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Applied and Environmental Microbiology, July 2005, p. 3556-3564, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3556-3564.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Assessment of Heat Resistance of Bacterial Spores from Food Product Isolates by Fluorescence Monitoring of Dipicolinic Acid Release

Remco Kort,1* Andrea C. O'Brien,1 Ivo H. M. van Stokkum,2 Suus J. C. M. Oomes,3 Wim Crielaard,1 Klaas J. Hellingwerf,1 and Stanley Brul1,3

Laboratory for Microbiology, Swammerdam Institute for Life Sciences, University of Amsterdam,1 Department of Physics and Astronomy, Faculty of Sciences, Vrije Universiteit, Amsterdam,2 Department of Microbiological Control, Unilever Research and Development, Vlaardingen, The Netherlands3

Received 1 September 2004/ Accepted 17 January 2005

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°C, 120°C, and 131°C, respectively. The estimated Z values were 6.3°C, 6.1°C, and 9.7°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°C, 121°C, and 131°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.

* Corresponding author. Mailing address: Laboratory for Microbiology, Room C-809, Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018WV Amsterdam, The Netherlands. Phone: 31 (0) 20 525 7062. Fax: 31 (0) 20 525 7056. E-mail: rkort{at}science.uva.nl.

Applied and Environmental Microbiology, July 2005, p. 3556-3564, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3556-3564.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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