The influence of thermal and nonthermal food preservation methodologies on the liberation and ultrastructure of bacterial endotoxins

Abstract

Consumer demands for fresh, microbiologically safe foods with high organoleptical and nutritional quality has led to the development of novel food preservation technologies as alternatives or enhancements to traditional preservation techniques. An example of these novel preservation technologies is high hydrostatic pressure (HHP) processing. It involves the applications of static pressure of 50 to 1 000 mega Pascals (MPa) to solid or packaged liquid foods, with varying holding times. The combination of factors to enhance preservation is increasingly being used in industry, e.g. the use of different temperatures and additives (hurdles) can enhance the preservative effect of HHP. In this study the influence of HHP on organism viability and growth response was assessed. The organisms evaluated included Escherichia coli O111, Listeria monocytogenes (UAFSBCC) and Staphylococcus aureus (ATCC 25923), in peptone water, which was subjected to HHP of 200 MPa for 15 minutes at 8 and 50 ºC respectively. Subsequent to the mentioned pressurisation, sub-culturing was performed and growth responses were evaluated at 0, 6, 18, 24, 30, 42 and 48 hours. Bacterial survival and growth response was measured by means of intact cell count, colony forming units and optical density. From the results it was eminent that bacterial cells were only sublethally injured and were able to repair within 48 hours of enriched sub-culturing. E. coli O111 proved to be most sensitive to HHP with Staphylococcus aureus (ATCC 25923) most resistant. This study also proved that bacterial concentration and inactivation rate are inversely proportionate to each other. Subsequent to growth and cell repair assessments, E. coli O111 was selected as a model to evaluate the effect of sublethal HHP on the liberation and toxicity of bacterial endotoxins (free and cell wall bound). It is also known that different extraction procedures extract different lipopolysaccharides (LPS) fractions and therefore LPS was extracted from the test broth by a combination method of Folch, Lees & Sloane-Stanley, and Venter and Ivanov. The extraction yielded a biphasic system, LPS with reduced lipid content in the upper phase (aqueous) and LPS with increased lipid content in the lower phase (organic). Following extraction the Limulus amebocyte lysate (LAL) test was performed to quantify the concentration (assumed) of LPS in the aqueous and organic phases. Free LPS was detected within six hours in the supernatant in the high and low bacterial loads, moreover the toxicity response of post HHP cell damage was more pronounced at 50 ºC (hurdle) than that observed for the treatments at 8 ºC (hurdle) and more so in the organic phases. The latter implied that HHP not only resulted in quantity LPS variation but also in structural change. However membrane repair was apparently complete after 48 hours, as differences in toxicity were no longer evident. Furthermore, the use of a porcine IL-6 ELISA assay was evaluated as an alternative for the customary LAL as a biomarker for pyrogenic substances in matrixes. Porcine whole blood was challenged for IL-6 production by LPS in the samples from the organic and aqueous systems. A porcine IL-6 enzyme-linked immunosorbent assay was used to assess IL-6 expression in whole blood after being challenged with LPS. From the results it emanated that HHP caused in a change in LPS structure which resulted in a decreased IL-6 expression in whole blood, indicating that structural adaptation of the cell membrane in response to HHP influenced the ability of LPS to stimulate macrophages and monocytes. Therefore, further research and development would be required to evaluate the influence of post HHP LPS on human IL-6 expression. When comparing the porcine IL-6 with the LAL no correlation in toxicity could be established in any of the treatment parameters.

Finally it can be concluded that HHP had an influence in the structural morphology of LPS. These structural changes could result in LPS being more toxic, it could also have an effect on the accuracy of immunological assessments, the ability to form biofilm, and susceptibility to phages.