Assessment of heat resistance of bacterial spores from food product isolates by fluorescence monitoring of dipicolinic acid release

Abstract

This study is aimed at the development and application of a convenient and rapid optical assay to monitor the wet-heat resistance of bacterial endospores occurring in food samples. We tested the feasibility of measuring the release of the abundant spore component dipicolinic acid (DPA) as a probe for heat inactivation. Spores were isolated from the laboratory type strain Bacillus subtilis 168 and from two food product isolates, Bacillus subtilis A163 and Bacillus sporothermodurans IC4. Spores from the lab strain appeared much less heat resistant than those from the two food product isolates. The decimal reduction times (D values) for spores from strains 168, A163, and IC4 recovered on Trypticase soy agar were 1.4, 0.7, and 0.3 min at 105 degrees C, 120 degrees C, and 131 degrees C, respectively. The estimated Z values were 6.3 degrees C, 6.1 degrees C, and 9.7 degrees C, respectively. The extent of DPA release from the three spore crops was monitored as a function of incubation time and temperature. DPA concentrations were determined by measuring the emission at 545 nm of the fluorescent terbium-DPA complex in a microtiter plate fluorometer. We defined spore heat resistance as the critical DPA release temperature (Tc), the temperature at which half the DPA content has been released within a fixed incubation time. We found Tc values for spores from Bacillus strains 168, A163, and IC4 of 108 degrees C, 121 degrees C, and 131 degrees C, respectively. On the basis of these observations, we developed a quantitative model that describes the time and temperature dependence of the experimentally determined extent of DPA release and spore inactivation. The model predicts a DPA release rate profile for each inactivated spore. In addition, it uncovers remarkable differences in the values for the temperature dependence parameters for the rate of spore inactivation, DPA release duration, and DPA release delay.

FIG. 1.

Wet-heat inactivation of Bacillus spores. All data points in each of the three curves have been fitted with a constant Z value. The lines represent the fit with equations 1 and 3. Fit parameters are shown in Table 2. (A) Heat inactivation of spores from strain 168 (cultured in modified MOPS medium with 1.5 mM Ca²⁺). (B) Heat inactivation of spores from strain A163 (cultured in MOPS medium). (C) Heat inactivation of spores from strain IC4 (cultured in modified MOPS medium with 1.5 mM Ca²⁺).

FIG. 2.

Fluorescence emission of the (Tb-DPA)⁺ complex. (A) Fluorescence emission spectra were recorded from 475 to 600 nm with an Aminco-Bowman series 2 luminescence spectrometer, with excitation at 270 nm and sensitivity at 565 V. The dipicolinic acid concentrations used for the emission spectra were 0 (dashed line) and 100 (solid line) μM in 100 μM terbium(III) chloride, 20 mM Tris buffer, pH 7.5. (B) Calibration curve for DPA concentrations from 0 to 10 μM monitored at 545 nm in a microplate fluorometer. The inset for DPA concentrations from 0 to 100 nM was used to determine the detection limit of 15 nM (see the text for more details). The experimental settings for the calibration curve were identical to those used for all further DPA measurements (see Materials and Methods).

FIG. 3.

Heat-induced dipicolinic acid release by Bacillus spores. DPA release is expressed as the percentage of the total content in spores from strains 168 (squares), A163 (circles), and IC4 (triangles). Spore suspensions were incubated for 1.5 min at the indicated temperatures, followed by determination of the amount of released DPA by 545-nm fluorescence emission measurements at 270-nm excitation of the (Tb-DPA)⁺ complex in a microplate fluorometer.

FIG. 4.

Kinetics of DPA release. (A) DPA release (expressed in relative fluorescence units) in spores isolated from the laboratory strain 168 during heat inactivation at 98°C (squares), 105°C (triangles), and 111°C (circles). Lines indicate DPA release as described by the model (equation 8). (B) DPA release rate profiles (expressed in arbitrary units) of an inactivated spore of B. subtilis 168 at 98°C (solid line), 105°C (dashed line), and 111°C (dotted line), as calculated from the proposed model (equation 5). The black square indicates m₉₈, the DPA release delay parameter at 98°C (expressed in minutes), and the double-headed arrow indicates Δ₉₈, the DPA release duration parameter at 98°C (expressed in minutes). (C) Measured (circles) (see also Fig. 3) and model-predicted (equation 9) (solid line) DPA release of spores from B. subtilis 168 during a 1.5-min incubation period.