Expression of clpL1 and clpL2 genes in Lactobacillus rhamnosus VTT E-97800 after exposure to acid and heat stress treatments or to freeze-drying

Abstract

The aim of the study was to evaluate the potential of utilising the information on expression levels of selected stress genes in assessing the quality of probiotic products. For this purpose RT-qPCR methods were developed to study the expression of clpL1 and clpL2 stress genes in Lactobacillus rhamnosus VTT E-97800 (E800) cells after exposure to processing-related stress conditions or to freeze-drying. Heat treatments in laboratory scale were performed with E800 cells incubated at 47 °C or 50 °C for 60 min. Acid treatments were performed both at laboratory and fermenter scale. At laboratory scale E800 cells were inoculated into General Edible Medium (GEM) adjusted to pH 4.0 and pH 3.5 and incubated at 37 °C for 180 min, whereas fermenter-grown cells were exposed to pH 4.0 for 60 min at the end of the fermentation. RNA from fresh cells and freeze-dried powders was reverse transcribed after isolation, quantification and standardisation. clpL1 and clpL2 transcripts were analysed by RT-qPCR with SYBR Green I. clpL1 was induced in L. rhamnosus E800 cells exposed to 50 °C and to a much lesser extent to 47 °C. No induction was observed for clpL2 in E800 cells during either acid or heat treatment, in any of the conditions applied. RNA isolation from freeze-dried powders was unsuccessful although several attempts were made with high quality products. In conclusion, our results suggest that developing quality indicators for probiotic products based on differences in the expression of stress genes is a challenging task for several reasons: at least with some genes (like in the present study with clpL) quite harsh conditions are needed to detect differences in the gene expression; mRNA isolation from freeze-dried powders was unsuccessful which hampers the quality analysis of large proportion of probiotic products; and furthermore RT-qPCR proved to be a too laborious procedure for routine use.