Inactivation of Vibrio parahaemolyticus, Vibrio vulnificus and noroviruses in oysters by high-hydrostatic pressure, mild heat and cold storage

Raw oysters have become a major "gastronomic" delicacy and are widely appreciated in various parts of the world. Unfortunately, raw or undercooked oysters pose a health risk due to their association with human pathogens including Vibrio spp. and human norovirus (NoV). The objective of this study was to determine the effect of high-hydrostatic pressure (HHP) alone, or in combination with other hurdles such as mild heat and cold storage to inactivate V. parahaemolyticus, V. vulnificus and NoV in oysters.;In the first study, the influence of growth conditions on pressure resistance of V. parahaemolyticus in oysters was determined. Of the growth temperatures evaluated (15, 20, 25, 30, 35 and 40°C), growth temperatures of 20 and 40°C yielded the greatest pressure resistance in V. parahaemolyticus. Post-pressure treatment recovery conditions including salt concentration, H2O2-degrading compounds, incubation temperature and gas atmosphere on the recovery of V.parahaemolyticus after pressure treatment were also determined. For recovery of pressure-injured cells, 0.1% peptone water with 1% NaCl and TSA with 0.5% NaCl was the best diluent and plating medium, respectively. Addition of sodium pyruvate (0.05 to 0.2%) or catalase (8 to 32 U/ml) did not increase the recovery of V. parahaemolyticus after pressure treatment. A 30°C incubation temperature was optimum for recovery of V. parahaemolyticus in pressure-treated oysters. The results of this study indicated that the growth conditions for V. parahaemolyticus before and after high hydrostatic pressure treatment should be taken into consideration when assessing the efficacy of pressure inactivation.;Next, the effects of HHP combined with mild heat and cold storage on the inactivation of V. parahaemolyticus and V. vulnificus in live oysters were investigated. Pressurization at 200 to 300 MPa for 2 min resulted in various degrees of inactivation, from 1.2 to >7 log MPN/g reductions. HHP and mild heat had synergistic effects. HHP at ≥275 MPa for 2 min followed by heat treatment at 45°C for 20 min and HHP at ≥200 MPa for 2 min followed by heat treatment at 50°C for 15 min completely eliminated both pathogens in oysters (negative enrichment results). Cold storage at -18, 4 and 10°C, prior to HHP, decreased V. parahaemolyticus or V. vulnificus populations by 1.5-3.0 log MPN/g, but did not increase their sensitivity to subsequent HHP treatments. HPP at 300 MPa for 2 min at 21°C, followed by 5-day ice storage or 7-day frozen storage, and HPP at 250 MPa for 2 min at 21°C, followed by 10-day ice or 7-day frozen storage, completely inactivated V. parahaemolyticus in oysters. The combination of HHP at a relatively low pressures followed by mild heat treatment or short term frozen storage could potentially be applied by the shellfish industry as a post-harvest process to eliminate V. parahaemolyticus and V. vulnificus in oysters.;Finally, the efficacy of HHP on inactivating human norovirus (NoV) and its two surrogates, Tulane virus (TV) and murine norovirus (MNV-1), in oysters was evaluated. TV was more sensitive to HHP than MNV-1 in oysters. A treatment at 350 MPa at 4 and 21°C reduced TV to below the detection limit (>4.5 log PFU/g reductions) while HHP at 350 MPa at 4°C resulted in a 4.3 log PFU/g reduction of MNV-1. For NoV, after binding to porcine gastric mucin (PGM)-conjugated magnetic beads (PGM-MBs), RT-PCR detectable NoV RNA was reduced by 0.4 to >4.1 log by HHP at 300 to 600 MPa for 5 min in oysters. Genogroup I.1 NoV was more resistant to HHP than the Genogroup II.4 strain. A treatment temperature of 6°C caused higher inactivation of NoV than a temperature of 25°C and for the treatment at 300 MPa, the difference was statistically significant (P < 0.05). These results showed that HHP could be applied as potential intervention for inactivating NoV in raw oysters. Binding to PGM-MB followed by RT-PCR detection can be explored as a practical means of evaluating the infectious and non-infectious states of NoV..