The release of dipicolinic acid--the rate-limiting step of Bacillus endospore inactivation during the high pressure thermal sterilization process

Abstract

High pressure combined with elevated temperatures can produce low acid, commercially sterile and shelf-stable foods. Depending on the temperature and pressure levels applied, bacterial endospores pass through different pathways, which can lead to a pressure-induced germination or inactivation. Regardless of the pathway, Bacillus endospores first release pyridine-2,6-dicarboxylic acid (DPA), which contributes to the low amount of free water in the spore core and is consequently responsible for the spore's high resistance against wet and dry heat. This is therefore the rate-limiting step in the high pressure sterilization process. To evaluate the impact of a broad pressure, temperature and time domain on the DPA release, Bacillus subtilis spores were pressure treated between 0.1 and 900 MPa at between 30 and 80 °C under isothermal isobaric conditions during dwell time. DPA quantification was assessed using HPLC, and samples were taken both immediately and 2 h after the pressure treatment. To obtain a release kinetic for some pressure-temperature conditions, samples were collected between 1s and 60 min after decompression. A multiresponse kinetic model was then used to derive a model covering all kinetic data. The isorate lines modeled for the DPA release in the chosen pressure-temperature landscape enabled the determination of three distinct zones. (I) For pressures <600 MPa and temperatures >50 °C, a 90% DPA release was achievable in less than 5 min and no difference in the amount of DPA was found immediately 2 h after pressurization. This may indicate irreversible damage to the inner spore membrane or membrane proteins. (II) Above 600 MPa the synergism between pressure and temperature diminished, and the treatment temperature alone dominated DPA release. (III) Pressures <600 MPa and temperatures <50 °C resulted in a retarded release of DPA, with strong increased differences in the amount of DPA released after 2 h, which implies a pressure-induced physiological like germination with cortex degradation, which continues after pressure release. Furthermore, at 600 MPa and 40 °C, a linear relationship was found for the DPA release rate constants ln(k(DPA)) between 1 and 30 min.