Targeting the vanillic acid decarboxylase gene for Alicyclobacillus acidoterrestris quantification and guaiacol assessment in apple juices using real time PCR.

Alicyclobacillus spp. has recently received much attention due to its implication in the spoilage of pasteurized fruit juices, which is characterized by the formation of guaiacol. Previous researches indicate that not all Alicyclobacillus spp. are able to produce guaiacol. The aim of this study was to identify possible differences in the vanillic acid decarboxylase gene involved in guaiacol biosynthesis and then develop specific detection methods for guaiacol producing Alicyclobacillus. Agarose gel electrophoresis results showed that the partial vdcC gene was present in all the guaiacol producing Alicyclobacillus, but absent in non-guaicaol producing strains apart from A. fastidiosus DSM 17978. On the basis of the vdcC gene sequence, a primer pair specific to A. acidoterrestris was designed; then a SYBR Green I real time PCR was established for the direct quantification of A. acidoterrestris in apple juice, and the detection limit was 2.6 × 10 CFU/mL. The developed real time PCR system was used to detect A. acidoterrestris in 36 artificially contaminated apple juice samples and guaiacol production in the sample was also analyzed by GC-MS. The Gompertz model was employed to describe the relationship between A. acidoterrestris cell concentration and guaiacol content, and the value of R was 0.854. This work provides an alternative to conventional methods of guaiacol quantification and A. acidoterrestris detection and could be very useful for the early recognition of A. acidoterrestris contamination in fruit juices.