Effects of pre- or post-processing storage conditions on high-hydrostatic pressure inactivation of Vibrio parahaemolyticus and V. vulnificus in oysters

International Journal of Food Microbiology 163 (2013) 146–152

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Effects of pre- or post-processing storage conditions on high-hydrostatic pressure inactivation of Vibrio parahaemolyticus and V. vulnificus in oysters Mu Ye a, Yaoxin Huang a, Joshua B. Gurtler b, Brendan A. Niemira b, Joseph E. Sites b, Haiqiang Chen a,⁎ a b

Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716, USA USDA-ARS, Eastern Regional Research Center, Wyndmoor, PA 19038, USA

a r t i c l e

i n f o

Article history: Received 16 November 2012 Received in revised form 17 February 2013 Accepted 18 February 2013 Available online 5 March 2013 Keywords: High-hydrostatic pressure Vibrio parahaemolyticus Vibrio vulnificus Cold storage Oyster

a b s t r a c t The effects of storage conditions on subsequent high-hydrostatic pressure (HHP) inactivation of Vibrio parahaemolyticus and Vibrio vulnificus in oysters were investigated. Live oysters were inoculated with V. parahaemolyticus or V. vulnificus to ca. 7–8 log MPN/g by feeding and stored at varying conditions (i.e., 21 or 35 °C for 5 h, 4 or 10 °C for 1 and 2 days and −18 °C for 2 weeks). Oyster meats were then treated at 225–300 MPa for 2 min at 4, 21 or 35 °C. HHP at 300 MPa for 2 min achieved a >5-log MPN/g reduction of V. parahaemolyticus, completely inactivating V. vulnificus (negative by enrichment) in oysters. Treatment temperatures of 4, 21 and 35 °C did not significantly affect pressure inactivation of V. parahaemolyticus or V. vulnificus (P > 0.05). 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. The effects of cold storage after HHP on inactivation of V. parahaemolyticus in oysters were also determined. Oysters were inoculated with V. parahaemolyticus and stored at 21 °C for 5 h or 4 °C for 1 day. Oyster meats were then treated at 250–300 MPa for 2 min at 21 or 35 °C and stored for 15 days in ice or in a freezer. V. parahaemolyticus populations in HHP-treated oysters gradually decreased during post-HHP ice or frozen storage. A validation study using whole-shell oysters was conducted to determine whether the presence of oyster shells influenced HHP inactivation of V. parahaemolyticus. No appreciable differences in inactivation between shucked oyster meat and whole-shell oysters were observed. 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 whole-shell oysters (>7 log reductions). The combination of HHP at a relatively low pressure (e.g., 250 MPa) followed by short-term frozen storage (7 days) could potentially be applied by the shellfish industry as a post-harvest process to eliminate V. parahaemolyticus in oysters. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Vibrio parahaemolyticus and Vibrio vulnificus are important foodborne pathogens of public health concern. Outbreaks with these pathogens have mainly been associated with consumption of raw or undercooked oysters (CDC, 1998, 1999, 2006). V. vulnificus can induce primary septicemia with mortality rates up to 50% (Linkous and Oliver, 1999) and is responsible for 95% of all seafood-related deaths in the United States (Oliver and Kaper, 2001). V. parahaemolyticus is a leading cause of acute human gastroenteritis from the consumption of contaminated seafood, with an estimated 35,000 annual cases in the United States (Scallan et al., 2011). The consumption of shellfish, including oysters, led to a 2006 outbreak of gastrointestinal illnesses in which 177 cases (72 culture-confirmed) were reported in three states (CDC, 2006). To mitigate foodborne illness risks associated with Vibrio spp., harvesting and post-harvest handling of oysters should meet specified ⁎ Corresponding author. Tel.: +1 302 831 1045; fax: +1 302 831 2822. E-mail address: [email protected] (H. Chen). 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.02.019

criteria to prevent multiplication of Vibrio to potentially hazardous levels. In addition, the use of a post-harvest process (PHP) to reduce Vibrio bacteria is also recommended. The National Shellfish Sanitation Program (NSSP) established a “Guide for Control of Molluscan Shellfish” which requires a PHP to reduce Vibrio by >3.52 log to non-detectable levels (b 30 MPN/g) (FDA, 2009). Examples of acceptable PHPs include depuration (Kelly and Dinuzzo, 1985), cold treatments (Cook and Ruple, 1992; Melody et al., 2008), mild-heat treatment (Andrews et al., 2000; Cook and Ruple, 1992), irradiation (Jakabi et al., 2003; Mahmoud, 2009) and high-hydrostatic pressure (HHP) (Calik et al., 2002; Cook, 2003; Kural and Chen, 2008). HHP can inactivate microorganisms and sometimes enzymes with only minor deleterious changes to flavor, color, and nutritional quality (San Martin et al., 2002). HHP is becoming increasingly acknowledged as the processing method of choice for oysters by virtue of its overriding safety, quality and economic benefits (He et al., 2002; Martin and Hall, 2006). With HHP, a satisfactory kill can be achieved due to the relatively high sensitivity of V. parahaemolyticus and V. vulnificus to pressure (Ye et al., 2012). Cold storage is commonly used to limit the growth of Vibrio spp. in shellfish

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following harvest. Numerous studies have reported that refrigeration or frozen storage is capable of achieving certain reductions of V. parahaemolyticus and V. vulnificus in seafood (Liu et al., 2009; Oliver, 1981; Parker et al., 1994; Thompson et al., 1976). The NSSP has established time–temperature matrices, requiring strict temperature control of oysters during transportation, processing and storage. In the V. parahaemolyticus control plan, the time oysters spend between harvest and refrigeration temperatures is not to exceed 5 h (FDA, 2009). The Interstate Shellfish Sanitation Conference (ISSC) has adopted freezing combined with frozen storage as an acceptable postharvest treatment to control V. vulnificus and V. parahaemolyticus in oysters. However, it is generally agreed that the inactivation effects of cold storage are limited, and complete elimination of both pathogens in oysters during cold storage is unlikely (Cook and Ruple, 1992; Johnson et al., 1973; Liu et al., 2009; Parker et al., 1994). Depending on the harvest season and methods of transportation and storage, oysters can be exposed to a variety of temperatures prior to pressure treatment (Cook et al., 2002). The harvest water temperature can vary from b 0 °C to > 30 °C, depending on the time and location of harvest. Oysters may be left on harvest vessels exposed to the sun without cooling for several hours or until the vessel docks. Moreover, oysters may also be held in cold seawater for up to 1 day in processing plants prior to processing. However, there is no information available regarding whether varying storage conditions before HHP could affect the subsequent pressure inactivation of Vibrio. Furthermore, to our knowledge, the fate of Vibrio survival in oysters during post-HHP cold storage has not been addressed. The objectives of this study were to 1) determine the effect of storage conditions on the subsequent pressure inactivation of V. parahaemolyticus and V. vulnificus in oysters, 2) determine the fate of Vibrio survivors during post-HHP cold storage as well as 3) evaluate whether frozen storage before and after HHP treatment could be used as an additional hurdle to enhance HHP inactivation of Vibrio. 2. Materials and methods 2.1. Cultures Two pressure-resistant Vibrio strains (V. parahaemolyticus ATCC 43996 and V. vulnificus MLT 403) were used in this study (Kural and Chen, 2008; Kural et al., 2008). Stock cultures of V. parahaemolyticus and V. vulnificus were maintained on tryptic soy agar (Difco Laboratories, Sparks, MD) (TSA) plus 0.5% NaCl (Fisher Scientific, Fair Lawn, NJ) (TSAS0.5%) at room temperature (ca. 21 °C). To prepare working cultures, a loopful of V. parahaemolyticus or V. vulnificus was transferred from a TSAS0.5% plate to a tube of 10 ml tryptic soy broth (Difco Laboratories) plus 0.5% NaCl (TSBS0.5%) and incubated at 35 °C for 9 h, allowing cells to reach stationary phase, as previously validated in Ye et al. (2011). A loopful of culture was then transferred to 100 ml of fresh TSBS0.5% and incubated at 35 °C for 9 h, to produce a cell density of ca. 10 8–10 9 CFU/g. 2.2. Oysters Live oysters (Crassostrea virginica) were obtained from the College of Earth, Ocean, and Environment at the University of Delaware. Oysters were cleaned, placed and maintained in an aerated, circulating seawater tank at room temperature and fed with algae (Reed Mariculture Inc., San Jose, CA). The salinity of the seawater was maintained at an optimal level of 1.5–2%.

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tray filled with 4 l of fresh seawater. To inoculate, 80 ml of the culture of V. parahaemolyticus or V. vulnificus was poured into the tray and mixed well. The tray was covered with a piece of aluminum foil and kept at room temperature for 24 h. 2.4. Effect of storage conditions before HHP on inactivation of V. parahaemolyticus or V. vulnificus in oysters Inoculated oysters were removed from seawater and subjected to different storage conditions: 1) 21 or 35 °C for 5 h in air, 2) 4 °C for 1 and 2 days or 10 °C for 1 day in seawater, or 3) − 18 °C in a freezer for 2 weeks. Oysters were then shucked. For frozen oysters that were used, they were shucked and thawed. The contents (meat and juice) were placed into sterile plastic pouches (polyethylene; Fisher Scientific, Pittsburg, PA). Pouches were double-sealed and doublebagged. Samples were pressure-treated at 225, 250, 275 or 300 MPa for 2 min using a high-pressure unit with water as the hydrostatic medium (model Avure PT-1; Avure Technologies, Kent, WA). Experiments were conducted at initial sample temperatures of 4, 21 or 35 °C. The pressure come-up rate was ca. 22 MPa/s and the pressure release time was b4 s. The compression heating factors were 1.9, 2.2 and 2.9 °C/100 MPa with initial sample temperatures of 4, 21 and 35 °C, respectively. Pressurization time reported in this study did not include the pressure come-up or release times. Populations of V. parahaemolyticus and V. vulnificus in oysters before storage, after storage, and after pressure treatment were determined. 2.5. Effects of cold storage after HHP on inactivation of V. parahaemolyticus in oysters Oysters were inoculated with V. parahaemolyticus as described above. Inoculated samples were stored at 21 °C for 5 h in air or at 4 °C for 1 day in seawater. Oysters were then shucked and the contents pressurized with 250 MPa at 21 and 35 °C, or with 300 MPa at 21 °C for 2 min. Inoculated oysters with or without HHP treatment were stored either in a cooler covered with ice or in a freezer at − 18 °C for up to 15 days. V. parahaemolyticus populations in samples were determined 1) following inoculation, 2) just prior to HHP, 3) immediately after HHP and 4) at selected time intervals during cold storage. 2.6. Validation of HHP on whole-shell live oysters 2.6.1. Effects of HHP on inactivation of V. parahaemolyticus in whole-shell oysters Whole-shell oysters were inoculated with V. parahaemolyticus as described above. Oysters were then banded, packaged, and treated with 250 or 300 MPa at room temperature (22–24 °C) for 2 min using a 2 l HHP unit (Avure Technologies, Kent, WA) with water as a hydrostatic medium at the Eastern Regional Research Center of the U.S. Department of Agriculture. The 2 l HHP unit used for these experiments is larger than the PT-1 HHP unit used for the experiments described above. The increased capacity allowed treatment of intact, whole-shell oysters, rather than oyster meat only. The pressure come-up rate for the 2-l HHP unit was ca. 20 MPa/s and the pressure release time was b4 s. Inoculated oysters with or without HHP treatment were stored either in a cooler covered with ice or in a freezer at − 18 °C for up to 15 days. Counts of V. parahaemolyticus in the samples were determined after inoculation, just prior to HHP, following HHP, and at selected time intervals during cold storage. Each sample consisted of two whole-shell oysters.

2.3. Inoculation of oysters with V. parahaemolyticus or V. vulnificus Live oysters were inoculated with V. parahaemolyticus or V. vulnificus as described by Kural and Chen (2008). Briefly, ca. 40 live oysters were removed from the seawater tank, placed into an autoclavable plastic

2.6.2. Microbial shelf life of HHP-treated whole-shell oysters Un-inoculated live whole-shell oysters were packaged and treated at 250 or 300 MPa at room temperature (22–24 °C) for 2 min using the 2-l HHP unit. Oysters with or without HHP treatment were either

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stored in a cooler covered with ice or in a freezer at − 18 °C for up to 15 days. Total aerobic plate counts (APC) and psychrotrophic plate counts (PPC) in the samples were determined before HHP, after HHP, and at selected time intervals during cold storage. Each sample consisted of three whole-shell oysters. 2.7. Microbiological analysis

Table 1 Effect of storage conditions on subsequent HHP inactivation of V. parahaemolyticus in oyster meat. Oysters were inoculated with V. parahaemolyticus to 7.8 ± 0.3 log MPN/g by feeding and stored at different conditions before being shucked. Oyster meats were then treated at 225–300 MPa for 2 min at initial sample temperatures of 4, 21 and 35 °C. Data represent mean log survivors (MPN/g) ± standard deviation. For each of six storage conditions, data in the same column having the same lower case letter are not significantly different (P > 0.05). The detection limit by plating was 3 MPN/g. Pre-HHP storage

2.7.1. Enumeration of V. parahaemolyticus and V. vulnificus Counts of V. parahaemolyticus and V. vulnificus in un-treated and treated samples were determined using the most probable number (MPN) method as described in the FDA Bacteriological Analytical Manual (FDA, 2004) with slight modifications. Briefly, samples were serially diluted with phosphate buffered saline (PBS, pH = 7.4) in ten-fold dilutions. Three × 1 ml portions of each dilution were inoculated into 3 tubes containing 10-ml alkaline peptone water (APW, pH = 8.5). APW tubes for V. parahaemolyticus-inoculated samples were incubated at 30 °C overnight, which was determined to be the optimal recovery temperature for V. parahaemolyticus following pressurization (Ye et al., 2011). A loop of enriched APW from the top 1.0 cm of a turbid tube was streaked onto thiosulfate citrate bile salts sucrose (TCBS) agar and incubated at 35 °C overnight. APW tubes for V. vulnificus samples were incubated at 35 °C overnight and a loop of the APW enrichment was streaked onto cellobiose colistin (CC) agar and incubated at 40 °C overnight to inhibit the growth of many other marine bacteria. After incubation, plates were evaluated for typical colonies to confirm the presence of V. parahaemolyticus or V. vulnificus in MPN tubes. Positive tubes were counted and the 3-tube-MPN tables were employed for final enumeration of the pathogens. Enrichment of undiluted samples was conducted for treatments where low counts were expected. For enrichments, 100 ml of APW was added into the remaining meats and incubated overnight at 35 °C along with the tubes. Enrichments were streaked onto TCBS or CC agar plates and the plates were evaluated for typical colonies to confirm the presence of V. parahaemolyticus or V. vulnificus after incubation, as described above. 2.7.2. APC and PPC At each sampling point, 3 oysters were shucked and homogenized, representing one sample. Samples were serially diluted with PBS in ten-fold dilutions. Bacterial counts in oyster samples were determined by spread-plating onto TSA. The APC and PPC were determined by incubating TSA plates at 35 °C for 48 h and 7 °C for 10 days, respectively (Cousin et al., 2001). 2.8. Statistical analysis Three independent trials were conducted for each treatment. Colony counts were converted to log MPN/g and means and standard deviations were calculated. Statistical analyses were conducted using JMP (SAS Institute, Cary, NC). Tukey's one-way multiple comparisons were used to determine significant differences among treatments (P b 0.05). 3. Results 3.1. Effects of storage conditions before HHP on inactivation of V. parahaemolyticus and V. vulnificus in oysters Effects of storage conditions before HHP on inactivation of V. parahaemolyticus and V. vulnificus in oysters are shown in Tables 1 and 2, respectively. Initial counts of V. parahaemolyticus and V. vulnificus in the untreated oysters were 7.8 and 8.4 log MPN/g, respectively. After storage at 21 or 35 °C for 5 h, populations of V. parahaemolyticus and V. vulnificus remained practically unchanged. V. parahaemolyticus proved to be cold-sensitive, as refrigerated storage at 4 or 10 °C for 1

Stored at 21 °C for 5 h (7.3 ± 0.3)⁎

HHP @ 225 MPa

4 21 35 Stored at 35 °C for 4 21 5 h (7.7 ± 0.3)⁎ 35 Stored at 4 °C for 4 21 1 day (6.3 ± 0.2)⁎ 35 Stored at 4 °C for 4 2 days (6.1 ± 0.7)⁎ 21 35 Stored at 10 °C for 4 ⁎ 21 1 day (6.2 ± 0.5) 35 Stored at −18 °C for 4 2 weeks (5.6 ± 0.4)⁎ 21 35

°C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C

5.2 5.7 5.2 5.9 6.0 5.1 3.7 5.2 4.0 4.0 5.0 3.7 4.9 5.3 3.7 3.5 4.4 3.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2a 0.3a 0.2a 0.2a 0.4a 0.4b 0.3a 0.2b 0.4a 0.6ab 0.7a 0.5b 0.3a 0.7a 0.3b 1.1ab 0.4a 0.7b

250 MPa 4.4 4.4 4.3 4.1 4.6 3.6 2.8 3.7 3.0 2.7 3.2 2.9 2.7 3.3 2.8 2.8 2.3 1.8

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

275 MPa

0.2a 0.4a 0.4a 0.9a 0.5a 0.9a 0.8a 0.3a 0.3a 0.2a 0.1b 0.4ab 0.5a 0.8a 0.5a 0.4a 1.0ab 0.5b

3.0 3.4 2.9 2.1 3.4 2.8 2.3 2.5 2.2 1.9 2.4 1.4 2.0 2.0 1.6 1.7 2.1 1.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2a 0.2a 0.5a 0.2a 0.5b 0.7ab 0.3a 0.5a 0.4a 0.5a 0.5a 0.7a 0.4a 0.5a 0.6a 0.3a 0.9a 0.1b

300 MPa 1.3 1.6 0.8 1.1 2.0 1.3 1.0 1.2 1.0 1.1 1.2 0.9 1.1 1.1 0.8 0.6 0.7 0.6

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.3ab 0.2a 0.2b 0.5a 0.4b 0.7ab 0.4a 0.6a 0.4a 0.4a 0.1a 0.6a 0.2a 0.5a 0.5a 0.0a 0.3a 0.0a

⁎ The numbers included in the “pre-HPP storage” column are MPN counts after storage and prior to HPP.

or 2 days decreased the populations of V. parahaemolyticus by 1.5– 1.7 log MPN/g. The strain of V. vulnificus was even more sensitive to cold storage, as one-day storage at 4 °C or 10 °C reduced its population by 3.0 and 1.9 log, respectively. Frozen storage for 2 weeks reduced V. parahaemolyticus and V. vulnificus by 2.2 and 2.9 log MPN/g, respectively. The V. vulnificus strain was more sensitive to HHP than the V. parahaemolyticus strain used in the present study. For example, after oysters were stored at 21 °C for 5 h, pressure treatments of 225, 250 and 275 MPa for 2 min at 21 °C reduced V. vulnificus by 4.3, 5.5 and 7.3 log MPN/g, respectively; whereas the same Table 2 Effect of storage conditions on subsequent HHP inactivation of V. vulnificus in oyster meat. Oysters were inoculated with V. vulnificus to a level of 8.4 ± 0.6 log MPN/g by feeding and stored at different conditions before being shucked. Oyster meats were then treated at 225–300 MPa for 2 min at initial sample temperatures of 4, 21 and 35 °C. Data represent mean log survivors (MPN/g) ± standard deviation. Numbers in fractions represent the number of samples testing positive after enrichment out of a total of 3 trials. For each of six storage conditions, data in the same column having the same lower case letter are not significantly different (P > 0.05). The detection limit by plating was 3 MPN/g. The detection limit by enrichment was 0.1 MPN/g. Pre-HHP storage Stored at 21 °C for 5 h (8.2 ± 0.7)⁎ Stored at 35 °C for 5 h (8.1 ± 0.2)⁎ Stored at 4 °C for 1 day (5.4 ± 0.2)⁎ Stored at 4 °C for 2 days (6.0 ± 0.5)⁎ Stored at 10 °C for 1 day (6.5 ± 0.4)⁎ Stored at −18 °C for 2 weeks (5.5 ± 0.5)⁎

HHP @ 4 21 35 4 21 35 4 21 35 4 21 35 4 21 35 4 21 35

°C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C

225 MPa 3.2 3.9 3.0 3.7 3.2 3.0 1.7 2.7 2.4 2.2 1.8 1.7 3.3 3.7 1.8 0.7 1.4 0.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

250 MPa a

0.7 0.2a 0.7a 0.4a 0.4a 0.8a 0.2a 0.4b 0.7b 0.5a 0.8a 0.7a 0.4a 0b 0.3c 0.2a 0.2b 0.2a

275 MPa a

2.0 ± 0.4 2.7 ± 0.3a 1.1 ± 0.9a 1.6 ± 0.6ab 2.3 ± 0.4a 1.1 ± 0.4b 1.1 ± 0.2a 1.1 ± 0.2a 1.0 ± 0.0a 0.7 ± 0.2a 1.1 ± 0.4a 0.7 ± 0.2a 1.7 ± 0.3a 2.1 ± 0.5a 0.8 ± 0.5b 0.7 ± 0.3a 0.9 ± 0.2a b0.5 (2/3)

0.7 ± 0.3a 0.9 ± 0.5a b0.5 (2/3) 0.9 ± 0.5a 1.0 ± 0.4a 0.5 ± 0.5a b0.5 (1/3) b0.5 (2/3) b0.5 (0/3) b0.5 (2/3) b0.5 (2/3) b0.5 (0/3) b0.5 (1/3) b0.5 (3/3) b0.5 (0/3) b0.5 (1/3) b0.5 (2/3) b0.5 (0/3)

⁎ The numbers included in the “pre-HPP storage” column are MPN counts after storage and prior to HPP.

M. Ye et al. / International Journal of Food Microbiology 163 (2013) 146–152

treatments only resulted in 1.6, 2.9 and 3.9 log MPN/g reductions of V. parahaemolyticus, respectively. The combinations of prior cold storage at 4, 10 or − 18 °C and HHP treatment of 275 MPa for 2 min at 35 °C completely eliminated V. vulnificus (negative enrichment results). However, none of the 300 MPa treatments inactivated all V. parahaemolyticus cells in oysters, even following prior cold storage. Under the same storage conditions, HHP conducted at 35 and 4 °C achieved higher reductions of both V. parahaemolyticus and V. vulnificus than at 21 °C for the majority of HHP treatments. Nevertheless, in most cases, the differences in log reduction among the three HHP treatment temperatures (viz., 4, 21 and 35 °C) were not statistically significant (P > 0.05). Although both V. parahaemolyticus and V. vulnificus strains were cold sensitive and cold storage reduced their counts (1.5–2.2 log for V. parahaemolyticus and 1.9–3.0 log for V. vulnificus), cold storage did not increase their sensitivity to subsequent HHP treatments. For example, reductions achieved by HHP alone (= counts after storage − counts after HHP) at 250 MPa and 21 °C were 2.9, 3.1, 2.6, 2.9 and 3.3 log when V. parahaemolyticus was stored at 21, 35, 4, 10 and −18 °C, respectively, while there was no statistical difference in inactivation results among these storage conditions. For V. vulnificus, it seems that prior refrigerated storage at 4 and 10 °C increased its resistance to subsequent HHP treatments. Inactivation of V. vulnificus by HHP alone at 225–275 MPa was lower for oysters stored at 4 and 10 °C than for oysters stored at 21 and 35 °C. For example, reductions achieved by HHP alone at 225 MPa and 21 °C were 4.3, 4.9, 2.7, 4.2 and 2.8 log when V. vulnificus was stored at 21 °C, 35 °C, 4 °C for 1 day, 4 °C for 2 days, and 10 °C, respectively. 3.2. Effect of cold storage after HHP on inactivation of V. parahaemolyticus in oysters Since V. parahaemolyticus was more resistant to both cold temperature and HHP than V. vulnificus, only V. parahaemolyticus was examined for response to post-processing as well as in the whole-shell oyster study. Populations of V. parahaemolyticus in oysters stored at room temperature for 5 h followed by HHP at 250 MPa or 300 MPa were reduced to ca. 3 log and 0.7 MPN/g, respectively (Table 3). During subsequent ice and frozen storages, populations of V. parahaemolyticus in HHP-treated and un-treated oysters were further reduced. V. parahaemolyticus counts in unpressurized samples decreased to 3.7 and 3.5 log MPN/g after 15 days ice and frozen storages, respectively. Ice and frozen storages of the HHP-treated samples resulted in up to 2.7 log greater reductions when compared with inactivation assessed immediately following HHP treatments. During the ice and frozen storages following HHP treatment, similar results were obtained at each sampling time point for the pressure treatment of 250 MPa conducted at two initial sample temperatures of 21 and 35 °C. The two 250 MPa treatments were

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capable of completely eliminating V. parahaemolyticus after 15 days of ice or frozen storage. The 300 MPa treatment was more effective than the two 250 MPa treatments, eliminating V. parahaemolyticus after 5 days of ice storage or after 7 days of frozen storage. Although HHP treatments with prior cold storage at 4 °C for 1 day resulted in lower counts of V. parahaemolyticus immediately following pressure treatments (data for day 0) than the corresponding HHP treatments with 5 h of prior storage at 21 °C, the benefits of prior cold storage to enhance V. parahaemolyticus inactivation disappeared during subsequent ice and frozen storages. That is, at each sampling time point during subsequent cold storage, there were no significant differences in populations of V. parahaemolyticus in oysters subjected to the same pressure treatment when comparing oysters exposed to prior warm storage at 21 °C and prior cold storage at 4 °C (P > 0.05). Therefore, in the following validation study, oysters were only exposed to warm storage at 21 °C prior to HHP. 3.3. Validation of HHP with whole-shell oysters 3.3.1. Effect of HHP on inactivation of V. parahaemolyticus in whole-shell oysters A validation study using whole-shell oysters was conducted to determine whether HHP had the same inactivation effect of V. parahaemolyticus in oyster meat as in whole-shell oysters. Results for whole-shell oysters (Table 4) were very comparable to that of oyster meat (Table 3). V. parahaemolyticus populations were slightly lower in whole-shell oysters than in oyster meat, probably due to the longer pressure come-up time of the 2-l unit; however, differences were not statistically significant for any sampling point (P > 0.05). HHP treatments at 1) 250 MPa followed by 10-day ice storage, 2) at 300 MPa followed by 5-day ice storage, and 3) at both pressure levels followed by 7-day frozen storage completely eliminated V. parahaemolyticus in whole-shell oysters. 3.3.2. Fluctuations in bacterial populations during storage The total aerobic mesophilic and psychrotrophic bacterial counts in pressure-treated and un-treated whole-shell oysters during storage in ice are shown in Fig. 1. Live oysters had initial APC counts of 4.6 log CFU/g and slightly higher PPC levels at 5.0 log CFU/g, which were indicative of the high quality of oysters, as fresh bivalve molluscs are generally considered of good quality with APC counts of b5 × 10 5 (i.e., 5.7 log) CFU/g (ICMSF, 1980). APC counts in oysters were significantly reduced by ≥2 log CFU/g following pressure treatments (P b 0.05). Treatments at 250 and 300 MPa for 2 min reduced the PPC to 3.3 and 1.5 log CFU/g, respectively. Both APC and PPC gradually increased during ice storage. At the end of 15-days of storage, APC and PPC counts in HHP-treated and un-treated oysters were b7 log CFU/g. Since oysters are generally considered spoiled when

Table 3 Effect of cold storage after HHP on inactivation of V. parahaemolyticus in oyster meat. Oysters were inoculated with V. parahaemolyticus by feeding and stored at 21 °C for 5 h or 4 °C for 1 day before being shucked. Oyster meats were then treated at 250–300 MPa for 2 min at 21 or 35 °C and stored for 15 days in ice or in a freezer. Data represent mean log survivors (MPN/g) ± standard deviation. Numbers in fractions represent the number of samples testing positive after enrichment out of a total of 3 trials. The initial count of V. parahaemolyticus in oysters after inoculation was 7.2 ± 0.2 log MPN/g. The detection limit by plating was 3 MPN/g. The detection limit by enrichment was 0.1 MPN/g. Day 0

Ice

Frozen

Day 1 Stored at 21 °C No HHP 250 MPa at 21 250 MPa at 35 300 MPa at 21

for 5 h prior to HHP 6.8 ± 0.2 °C 3.0 ± 0.2 °C 3.2 ± 0.1 °C 0.7 ± 0.2

Stored at 4 °C for 1 day prior to HHP No HHP 5.6 ± 0.4 250 MPa at 21 °C 2.2 ± 0.2 250 MPa at 35 °C 2.1 ± 0.4 300 MPa at 21 °C b0.5 (1/3)

5.3 1.8 1.7 0.3

± ± ± ±

0.6 0.3 0.3 0.6

5.3 ± 0.1 1.6 ± 0.2 1.7 ± 0.3 b0.5 (1/3)

Day 2

Day 5

Day 10

Day 15

Day 7

Day 15

4.7 ± 0.4 1.5 ± 0.3 0.9 ± 0.4 b0.5 (1/3)

4.7 ± 0.1 0.8 ± 0.4 0.7 ± 0.2 b0.5 (0/3)

3.3 ± 0.3 b0.5 (0/3) b0.5 (1/3) b0.5 (0/3)

3.7 ± 0.6 b0.5 (0/3) b0.5 (0/3) b0.5 (0/3)

4.0 ± 0.4 b0.5 (1/3) b0.5 (1/3) b0.5 (0/3)

3.5 ± 0.1 b0.5 (0/3) b0.5 (0/3) b0.5 (0/3)

4.5 ± 0.6 1.0 ± 0.4 1.0 ± 0.4 b0.5 (1/3)

5.0 ± 0.4 0.7 ± 0.2 0.5 ± 0.1 b0.5 (0/3)

4.2 ± 0.8 b0.5 (0/3) b0.5 (0/3) b0.5 (0/3)

3.4 ± 0.9 b0.5 (0/3) b0.5 (0/3) b0.5 (0/3)

3.4 ± 0.2 b0.5 (1/3) b0.5 (1/3) b0.5 (0/3)

3.6 ± 0.2 b0.5 (0/3) b0.5 (0/3) b0.5 (0/3)

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Table 4 Effect of cold storage after HHP on inactivation of V. parahaemolyticus in whole-shell oysters. Oysters were inoculated with V. parahaemolyticus by feeding and stored at 21 °C for 5 h. They were then treated at 250–300 MPa for 2 min at 22–24 °C and stored for 15 days in ice or in a freezer. Data represent mean log survivors (MPN/g) ± standard deviation. Numbers in fractions represent the number of samples testing positive after enrichment out of 3 trials. The detection limit by plating was 3 MPN/g. The detection limit by enrichment was 0.1 MPN/g.

No HHP 250 MPa 300 MPa

Day 0

Ice

Frozen

Day 1

Day 5

Day 10

Day 15

Day 7

Day 15

7.0 ± 0.4 2.8 ± 0.4 0.6 ± 0.1

5.5 ± 0.2 2.0 ± 0.5 b0.5 (2/3)

5.0 ± 0 0.5 ± 0.5 b0.5 (0/3)

4.4 ± 0.2 b0.5 (0/3) b0.5 (0/3)

4.4 ± 0.1 b0.5 (0/3) b0.5 (0/3)

5.1 ± 0.2 b0.5 (0/3) b0.5 (0/3)

3.6 ± 0.3 b0.5 (0/3) b0.5 (0/3)

APC increases to >10 7 CFU/g (Kim et al., 2002), the HHP-treated and un-treated oysters had a shelf life of at least 15 days when stored in ice. When HHP-treated and un-treated oysters were kept frozen during storage, APC and PPC counts were significantly lower than those in oysters stored in ice after 15 days (P b 0.05) (Table 5). 4. Discussion The effectiveness of HHP on inactivating Vibrio depends on processing parameters such as pressure level, holding time, temperature and the physiological state of the bacteria (Berlin et al., 1999; Calik et al., 2002; Cook, 2003; Koo et al., 2006; Kural and Chen, 2008; Kural et al., 2008; Ma and Su, 2011; Ye et al., 2012). Generally speaking, Vibrio spp. are sensitive to HHP and can be inactivated by pressures ranging from 200 to 350 MPa. Cook (2003) found that a pressure treatment of 250 MPa for 2 min at 27–29 °C reduced V. vulnificus in oysters by ca. 6 log. However, V. parahaemolyticus serotype O3:K6 in oysters required a pressure of 300 MPa for 3 min at 24–25 °C for a 5-log reduction. Kural and Chen (2008) reported that pressure treatment needs to

be conducted at levels of ≥ 250 MPa to achieve a 5-log reduction of V. vulnificus in live oysters. They later reported that achieving a 5-log reduction of V. parahaemolyticus in live oysters requires ≥350 MPa for 2 min at temperatures between 1 and 35 °C (Kural et al., 2008). Our results agree well with those of previous studies. Treatment temperature is known to influence the effectiveness of HHP on microbial inactivation. It has been demonstrated that slightly elevated temperatures enhance pressure inactivation of Escherichia coli O157:H7 (Neetoo and Chen, 2010), Salmonella (Neetoo and Chen, 2010), Listeria monocytogenes (Chen, 2007), and V. parahaemolyticus (Kural et al., 2008). Kural and Chen (2008) reported that pressure inactivation of V. vulnificus was enhanced as the temperature decreased below 20 °C and increased above 30 °C. In the present study, between the treatment temperatures of 4, 21, and 35 °C, there were no substantial differences in HHP inactivation of V. parahaemolyticus and V. vulnificus. In previous studies reported in the literature, the effects of storage conditions prior to HHP treatment on the pressure inactivation of Vibrio was not taken into consideration. In studies conducted by Calik et al. (2002), Cook (2003), Hu et al. (2005), Koo et al. (2006), Kural et al. (2008), and Kural and Chen (2008), oysters were inoculated with V. parahaemolyticus or V. vulnificus through feeding; however, different storage conditions for inoculated oysters prior to HHP were used for each study. Prior to HHP treatment, inoculated oysters were stored at 10 °C (length of time unreported) by Calik et al. (2002), at room temperature for 24 h by Cook (2003), at refrigerated temperature (length of time unreported) by Hu et al. (2005), and at refrigerated temperature for b24 h by Koo et al. (2006). Kural et al. (2008) and Kural and Chen (2008) treated oysters by HHP immediately after inoculation. In reality, oysters could be exposed to various storage temperatures after contamination and prior to HHP treatment. Oyster harvest water temperatures can vary from b0 °C to > 30 °C, depending on the time and location of harvest. Oysters are known to be left on decks of harvest vessels in the sun without cooling for several hours or held in cold seawater for up to 1 day in processing plants before HHP. In the present study, different storage conditions were applied to oysters before HHP to simulate oysters being left on decks of harvest vessels without cooling for several hours, or held in cold seawater. Although cold storage at 4, and 10 °C resulted in lower Vibrio counts in oysters than warm storage at 21 and 35 °C, it did not affect the sensitivity of V. parahaemolyticus to subsequent HHP treatments and even increased the pressure resistance of Table 5 Effect of HHP and frozen storage on APC and PPC in whole-shell oysters. Un-inoculated oysters were treated at 250–300 MPa for 2 min at 22–24 °C and stored for 15 days in a freezer. Data represent mean log survivors (CFU/g) ± standard deviation. Data in the same row having the same upper case letter are not significantly different (P > 0.05). Data in the same column having the same lower case letter are not significantly different (P > 0.05). APC

Fig. 1. Effect of HHP and ice storage on total aerobic plate counts (APC) (a) and psychrotrophic plate counts (PPC) (b) in whole-shell oysters. Un-inoculated wholeshell oysters were treated at 250–300 MPa for 2 min at 22–24 °C and stored for 15 days in ice. Data represent mean log survivors (CFU/g). Error bars represent 1 standard deviation.

PPC

Initial No HHP 250 MPa 300 MPa

Frozen Aa

4.6 ± 0.4 2.6 ± 0.5Ab 2.1 ± 0.6Ab

Initial Ba

3.8 ± 0.1 3.3 ± 0.2Bb 2.6 ± 0.0Bc

Frozen Aa

5.0 ± 0.1 3.3 ± 0.3Ab 1.5 ± 0.2Ac

4.3 ± 0.1Ba 4.1 ± 0.4Bb 3.6 ± 0.1Bc

M. Ye et al. / International Journal of Food Microbiology 163 (2013) 146–152

V. vulnificus. The reason for this increased pressure resistance of V. vulnificus with prior cold storage is unknown. It is possible that exposure to cold temperatures before processing increases the percentage of polyunsaturated fatty acid in cell membranes, and therefore the resistance to HHP processing. Oysters could potentially be contaminated with high populations of naturally occurring V. parahaemolyticus (> 6 log MPN/g) upon exposure to elevated temperatures, producing potentially greater health hazards (Gooch et al., 2001; Lorca et al., 2001). It has been demonstrated that the current industry practice of HHP (≤300 MPa) is not capable of completely inactivating V. parahaemolyticus in oysters when the contamination level is high (Ye et al., 2012). Therefore, it is important to know the fate of V. parahaemolyticus survivors after HHP to understand the risks associated with excessive pathogen levels in oysters. In the present study, icing was chosen as the storage condition following HHP treatment to model a common retail market practice for oysters after harvest or processing. We found that following HHP, V. parahaemolyticus populations gradually decreased during ice storage. HHP at 250 MPa, followed by 10-day ice storage, or 300 MPa treatment followed by 5-day ice storage were sufficient to completely eliminate V. parahaemolyticus in whole-shell oysters (>7 log reduction). It is possible that cold storage after HHP might capitalize on cell damage induced by pressure treatment or could inhibit the recovery of the sub-lethally injured cells, leading to subsequent death. Although the combination of prior cold storage and HHP resulted in greater inactivation of V. parahaemolyticus than the combination of prior warm storage and HHP, there was no statistically significant difference in populations of V. parahaemolyticus between those two treatments during post-HHP cold storage. Therefore, storage conditions prior to HHP might not be a concern when designing HHP processing parameters, which would allow significant flexibility for oyster harvest, processing and storage. A number of oyster producers use frozen storage to provide high quality frozen oysters to consumers for raw consumption. The common practice is to hold oysters at − 21 ± 2 °C for 3 to 6 months before the oysters are distributed to consumers (Drake et al., 2007; Liu et al., 2009). In the present study, frozen storage before or after HHP was used as a means to help achieve complete inactivation of V. parahaemolyticus in oysters. While using higher pressures may guarantee safer products, it is not always desirable since it would increase processing costs and might adversely affect the sensory quality of oysters. Although frozen storage at − 18 °C for 2 weeks reduced V. parahaemolyticus populations by 2.2 log, the combination of frozen storage followed by 300 MPa did not completely inactivate it. In contrast, frozen storage after HHP enhanced the inactivation of V. parahaemolyticus in oysters. HHP at 250 or 300 MPa for 2 min at 22–24 °C, followed by 7-day frozen storage, were able to completely eliminate V. parahaemolyticus in whole-shell oysters (Table 4). Similarly, Black et al. (2010) investigated the fate of E. coli O157:H7 in ground beef during post-HHP frozen storage. HHP at 400 MPa for 10 min at 20 °C reduced E. coli O157:H7 in ground beef by 3 log CFU/g, while an extra 2–3 log reduction was observed during the subsequent 30-day frozen storage. A recent study in our laboratory also demonstrated that post-HHP frozen storage substantially enhanced the inactivation of E. coli O157:H7 and Salmonella spp. in strawberry puree (Huang et al., 2013). The inactivation of V. parahaemolyticus in oysters by HHP was studied with shucked oyster meat and validated with whole-shell oysters. Since pressure is transmitted instantaneously and uniformly throughout the pressure chamber, the process is independent of food shape, size or geometry (Farr, 1990; Knorr, 1999). Thus, no appreciable differences of inactivation between shucked oyster meat and whole-shell oysters were expected. This was confirmed by our results, which showed that the presence of shells did not influence HHP inactivation of V. parahaemolyticus within oysters. It is advantageous to use shucked oyster meat to run HHP experiments for inoculation studies since shucked meat is easier to handle and package. In

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addition, using shucked meat would minimize the possibility of equipment contamination by pathogens as a result of sharp oyster shells puncturing packages leading to leakage of contaminated contents. The shelf life of oysters stored at refrigeration temperatures varies and is dependent on the initial bacterial load. A decrease (2–3 log CFU/g) in total microflora after HHP treatment of oysters in the range of 200–600 MPa has previously been reported, and HHP appeared to delay the onset of bacterial growth during subsequent refrigerated storage (He et al., 2002; Linton et al., 2003; Lopez-Caballero et al., 2000). It has been reported that HHP at 207–310 MPa for 1–2 min could extend the shelf life of oysters stored on ice from 9 to 20 days (He et al., 2002), while oysters processed at 400 MPa for 5 min at 20 °C and stored on ice had a shelf life of 21 days (Cruz-Romero et al., 2008). Our results agree with previous studies, which indicated that HHP significantly reduced background microorganisms and that the microbial shelf life of HHP oysters was maintained for at least 15 days when stored in ice. In summary, the present study showed that HHP was an effective oyster PHP for the control of V. parahaemolyticus and V. vulnificus and prior cold storage did not increase the sensitivity of V. parahaemolyticus or V. vulnificus to subsequent HHP treatments. HHP at 300 MPa for 2 min at 21 °C, followed by 5-day ice storage or 7-day frozen storage, completely eliminated V. parahaemolyticus in whole-shell oysters. HHP-treated oysters maintained a microbial shelf life of at least 15 days when stored in ice. HHP followed by ice storage could be applied by the seafood industry as a PHP to inactivate V. parahaemolyticus and V. vulnificus in raw oysters and HHP combined with frozen storage could improve the microbial safety of frozen oysters.

Acknowledgment This project was supported by the Agriculture and Food Research Initiative Competitive Grants Program of the USDA National Institute of Food and Agriculture, NIFA award no: 2011-68003-30005. The authors are grateful to Dr. Peggy M. Tomasula for the use of the HHP unit at the USDA-ERRC.

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