Abstract/Summary

High hydrostatic pressure (HHP) food processing is a novel minimal technology that involves hydrostatic pressure in the inactivation of foodborne pathogens towards achieving stable food shelf life, microbial safety and nutritious food products. Escherichia coli is an emerging foodborne pathogen sensitive to lethal levels of HHP, but some pathogenic strains have developed piezotolerance compromising HHP efficiency and presenting a potential risk to food safety. Gene composition and regulation are essential in bacterial adaptation to food processing conditions. Genetic information that enabled piezotolerance in foodborne bacteria is inadequate. Identifying gene and molecular mechanisms that play significant roles in piezotolerance could enable the design of efficient HHP treatment and tackle microbial food safety issues. The conventional bagging method majorly applied in HHP is challenging. Designing techniques for rapid HHP assessment of bacterial cultures will encourage the unravelling of many genes relevant to piezotolerance. The research aimed to apply functional genomics in conventional microbiology studies to: identify genes and molecular mechanisms relevant to piezotolerance, design hurdle technology of synergistic combination of HHP and supercritical carbon dioxide and adopt the technology in designing conditions for bacterial inactivation in ready-to-eat salmon. Bagging of E. coli using bubble wrap was investigated. Three needle sizes were determined for making holes in bubble wraps. Moreso, nail polish, chemical sealants, and a physical sealant, Grey septum were evaluated for sealing holes made by the selected needle. The counting of E. coli colonies in agar containing tetrazolium salt in bubble wrap in HHP studies was investigated. Conventional dilution of E. coli in agar and broth before HHP and plating afterwards was evaluated. E. coli K-12 (WT) and its mutants were screened in HHP at 300 MPa for 5 min using the bubble wrap-septum technique, and viability was determined with the conventional diluting and plating method. Twenty-six presumptive piezosensitive and twenty-four presumptive piezotolerant mutants were selected. Further assessment of these mutants in HHP revealed that twenty-eight and eleven genes confer piezotolerance and piezosensitivity, respectively, with associated molecular mechanisms. E. coli K-12 and selected mutants were treated in the designed hurdle technology of synergistic combination of Supercritical carbon dioxide (Sc-CO2) and HHP of 2.3X volume of CO2 at 150 MPa for 10 min at 35 oC. Ten genes relevant to Sc-CO2 tolerance were revealed, including zntA, yaiS, ybiA, yafX, cpxA, ylaC, rseA, ydcE, ynfC and rpoS. The WT inactivation was higher in combined treatment than in HHP only. Similarly, its inactivation increased at 150 to 300 MPa, and in increased holding times, 10 to 20 min, p<0.05. The concentration of WT, 8 log CFU mL-1 contaminated on 1 to 3g salmon weights reduced significantly with the designed hurdle of Sc-CO2 and HHP than HHP only. The logarithm concentration reduction of E. coli was highest on 3g salmon, 5.6 log CFU mL-1 in the combined treatment after 20 min at 300 MPa, p<0.05. Similarly, the inactivation of L. monocytogenes contaminated on 1 to 3g of salmon was higher using the combined treatment than HHP only. The logarithm concentration reduction of L. monocytogenes was highest on 3g salmon, > 5.0 log CFU mL-1 in the combined treatment after 20 min at 300 MPa. The designed hurdle method is promising for fish processing. However, the process needs modification to preserve the fish’s natural colour, enhancing its organoleptic acceptability. This study can provide a standard guideline to gain information on all genes in E. coli and other foodborne pathogens relevant to HHP and to design efficient hurdle technology of Sc-CO2+HHP for their control, ensuring the safety of ready-to-eat food products.