Low-temperature depuration and low-temperature high hydrostatic pressure processing as post-harvest interventions for diminishing Vibrio parahaemolyticus contamination in oysters

Graduation date: 2014Access restricted to the OSU Community, at author's request, from June 7, 2014 - Dec. 7, 2014Vibrio parahaemolyticus is a leading cause of acute gastroenteritis associated with consumption of seafood, particularly raw oysters. The United States Centers for Disease Control and Prevention (CDC) estimated that 45,000 cases of V. parahaemolyticus infection occur each year in the U.S. A recent CDC report revealed that the incidence of V. parahaemolyticus infection increased 76% when comparing 2011 level to 1996-1998 level. The aim of this research was to develop low-temperature depuration and low-temperature high pressure processing (HPP) as post-harvest means for decreasing V. parahaemolyticus contamination in oysters for safe consumption. Fresh Pacific oysters (Crassostrea gigas) were inoculated with a mixed culture (10⁵ MPN/g) of five clinical V. parahaemolyticus strains and depurated with UV-sterilized artificial seawater in a laboratory-recirculating system at refrigeration temperatures (2, 3, 7, 10, 12.5, and 15 °C) for 4 to 6 days. Depuration of oysters at 2 or 3 °C for 4 days did not result in significant reductions (P > 0.05) of V. parahaemolyticus in the oysters. However, depuration of oysters in 30-ppt seawater at 7 to 15 ºC for 5 days decreased populations of V. parahaemolyticus in oysters by >3.0 log MPN/g with no loss of oysters. Further studies revealed that the efficacy of depuration in reducing V. parahaemolyticus in oysters was influenced by water salinity with an optimum range of 20 - 30 ppt, but not types (diploid vs triploid) or sizes of oysters. The low-temperature depuration (10 - 12.5 °C, 25 ppt) can be applied as a simple and cost-effective treatment for reducing V. parahaemolyticus contamination in oysters. Investigation of the efficacy of low-temperature HPP in inactivating V. parahaemolyticus was conducted with clinical and environmental strains of V. parahaemolyticus strains in 2% NaCl solution or oyster homogenates subjected to pressure treatments (200, 250 and 300 MPa for 5 and 10 min) at 20, 15, 5 and 1.5 °C. Inactivation of V. parahaemolyticus cells by HPP was greatly enhanced by lowering processing temperature from 20 to 5 and 1.5 °C. A treatment of 250 MPa for 5 min at 1.5 °C resulted in complete inactivation (>6.4 log CFU/ml) of V. parahaemolyticus in culture suspension and oyster homogenates. This low-temperature HPP can be applied as a post-harvest process for complete elimination of V. parahaemolyticus from raw oysters for safe consumption. Studies of the mechanism of HPP for inactivating V. parahaemolyticus cells observed cell disruption, indicated by increased leakage of intracellular materials, after pressure treatments of 200 to 300 MPa for 5 and 10 min. Scanning electron microscopy imaging revealed a clear disruption of V. parahaemolyticus cells after a treatment of 300 MPa for 5 min. In addition, HPP caused protein denaturation as evidenced by decreases in protein and sulfhydryl contents of cellular proteins extracted from V. parahaemolyticus cells after pressure treatments. SDS-PAGE analysis of cellular proteins also demonstrated changes in protein profiles of the cells after pressure treatments. These observations suggest that inactivation of V. parahaemolyticus cells by HPP is associated with cell membrane damage and protein denaturation, which appear to be dependent on the strain and pressure applied in a treatment