280: Unlocking the Power of qPCR: A New Frontier in Postbiotic Quantification

Introduction

Postbiotics refers to a preparation of inanimate microorganisms and/or their components that confer a health benefit on the host. Unlike probiotics, postbiotics are dead cells, that are able to interact with the host to achieve potential health benefits. Postbiotics have higher stability and longer shelf life compared to probiotics, which led to a growing interest in this category. While culture-based methods are the current standard method for probiotic enumeration, these methods are not applicable for the enumeration of postbiotics. Culture-independent methods such as PCR based methods have higher potential in enumerating this category. PCR based methods are targeted methods that enable species-specific or strain-specific quantification, based on assay design. Here, we explore the potential of PCR based methods in enumerating heat-killed postbiotics of strain Lacticaseibacillus rhamnosus GG.

Methods

A strain-specific real-time PCR assay was previously validated for the viable count determination of L. rhamnosus GG. Here, the potential of the method to quantify heat-killed cells (postbiotics) of L. rhamnosus GG was evaluated. Five samples were heat-killed, then non-heated and heat-killed cells were tested using the strain specific enumeration method with and without a viability dye to determine viable count and total count, respectively. The cell counts were interpolated from the standard curve based on Cq values. The dead cell count was determined as the difference between total count and viable count.

Results

When the method was used without a viability dye, it determined total cell count and when used with a viability dye, it determined viable cell count. The difference between total count and viable count was calculated to determine dead cell count. The method was able to successfully determine the total cell, viable cell and dead cell counts, proving the ability of the method to quantify postbiotics (heat-killed or tyndallized cells).

Significance

This method has huge potential for the quantification of heat-killed cells, which are otherwise not detectable using culture-based methods. It’s a strain specific approach, which makes it suitable for both mono-strain and multi-strain finished products in a variety of matrixes. The method also provides rapid results offering significant value in ensuring quality within this emerging category.

Authors: Hanan R. Shehata, Basma Hassane, Amber Thelen, Marie-Eve Boyte