.
Significance
The results demonstrate flow cytometry can be a powerful tool to establish and confirm concentration of postbiotic materials, which is the first, critical step in ensuring product quality and delivering to consumers the benefits they expect.
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