Detection of Alicyclobacillus spp. and Identification of Guaiacol Production Using the GENE-UP? PRO ACB, IFU Method No. 12, and Cosmo Bio Assays

Some species of Alicyclobacillus spoil beverages by producing guaiacol. Current culture-based methods detect the presence of Alicyclobacillus spp. and a subsequent peroxidase assay determines if the isolate can produce guaiacol. However, these methods are time-consuming and can yield false negatives due to differences in growth optima among species. The purpose of this study was to compare a RT-PCR-based method, the GENE-UP (R) PRO ACB assay, to the IFU Method No. 12 Enumeration and Enrichment methods. Ten species of Alicyclobacillus were detected using the tested RT-PCR assay, while A. dauci and A. kakegewensis were not detected using either IFU protocol. Low concentrations (1-10, 10-100, and 100-1,000 CFU/10 mL) of A. aci-doterrestris, A. suci, and A. acidocaldarius were tested in five matrices. The proportion of positive samples iden-tified using the tested RT-PCR assay (62/84) or the IFU Enrichment protocol (62/84) did not differ significantly from the proportion of inoculated samples (63/84). However, the IFU Enumeration method (32/84) detected statistically fewer positives. Additionally, methods identifying guaiacol production were compared. The pro-portion of correctly identified guaiacol producers using the tested RT-PCR assay (51/63) was not significantly different than those identified using the 3 h Cosmo Bio assay (54/63). Finally, four commercial samples of orange juice and sucrose solution were tested. Alicyclobacillus spp. were identified in all four samples using the IFU Enrichment method and in two samples using the tested RT-PCR assay. However, Alicyclobacillus was not detected in any sample using the IFU Enumeration method. Overall, this study showed consistent detection of Alicyclobacillus spp. using either the IFU Enrichment protocol or the tested RT-PCR assay, which both outperformed the IFU Enumeration protocol. Both the 3 h guaiacol bioassay and the tested RT-PCR assays consistently differentiated guaiacol-producing and nonproducing strains.