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Conditions for high pressure inactivation of Vibrio parahaemolyticus in oysters
International Journal of Food Microbiology 127 (2008) 1–5
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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Conditions for high pressure inactivation of Vibrio parahaemolyticus in oysters Ayse G. Kural a, Adrienne E.H. Shearer a, David H. Kingsley b, Haiqiang Chen a,⁎ a b
Department of Animal & Food Sciences, University of Delaware, Newark, DE 19716-2150, United States U.S. Department of Agriculture, Agricultural Research Service, Microbial Food Safety Research Unit, W. W. Baker Center, Delaware State University, Dover, DE 19901, United States
A R T I C L E
I N F O
Article history: Received 12 March 2008 Received in revised form 2 May 2008 Accepted 2 May 2008 Keywords: High pressure processing Vibrio parahaemolyticus Oysters Treatment temperature Treatment time
A B S T R A C T The objective of this study was to identify the high pressure processing conditions (pressure level, time, and temperature) needed to achieve a 5-log reduction of Vibrio parahaemolyticus in live oysters (Crassostrea virginica). Ten strains of V. parahaemolyticus were separately tested for their resistances to high pressure. The two most pressure-resistant strains were then used as a cocktail to represent baro-tolerant environmental strains. To evaluate the effect of temperature on pressure inactivation of V. parahaemolyticus, Vibrio-free oyster meats were inoculated with the cocktail of V. parahaemolyticus and incubated at room temperature (approximately 21 °C) for 24 h. Oyster meats were then blended and treated at 250 MPa for 5 min, 300 MPa for 2 min, and 350 MPa for 1 min. Pressure treatments were carried out at −2, 1, 5, 10, 20, 30, 40, and 45 °C. Temperatures ≥30 °C enhanced pressure inactivation of V. parahaemolyticus. To achieve a 5-log reduction of V. parahaemolyticus in live oysters, pressure treatment needed to be ≥350 MPa for 2 min at temperatures between 1 and 35 °C and ≥300 MPa for 2 min at 40 °C. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Bivalve mollusks are filter-feeders that obtain food from the environment by filtering seawater through their gills. In this process they may concentrate pathogens from polluted water. Among bivalves, the oyster predominates as a disease vector in the USA, UK, and Australia (Cliver, 1995; Lees, 2000). Several recent outbreaks of Vibrio parahaemolyticus associated with oysters have heightened concerns about the safety of raw oyster consumption. In 2006, an outbreak of V. parahaemolyticus infections resulted in 177 cases and was linked to the consumption of contaminated raw shellfish including oysters (Balter et al., 2006). In 1998, the largest V. parahaemolyticus outbreak reported to date in the USA involving 416 cases was linked to consumption of raw oysters (DePaola et al., 2000). To control V. parahaemolyticus infections, the Interstate Shellfish Sanitation Conference (ISSC) proposed post-harvest treatment of shellfish using interventions such as pasteurization. The standard set by the ISSC is a 5-log reduction of V. parahaemolyticus levels with an endpoint of nondetectable at the level of b10 CFU/g (Cook, 2003). Post-harvest treatments including mild heat and irradiation have been proposed to control pathogens in shellfish. However, these treatments are of limited utility since they adversely affect the sensory qualities of shellfish (DiGirolamo et al., 1972; Cook and Ruple, 1992;
⁎ Corresponding author. Tel.: +1 302 831 1045; fax: +1 302 831 2822. E-mail address: [email protected] (H. Chen). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.05.003
Harewood et al., 1994). High pressure processing has been used commercially in the USA to facilitate the shucking of raw oysters for several years. The additional advantage of this technology is that it can inactivate V. parahaemolyticus and Vibrio vulnificus in oysters without compromising their sensory attributes (Lopez-Caballero et al., 2000; He et al., 2002; Cook, 2003). A pressure range of 205–275 MPa at temperatures ranging from 10 to 30 °C, and treatment times of 1 to 3 min are typically used. To our knowledge, studies involving pressure inactivation of V. parahaemolyticus have been conducted only at temperatures between 20 and 25 °C (Styles et al., 1991; Berlin et al., 1999; Calik et al., 2002; Cook, 2003; Koo et al., 2006). It is well documented that the temperature of food during pressurization plays a significant role in inactivation of microorganisms. Temperatures below and slightly above room temperature can enhance pressure inactivation of bacteria. To give examples, Chen (2007a) found that Listeria monocytogenes was most resistant to pressure at temperatures between 10 and 30 °C; Carlez et al. (1993) found that the rates of pressure inactivation of Pseudomonas fluorescens and Listeria innocua in minced beef muscle were much lower at room temperatures than at 4 °C; and a recent study in our laboratory demonstrated that temperatures b20 °C or N30 °C substantially increased pressure inactivation of V. vulnificus in oysters (Kural and Chen, 2008). Therefore, the effect of temperature on pressure inactivation of V. parahaemolyticus in oysters warranted further study. It is economically beneficial to use lower levels of pressure in combination with optimum treatment temperatures to obtain the desired target levels of pathogen inactivation. From a food
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quality point of view, it is better to reduce pressure levels and treatment times since over processing by pressure might adversely affect the sensory qualities of oysters. The objectives of this study were to determine the effect of treatment temperature and pressure levels on inactivation of V. parahaemolyticus and to identify the pressure level, time, and temperature parameters needed to achieve a 5-log reduction of V. parahaemolyticus in live oysters. 2. Materials and methods 2.1. Identification of pressure-resistant strains of V. parahaemolyticus Ten strains of V. parahaemolyticus were tested for their relative sensitivity to high pressure. The strains were ATCC 17802, ATCC 35118, ATCC 17803, ATCC 43996, ATCC 27519, ATCC 49529, ATCC 33845, ATCC 33846, DIE12-052499, and DAL 1094. The last two strains were kindly provided by Dr. Gary Richards from the USDA and the other strains by Dr. Jingkun Li from Strategic Diagnostics, Inc. (Newark, DE). The methods for preparation of cultures and pressure treatment as described by Kural and Chen (2008) were used. Briefly, stock cultures were maintained on plates of tryptic soy agar (TSA; Difco; Becton Dickinson, Sparks, MD, USA) with 2.5% NaCl (TSA-2.5%S) and working cultures of individual strains were prepared in tryptic soy broth (TSB; Difco) with 2.5% NaCl (TSB-2.5%S). A 2.5 ml portion of each culture was pressure-treated at 250, 300, 350, and 400 MPa for 2 min at 21 °C. Pressurization time reported in this study does not include the pressure come-up or release times. Before and after pressurization, numbers of V. parahaemolyticus were determined by preparing serial ten-fold dilutions of treated and un-treated cultures in 0.1% peptone water supplemented with 3% NaCl (P-3%S) and preparing pour plates of suitable dilutions using TSA-2.5%S. Plates were incubated at 37 °C for 72 h prior to counting colonies to allow injured cells to form visible colonies. Based on the results obtained, the two most pressure-resistant V. parahaemolyticus strains, ATCC 43996 and DIE12-052499, were selected for use as a 2-strain cocktail for subsequent experiments. 2.2. Plating media and salt concentration for recovery of pressure-injured cells The two most pressure-resistant strains of V. parahaemolyticus were grown individually in TSB-2.5%S at 37 °C for 24 h. Equal volumes of the two cultures were mixed to form a cocktail. The cocktail was treated at 350 MPa for 2 min at 21 °C. After pressurization, serial dilutions of the cocktail in P-3%S were prepared and numbers of V. parahaemolyticus were determined by spread plating suitable dilutions using TSA plus 0.5% NaCl (TSA-0.5%S), TSA-2.5%S, TSA-0.5% S overlayed with Thiosulfate Citrate Bile Salts Sucrose (TCBS; Difco), which is a selective medium for Vibrio (TSA-0.5%S/TCBS), TSA-2.5%S overlayed with TCBS (TSA-2.5%S/TCBS), and TCBS. Plates were incubated for 5 h before being overlayed with TCBS, as described by Kural and Chen (2008). 2.3. Effects of temperature on pressure inactivation of V. parahaemolyticus in oyster homogenates Medium to large market-size live oysters (Crassostrea virginica), obtained from the College of Marine Studies at the University of Delaware, Lewes, DE, were maintained in an aerated, circulating seawater tank kept at room temperature. The salinity of the seawater was maintained at between 1.5 and 2%. V. parahaemolyticus ATCC 43996 and V. parahaemolyticus DIE12-052499 were grown individually in TSB plus 0.5% NaCl (TSB-0.5%S) at 37 °C for 24 h with shaking, and equal volume of each culture was mixed to form a cocktail. Fifteen live oysters were shucked, and the meat was treated at 450 MPa for 2 min at 21 °C to completely inactivate naturally-present Vibrio (Cook, 2003). Oyster
meat samples were then individually inoculated with 0.1 ml of the V. parahaemolyticus cocktail, incubated at room temperature for 24 h, and homogenized as described by Kural and Chen (2008). Five-gram portions of the homogenate were treated at 250 MPa for 5 min, 300 MPa for 2 min, or 350 MPa for 1 min. The treatment times were selected to give partial inactivation of V. parahaemolyticus so that the temperature effect could be compared. Pressure treatments were carried out with samples at the following initial temperatures: −2, 1, 5, 10, 20, 30, 40, and 45 °C. Water was used as the hydrostatic medium for treatment temperatures above 0 °C, and a mixture of water and propylene glycol (1:1, vol:vol) was used for pressure treatment at −2 °C. The temperatures for the water bath and samples inside the chamber during pressurization were monitored every 2 s using K-type thermocouples (DASYTEC USA, Bedford, NH, USA). After pressure treatments, counts of V. parahaemolyticus in the samples were determined using the overlay method with plates being incubated for 4 h before they were overlayed with TCBS (Kural and Chen, 2008). 2.4. Variations in uptake of V. parahaemolyticus by live oysters V. parahaemolyticus ATCC 43996 and V. parahaemolyticus DIE12052499 were grown individually in TSB-0.5%S at 37 °C, overnight, with shaking. A 1-ml portion of each culture was transferred into 60 ml of TSB-0.5%S. The two cultures were incubated for 24 h at 37 °C and mixed to form a cocktail. Five live oysters were placed into an autoclavable plastic tray filled with 3 L of fresh seawater of 1.5 to 2% salinity and held at room temperature. An air pump (Tetra, Whisper Air Pump, Blacksburg, VA, USA) was used to provide air to the seawater and oysters were fed with algae (Reed Mariculture Inc., San Jose, CA, USA) before they were exposed to V. parahaemolyticus. The 60 ml of V. parahaemolyticus cocktail was poured into the tray and mixed well with the seawater. The tray was then covered with aluminum foil and left at a room temperature of about 21 °C for 24 h. Following V. parahaemolyticus uptake, the oysters were shucked and the meat from each oyster was placed in a sterile stomacher bag. Each portion of oyster meat was stomached for 2 min with two parts of P-1%S. Serial dilutions were made using P-1%S as a diluent, and counts of V. parahaemolyticus were determined using the overlay method. 2.5. Processing parameters needed for a 5-log reduction of V. parahaemolyticus in oysters Twenty-seven live market-size oysters were exposed to a cocktail of V. parahaemolyticus during feeding, as previously described, using 120 ml of the cocktail added to 6 L of seawater. After 24 h, oysters were shucked, and the meat and fluid from each shell were placed in a separate sterile plastic pouch. The pouches were sealed and submerged in the hydrostatic medium surrounding the pressure vessel for 10 min to reach the treatment temperature of 1, 20, 35, or 40 °C. Samples were pressure-treated for 2 min at 250, 300, 350, 400, or 450 MPa. P-1%S was added to each portion of un-treated or pressure-treated oyster meat in a ratio of 1:1 (vol:wt) and the portions were stomached for 2 min. Counts of V. parahaemolyticus were determined using the Most Probable Number (MPN) method in the U.S. FDA Bacteriological Analytical Manual (USFDA, 2006). For treatments in which low counts were expected, 100 ml of alkaline peptone water was added to the contents of the stomacher bag and the bags were incubated for 24 h at 37 °C. Loopful of the bag contents were then streaked onto TCBS agar. Colonies on TCBS plates from the MPN and enrichment experiments were confirmed to be V. parahaemolyticus using a PCR method (Bej et al., 1999). 2.6. Statistical analyses At least three independent trials were conducted for each experiment. Statistical analyses were conducted using Minitab® Release
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14.1 (Minitab Inc., University Park, PA, USA). One-way analysis of variance (ANOVA) was used to decide the significance of differences between treatments (P b 0.05). For multiple comparisons, Tukey's OneWay Multiple Comparisons Test was used (family error rate = 0.05). 3. Results 3.1. Identification of pressure-resistant strains of V. parahaemolyticus Data for the two most pressure-resistant strains (ATCC 43996 and DIE12-052499), one pressure-sensitive strain (ATCC 49529), and one moderately pressure-resistant strain (ATCC 17803) are shown in Fig. 1. Inactivation of V. parahaemolyticus was observed at 250 MPa for all strains, with reductions varying from 0.2 to 3.5 logs. A 2-min treatment at 400 MPa and 21 °C was sufficient to reduce the number of all strains of V. parahaemolyticus to b1 CFU/ml. Substantial differences in pressure resistance were observed among the V. parahaemolyticus strains in the pressure range of 250–350 MPa. Thus, a 2-min treatment at 350 MPa reduced the number of ATCC 43996 by 5.0 and ATCC 49529 by 8.7 log units. 3.2. Plating media and salt concentration for recovery of pressure-injured cells The counts of V. parahaemolyticus after pressure treatment were 6.7, 6.5, 7.0, 6.5 and 3.5 log units on TSA-0.5%S, TSA-2.5%S, TSA-0.5%S/TCBS, TSA-2.5%S/TCBS, and TCBS, respectively. The number of colonies recovered on the overlay plates was consistently higher with TSA supplemented with 0.5% NaCl than with TSA supplemented with 2.5% NaCl, although this difference was not significant (P N 0.05). Therefore, 0.5% NaCl was used to supplement TSA or TSB and 1% NaCl was used to supplement 0.1% peptone water for the subsequent experiments. 3.3. Effect of temperature on pressure inactivation of V. parahaemolyticus in oyster homogenates Studies in our laboratory showed that there was no significant difference (P N 0.05) in recovery of V. parahaemolyticus between 4-h and 5-h incubation before overlaying of TCBS. Therefore, the overlay method with 4-h incubation before the TCBS overlay was used. Fig. 2 shows the effect of temperature on pressure inactivation of the cocktail of the two most pressure-resistant V. parahaemolyticus strains. Temperatures
Fig. 2. Effect of temperature on pressure inactivation of a cocktail of Vibrio parahaemolyticus ATCC 43996 and DIE12-052499 inoculated into oyster meat. The initial counts were approximately 2 × 108 CFU/g. Error bars represent ± one standard deviation. N0 = numbers recovered from un-treated samples or initial numbers: N = numbers recovered from pressure-treated samples.
≥30 °C considerably enhanced pressure inactivation of V. parahaemolyticus. Thus, treatment at 350 MPa for 1 min at temperatures of 20, 30, 40, and 45 °C reduced the counts by 4.7, 5.9, 7.4, and 7.7 log units, respectively. The effect of cold temperature on pressure inactivation of V. parahaemolyticus varied with the pressure applied. When treated at 250 MPa, V. parahaemolyticus was most resistant to pressure at 20 °C. Temperatures above and below 20 °C enhanced pressure inactivation. Thus, with 250 MPa-treatments at −2, 20 or 40 °C numbers were reduced by 5.9, 4.0, and 7.7 log units, respectively . When treated at 300 MPa, V. parahaemolyticus was most resistant to pressure between 1 and 20 °C, with reductions varying from 3.5 to 3.7 log units. When treated at 350 MPa, temperatures between −2 and 10 °C increased the pressure resistance of V. parahaemolyticus, whereas temperatures of ≥20 °C enhanced its sensitivity to pressure. It is important to note that temperature of a food sample increases during pressurization due to adiabatic heating. With an initial sample temperature of 45 °C, the highest temperatures reached by the oyster samples during pressurization due to adiabatic heating were 54 °C at 250-MPa, 55 °C at 300-MPa, and 57 °C at 350-MPa. With an initial sample temperature of 40 °C, the highest temperatures reached were 48 °C at 250-MPa, 50 °C at 300-MPa, and 51 °C at 350-MPa. With initial sample temperatures of ≤30 °C, the highest temperatures reached were ≤40 °C for all the pressure treatments.
Table 1 Reduction in the log numbers of a two-strain cocktail of Vibrio parahaemolyticus accumulated within live oysters by feeding resulting from treatment of the oysters at various pressures and temperaturesa
Fig. 1. Pressure resistance data for Vibrio parahaemolyticus suspended in TSB + 2.5% NaCl. Pressure treatments were carried out at 21 °C for 2 min. The initial numbers were approximately 5 × 108 CFU/ml. N0 = numbers recovered from un-treated samples or initial numbers: N = numbers recovered from pressure-treated samples. At each pressure, columns labeled with different letters are significantly different (P b 0.05). Error bars represent ± one standard deviation.
Pressure
Temperature (°C)
(MPa)
1
20
35
40
250 300 350 400 450
3.1 ± 0.5 4.9 ± 0.5 5.4 ± 0.9 5.8 ± 0.2 N6.5 ± 0.4 (2/3)
2.1 ± 0.2 3.9 ± 1.2 5.3 ± 1.5 N 5.9 ± 1.4 (2/3) N 6.5 ± 0.4 (0/3)
3.5 ± 0.5 4.9 ± 0.3 N6.5 ± 0.4 (2/3) N6.5 ± 0.4 (0/3) N6.5 ± 0.4 (0/3)
3.5 ± 0.8 5.4 ± 1.3 N 6.5 ± 0.4 (0/3) N 6.5 ± 0.4 (0/3) N 6.5 ± 0.4 (0/3)
a
The initial counts were approximately 6.3 log MPN/g. Data are the means of log reductions ± one standard deviation (number of positive samples from enrichment/total trials). The unit for the data was log MPN/g. When the count of Vibrio parahaemolyticus in a sample was b0.6 MPN/g, it was assumed to be 0.6 MPN/g in calculation of means and standard deviations.
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3.4. Processing parameters needed to achieve a minimum 5-log reduction of V. parahaemolyticus in oysters After 24 h of feeding, the numbers of V. parahaemolyticus in oysters were ≥4.8 log CFU/g with a mean of 6.7 log CFU/g and standard deviations ≤1.0 log (within each replicate) for the data from all three replicates. The results for pressure inactivation of the cocktail of V. parahaemolyticus accumulated by live oysters are shown in Table 1. As the treatment pressure increased, the numbers of survivors were reduced. Temperatures of 35 and 40 °C enhanced pressure inactivation considerably. The effect of cold temperatures on pressure inactivation of V. parahaemolyticus depended on the pressure used. Treatment at 250 MPa was significantly more effective (P b 0.05) for inactivating V. parahaemolyticus at 1 °C than at 20 °C. When oysters were treated at 300 MPa, greater inactivation was observed with treatment at 1 °C than at 20 °C; but this difference was not significant (P N 0.05). No significant differences (P N 0.05) in reductions at 1 or 20 °C were observed for treatments at ≥350 MPa. 4. Discussion The difference in pressure susceptibility between the pressureresistant and pressure-sensitive strains was substantial. Cook (2003) also observed variation in resistance to pressure among strains of V. parahaemolyticus. V. parahaemolyticus requires NaCl for growth (Oliver, 1981; USFDA, 2006). In this study, un-treated or un-injured cells grew equally well in TSB-2.5%S and TSB-0.5%S (data not shown); however, it appears that pressure-treated or injured cells grew better in TSA supplemented with 0.5% NaCl than in TSA supplemented with 2.5% NaCl. This result indicates that the membranes of V. parahaemolyticus were probably damaged during pressurization. It is known that low temperatures induce membrane damage of V. parahaemolyticus, leading to a greater sensitivity to salt (Oliver, 1981). Membrane damage caused by high pressure has also been reported for Salmonella enterica serovar Typhimurium (Ritz et al., 2000). Since the pressure chamber used in this study was not large enough to hold a whole shell oyster, shucked oyster meat instead of whole shell oysters were used for pressurization. No appreciable differences of inactivation between shucked oyster meat and whole shell oysters were expected since pressure acts instantaneously and uniformly throughout a pressure chamber (Farkas and Hoover, 2000) and Cook (2003) found similar reductions in the counts of V. vulnificus in shell oysters and oyster meat homogenates subjected to pressure treatments. Elevated temperatures above 30 °C enhanced pressure inactivation of V. parahaemolyticus in oysters (Fig. 2). This is in agreement with findings reported for L. monocytogenes (Chen, 2007a), Staphylococcus aureus (Chen, 2007b), E. coli (Ponce et al., 1998), and Salmonella enterica serovar Enteritidis (Ponce et al., 1999). V. parahaemolyticus is relatively sensitive to high temperatures with temperatures ≥50 °C being lethal (Andrews et al., 2000, 2003). Therefore, in addition to the inactivation caused by high pressure, the high temperatures achieved during pressurization, due to adiabatic heating, could also contribute to the inactivation of V. parahaemolyticus. Since an initial sample temperature ≥ 30 °C could enhance pressure inactivation of V. parahaemolyticus, initial temperatures of 35 or 40 °C were used for the studies with live oysters. Temperatures N53 °C affect the quality of oyster meats (Andrews et al., 2003). Therefore, an initial temperature of 45 °C was not used in the studies with live oysters since the temperatures reached during pressurization would be high enough to adversely affect the sensory quality of oysters. Although pressure treatment at 1 °C was slightly better than treatment at 20 °C for inactivation of V. parahaemolyticus in live oysters, it seems that V. parahaemolyticus survived better at 1 °C than at 20 °C after pressure treatment, since two out of three oyster samples treated at 450 MPa and 1 °C were positive for V. parahaemolyticus after enrichment whereas all three oyster samples treated at 450 MPa and 20 °C were
negative after enrichment. V. parahaemolyticus is known to be coldsensitive and does not survive well at refrigeration temperatures (Gooch et al., 2002). The apparent difference in the sensitivity of V. parahaemolyticus to pressure at 1 and 20 °C might then be due to the large variability in uptake of V. parahaemolyticus by individual oysters. Our results for pressure inactivation of V. parahaemolyticus in live oysters are in agreement with the findings of others (Calik et al., 2002; Cook, 2003). Moreover, Cook (2003) found that V. parahaemolyticus, which accumulated within oysters through feeding, appeared to possess higher levels of pressure resistance than did strain inoculated into oyster homogenates. Our findings did not support this. To give an example, when oysters were treated at 300 MPa for 2 min at 1 °C, the numbers of V. parahaemolyticus accumulated within oysters through feeding were reduced by 4.9 log units, while the numbers of V. parahaemolyticus inoculated directly into oyster meats were reduced by 3.6 log units. Our findings demonstrated that pressure processing treatments of 205–275 MPa at 10–30 °C for 1–3 min currently used in the oyster industry would not give a 5-log reduction of V. parahaemolyticus. For that, pressure treatment apparently needs to be ≥350 MPa at 1–35 °C for 2 min or ≥300 MPa at 40 °C for 2 min. Such treatments would also give a N5-log reduction of V. vulnificus in oysters (Kural and Chen, 2008). Treatment times could be extended to N2 min to achieve a N5-log reduction at lower pressures. Since the numbers of V. parahaemolyticus found in oysters at harvest are typically b103 CFU/g (Gooch et al., 2002), a treatment giving a 5-log reduction should be adequate. Acknowledgments The authors wish to thank Drs. Gary Richards and Gulnihal Ozbay for their helpful discussions. The project was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number # 2005-35201-16349. References Andrews, L.S., Park, D.L., Chen, Y.P., 2000. Low temperature pasteurization to reduce the risk of vibrio infections from raw shell-stock oysters. Food Additives and Contaminants 17, 787–791. Andrews, L.S., DeBlanc, S., Veal, C.D., Park, D.L., 2003. Response of Vibrio parahaemolyticus 03:K6 to a hot water/cold shock pasteurization process. Food Additives and Contaminants 20, 331–334. Balter, S., Hanson, H., Kornstein, L., Lee, L., Reddy, V., Sahl, S., Stavinsky, F., Fage, M., Johnson, G., Bancroft, J., Keene, W., Koepsell, J., Williams, M., MacDonald, K., Napolilli, N., Hofmann, J., Bopp, C., Lynch, M., Moore, K., Painter, J., Puhr, N., Yu, P., 2006. Vibrio parahaemolyticus infections associated with consumption of raw shellfish — three states, 2006 (Reprinted from MMWR, vol 55, pg 854–856, 2006). Journal of the American Medical Association 296, 2309–2310. Bej, A.K., Patterson, D.P., Brasher, C.W., Vickery, M.C.L., Jones, D.D., Kaysner, C.A., 1999. Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh. Journal of Microbiological Methods 36, 215–225. Berlin, D.L., Herson, D.S., Hicks, D.T., Hoover, D.G., 1999. Response of pathogenic Vibrio species to high hydrostatic pressure. Applied and Environmental Microbiology 65, 2776–2780. Calik, H., Morrissey, M.T., Reno, P.W., An, H., 2002. Effect of high-pressure processing on Vibrio parahaemolyticus strains in pure culture and Pacific oysters. Journal of Food Science 67, 1506–1510. Carlez, A., Rosec, J.P., Richard, N., Cheftel, J.C., 1993. High pressure inactivation of Citrobacter freundii, Pseudomonas fluorescens and Listeria innocua in inoculated minced beef muscle. Food Science and Technology-Lebensmittel-Wissenschaft & Technologie 26, 357–363. Chen, H.Q., 2007a. Temperature-assisted pressure inactivation of Listeria monocytogenes in Turkey breast meat. International Journal of Food Microbiology 117, 55–60. Chen, H.Q., 2007b. Use of linear, Weibull, and log-logistic functions to model pressure inactivation of seven foodborne pathogens in milk. Food Microbiology 24, 197–204. Cliver, D.O., 1995. Detection and control of foodborne viruses. Trends in Food Science & Technology 6, 353–358. Cook, D.W., 2003. Sensitivity of Vibrio species in phosphate-buffered saline and in oysters to high-pressure processing. Journal of Food Protection 66, 2276–2282. Cook, D.W., Ruple, A.D., 1992. Cold storage and mild heat treatment as processing aids to reduce the numbers of Vibrio vulnificus in raw oysters. Journal of Food Protection 55, 985–989. DePaola, A., Kaysner, C.A., Bowers, J., Cook, D.W., 2000. Environmental investigations of Vibrio parahaemolyticus in oysters after outbreaks in Washington, Texas, and New York (1997 and 1998). Applied and Environmental Microbiology 66, 4649–4654.
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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Conditions for high pressure inactivation of Vibrio parahaemolyticus in oysters Ayse G. Kural a, Adrienne E.H. Shearer a, David H. Kingsley b, Haiqiang Chen a,⁎ a b
Department of Animal & Food Sciences, University of Delaware, Newark, DE 19716-2150, United States U.S. Department of Agriculture, Agricultural Research Service, Microbial Food Safety Research Unit, W. W. Baker Center, Delaware State University, Dover, DE 19901, United States
A R T I C L E
I N F O
Article history: Received 12 March 2008 Received in revised form 2 May 2008 Accepted 2 May 2008 Keywords: High pressure processing Vibrio parahaemolyticus Oysters Treatment temperature Treatment time
A B S T R A C T The objective of this study was to identify the high pressure processing conditions (pressure level, time, and temperature) needed to achieve a 5-log reduction of Vibrio parahaemolyticus in live oysters (Crassostrea virginica). Ten strains of V. parahaemolyticus were separately tested for their resistances to high pressure. The two most pressure-resistant strains were then used as a cocktail to represent baro-tolerant environmental strains. To evaluate the effect of temperature on pressure inactivation of V. parahaemolyticus, Vibrio-free oyster meats were inoculated with the cocktail of V. parahaemolyticus and incubated at room temperature (approximately 21 °C) for 24 h. Oyster meats were then blended and treated at 250 MPa for 5 min, 300 MPa for 2 min, and 350 MPa for 1 min. Pressure treatments were carried out at −2, 1, 5, 10, 20, 30, 40, and 45 °C. Temperatures ≥30 °C enhanced pressure inactivation of V. parahaemolyticus. To achieve a 5-log reduction of V. parahaemolyticus in live oysters, pressure treatment needed to be ≥350 MPa for 2 min at temperatures between 1 and 35 °C and ≥300 MPa for 2 min at 40 °C. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Bivalve mollusks are filter-feeders that obtain food from the environment by filtering seawater through their gills. In this process they may concentrate pathogens from polluted water. Among bivalves, the oyster predominates as a disease vector in the USA, UK, and Australia (Cliver, 1995; Lees, 2000). Several recent outbreaks of Vibrio parahaemolyticus associated with oysters have heightened concerns about the safety of raw oyster consumption. In 2006, an outbreak of V. parahaemolyticus infections resulted in 177 cases and was linked to the consumption of contaminated raw shellfish including oysters (Balter et al., 2006). In 1998, the largest V. parahaemolyticus outbreak reported to date in the USA involving 416 cases was linked to consumption of raw oysters (DePaola et al., 2000). To control V. parahaemolyticus infections, the Interstate Shellfish Sanitation Conference (ISSC) proposed post-harvest treatment of shellfish using interventions such as pasteurization. The standard set by the ISSC is a 5-log reduction of V. parahaemolyticus levels with an endpoint of nondetectable at the level of b10 CFU/g (Cook, 2003). Post-harvest treatments including mild heat and irradiation have been proposed to control pathogens in shellfish. However, these treatments are of limited utility since they adversely affect the sensory qualities of shellfish (DiGirolamo et al., 1972; Cook and Ruple, 1992;
⁎ Corresponding author. Tel.: +1 302 831 1045; fax: +1 302 831 2822. E-mail address: [email protected] (H. Chen). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.05.003
Harewood et al., 1994). High pressure processing has been used commercially in the USA to facilitate the shucking of raw oysters for several years. The additional advantage of this technology is that it can inactivate V. parahaemolyticus and Vibrio vulnificus in oysters without compromising their sensory attributes (Lopez-Caballero et al., 2000; He et al., 2002; Cook, 2003). A pressure range of 205–275 MPa at temperatures ranging from 10 to 30 °C, and treatment times of 1 to 3 min are typically used. To our knowledge, studies involving pressure inactivation of V. parahaemolyticus have been conducted only at temperatures between 20 and 25 °C (Styles et al., 1991; Berlin et al., 1999; Calik et al., 2002; Cook, 2003; Koo et al., 2006). It is well documented that the temperature of food during pressurization plays a significant role in inactivation of microorganisms. Temperatures below and slightly above room temperature can enhance pressure inactivation of bacteria. To give examples, Chen (2007a) found that Listeria monocytogenes was most resistant to pressure at temperatures between 10 and 30 °C; Carlez et al. (1993) found that the rates of pressure inactivation of Pseudomonas fluorescens and Listeria innocua in minced beef muscle were much lower at room temperatures than at 4 °C; and a recent study in our laboratory demonstrated that temperatures b20 °C or N30 °C substantially increased pressure inactivation of V. vulnificus in oysters (Kural and Chen, 2008). Therefore, the effect of temperature on pressure inactivation of V. parahaemolyticus in oysters warranted further study. It is economically beneficial to use lower levels of pressure in combination with optimum treatment temperatures to obtain the desired target levels of pathogen inactivation. From a food
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quality point of view, it is better to reduce pressure levels and treatment times since over processing by pressure might adversely affect the sensory qualities of oysters. The objectives of this study were to determine the effect of treatment temperature and pressure levels on inactivation of V. parahaemolyticus and to identify the pressure level, time, and temperature parameters needed to achieve a 5-log reduction of V. parahaemolyticus in live oysters. 2. Materials and methods 2.1. Identification of pressure-resistant strains of V. parahaemolyticus Ten strains of V. parahaemolyticus were tested for their relative sensitivity to high pressure. The strains were ATCC 17802, ATCC 35118, ATCC 17803, ATCC 43996, ATCC 27519, ATCC 49529, ATCC 33845, ATCC 33846, DIE12-052499, and DAL 1094. The last two strains were kindly provided by Dr. Gary Richards from the USDA and the other strains by Dr. Jingkun Li from Strategic Diagnostics, Inc. (Newark, DE). The methods for preparation of cultures and pressure treatment as described by Kural and Chen (2008) were used. Briefly, stock cultures were maintained on plates of tryptic soy agar (TSA; Difco; Becton Dickinson, Sparks, MD, USA) with 2.5% NaCl (TSA-2.5%S) and working cultures of individual strains were prepared in tryptic soy broth (TSB; Difco) with 2.5% NaCl (TSB-2.5%S). A 2.5 ml portion of each culture was pressure-treated at 250, 300, 350, and 400 MPa for 2 min at 21 °C. Pressurization time reported in this study does not include the pressure come-up or release times. Before and after pressurization, numbers of V. parahaemolyticus were determined by preparing serial ten-fold dilutions of treated and un-treated cultures in 0.1% peptone water supplemented with 3% NaCl (P-3%S) and preparing pour plates of suitable dilutions using TSA-2.5%S. Plates were incubated at 37 °C for 72 h prior to counting colonies to allow injured cells to form visible colonies. Based on the results obtained, the two most pressure-resistant V. parahaemolyticus strains, ATCC 43996 and DIE12-052499, were selected for use as a 2-strain cocktail for subsequent experiments. 2.2. Plating media and salt concentration for recovery of pressure-injured cells The two most pressure-resistant strains of V. parahaemolyticus were grown individually in TSB-2.5%S at 37 °C for 24 h. Equal volumes of the two cultures were mixed to form a cocktail. The cocktail was treated at 350 MPa for 2 min at 21 °C. After pressurization, serial dilutions of the cocktail in P-3%S were prepared and numbers of V. parahaemolyticus were determined by spread plating suitable dilutions using TSA plus 0.5% NaCl (TSA-0.5%S), TSA-2.5%S, TSA-0.5% S overlayed with Thiosulfate Citrate Bile Salts Sucrose (TCBS; Difco), which is a selective medium for Vibrio (TSA-0.5%S/TCBS), TSA-2.5%S overlayed with TCBS (TSA-2.5%S/TCBS), and TCBS. Plates were incubated for 5 h before being overlayed with TCBS, as described by Kural and Chen (2008). 2.3. Effects of temperature on pressure inactivation of V. parahaemolyticus in oyster homogenates Medium to large market-size live oysters (Crassostrea virginica), obtained from the College of Marine Studies at the University of Delaware, Lewes, DE, were maintained in an aerated, circulating seawater tank kept at room temperature. The salinity of the seawater was maintained at between 1.5 and 2%. V. parahaemolyticus ATCC 43996 and V. parahaemolyticus DIE12-052499 were grown individually in TSB plus 0.5% NaCl (TSB-0.5%S) at 37 °C for 24 h with shaking, and equal volume of each culture was mixed to form a cocktail. Fifteen live oysters were shucked, and the meat was treated at 450 MPa for 2 min at 21 °C to completely inactivate naturally-present Vibrio (Cook, 2003). Oyster
meat samples were then individually inoculated with 0.1 ml of the V. parahaemolyticus cocktail, incubated at room temperature for 24 h, and homogenized as described by Kural and Chen (2008). Five-gram portions of the homogenate were treated at 250 MPa for 5 min, 300 MPa for 2 min, or 350 MPa for 1 min. The treatment times were selected to give partial inactivation of V. parahaemolyticus so that the temperature effect could be compared. Pressure treatments were carried out with samples at the following initial temperatures: −2, 1, 5, 10, 20, 30, 40, and 45 °C. Water was used as the hydrostatic medium for treatment temperatures above 0 °C, and a mixture of water and propylene glycol (1:1, vol:vol) was used for pressure treatment at −2 °C. The temperatures for the water bath and samples inside the chamber during pressurization were monitored every 2 s using K-type thermocouples (DASYTEC USA, Bedford, NH, USA). After pressure treatments, counts of V. parahaemolyticus in the samples were determined using the overlay method with plates being incubated for 4 h before they were overlayed with TCBS (Kural and Chen, 2008). 2.4. Variations in uptake of V. parahaemolyticus by live oysters V. parahaemolyticus ATCC 43996 and V. parahaemolyticus DIE12052499 were grown individually in TSB-0.5%S at 37 °C, overnight, with shaking. A 1-ml portion of each culture was transferred into 60 ml of TSB-0.5%S. The two cultures were incubated for 24 h at 37 °C and mixed to form a cocktail. Five live oysters were placed into an autoclavable plastic tray filled with 3 L of fresh seawater of 1.5 to 2% salinity and held at room temperature. An air pump (Tetra, Whisper Air Pump, Blacksburg, VA, USA) was used to provide air to the seawater and oysters were fed with algae (Reed Mariculture Inc., San Jose, CA, USA) before they were exposed to V. parahaemolyticus. The 60 ml of V. parahaemolyticus cocktail was poured into the tray and mixed well with the seawater. The tray was then covered with aluminum foil and left at a room temperature of about 21 °C for 24 h. Following V. parahaemolyticus uptake, the oysters were shucked and the meat from each oyster was placed in a sterile stomacher bag. Each portion of oyster meat was stomached for 2 min with two parts of P-1%S. Serial dilutions were made using P-1%S as a diluent, and counts of V. parahaemolyticus were determined using the overlay method. 2.5. Processing parameters needed for a 5-log reduction of V. parahaemolyticus in oysters Twenty-seven live market-size oysters were exposed to a cocktail of V. parahaemolyticus during feeding, as previously described, using 120 ml of the cocktail added to 6 L of seawater. After 24 h, oysters were shucked, and the meat and fluid from each shell were placed in a separate sterile plastic pouch. The pouches were sealed and submerged in the hydrostatic medium surrounding the pressure vessel for 10 min to reach the treatment temperature of 1, 20, 35, or 40 °C. Samples were pressure-treated for 2 min at 250, 300, 350, 400, or 450 MPa. P-1%S was added to each portion of un-treated or pressure-treated oyster meat in a ratio of 1:1 (vol:wt) and the portions were stomached for 2 min. Counts of V. parahaemolyticus were determined using the Most Probable Number (MPN) method in the U.S. FDA Bacteriological Analytical Manual (USFDA, 2006). For treatments in which low counts were expected, 100 ml of alkaline peptone water was added to the contents of the stomacher bag and the bags were incubated for 24 h at 37 °C. Loopful of the bag contents were then streaked onto TCBS agar. Colonies on TCBS plates from the MPN and enrichment experiments were confirmed to be V. parahaemolyticus using a PCR method (Bej et al., 1999). 2.6. Statistical analyses At least three independent trials were conducted for each experiment. Statistical analyses were conducted using Minitab® Release
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14.1 (Minitab Inc., University Park, PA, USA). One-way analysis of variance (ANOVA) was used to decide the significance of differences between treatments (P b 0.05). For multiple comparisons, Tukey's OneWay Multiple Comparisons Test was used (family error rate = 0.05). 3. Results 3.1. Identification of pressure-resistant strains of V. parahaemolyticus Data for the two most pressure-resistant strains (ATCC 43996 and DIE12-052499), one pressure-sensitive strain (ATCC 49529), and one moderately pressure-resistant strain (ATCC 17803) are shown in Fig. 1. Inactivation of V. parahaemolyticus was observed at 250 MPa for all strains, with reductions varying from 0.2 to 3.5 logs. A 2-min treatment at 400 MPa and 21 °C was sufficient to reduce the number of all strains of V. parahaemolyticus to b1 CFU/ml. Substantial differences in pressure resistance were observed among the V. parahaemolyticus strains in the pressure range of 250–350 MPa. Thus, a 2-min treatment at 350 MPa reduced the number of ATCC 43996 by 5.0 and ATCC 49529 by 8.7 log units. 3.2. Plating media and salt concentration for recovery of pressure-injured cells The counts of V. parahaemolyticus after pressure treatment were 6.7, 6.5, 7.0, 6.5 and 3.5 log units on TSA-0.5%S, TSA-2.5%S, TSA-0.5%S/TCBS, TSA-2.5%S/TCBS, and TCBS, respectively. The number of colonies recovered on the overlay plates was consistently higher with TSA supplemented with 0.5% NaCl than with TSA supplemented with 2.5% NaCl, although this difference was not significant (P N 0.05). Therefore, 0.5% NaCl was used to supplement TSA or TSB and 1% NaCl was used to supplement 0.1% peptone water for the subsequent experiments. 3.3. Effect of temperature on pressure inactivation of V. parahaemolyticus in oyster homogenates Studies in our laboratory showed that there was no significant difference (P N 0.05) in recovery of V. parahaemolyticus between 4-h and 5-h incubation before overlaying of TCBS. Therefore, the overlay method with 4-h incubation before the TCBS overlay was used. Fig. 2 shows the effect of temperature on pressure inactivation of the cocktail of the two most pressure-resistant V. parahaemolyticus strains. Temperatures
Fig. 2. Effect of temperature on pressure inactivation of a cocktail of Vibrio parahaemolyticus ATCC 43996 and DIE12-052499 inoculated into oyster meat. The initial counts were approximately 2 × 108 CFU/g. Error bars represent ± one standard deviation. N0 = numbers recovered from un-treated samples or initial numbers: N = numbers recovered from pressure-treated samples.
≥30 °C considerably enhanced pressure inactivation of V. parahaemolyticus. Thus, treatment at 350 MPa for 1 min at temperatures of 20, 30, 40, and 45 °C reduced the counts by 4.7, 5.9, 7.4, and 7.7 log units, respectively. The effect of cold temperature on pressure inactivation of V. parahaemolyticus varied with the pressure applied. When treated at 250 MPa, V. parahaemolyticus was most resistant to pressure at 20 °C. Temperatures above and below 20 °C enhanced pressure inactivation. Thus, with 250 MPa-treatments at −2, 20 or 40 °C numbers were reduced by 5.9, 4.0, and 7.7 log units, respectively . When treated at 300 MPa, V. parahaemolyticus was most resistant to pressure between 1 and 20 °C, with reductions varying from 3.5 to 3.7 log units. When treated at 350 MPa, temperatures between −2 and 10 °C increased the pressure resistance of V. parahaemolyticus, whereas temperatures of ≥20 °C enhanced its sensitivity to pressure. It is important to note that temperature of a food sample increases during pressurization due to adiabatic heating. With an initial sample temperature of 45 °C, the highest temperatures reached by the oyster samples during pressurization due to adiabatic heating were 54 °C at 250-MPa, 55 °C at 300-MPa, and 57 °C at 350-MPa. With an initial sample temperature of 40 °C, the highest temperatures reached were 48 °C at 250-MPa, 50 °C at 300-MPa, and 51 °C at 350-MPa. With initial sample temperatures of ≤30 °C, the highest temperatures reached were ≤40 °C for all the pressure treatments.
Table 1 Reduction in the log numbers of a two-strain cocktail of Vibrio parahaemolyticus accumulated within live oysters by feeding resulting from treatment of the oysters at various pressures and temperaturesa
Fig. 1. Pressure resistance data for Vibrio parahaemolyticus suspended in TSB + 2.5% NaCl. Pressure treatments were carried out at 21 °C for 2 min. The initial numbers were approximately 5 × 108 CFU/ml. N0 = numbers recovered from un-treated samples or initial numbers: N = numbers recovered from pressure-treated samples. At each pressure, columns labeled with different letters are significantly different (P b 0.05). Error bars represent ± one standard deviation.
Pressure
Temperature (°C)
(MPa)
1
20
35
40
250 300 350 400 450
3.1 ± 0.5 4.9 ± 0.5 5.4 ± 0.9 5.8 ± 0.2 N6.5 ± 0.4 (2/3)
2.1 ± 0.2 3.9 ± 1.2 5.3 ± 1.5 N 5.9 ± 1.4 (2/3) N 6.5 ± 0.4 (0/3)
3.5 ± 0.5 4.9 ± 0.3 N6.5 ± 0.4 (2/3) N6.5 ± 0.4 (0/3) N6.5 ± 0.4 (0/3)
3.5 ± 0.8 5.4 ± 1.3 N 6.5 ± 0.4 (0/3) N 6.5 ± 0.4 (0/3) N 6.5 ± 0.4 (0/3)
a
The initial counts were approximately 6.3 log MPN/g. Data are the means of log reductions ± one standard deviation (number of positive samples from enrichment/total trials). The unit for the data was log MPN/g. When the count of Vibrio parahaemolyticus in a sample was b0.6 MPN/g, it was assumed to be 0.6 MPN/g in calculation of means and standard deviations.
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3.4. Processing parameters needed to achieve a minimum 5-log reduction of V. parahaemolyticus in oysters After 24 h of feeding, the numbers of V. parahaemolyticus in oysters were ≥4.8 log CFU/g with a mean of 6.7 log CFU/g and standard deviations ≤1.0 log (within each replicate) for the data from all three replicates. The results for pressure inactivation of the cocktail of V. parahaemolyticus accumulated by live oysters are shown in Table 1. As the treatment pressure increased, the numbers of survivors were reduced. Temperatures of 35 and 40 °C enhanced pressure inactivation considerably. The effect of cold temperatures on pressure inactivation of V. parahaemolyticus depended on the pressure used. Treatment at 250 MPa was significantly more effective (P b 0.05) for inactivating V. parahaemolyticus at 1 °C than at 20 °C. When oysters were treated at 300 MPa, greater inactivation was observed with treatment at 1 °C than at 20 °C; but this difference was not significant (P N 0.05). No significant differences (P N 0.05) in reductions at 1 or 20 °C were observed for treatments at ≥350 MPa. 4. Discussion The difference in pressure susceptibility between the pressureresistant and pressure-sensitive strains was substantial. Cook (2003) also observed variation in resistance to pressure among strains of V. parahaemolyticus. V. parahaemolyticus requires NaCl for growth (Oliver, 1981; USFDA, 2006). In this study, un-treated or un-injured cells grew equally well in TSB-2.5%S and TSB-0.5%S (data not shown); however, it appears that pressure-treated or injured cells grew better in TSA supplemented with 0.5% NaCl than in TSA supplemented with 2.5% NaCl. This result indicates that the membranes of V. parahaemolyticus were probably damaged during pressurization. It is known that low temperatures induce membrane damage of V. parahaemolyticus, leading to a greater sensitivity to salt (Oliver, 1981). Membrane damage caused by high pressure has also been reported for Salmonella enterica serovar Typhimurium (Ritz et al., 2000). Since the pressure chamber used in this study was not large enough to hold a whole shell oyster, shucked oyster meat instead of whole shell oysters were used for pressurization. No appreciable differences of inactivation between shucked oyster meat and whole shell oysters were expected since pressure acts instantaneously and uniformly throughout a pressure chamber (Farkas and Hoover, 2000) and Cook (2003) found similar reductions in the counts of V. vulnificus in shell oysters and oyster meat homogenates subjected to pressure treatments. Elevated temperatures above 30 °C enhanced pressure inactivation of V. parahaemolyticus in oysters (Fig. 2). This is in agreement with findings reported for L. monocytogenes (Chen, 2007a), Staphylococcus aureus (Chen, 2007b), E. coli (Ponce et al., 1998), and Salmonella enterica serovar Enteritidis (Ponce et al., 1999). V. parahaemolyticus is relatively sensitive to high temperatures with temperatures ≥50 °C being lethal (Andrews et al., 2000, 2003). Therefore, in addition to the inactivation caused by high pressure, the high temperatures achieved during pressurization, due to adiabatic heating, could also contribute to the inactivation of V. parahaemolyticus. Since an initial sample temperature ≥ 30 °C could enhance pressure inactivation of V. parahaemolyticus, initial temperatures of 35 or 40 °C were used for the studies with live oysters. Temperatures N53 °C affect the quality of oyster meats (Andrews et al., 2003). Therefore, an initial temperature of 45 °C was not used in the studies with live oysters since the temperatures reached during pressurization would be high enough to adversely affect the sensory quality of oysters. Although pressure treatment at 1 °C was slightly better than treatment at 20 °C for inactivation of V. parahaemolyticus in live oysters, it seems that V. parahaemolyticus survived better at 1 °C than at 20 °C after pressure treatment, since two out of three oyster samples treated at 450 MPa and 1 °C were positive for V. parahaemolyticus after enrichment whereas all three oyster samples treated at 450 MPa and 20 °C were
negative after enrichment. V. parahaemolyticus is known to be coldsensitive and does not survive well at refrigeration temperatures (Gooch et al., 2002). The apparent difference in the sensitivity of V. parahaemolyticus to pressure at 1 and 20 °C might then be due to the large variability in uptake of V. parahaemolyticus by individual oysters. Our results for pressure inactivation of V. parahaemolyticus in live oysters are in agreement with the findings of others (Calik et al., 2002; Cook, 2003). Moreover, Cook (2003) found that V. parahaemolyticus, which accumulated within oysters through feeding, appeared to possess higher levels of pressure resistance than did strain inoculated into oyster homogenates. Our findings did not support this. To give an example, when oysters were treated at 300 MPa for 2 min at 1 °C, the numbers of V. parahaemolyticus accumulated within oysters through feeding were reduced by 4.9 log units, while the numbers of V. parahaemolyticus inoculated directly into oyster meats were reduced by 3.6 log units. Our findings demonstrated that pressure processing treatments of 205–275 MPa at 10–30 °C for 1–3 min currently used in the oyster industry would not give a 5-log reduction of V. parahaemolyticus. For that, pressure treatment apparently needs to be ≥350 MPa at 1–35 °C for 2 min or ≥300 MPa at 40 °C for 2 min. Such treatments would also give a N5-log reduction of V. vulnificus in oysters (Kural and Chen, 2008). Treatment times could be extended to N2 min to achieve a N5-log reduction at lower pressures. Since the numbers of V. parahaemolyticus found in oysters at harvest are typically b103 CFU/g (Gooch et al., 2002), a treatment giving a 5-log reduction should be adequate. Acknowledgments The authors wish to thank Drs. Gary Richards and Gulnihal Ozbay for their helpful discussions. The project was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number # 2005-35201-16349. References Andrews, L.S., Park, D.L., Chen, Y.P., 2000. Low temperature pasteurization to reduce the risk of vibrio infections from raw shell-stock oysters. Food Additives and Contaminants 17, 787–791. Andrews, L.S., DeBlanc, S., Veal, C.D., Park, D.L., 2003. Response of Vibrio parahaemolyticus 03:K6 to a hot water/cold shock pasteurization process. Food Additives and Contaminants 20, 331–334. Balter, S., Hanson, H., Kornstein, L., Lee, L., Reddy, V., Sahl, S., Stavinsky, F., Fage, M., Johnson, G., Bancroft, J., Keene, W., Koepsell, J., Williams, M., MacDonald, K., Napolilli, N., Hofmann, J., Bopp, C., Lynch, M., Moore, K., Painter, J., Puhr, N., Yu, P., 2006. Vibrio parahaemolyticus infections associated with consumption of raw shellfish — three states, 2006 (Reprinted from MMWR, vol 55, pg 854–856, 2006). Journal of the American Medical Association 296, 2309–2310. Bej, A.K., Patterson, D.P., Brasher, C.W., Vickery, M.C.L., Jones, D.D., Kaysner, C.A., 1999. Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh. Journal of Microbiological Methods 36, 215–225. Berlin, D.L., Herson, D.S., Hicks, D.T., Hoover, D.G., 1999. Response of pathogenic Vibrio species to high hydrostatic pressure. Applied and Environmental Microbiology 65, 2776–2780. Calik, H., Morrissey, M.T., Reno, P.W., An, H., 2002. Effect of high-pressure processing on Vibrio parahaemolyticus strains in pure culture and Pacific oysters. Journal of Food Science 67, 1506–1510. Carlez, A., Rosec, J.P., Richard, N., Cheftel, J.C., 1993. High pressure inactivation of Citrobacter freundii, Pseudomonas fluorescens and Listeria innocua in inoculated minced beef muscle. Food Science and Technology-Lebensmittel-Wissenschaft & Technologie 26, 357–363. Chen, H.Q., 2007a. Temperature-assisted pressure inactivation of Listeria monocytogenes in Turkey breast meat. International Journal of Food Microbiology 117, 55–60. Chen, H.Q., 2007b. Use of linear, Weibull, and log-logistic functions to model pressure inactivation of seven foodborne pathogens in milk. Food Microbiology 24, 197–204. Cliver, D.O., 1995. Detection and control of foodborne viruses. Trends in Food Science & Technology 6, 353–358. Cook, D.W., 2003. Sensitivity of Vibrio species in phosphate-buffered saline and in oysters to high-pressure processing. Journal of Food Protection 66, 2276–2282. Cook, D.W., Ruple, A.D., 1992. Cold storage and mild heat treatment as processing aids to reduce the numbers of Vibrio vulnificus in raw oysters. Journal of Food Protection 55, 985–989. DePaola, A., Kaysner, C.A., Bowers, J., Cook, D.W., 2000. Environmental investigations of Vibrio parahaemolyticus in oysters after outbreaks in Washington, Texas, and New York (1997 and 1998). Applied and Environmental Microbiology 66, 4649–4654.
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