Abstract
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 EuCl3 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 Cu2+, Fe2+, Fe3+, Al3+, and Zn2+, 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 R2 = 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.
Highlights
Bacillus species have been extensively studied for use as probiotics during the past 20 years both for scientific exploration and commercial development (Hong et al 2005; Cutting 2011)
dipicolinic acid (DPA) standard fluorimetry assay The supplementation of chelating agents in DPA-Eu complexes greatly enhanced the production of fluorescence signals by ethylene glycol tetraacetic acid (EGTA), hydroxyethylethylenediamine triacetic acid (HEDTA), ethylenediaminetetraacetic acid (EDTA), and CyDTA (Fig. 1a)
The Tb-based DPA fluorimetry assay has been intensively studied for determining the number of endospores since the DPA-Tb complex shows the advantage of highly enhanced fluorescence and a long fluorescence lifetime (Rosen et al 1997; Hindle and Hall 1999; Pellegrino et al 2002)
Summary
Bacillus species have been extensively studied for use as probiotics during the past 20 years both for scientific exploration and commercial development (Hong et al 2005; Cutting 2011). Spore probiotics are widely used in humans for medical purposes as dietary supplements for preventing diarrhea and intestinal disorders. The strain of Bacillus licheniformis BL20386 used in this study is the main ingredient in probiotic Zhengchangsheng® capsule, a useful therapeutic agent for the treatment of diarrhea (Heo et al 2014). In the field of animal nutrition, spore probiotics enhance the growth performance and diseaseresistance of poultry, swine, and shrimps (Cutting 2011). The B. subtilis MA139 strain, used in this study, has been applied as feed probiotics, benefiting piglets’ growth and modifying intestinal microflora (Guo et al 2006). Except for non-spore Lactobacillus probiotics, endospores are extremely heat stable and acid resistant, which enables long-time storage without loss of viability during storage at room temperature and survival when passing through the gastric barrier
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