Behaviour of bacterial endospores and toxins as safety determinants in low acid pressurized food

The inactivation of bacterial endospores by hydrostatic pressure requires the combined application of heat and pressure. After analyzing the flora of carrots and mashed carrots, strains concerning food spoilage were isolated from the carrot habitat to obtain practice relevant “wild strains”. The resistance of spores of 14 food isolates and 5 laboratory strains to treatments with pressure and temperature in mashed carrots was determined. A large variation was observed in the pressure resistance of spores. The sporulation conditions further influenced their pressure resistance. The loss of dipicolinic acid (DPA) from spores varying in their pressure resistance was determined and spore sublethal injury was assessed by determination of the detection times of individual spores. Treatment of spores with pressure and temperature resulted in DPA-free, phase bright spores. These spores were sensitive to moderate heat and exhibited strongly increased detection times as judged by the time required for single spores to grow to visible turbidity of the growth medium. The role of DPA in heat and pressure resistance was further substantiated by the use of the DPA-deficient mutant strain. These results indicate that inactivation of spores by combined pressure / temperature processing is achieved by a two stage mechanism that does not involve germination. DPA is released predominantly by a physico-chemical rather than a physiological process, and the DPA-free spores are inactivated by moderate heat independent of the pressure level. Furthermore, the effect of pressurization on endospores of Clostridium botulinum, as the target organism concerning the safety of low acid, canned food, was investigated. The resistance of seven strains was compared to that of Bacillus cereus, B. subtilis, B. licheniformis, B. smithii, B. amyloliquefaciens, and Thermoanaerobacterium thermosaccharolyticum with respect to treatments with pressure and heat. A large variation was observed in the pressure resistance of C. botulinum spores. Spores of the proteolytic C. botulinum TMW 2.357 exhibited a greater resistance to pressure than spores from all other bacteria examined with the exception of B. amyloliquefaciens. The heat resistance of spores did not correlate with the pressure resistance, neither within strains of C. botulinum nor when compared to spores of T. thermosaccharolyticum. A quantitative release of DPA was observed from C. botulinum spores upon combined pressure / temperature treatments only after inactivation of > 99.999% of the spores. Thus, it was confirmed, that DPA is released by a physico-chemical rather than a physiological process. The resistance of spores to combined pressure / temperature treatments correlated with their ability to retain DPA. The behavior of spores of B. amyloliquefaciens TMW 2.479 and spores of C. botulinum TMW 2.357, as most resistant spores concerning spoilage and food safety, to combined pressure/ temperature treatment with isothermal holding times was further investigated. Both strains showed a pronounced pressure dependent tailing, which was absent at moderate pressure. Inactivation of the clostridia differed strongly to that of the bacilli, as latter showed between 800 and 1200 MPa almost no varying effect in respect of a faster spore reduction. As consequence, the order of the resistance of spores to combined pressure / temperature treatment is not fixed. This different behavior makes it impossible to suggest a generally valid target organism for the pressure processing of low acid foods. A closer look at the safety of novel food processing techniques enabled the evaluation of safety concepts, also for established (thermal) processes. Even though the inactivation of microorganisms by high pressure treatment is the subject of intense investigations, the effect of high pressure on bacterial toxins has not been studied so far. In this study, the influence of combined pressure / temperature treatment on bacterial enterotoxins from Staphylococcus aureus, Bacillus cereus, Vibrio cholerae and Escherichia coli (STa) was determined. Structural alterations were monitored in enzyme immunoassays (EIA’s). Cytotoxicity of the pressure treated supernatant of toxigenic B. cereus DSM 4384 was investigated in order to compare its toxicity with the results obtained in the immunoassay. Reduction of the immunochemical reactivity could be demonstrated, when high pressure was combined with heat. At lower temperatures, there was almost no effect of pressurization of up to 800 MPa in the respective assays. The biological activity of proteins does not necessarily correlate with their immuno reactivity. Likewise, heat and pressure resistance of bacterial toxins did not correlate. The results indicate that pressurization can increase inactivation observed by heat treatment, and combined treatments may be effective at lower temperatures and/or shorter incubation time. However, pressurization may not eliminate the toxins from food to the same extent as temperature treatment. Still, it must be emphasized that pressurization does not mask bacterial toxins, which admits their detection after pressure based food processing.