| Biological validation of traditional and alternative food processing technologies which result in commercially sterile products remains challenging. This is particularly true for multiphase food products, largely because of difficulties in proving that the fastest moving particles have been exposed to a sufficiently lethal treatment to inactivate the pathogen of concern, Clostridium botulinum. The purpose of this research was to investigate alternative approaches for application to biovalidation of commercial sterilization. In the first phase of the research, the spores of bacteria commonly used as surrogates in thermal inactivation studies, were immobilized in sodium alginate and the performance of the surrogates validated. The second study focused on developing a molecular-based method to rapidly quantify viable spores surviving thermal processing.;In the first study, spore crops for Geobacillus stearothermophilus and Bacillus subtilis were produced, suspended in a variety of media (water, alginate and salsa con queso), and their thermal inactivation kinetics determined. The D-values for G. stearothermophilus and B. subtilis at 121 °C were above the target D-value of 0.20 min for Clostridium botulinum; in most instances, suspension media did not affect D-values or resulting zD values in a statistically significant manner. The spores were immobilized in a 3% sodium alginate suspension which was used to produce "beads" of approximately 30 muL in volume. These beads could be easily manipulated, colored with dyes, and had consistently high concentrations of spores whose thermal inactivation kinetics did not differ from spore stock suspension. Although efforts were made to track and recover the beads in a model aseptic continuous microwave process, mechanical difficulties complicated their timely recovery, making it difficult to conclusively determine process lethality. However, the alginate-encapsulated spores maintained their physical integrity even after exposure to rigorous time and temperature combinations.;In the second study, a molecular method was developed for the detection and discrimination of viable and non-viable spores of Clostridium sporogenes. In the first phases of the research, a method to extract spore-associated DNA was developed, which included the combined steps of decoating and lysozyme digestion. The process resulted in recovery of high yields of quality DNA. A Sybr green-based quantitative real-time (qPCR) assay targeting the GerAB gene was also designed. When combined with the DNA extraction steps, the assay was log linear over a range of from 102-10 9 spores/mL, with a lower limit of detection of approximately 10 2 spores/mL. It was confirmed that exposure of spores to 121°C for as little as 2 min resulted in near complete degradation of the DNA and loss of PCR amplifiability, suggesting that under stringent heat treatment, the PCR-based method would be able to distinguish viable spores from those which had been killed. Under less stringent processing conditions, the DNA from non-viable spores remained detectable. In this case, the decoated spores were treated a 12.5 mug/ml concentration of propidium monoazide (PMA), a DNA intercalating agent, which selectively enters inactivated bacterial cells, binds to the DNA and makes it unavailable for amplification. Unfortunately, the PMA was not able to selectively inhibit the amplification of DNA derived from dead spores. This was evidenced by the fact that CT values obtained for live and thermally treated spores were nearly identical, regardless of PMA treatment status. Further options for the selective detection of DNA derived from viable spores are under investigation.;Taken together, this research demonstrates the feasibility, as well as hurdles, involved in the design and use of novel methods to evaluate thermal process efficacy as applied to particulate foods. Clearly, alginate-immobilization of spores is an effective method to produce large amounts of stable product for use in biovalidation. PCR-based detection methods to rapidly quantify process lethality have promise, especially when applied to stringent processes; application to less stringent processes requires further refinement. With additional research, it should be possible to move biovalidation forward to include emerging molecular-based technologies for rapid and reliable determination of process lethality. |
