Rapid and simple detection of endospore counts in probiotic Bacillus cultures using dipicolinic acid (DPA) as a marker

Spore counting in probiotic Bacillus cultures using dipicolinic acid (DPA) as a marker was studied for developing a rapid and simple detection method. The newly developed method is based on the fluorescence enhancement by a new chelating agent, which forms a complex with EuCl(3) and DPA. The results showed that 1,2-cyclohexanediamine-N,N,N′N′-tetraacetic acid (CyDTA) greatly enhanced the fluorescence intensity in all selected chelating agents. The optimal composition of the fluorescence complex DPA-Eu-CyDTA had a detection limit of 0.3 nM of DPA. Metal ions in high concentrations, including Cu(2+), Fe(2+), Fe(3+), Al(3+), and Zn(2+), might lower the detection sensitivity, which could be eliminated by diluting the sample with the metal ions below 10 μM. The maximum release of DPA was achieved by heating treatments at 121 °C for at least 10 min for two types of Bacillus endospores. The spore concentrations and corresponding released DPA fluorescence intensities were linearly associated (coefficient R(2) = 0.9993 and 0.9995 for Bacillus subtilis MA139 and Bacillus licheniformis BL20386, respectively). The detection limit for both strains reached approximately 6800 spores/mL. The verification results showed that the DPA fluorimetry assay developed in the present study was fully consistent with the plate-counting assay. The study shows that the fluorescence complex DPA-Eu-CyDTA can be reliably used for the detection of endospores in Bacillus fermentation for the production of probiotics.