Date Published: June 19, 2018
Publisher: Springer Berlin Heidelberg
Author(s): Xiao-Sheng Liang, Chun Liu, Zhu Long, Xiao-Hua Guo.
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.
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 disease-resistance of poultry, swine, and shrimps (Cutting 2011). The B. subtilis MA139 strain, also 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.
Lanthanide ions chelated by DPA show enhanced fluorescence by energy transference. Their unique fluorescent characteristic is often adapted for the determination of DPA from spore-formers in environmental samples (Fichtel et al. 2007). 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). However, in the study by Hindle and Hall (1999), the desired LOD for DPA often relied on excess Tb compared with DPA, and different concentrations of Tb could result in different calibration curves corresponding to standard DPA concentrations. Moreover, unchelated Tb could produce too high background fluorescence (Hindle and Hall 1999), which was in line with the result in Fig. 1b. Therefore, the LOD may depend on desirable concentrations of TbCl3, and the concentrations of reagents are often adjusted before detection (Hindle and Hall 1999). However, in fermentation for spore production, the spore concentration often relies on the culture time and fermentation conditions, and many analytes with different spore concentrations produced in spore optimization designs must be detected simultaneously (Ren et al. 2018). Therefore, the DPA-Tb fluorimetry assay cannot be used as a general analytical method for rapid endospore quantification.