Graduate Thesis Or Dissertation
 

Application of Antimicrobial Substance in Depuration for Decreasing Vibrio Parahaemolyticus Contamination in the Pacific Oysters (Crassostrea gigas)

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  • Vibrio parahaemolyticus is the leading cause of foodborne illnesses associated with seafood consumption. Consuming raw or undercooked seafood contaminated with V. parahaemolyticus can result in development of acute gastroenteritis with symptoms of nausea, vomiting, abdominal cramping and watery diarrhea within 24 hours of infection. Illness is usually self-limited and lasts for about 3 days. The United States Centers for Disease Control and Prevention estimated that 45,000 cases of V. parahaemolyticus occur annually in the U.S. Depuration is a process which holds shellfish in clean seawater to allow them to purge sand and bacteria. Recently, we developed a refrigerated seawater (7 to 15 °C) depuration capable of decreasing V. parahaemolyticus populations in oysters by >3.0 log MPN/g after 5 days of the process with no adverse effect on oysters. However, the process needs to be shorted to allow application by the shellfish industry. This study investigated the bactericidal activity of natural antimicrobial substances, including rosemary, marjoram, clove, and grape seed extract, against five clinical V. parahaemolyticus strains (10290, 10292, 10293, BE 98-2029, O27-1c1) for their potential applications in depuration to increase its efficacy in reducing V. parahaemolyticus populations in contaminated oysters. Marjoram and rosemary contain low levels of phenolic compounds and had very little antimicrobial activity against V. parahaemolyticus. However, clove and grape seed extract exhibited strong bactericidal effects against clinical strains of V. parahaemolyticus. Populations of V. parahaemolyticus in tryptic soy broth supplemented with 1.5% NaCl (TSB-salt) containing 1.5% clove extract decreased rapidly by >3.88 - >4.81 log CFU/ml within 1 h of incubation at 37 °C. Similarly, populations of V. parahaemolyticus in TSB-salt containing 1% grape seed extract all declined to non-detectable (>4.69-log reductions) after 2 h. Further studies confirmed that both 1.5% of clove extract and 1.0% grade seed extract in TSB-salt reduced a mixed culture of 5 clinical V. parahaemolyticus strains from 5.42 log CFU/ml to non-detectable (<10 CFU/ml) in 1 h at 37 °C. Both clove and grape seed extract exhibited strong antimicrobial activity against clinical strains of V. parahaemolyticus and might be utilized as natural antimicrobial agents to reduce V. parahaemolyticus contamination in seafood. Studies of application of grape seed extract (GSE) in oyster depuration revealed that addition of GSE in artificial seawater (ASW) for depuration increased the efficacy of depuration for reducing V. parahaemolyticus populations in the Pacific oysters (Crassostrea gigas). The populations of V. parahaemolyticus in laboratory inoculated oysters decreased by 3.12 log MPN/g after 3 days of depuration in ASW at 12.5 °C. A greater reduction (3.61 log MPN/g) of V. parahaemolyticus populations in oysters was observed after 3 days of depuration at 12.5 °C with ASW containing 1.0% GSE containing 1.8 mg/ml total phenolic contents as gallic acid equivalents. Increasing the concentration of GSE in ASW from 1.0 to 1.5% greatly enhanced the efficacy of the depuration in decontaminating V. parahaemolyticus in oysters. Populations of V. parahaemolyticus in oysters decreased by 3.77 and 4.18 log MPN/g after one and two days of depuration in ASW containing 1.5% GSE (3.1 mg/ml total phenolic contents as gallic acid equivalents), respectively. However, it required 5 days of depuration in ASW at 12.5 °C to decrease V. parahaemolyticus populations in oysters by 3.71 log MPN/g. Addition of GSE in ASW greatly enhanced depuration process for decontaminating V. parahaemolyticus in oysters. Depuration at 12.5 °C with ASW containing 1.5% GSE was capable of achieving >3.52 log MPN/g reduction of V. parahaemolyticus in Pacific oysters in two days. Further studies are needed to validate the efficacy of this depuration process for decontaminating naturally accumulated V. parahaemolyticus in Pacific oysters.  
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