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Effect of growth and recovery temperatures on pressure resistance of Listeria monocytogenes
International Journal of Food Microbiology 136 (2010) 359–363
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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
Effect of growth and recovery temperatures on pressure resistance of Listeria monocytogenes Adrienne E.H. Shearer, Hudaa S. Neetoo, Haiqiang Chen ⁎ University of Delaware, Department of Animal and Food Sciences, Newark, DE 19716, USA
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Article history: Received 27 May 2009 Received in revised form 23 October 2009 Accepted 31 October 2009 Keywords: Listeria monocytogenes High hydrostatic pressure Growth temperature Recovery temperature Resistance Food
a b s t r a c t Experimental conditions can affect the outcome of bacterial stress-tolerance assays. Growth conditions that optimize microbial recovery should be established to help evaluate the effectiveness of treatment conditions for food safety. The objectives of this study were to determine the effects of growth and recovery temperatures on pressure resistance of early stationary-phase Listeria monocytogenes in milk. The tested conditions were the following: (1) L. monocytogenes was grown at various temperatures (10, 15, 20, 25, 30, 35, 40 and 43 °C), suspended in ultra-high temperature (UHT) -processed whole milk, pressure-treated at 400 MPa for 2 min at 21 °C and recovered on Tryptic Soy Agar supplemented with 0.6% yeast extract (TSAYE) at 35 °C; (2) L. monocytogenes was grown at 35 and 43 °C, pressure treated in milk (400 and 500 MPa, respectively, for 2 min at 21 °C) and recovered on TSAYE at various temperatures (4, 10, 15, 20, 25, 30, 35 and 40 °C); (3) L. monocytogenes originally grown at 35 °C, was pressure treated in milk (400 or 450 MPa for 2 min at 21 °C), and recovered on TSAYE at 10 °C for various time intervals (1, 2, 3, 6, 9 and 12 days) then at 35 °C for 5 days. There was no significant difference (P N 0.05) in pressure-resistance of L. monocytogenes grown at 10 to 25 °C with approximately 6.5log CFU/ml population reductions. At growth temperatures greater than 25 °C, pressure resistance increased with less than 1-log CFU/ml reduction observed for L. monocytogenes originally grown at 43 °C. After pressure treatment, regardless of growth temperature and pressure treatment, the greatest recovery of L. monocytogenes was within the 4 to 20 °C range; maximum recovery at 10 °C required approximately 24 days. The time for comparable post-pressure treatment recovery could be reduced by incubation at 10 °C for at least 2 days followed by incubation at 35 °C for 5 days. The findings of the present study indicate that growth and recovery temperatures affect the pressure resistance of L. monocytogenes and should, therefore, be taken into account when assessing the adequacy of inactivation treatments. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Ingestion of food contaminated with Listeria monocytogenes can result in serious illness affecting the gastrointestinal and nervous systems and can lead to miscarriage in pregnant women (Gandhi and Chikindas, 2007). L. monocytogenes is particularly problematic for food safety assurance due to its tolerance of environmental hurdles including its ability to grow at refrigeration temperatures (Gandhi and Chikindas, 2007; Wemekamp-Kamphuis et al., 2002) commonly used to prevent the growth of organisms of significance for food quality and public health. The pathogen has complicated a number of different food systems including those that do not receive a processing treatment lethal to L. monocytogenes as well as foods that have been inadvertently contaminated after processing. Milk and dairy products are among the foods that have been associated with Listeria contamination (Gandhi ⁎ Corresponding author. Department of Animal and Food Sciences, University of Delaware, 020 Townsend Hall, Newark, DE 19716-2150, USA. Tel.: +1 302 831 1045; fax: +1 302 831 2822. E-mail address: [email protected] (H. Chen). 0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2009.10.034
and Chikindas, 2007). This can arise from cows with udder infections or contaminated equipment throughout collection and processing (Linton et al., 2008). Inactivation of L. monocytogenes in milk can be achieved with heat pasteurization in conjunction with good handling practices (Farber et al., 1992). High hydrostatic pressure has been evaluated as an alternative method for processing milk and other dairy products (Linton et al., 2008). A notable degree of inactivation of L. monocytogenes (Chen, 2007a; Dogan and Erkmen, 2004; Erkmen and Dogan, 2004; Simpson and Gilmour, 1997), and other pathogens (Chen, 2007a) in milk has been demonstrated with high pressure, although some pathogenic bacteria have demonstrated considerable barotolerance in milk (Patterson et al., 1995). Pressure inactivation of L. monocytogenes in milk is affected by a number of factors including bacterial strain (Chen et al., 2009), stage of growth (Hayman et al., 2007; McClements et al., 2001; Wen et al., 2009), growth conditions (Bull et al., 2005; Hayman et al., 2007; McClements et al., 2001), pressure level (Erkmen and Dogan, 2004), treatment time (Erkmen and Dogan, 2004), imposition of other stresses (Hayman et al., 2008; Simpson and Gilmour, 1997; Wemekamp-Kamphuis et al., 2002), and recovery conditions (Bozoglu et al.,
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2004; Bull et al., 2005; Koseki et al., 2008). Unless the conditions for maximal barotolerance and full recovery of injured bacteria are established for treatment of a food system of interest, survivors may not be detected and process requirements underestimated. With particular interest to growth and recovery temperature effects, Bull et al. (2005) reported that prior growth temperature affects pressure resistance, but interactions of temperature, milk type, and pressure confounded interpretation of the effects. Hayman et al. (2007) reported that L. monocytogenes grown at the upper limit of its growth range (43 °C) was more pressure-resistant than when grown at optimal (35 °C) or reduced temperature (4 °C). After growth at temperatures closer to optimal, both short-term cold- (Wemekamp-Kamphuis et al., 2002) and heat-shock (Hayman et al., 2008) treatments increased the barotolerance of L. monocytogenes in milk. Temperature during recovery after pressure treatment of milk is also an important consideration as Bull et al. (2005) reported that, of three temperatures evaluated, 15 °C provided better recovery of L. monocytogenes than 4 °C or 30 °C, though the specific number of survivors was not determined. Bozoglu et al. (2004) found that detection of injured L. monocytogenes occurred more rapidly at 30 °C than 4 °C, though the percent recovery was not determined. Prolonged storage of pressure-treated milk supported the recovery and growth of L. monocytogenes better at 4 °C and 25 °C than at 37 °C (Koseki et al., 2008). These studies indicate the importance of growth and recovery temperatures and time in the determination of adequate pressure processing parameters. Although trends are apparent, the variations in findings and approaches also demonstrate the value of a study that compares the magnitude of post-pressure treatment recovery over prolonged time as affected by the broad range of temperatures that support growth and recovery of L. monocytogenes. The study presented herein builds on the findings of previous reports with specific objectives to (1) determine the relationship between pressure resistance and temperature of growth of L. monocytogenes throughout its growth range prior to pressure treatment, (2) determine the optimum incubation temperature throughout its growth range for maximal recovery of L. monocytogenes after pressure treatment, and (3) minimize incubation time through temperature shift after pressure treatment for maximum recovery of L. monocytogenes. The pressure conditions utilized permit L. monocytogenes survival such that differences among growth and recovery temperatures could be evaluated and compared to studies previously conducted. 2. Materials and methods 2.1. Determination of time to stationary phase L. monocytogenes ATCC 19115 was grown on Tryptic Soy Agar (Becton Dickinson and Co., Sparks, MD) supplemented with 0.6% yeast extract (Becton Dickinson and Co., Franklin Lakes, NJ, USA) (TSAYE) for approximately 17 h at 37 °C. A colony was transferred to 10 ml Tryptic Soy Broth (containing dextrose, Becton Dickinson) supplemented with 0.6% yeast extract (TSBYE) and incubated at 35 °C for approximately 17 h. A loopful (10 µl) of the overnight culture was subcultured in fresh TSBYE (100 ml) and incubated at 4, 10, 15, 20, 25, 30, 35, 40 and 43 °C. The time to reach early stationary phase at each temperature was determined by periodic sampling with serial dilution in 0.1% peptone water (BD/Difco Laboratories, Detroit, MI), spread-plating on TSAYE and incubating plates at 35 °C for 72 h. 2.2. Effect of growth temperature on pressure resistance L. monocytogenes was grown at 4, 10, 15, 20, 25, 30, 35, 40 or 43 °C to early stationary phase in TSBYE. Thirty-five milliliters were centrifuged for 15 min at 6318 ×g (IEC Centra-4B Centrifuge, International Equipment Co.), washed once with 0.1 M sodium phosphate buffer, pH 7.0 (Fisher Scientific, Fair Lawn, NJ), and resuspended in 35 ml of ultrahigh temperature (UHT) processed whole milk. The initial count of L.
monocytogenes in milk was approximately 109 CFU/ml. Milk (12 ml) was transferred to a sterile 3-mil thick polypropylene pouch (VWR International, West Chester, PA, USA), heat-sealed, and sealed in a secondary pouch for added safety. Samples were treated in an Avure PT1 laboratory-scale pressure unit (Avure Technologies Inc., Kent, WA, USA) monitored with DASYLab ® 7.0 software (DASYTEC USA, Bedford, NH). Samples were treated at 400 MPa for 2 min (not inclusive of comeup time of less than 30 s with almost instantaneous depressurization) at 21 °C with water as the surrounding pressure-transmitting medium. Enumeration of survivors was conducted by duplicate spread plating of appropriate serial dilutions (in 0.1% peptone water) on TSAYE and incubating at 35 °C for 72 h. Three independent trials were conducted. 2.3. Effect of temperature on recovery of L. monocytogenes from pressure-treated samples L. monocytogenes was grown at two temperatures (35 and 43 °C) to early stationary phase in TSBYE, centrifuged, washed and resuspended in UHT whole milk as previously described. Milk samples were treated at 400 MPa and 500 MPa for cultures grown at 35 and 43 °C respectively, for 2 min at 21 °C. Enumeration of survivors was conducted by duplicate spread plating of appropriate serial dilutions (in 0.1% peptone water) on TSAYE and incubating at 4, 10, 15, 20, 25, 30, 35 and 40 °C until no change in counts was observed. Colonies that developed during prolonged incubation on TSAYE were periodically streaked onto modified Oxford agar (Difco Laboratories, Sparks, MD, USA) to check for growth characteristic of L. monocytogenes. Three to four independent trials were conducted. 2.4. Effect of incubation temperature shift on recovery time L. monocytogenes was grown to early stationary phase at 35 °C, centrifuged, washed, and resuspended in UHT whole milk as previously described. Milk samples were treated at 400 and 450 MPa for 2 min at 21 °C. Survivors were enumerated on TSAYE incubated at 10 °C for various time intervals (1, 2, 3, 6, 9 and 12 d), followed by incubation at 35 °C for 5 d. Three independent trials were conducted. 2.5. Statistical analyses Statistical analyses were conducted using Minitab® Release 15 (Minitab Inc., University Park, PA, USA). One-way analysis of variance (ANOVA) was used to determine the significance of differences between treatments (P b 0.05). For multiple comparisons, Tukey's One-Way Multiple Comparisons Test was used (family error rate = 0.05). 3. Results 3.1. Determination of time to stationary phase The time to reach early stationary phase with approximately 109 CFU/ml at 4, 10, 15, 20, 25, 30, 35, 40 and 43 °C was 384, 96, 48, 36, 18, 12, 12, 12 and 16.25 h, respectively. These incubation times were used for all subsequent experiments. 3.2. Effect of growth temperature on pressure resistance There was no significant difference (P N 0.05) in pressure-resistance of L. monocytogenes grown within the range of 10 to 25 °C. With these growth temperatures, approximately 6.5-log CFU/ml population reduction after treatment at 400 MPa for 2 min at 21 °C and recovery at 35 °C (Fig. 1) was observed. Pressure resistance increased as the growth temperatures increased from 25 to 43 °C. Less than a 1-log CFU/ml reduction was observed after pressure treatment when L. monocytogenes was grown at 43 °C (Fig. 1).
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Fig. 1. Effect of growth temperature prior to pressure treatment on resistance of L. monocytogenes (initial count of approximately 109 CFU/ml in UHT whole milk) to pressure (400 MPa for 2 min at 21 °C). L. monocytogenes was enumerated on TSAYE after incubation at 35 °C for 72 h. Error bars represent ± 1 standard deviation. Bars labeled with different letters are significantly different (P b 0.05).
3.3. Effect of temperature on recovery of L. monocytogenes from pressure-treated samples Regardless of prior growth temperature (35 or 43 °C) and pressure treatment (400 or 500 MPa for 2 min at 21 °C), the greatest recovery of pressure-treated L. monocytogenes was obtained when TSAYE plates were incubated between 4 and 20 °C after pressure treatment (Fig. 2). Incubation at 10 °C provided the highest recovery by as much as 2 logs CFU/ml, although not statistically different (P N 0.05) from recovery at 4, 15, or 20 °C. The time for maximum recovery at 10 °C was 24 days. The incubation times for maximum recovery at the other tested temperatures were approximately 50, 15, and 10 days at 4, 15, and 20 °C, respectively, and approximately 5 days for 25 to 40 °C. 3.4. Effect of incubation temperature shift on recovery time Incubation of L. monocytogenes for at least 2 days at 10 °C followed by incubation at 35 °C for 5 days resulted in the statistically same recovery as that achieved by incubation at 10 °C for 24 days. Thus, the recovery period for L. monocytogenes could be shortened from 24 to 7 days by applying the above incubation temperature shift (Fig. 3). 4. Discussion There are a number of variables, including growth phase (Hayman et al., 2007; McClements et al., 2001), medium (Dogan and Erkmen, 2004) and temperature (Wemekamp-Kamphuis et al., 2002), that can affect bacterial resistance to subsequent stresses. The extent of recovery after a stress can also be affected by recovery medium and temperature (Bull et al., 2005). The focus of this study was high hydrostatic pressure resistance of stationary-phase L. monocytogenes as affected by temperature, both during growth prior to treatment and recovery after treatment. Populations of L. monocytogenes in stationary phase have been reported to exhibit greater barotolerance in milk than exponentiallygrowing cells (Hayman et al., 2007; McClements et al., 2001); and thus, early stationary-phase cells were used for the present study. Subsequent studies have demonstrated even greater barotolerance over L. monocytogenes in stationary phase by cells in a long-term-survival phase, which can occur after an initial death phase under prolonged storage and without addition of fresh nutrients (Wen et al., 2009). This phase of survival further emphasizes the importance of meticulous cleaning of processing equipment to prevent harborage and potential release of viable and resistant L. monocytogenes (Wen et al., 2009) in processed
milk, though the prevalence of such cells in freshly-collected milk is not known. Combinations of stresses applied simultaneously may also affect resistance to subsequent stresses. For example, a pH decrease during incubation of L. monocytogenes in the presence of glucose has been reported to adapt cells to subsequent acid stress (Koutsoumanis et al., 2003). The extent of this adaptation may also be dependent on the temperature during acid exposure. Some acid-adapted bacteria also have increased barotolerance (Scheyhing et al., 2004). L. monocytogenes would have had some exposure to reduced pH as a normal course of growth in this study and thus may have different, presumably increased, pressure resistance than had neutral conditions been artificially maintained. This may better reflect potential pressure resistance of bacterial contaminants that could be introduced at various points during food handling that may have varying history of exposures to prior stresses. Previous studies have demonstrated the importance of temperature exposure prior to, and after, high hydrostatic pressure treatment on the baroresistance of L. monocytogenes. From collection through processing and storage, milk temperatures can potentially span most of the growth temperature range of L. monocytogenes. If milk is contaminated with L. monocytogenes during or shortly after collection while still warm or if the temperature is permitted to increase, growth at such elevated temperatures may increase the resistance of L. monocytogenes to subsequent inactivation by high hydrostatic pressure. We observed approximately a 6-log difference in the number of L. monocytogenes that survived treatment of 400 MPa for 2 min at 21 °C when grown at 43 °C as compared to when grown in the range of 10 to 25 °C. These results are generally in agreement with those reported by Hayman et al. (2007) who demonstrated that L. monocytogenes grown at 43 °C was more baroresistant than when grown at 15 °C. The inactivation observed with 15 °C growth in the present study was greater however by approximately 2 logs, and we also observed no statistical difference in baroresistance among L. monocytogenes grown within the full range of 10 to 30 °C, as evaluated in 5-degree increments. Interestingly, a few degree difference above the optimal growth temperature markedly increased barotolerance of L. monocytogenes. The implications of this and the previous study (Hayman et al., 2007) are similar such that L. monocytogenes with prior growth at temperatures above optimal may need increased pressure and/or other type of stress for complete inactivation, or low temperature storage of milk prior to pressure treatment may be a critical parameter for process control. The length of time at reduced temperatures and the extent of growth prior to temperature reduction may also be critical. Wemekamp-
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Fig. 2. Effect of incubation temperature on recovery of L. monocytogenes after pressure treatment. L. monocytogenes was grown at 35 °C (initial count in UHT whole milk 2 × 109 CFU/ml) and pressure treated at 400 MPa for 2 min at 21 °C or grown at 43 °C (initial count in UHT whole milk 2 × 107 CFU/ml) and pressure treated at 500 MPa for 2 min at 21 °C. Error bars represent ± 1 standard deviation. Bars labeled with different letters are significantly different (P b 0.05). Statistical comparisons were made among recovery temperatures for each pressure treatment; comparisons were not made between the two pressure treatments.
Kamphuis et al. (2002) demonstrated that growth at 37 °C followed by cold shock at 10 °C for 4 h increased L. monocytogenes barotolerance as compared to cells growing exponentially at 37 °C. This was accompa-
nied by an increased production of certain cold-shock proteins (CSPs). With extended incubation of L. monocytogenes at 10 °C, the levels of CSPs increased as growth resumed with even greater production
Fig. 3. Effect of time of temperature shift from 10 °C to 35 °C on recovery of L. monocytogenes (initial count in UHT whole milk 3 × 109 CFU/ml) pressure treated at 400 or 450 MPa for 2 min at 21 °C. With the exception of the 24-day sample which was held at 10 °C for the entire incubation period as a control, samples were incubated at 10 °C for the length of time indicated on the x-axis and then transferred to 35 °C for 5 days for the final count. Error bars represent ± 1 standard deviation. Bars labeled with different letters are significantly different (P b 0.05). Statistical comparisons were made among recovery temperatures for each pressure treatment; comparisons were not made between the two pressure treatments.
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observed during stationary phase after cold shock. One might speculate an additional increase of CSPs could further increase barotolerance, but this was not evaluated (Wemekamp-Kamphuis et al., 2002). In the present study, growth of L. monocytogenes at 10 °C for 96 h resulted in one of the lowest pressure resistances observed among the growth temperatures tested. However, as discussed in more detail later, lower incubation temperatures (4 to 20 °C) after pressure treatment provided better recovery than 35 °C as determined in subsequent experiments, and thus the number of L. monocytogenes recovered in this phase of the study may not reflect the maximum possible recovery after pressure treatment. It would be interesting to evaluate the combined effect of growth at lower temperatures prior to pressure treatment with incubation for recovery after pressure treatment also at reduced temperatures to determine the extent of pressure-induced injury of these cells. Temperature shock in the opposite direction by heat-shocking at 48 °C for up to 30 min has also been reported to increase L. monocytogenes barotolerance (Hayman et al., 2008); and thus, while the simultaneous application of heat during pressure has enhanced inactivation (Chen, 2007b), the timing of heat exposure may also be critical in the determination of process needs. Farber et al. (1992) reported that L. monocytogenes grown at 43 °C also has greater thermotolerance than that grown at 30 °C and maintains an increased tolerance for at least 24h after refrigerated storage. The effect of similar handling conditions on L. monocytogenes barotolerance warrants study. For researchers and processors attempting to establish appropriate process treatments for specific products and operations, the temperature(s) of maximum tolerance should be used in such laboratory studies if there is any chance of L. monocytogenes exposure to stress-adaptive conditions prior to processing including growth at a sustained elevated temperature or temperature fluctuations. Also worthy of evaluation is how the temperature of growth in raw milk affects L. monocytogenes barotolerance as directly compared to growth in microbiological media which was used for our study as well as by Hayman et al. (2007) and Wemekamp-Kamphuis et al. (2002). The other focus of this study was to determine how temperature after high pressure treatment affected the extent and rate of recovery of L. monocytogenes. A previous study (Bull et al., 2005) demonstrated that pressure-treated L. monocytogenes recovered more quickly and over a longer storage period in milk at 15 °C than 4 or 30 °C by a presence/ absence approach. In the present study, L. monocytogenes was recovered at eight temperatures within its growth range, and numbers of survivors were determined on non-selective media to help characterize the magnitude of difference in recovery. Using this approach, 10 °C provided the greatest recovery by as much as 2-logs over higher temperatures regardless of prior growth temperature (35 or 43 °C) and pressure level (400 or 500 MPa, respectively), although the difference in recovery within the 4 to 20 °C recovery range was not statistically significant (P N 0.05). In the Bull et al. (2005) study, it was noted that storage at 4 °C did not allow for full recovery of pressure-injured L. monocytogenes, but a switch from 4 °C to 30 °C provided better recovery, and such conditions may be encountered in situations of temperature abuse during milk storage. They speculated that L. monocytogenes recovers in two stages, and the stress of 4 °C and 30 °C storage does not permit complete repair for extended storage. In the present study, L. monocytogenes required 24 days to reach maximum recovery at 10 °C. Statisticallycomparable recovery could be obtained by incubating plates at 10 °C for at least 2 days and then incubating at 35 °C. Presumably, the temperature switch permitted more rapid colony formation that was visually detectable; it is unclear if the switch to 35 °C also accelerated a specific stage of an injury recovery process. This work corroborates prior reports of the influence of growth and recovery temperatures on baroresistance of early stationary phase L. monocytogenes. The studies support previous findings that growth at the upper range of growth temperatures prior to pressure treatment increases baroresistance, and incubation at lower temperatures after
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pressure treatment increases recovery. Further, the studies provide the magnitude of survival and the temperatures at which the number of survivors becomes significantly different throughout the growth temperature range as evaluated in small temperature increments. The study also demonstrates the adequacy of short-term recovery after pressure treatment by incubation at 10 °C followed by incubation at 35 °C to expedite enumeration of survivors under the conditions evaluated. While this study demonstrates the importance of growth and recovery conditions on barotolerance at a single treatment time, further studies are needed to establish the influence of these same conditions on the complete inactivation profile of L. monocytogenes in milk. Acknowledgement This study was supported by start-up funds from the University of Delaware Department of Animal and Food Sciences. References Bozoglu, F., Alpas, H., Kaletunç, G., 2004. Injury recovery of foodborne pathogens in high hydrostatic pressure treated milk during storage. FEMS Immunology and Medical Microbiology 40, 243–247. Bull, M.K., Hayman, M.M., Stewart, C.M., Szabo, E.A., Knabel, S.J., 2005. Effect of prior growth temperature, type of enrichment medium, and temperature and time of storage on recovery of Listeria monocytogenes following high pressure processing of milk. International Journal of Food Microbiology 101, 53–61. Chen, H.Q., 2007a. Use of linear, Weibull, and log-logistic functions to model pressure inactivation of seven foodborne pathogens in milk. Food Microbiology 24, 197–204. Chen, H.Q., 2007b. Temperature-assisted pressure inactivation of Listeria monocytogenes in Turkey breast meat. International Journal of Food Microbiology 117, 55–60. Chen, H.Q, Neetoo, H., Ye, M., Joerger, R.D., 2009. Differences in pressure tolerance of Listeria monocytogenes strains are not correlated with other stress tolerances and are not based on differences in CtsR. Food Microbiology 26, 404–408. Dogan, C., Erkmen, O., 2004. High pressure inactivation kinetics of Listeria monocytogenes inactivation in broth, milk, and peach and orange juices. Journal of Food Engineering 62, 47–52. Erkmen, O., Dogan, C., 2004. Effects of ultra high hydrostatic pressure on Listeria monocytogenes and natural flora in broth, milk and fruit juices. International Journal of Food Science and Technology 39, 91–97. Farber, J.M., Daley, E., Coates, F., Emmons, D.B., McKellar, R., 1992. Factors influencing survival of Listeria monocytogenes in milk in a high-temperature short-time pasteurizer. Journal of Food Protection 55 (12), 946–951. Gandhi, M., Chikindas, M.L., 2007. Listeria: A foodborne pathogen that knows how to survive. International Journal of Food Microbiology 113, 1–15. Hayman, M.M., Anantheswaran, R.C., Knabel, S.J., 2008. Heat shock induces barotolerance in Listeria monocytogenes. Journal of Food Protection 71 (2), 426–430. Hayman, M.M., Anantheswaran, R.C., Knabel, S.J., 2007. The effects of growth temperature and growth phase on the inactivation of Listeria monocytogenes in whole milk subject to high pressure processing. International Journal of Food Microbiology 115, 220–226. Koutsoumanis, K.P., Kendall, P.A., Sofos, J.N., 2003. Effect of food processing-related stresses on acid tolerance of Listeria monocytogenes. Applied and Environmental Microbiology 69 (12), 7514–7516. Koseki, S., Mizuno, Y., Yamamoto, K., 2008. Use of mild-heat treatment following highpressure processing to prevent recovery of pressure-injured Listeria monocytogenes in milk. Food Microbiology 25, 288–293. Linton, M., Mackle, A.B., Upadhyay, V.K., Kelly, A.L., Patterson, M.F., 2008. The fate of Listeria monocytogenes during the manufacture of Camembert-type cheese: A comparison between raw milk and milk treated with high hydrostatic pressure. Innovative Food Science and Emerging Technologies 9, 423–428. McClements, J.M.C., Patterson, M.F., Linton, M., 2001. The effect of growth stage and growth temperature on high hydrostatic pressure inactivation of some psychrotrophic bacteria in milk. Journal of Food Protection 64 (4), 514–522. Patterson, M.F., Quinn, M., Simpson, R., Gilmour, A., 1995. Sensitivity of vegetative pathogens to high hydrostatic pressure treatment in phosphate-buffered saline and foods. Journal of Food Protection 58, 24–529. Scheyhing, C.H., Hörmann, S., Ehrmann, M.A., Vogel, R.F., 2004. Barotolerance is inducible by preincubation under hydrostatic pressure, cold-, osmotic- and acid-stress conditions in Lactobacillus sanfranciscensis DSM 20451T. Letters in Applied Microbiology 39, 284–289. Simpson, R.K., Gilmour, A., 1997. The resistance of Listeria monocytogenes to high hydrostatic pressure in foods. Food Microbiology 14, 567–573. Wemekamp-Kamphuis, H.H., Karatzas, A.K., Wouters, J.A., Abee, T., 2002. Enhanced levels of cold shock proteins in Listeria monocytogenes LO28 upon exposure to low temperature and high hydrostatic pressure. Applied and Environmental Microbiology 68 (2), 456–463. Wen, J., Anantheswaran, R.C., Knabel, S.J., 2009. Changes in barotolerance, thermotolerance, and cellular morphology throughout the life cycle of Listeria monocytogenes. Applied and Environmental Microbiology 75 (6), 1581–1588.
Contents lists available at ScienceDirect
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
Effect of growth and recovery temperatures on pressure resistance of Listeria monocytogenes Adrienne E.H. Shearer, Hudaa S. Neetoo, Haiqiang Chen ⁎ University of Delaware, Department of Animal and Food Sciences, Newark, DE 19716, USA
a r t i c l e
i n f o
Article history: Received 27 May 2009 Received in revised form 23 October 2009 Accepted 31 October 2009 Keywords: Listeria monocytogenes High hydrostatic pressure Growth temperature Recovery temperature Resistance Food
a b s t r a c t Experimental conditions can affect the outcome of bacterial stress-tolerance assays. Growth conditions that optimize microbial recovery should be established to help evaluate the effectiveness of treatment conditions for food safety. The objectives of this study were to determine the effects of growth and recovery temperatures on pressure resistance of early stationary-phase Listeria monocytogenes in milk. The tested conditions were the following: (1) L. monocytogenes was grown at various temperatures (10, 15, 20, 25, 30, 35, 40 and 43 °C), suspended in ultra-high temperature (UHT) -processed whole milk, pressure-treated at 400 MPa for 2 min at 21 °C and recovered on Tryptic Soy Agar supplemented with 0.6% yeast extract (TSAYE) at 35 °C; (2) L. monocytogenes was grown at 35 and 43 °C, pressure treated in milk (400 and 500 MPa, respectively, for 2 min at 21 °C) and recovered on TSAYE at various temperatures (4, 10, 15, 20, 25, 30, 35 and 40 °C); (3) L. monocytogenes originally grown at 35 °C, was pressure treated in milk (400 or 450 MPa for 2 min at 21 °C), and recovered on TSAYE at 10 °C for various time intervals (1, 2, 3, 6, 9 and 12 days) then at 35 °C for 5 days. There was no significant difference (P N 0.05) in pressure-resistance of L. monocytogenes grown at 10 to 25 °C with approximately 6.5log CFU/ml population reductions. At growth temperatures greater than 25 °C, pressure resistance increased with less than 1-log CFU/ml reduction observed for L. monocytogenes originally grown at 43 °C. After pressure treatment, regardless of growth temperature and pressure treatment, the greatest recovery of L. monocytogenes was within the 4 to 20 °C range; maximum recovery at 10 °C required approximately 24 days. The time for comparable post-pressure treatment recovery could be reduced by incubation at 10 °C for at least 2 days followed by incubation at 35 °C for 5 days. The findings of the present study indicate that growth and recovery temperatures affect the pressure resistance of L. monocytogenes and should, therefore, be taken into account when assessing the adequacy of inactivation treatments. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Ingestion of food contaminated with Listeria monocytogenes can result in serious illness affecting the gastrointestinal and nervous systems and can lead to miscarriage in pregnant women (Gandhi and Chikindas, 2007). L. monocytogenes is particularly problematic for food safety assurance due to its tolerance of environmental hurdles including its ability to grow at refrigeration temperatures (Gandhi and Chikindas, 2007; Wemekamp-Kamphuis et al., 2002) commonly used to prevent the growth of organisms of significance for food quality and public health. The pathogen has complicated a number of different food systems including those that do not receive a processing treatment lethal to L. monocytogenes as well as foods that have been inadvertently contaminated after processing. Milk and dairy products are among the foods that have been associated with Listeria contamination (Gandhi ⁎ Corresponding author. Department of Animal and Food Sciences, University of Delaware, 020 Townsend Hall, Newark, DE 19716-2150, USA. Tel.: +1 302 831 1045; fax: +1 302 831 2822. E-mail address: [email protected] (H. Chen). 0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2009.10.034
and Chikindas, 2007). This can arise from cows with udder infections or contaminated equipment throughout collection and processing (Linton et al., 2008). Inactivation of L. monocytogenes in milk can be achieved with heat pasteurization in conjunction with good handling practices (Farber et al., 1992). High hydrostatic pressure has been evaluated as an alternative method for processing milk and other dairy products (Linton et al., 2008). A notable degree of inactivation of L. monocytogenes (Chen, 2007a; Dogan and Erkmen, 2004; Erkmen and Dogan, 2004; Simpson and Gilmour, 1997), and other pathogens (Chen, 2007a) in milk has been demonstrated with high pressure, although some pathogenic bacteria have demonstrated considerable barotolerance in milk (Patterson et al., 1995). Pressure inactivation of L. monocytogenes in milk is affected by a number of factors including bacterial strain (Chen et al., 2009), stage of growth (Hayman et al., 2007; McClements et al., 2001; Wen et al., 2009), growth conditions (Bull et al., 2005; Hayman et al., 2007; McClements et al., 2001), pressure level (Erkmen and Dogan, 2004), treatment time (Erkmen and Dogan, 2004), imposition of other stresses (Hayman et al., 2008; Simpson and Gilmour, 1997; Wemekamp-Kamphuis et al., 2002), and recovery conditions (Bozoglu et al.,
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2004; Bull et al., 2005; Koseki et al., 2008). Unless the conditions for maximal barotolerance and full recovery of injured bacteria are established for treatment of a food system of interest, survivors may not be detected and process requirements underestimated. With particular interest to growth and recovery temperature effects, Bull et al. (2005) reported that prior growth temperature affects pressure resistance, but interactions of temperature, milk type, and pressure confounded interpretation of the effects. Hayman et al. (2007) reported that L. monocytogenes grown at the upper limit of its growth range (43 °C) was more pressure-resistant than when grown at optimal (35 °C) or reduced temperature (4 °C). After growth at temperatures closer to optimal, both short-term cold- (Wemekamp-Kamphuis et al., 2002) and heat-shock (Hayman et al., 2008) treatments increased the barotolerance of L. monocytogenes in milk. Temperature during recovery after pressure treatment of milk is also an important consideration as Bull et al. (2005) reported that, of three temperatures evaluated, 15 °C provided better recovery of L. monocytogenes than 4 °C or 30 °C, though the specific number of survivors was not determined. Bozoglu et al. (2004) found that detection of injured L. monocytogenes occurred more rapidly at 30 °C than 4 °C, though the percent recovery was not determined. Prolonged storage of pressure-treated milk supported the recovery and growth of L. monocytogenes better at 4 °C and 25 °C than at 37 °C (Koseki et al., 2008). These studies indicate the importance of growth and recovery temperatures and time in the determination of adequate pressure processing parameters. Although trends are apparent, the variations in findings and approaches also demonstrate the value of a study that compares the magnitude of post-pressure treatment recovery over prolonged time as affected by the broad range of temperatures that support growth and recovery of L. monocytogenes. The study presented herein builds on the findings of previous reports with specific objectives to (1) determine the relationship between pressure resistance and temperature of growth of L. monocytogenes throughout its growth range prior to pressure treatment, (2) determine the optimum incubation temperature throughout its growth range for maximal recovery of L. monocytogenes after pressure treatment, and (3) minimize incubation time through temperature shift after pressure treatment for maximum recovery of L. monocytogenes. The pressure conditions utilized permit L. monocytogenes survival such that differences among growth and recovery temperatures could be evaluated and compared to studies previously conducted. 2. Materials and methods 2.1. Determination of time to stationary phase L. monocytogenes ATCC 19115 was grown on Tryptic Soy Agar (Becton Dickinson and Co., Sparks, MD) supplemented with 0.6% yeast extract (Becton Dickinson and Co., Franklin Lakes, NJ, USA) (TSAYE) for approximately 17 h at 37 °C. A colony was transferred to 10 ml Tryptic Soy Broth (containing dextrose, Becton Dickinson) supplemented with 0.6% yeast extract (TSBYE) and incubated at 35 °C for approximately 17 h. A loopful (10 µl) of the overnight culture was subcultured in fresh TSBYE (100 ml) and incubated at 4, 10, 15, 20, 25, 30, 35, 40 and 43 °C. The time to reach early stationary phase at each temperature was determined by periodic sampling with serial dilution in 0.1% peptone water (BD/Difco Laboratories, Detroit, MI), spread-plating on TSAYE and incubating plates at 35 °C for 72 h. 2.2. Effect of growth temperature on pressure resistance L. monocytogenes was grown at 4, 10, 15, 20, 25, 30, 35, 40 or 43 °C to early stationary phase in TSBYE. Thirty-five milliliters were centrifuged for 15 min at 6318 ×g (IEC Centra-4B Centrifuge, International Equipment Co.), washed once with 0.1 M sodium phosphate buffer, pH 7.0 (Fisher Scientific, Fair Lawn, NJ), and resuspended in 35 ml of ultrahigh temperature (UHT) processed whole milk. The initial count of L.
monocytogenes in milk was approximately 109 CFU/ml. Milk (12 ml) was transferred to a sterile 3-mil thick polypropylene pouch (VWR International, West Chester, PA, USA), heat-sealed, and sealed in a secondary pouch for added safety. Samples were treated in an Avure PT1 laboratory-scale pressure unit (Avure Technologies Inc., Kent, WA, USA) monitored with DASYLab ® 7.0 software (DASYTEC USA, Bedford, NH). Samples were treated at 400 MPa for 2 min (not inclusive of comeup time of less than 30 s with almost instantaneous depressurization) at 21 °C with water as the surrounding pressure-transmitting medium. Enumeration of survivors was conducted by duplicate spread plating of appropriate serial dilutions (in 0.1% peptone water) on TSAYE and incubating at 35 °C for 72 h. Three independent trials were conducted. 2.3. Effect of temperature on recovery of L. monocytogenes from pressure-treated samples L. monocytogenes was grown at two temperatures (35 and 43 °C) to early stationary phase in TSBYE, centrifuged, washed and resuspended in UHT whole milk as previously described. Milk samples were treated at 400 MPa and 500 MPa for cultures grown at 35 and 43 °C respectively, for 2 min at 21 °C. Enumeration of survivors was conducted by duplicate spread plating of appropriate serial dilutions (in 0.1% peptone water) on TSAYE and incubating at 4, 10, 15, 20, 25, 30, 35 and 40 °C until no change in counts was observed. Colonies that developed during prolonged incubation on TSAYE were periodically streaked onto modified Oxford agar (Difco Laboratories, Sparks, MD, USA) to check for growth characteristic of L. monocytogenes. Three to four independent trials were conducted. 2.4. Effect of incubation temperature shift on recovery time L. monocytogenes was grown to early stationary phase at 35 °C, centrifuged, washed, and resuspended in UHT whole milk as previously described. Milk samples were treated at 400 and 450 MPa for 2 min at 21 °C. Survivors were enumerated on TSAYE incubated at 10 °C for various time intervals (1, 2, 3, 6, 9 and 12 d), followed by incubation at 35 °C for 5 d. Three independent trials were conducted. 2.5. Statistical analyses Statistical analyses were conducted using Minitab® Release 15 (Minitab Inc., University Park, PA, USA). One-way analysis of variance (ANOVA) was used to determine the significance of differences between treatments (P b 0.05). For multiple comparisons, Tukey's One-Way Multiple Comparisons Test was used (family error rate = 0.05). 3. Results 3.1. Determination of time to stationary phase The time to reach early stationary phase with approximately 109 CFU/ml at 4, 10, 15, 20, 25, 30, 35, 40 and 43 °C was 384, 96, 48, 36, 18, 12, 12, 12 and 16.25 h, respectively. These incubation times were used for all subsequent experiments. 3.2. Effect of growth temperature on pressure resistance There was no significant difference (P N 0.05) in pressure-resistance of L. monocytogenes grown within the range of 10 to 25 °C. With these growth temperatures, approximately 6.5-log CFU/ml population reduction after treatment at 400 MPa for 2 min at 21 °C and recovery at 35 °C (Fig. 1) was observed. Pressure resistance increased as the growth temperatures increased from 25 to 43 °C. Less than a 1-log CFU/ml reduction was observed after pressure treatment when L. monocytogenes was grown at 43 °C (Fig. 1).
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Fig. 1. Effect of growth temperature prior to pressure treatment on resistance of L. monocytogenes (initial count of approximately 109 CFU/ml in UHT whole milk) to pressure (400 MPa for 2 min at 21 °C). L. monocytogenes was enumerated on TSAYE after incubation at 35 °C for 72 h. Error bars represent ± 1 standard deviation. Bars labeled with different letters are significantly different (P b 0.05).
3.3. Effect of temperature on recovery of L. monocytogenes from pressure-treated samples Regardless of prior growth temperature (35 or 43 °C) and pressure treatment (400 or 500 MPa for 2 min at 21 °C), the greatest recovery of pressure-treated L. monocytogenes was obtained when TSAYE plates were incubated between 4 and 20 °C after pressure treatment (Fig. 2). Incubation at 10 °C provided the highest recovery by as much as 2 logs CFU/ml, although not statistically different (P N 0.05) from recovery at 4, 15, or 20 °C. The time for maximum recovery at 10 °C was 24 days. The incubation times for maximum recovery at the other tested temperatures were approximately 50, 15, and 10 days at 4, 15, and 20 °C, respectively, and approximately 5 days for 25 to 40 °C. 3.4. Effect of incubation temperature shift on recovery time Incubation of L. monocytogenes for at least 2 days at 10 °C followed by incubation at 35 °C for 5 days resulted in the statistically same recovery as that achieved by incubation at 10 °C for 24 days. Thus, the recovery period for L. monocytogenes could be shortened from 24 to 7 days by applying the above incubation temperature shift (Fig. 3). 4. Discussion There are a number of variables, including growth phase (Hayman et al., 2007; McClements et al., 2001), medium (Dogan and Erkmen, 2004) and temperature (Wemekamp-Kamphuis et al., 2002), that can affect bacterial resistance to subsequent stresses. The extent of recovery after a stress can also be affected by recovery medium and temperature (Bull et al., 2005). The focus of this study was high hydrostatic pressure resistance of stationary-phase L. monocytogenes as affected by temperature, both during growth prior to treatment and recovery after treatment. Populations of L. monocytogenes in stationary phase have been reported to exhibit greater barotolerance in milk than exponentiallygrowing cells (Hayman et al., 2007; McClements et al., 2001); and thus, early stationary-phase cells were used for the present study. Subsequent studies have demonstrated even greater barotolerance over L. monocytogenes in stationary phase by cells in a long-term-survival phase, which can occur after an initial death phase under prolonged storage and without addition of fresh nutrients (Wen et al., 2009). This phase of survival further emphasizes the importance of meticulous cleaning of processing equipment to prevent harborage and potential release of viable and resistant L. monocytogenes (Wen et al., 2009) in processed
milk, though the prevalence of such cells in freshly-collected milk is not known. Combinations of stresses applied simultaneously may also affect resistance to subsequent stresses. For example, a pH decrease during incubation of L. monocytogenes in the presence of glucose has been reported to adapt cells to subsequent acid stress (Koutsoumanis et al., 2003). The extent of this adaptation may also be dependent on the temperature during acid exposure. Some acid-adapted bacteria also have increased barotolerance (Scheyhing et al., 2004). L. monocytogenes would have had some exposure to reduced pH as a normal course of growth in this study and thus may have different, presumably increased, pressure resistance than had neutral conditions been artificially maintained. This may better reflect potential pressure resistance of bacterial contaminants that could be introduced at various points during food handling that may have varying history of exposures to prior stresses. Previous studies have demonstrated the importance of temperature exposure prior to, and after, high hydrostatic pressure treatment on the baroresistance of L. monocytogenes. From collection through processing and storage, milk temperatures can potentially span most of the growth temperature range of L. monocytogenes. If milk is contaminated with L. monocytogenes during or shortly after collection while still warm or if the temperature is permitted to increase, growth at such elevated temperatures may increase the resistance of L. monocytogenes to subsequent inactivation by high hydrostatic pressure. We observed approximately a 6-log difference in the number of L. monocytogenes that survived treatment of 400 MPa for 2 min at 21 °C when grown at 43 °C as compared to when grown in the range of 10 to 25 °C. These results are generally in agreement with those reported by Hayman et al. (2007) who demonstrated that L. monocytogenes grown at 43 °C was more baroresistant than when grown at 15 °C. The inactivation observed with 15 °C growth in the present study was greater however by approximately 2 logs, and we also observed no statistical difference in baroresistance among L. monocytogenes grown within the full range of 10 to 30 °C, as evaluated in 5-degree increments. Interestingly, a few degree difference above the optimal growth temperature markedly increased barotolerance of L. monocytogenes. The implications of this and the previous study (Hayman et al., 2007) are similar such that L. monocytogenes with prior growth at temperatures above optimal may need increased pressure and/or other type of stress for complete inactivation, or low temperature storage of milk prior to pressure treatment may be a critical parameter for process control. The length of time at reduced temperatures and the extent of growth prior to temperature reduction may also be critical. Wemekamp-
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Fig. 2. Effect of incubation temperature on recovery of L. monocytogenes after pressure treatment. L. monocytogenes was grown at 35 °C (initial count in UHT whole milk 2 × 109 CFU/ml) and pressure treated at 400 MPa for 2 min at 21 °C or grown at 43 °C (initial count in UHT whole milk 2 × 107 CFU/ml) and pressure treated at 500 MPa for 2 min at 21 °C. Error bars represent ± 1 standard deviation. Bars labeled with different letters are significantly different (P b 0.05). Statistical comparisons were made among recovery temperatures for each pressure treatment; comparisons were not made between the two pressure treatments.
Kamphuis et al. (2002) demonstrated that growth at 37 °C followed by cold shock at 10 °C for 4 h increased L. monocytogenes barotolerance as compared to cells growing exponentially at 37 °C. This was accompa-
nied by an increased production of certain cold-shock proteins (CSPs). With extended incubation of L. monocytogenes at 10 °C, the levels of CSPs increased as growth resumed with even greater production
Fig. 3. Effect of time of temperature shift from 10 °C to 35 °C on recovery of L. monocytogenes (initial count in UHT whole milk 3 × 109 CFU/ml) pressure treated at 400 or 450 MPa for 2 min at 21 °C. With the exception of the 24-day sample which was held at 10 °C for the entire incubation period as a control, samples were incubated at 10 °C for the length of time indicated on the x-axis and then transferred to 35 °C for 5 days for the final count. Error bars represent ± 1 standard deviation. Bars labeled with different letters are significantly different (P b 0.05). Statistical comparisons were made among recovery temperatures for each pressure treatment; comparisons were not made between the two pressure treatments.
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observed during stationary phase after cold shock. One might speculate an additional increase of CSPs could further increase barotolerance, but this was not evaluated (Wemekamp-Kamphuis et al., 2002). In the present study, growth of L. monocytogenes at 10 °C for 96 h resulted in one of the lowest pressure resistances observed among the growth temperatures tested. However, as discussed in more detail later, lower incubation temperatures (4 to 20 °C) after pressure treatment provided better recovery than 35 °C as determined in subsequent experiments, and thus the number of L. monocytogenes recovered in this phase of the study may not reflect the maximum possible recovery after pressure treatment. It would be interesting to evaluate the combined effect of growth at lower temperatures prior to pressure treatment with incubation for recovery after pressure treatment also at reduced temperatures to determine the extent of pressure-induced injury of these cells. Temperature shock in the opposite direction by heat-shocking at 48 °C for up to 30 min has also been reported to increase L. monocytogenes barotolerance (Hayman et al., 2008); and thus, while the simultaneous application of heat during pressure has enhanced inactivation (Chen, 2007b), the timing of heat exposure may also be critical in the determination of process needs. Farber et al. (1992) reported that L. monocytogenes grown at 43 °C also has greater thermotolerance than that grown at 30 °C and maintains an increased tolerance for at least 24h after refrigerated storage. The effect of similar handling conditions on L. monocytogenes barotolerance warrants study. For researchers and processors attempting to establish appropriate process treatments for specific products and operations, the temperature(s) of maximum tolerance should be used in such laboratory studies if there is any chance of L. monocytogenes exposure to stress-adaptive conditions prior to processing including growth at a sustained elevated temperature or temperature fluctuations. Also worthy of evaluation is how the temperature of growth in raw milk affects L. monocytogenes barotolerance as directly compared to growth in microbiological media which was used for our study as well as by Hayman et al. (2007) and Wemekamp-Kamphuis et al. (2002). The other focus of this study was to determine how temperature after high pressure treatment affected the extent and rate of recovery of L. monocytogenes. A previous study (Bull et al., 2005) demonstrated that pressure-treated L. monocytogenes recovered more quickly and over a longer storage period in milk at 15 °C than 4 or 30 °C by a presence/ absence approach. In the present study, L. monocytogenes was recovered at eight temperatures within its growth range, and numbers of survivors were determined on non-selective media to help characterize the magnitude of difference in recovery. Using this approach, 10 °C provided the greatest recovery by as much as 2-logs over higher temperatures regardless of prior growth temperature (35 or 43 °C) and pressure level (400 or 500 MPa, respectively), although the difference in recovery within the 4 to 20 °C recovery range was not statistically significant (P N 0.05). In the Bull et al. (2005) study, it was noted that storage at 4 °C did not allow for full recovery of pressure-injured L. monocytogenes, but a switch from 4 °C to 30 °C provided better recovery, and such conditions may be encountered in situations of temperature abuse during milk storage. They speculated that L. monocytogenes recovers in two stages, and the stress of 4 °C and 30 °C storage does not permit complete repair for extended storage. In the present study, L. monocytogenes required 24 days to reach maximum recovery at 10 °C. Statisticallycomparable recovery could be obtained by incubating plates at 10 °C for at least 2 days and then incubating at 35 °C. Presumably, the temperature switch permitted more rapid colony formation that was visually detectable; it is unclear if the switch to 35 °C also accelerated a specific stage of an injury recovery process. This work corroborates prior reports of the influence of growth and recovery temperatures on baroresistance of early stationary phase L. monocytogenes. The studies support previous findings that growth at the upper range of growth temperatures prior to pressure treatment increases baroresistance, and incubation at lower temperatures after
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pressure treatment increases recovery. Further, the studies provide the magnitude of survival and the temperatures at which the number of survivors becomes significantly different throughout the growth temperature range as evaluated in small temperature increments. The study also demonstrates the adequacy of short-term recovery after pressure treatment by incubation at 10 °C followed by incubation at 35 °C to expedite enumeration of survivors under the conditions evaluated. While this study demonstrates the importance of growth and recovery conditions on barotolerance at a single treatment time, further studies are needed to establish the influence of these same conditions on the complete inactivation profile of L. monocytogenes in milk. Acknowledgement This study was supported by start-up funds from the University of Delaware Department of Animal and Food Sciences. References Bozoglu, F., Alpas, H., Kaletunç, G., 2004. Injury recovery of foodborne pathogens in high hydrostatic pressure treated milk during storage. FEMS Immunology and Medical Microbiology 40, 243–247. Bull, M.K., Hayman, M.M., Stewart, C.M., Szabo, E.A., Knabel, S.J., 2005. Effect of prior growth temperature, type of enrichment medium, and temperature and time of storage on recovery of Listeria monocytogenes following high pressure processing of milk. International Journal of Food Microbiology 101, 53–61. Chen, H.Q., 2007a. Use of linear, Weibull, and log-logistic functions to model pressure inactivation of seven foodborne pathogens in milk. Food Microbiology 24, 197–204. Chen, H.Q., 2007b. Temperature-assisted pressure inactivation of Listeria monocytogenes in Turkey breast meat. International Journal of Food Microbiology 117, 55–60. Chen, H.Q, Neetoo, H., Ye, M., Joerger, R.D., 2009. Differences in pressure tolerance of Listeria monocytogenes strains are not correlated with other stress tolerances and are not based on differences in CtsR. Food Microbiology 26, 404–408. Dogan, C., Erkmen, O., 2004. High pressure inactivation kinetics of Listeria monocytogenes inactivation in broth, milk, and peach and orange juices. Journal of Food Engineering 62, 47–52. Erkmen, O., Dogan, C., 2004. Effects of ultra high hydrostatic pressure on Listeria monocytogenes and natural flora in broth, milk and fruit juices. International Journal of Food Science and Technology 39, 91–97. Farber, J.M., Daley, E., Coates, F., Emmons, D.B., McKellar, R., 1992. Factors influencing survival of Listeria monocytogenes in milk in a high-temperature short-time pasteurizer. Journal of Food Protection 55 (12), 946–951. Gandhi, M., Chikindas, M.L., 2007. Listeria: A foodborne pathogen that knows how to survive. International Journal of Food Microbiology 113, 1–15. Hayman, M.M., Anantheswaran, R.C., Knabel, S.J., 2008. Heat shock induces barotolerance in Listeria monocytogenes. Journal of Food Protection 71 (2), 426–430. Hayman, M.M., Anantheswaran, R.C., Knabel, S.J., 2007. The effects of growth temperature and growth phase on the inactivation of Listeria monocytogenes in whole milk subject to high pressure processing. International Journal of Food Microbiology 115, 220–226. Koutsoumanis, K.P., Kendall, P.A., Sofos, J.N., 2003. Effect of food processing-related stresses on acid tolerance of Listeria monocytogenes. Applied and Environmental Microbiology 69 (12), 7514–7516. Koseki, S., Mizuno, Y., Yamamoto, K., 2008. Use of mild-heat treatment following highpressure processing to prevent recovery of pressure-injured Listeria monocytogenes in milk. Food Microbiology 25, 288–293. Linton, M., Mackle, A.B., Upadhyay, V.K., Kelly, A.L., Patterson, M.F., 2008. The fate of Listeria monocytogenes during the manufacture of Camembert-type cheese: A comparison between raw milk and milk treated with high hydrostatic pressure. Innovative Food Science and Emerging Technologies 9, 423–428. McClements, J.M.C., Patterson, M.F., Linton, M., 2001. The effect of growth stage and growth temperature on high hydrostatic pressure inactivation of some psychrotrophic bacteria in milk. Journal of Food Protection 64 (4), 514–522. Patterson, M.F., Quinn, M., Simpson, R., Gilmour, A., 1995. Sensitivity of vegetative pathogens to high hydrostatic pressure treatment in phosphate-buffered saline and foods. Journal of Food Protection 58, 24–529. Scheyhing, C.H., Hörmann, S., Ehrmann, M.A., Vogel, R.F., 2004. Barotolerance is inducible by preincubation under hydrostatic pressure, cold-, osmotic- and acid-stress conditions in Lactobacillus sanfranciscensis DSM 20451T. Letters in Applied Microbiology 39, 284–289. Simpson, R.K., Gilmour, A., 1997. The resistance of Listeria monocytogenes to high hydrostatic pressure in foods. Food Microbiology 14, 567–573. Wemekamp-Kamphuis, H.H., Karatzas, A.K., Wouters, J.A., Abee, T., 2002. Enhanced levels of cold shock proteins in Listeria monocytogenes LO28 upon exposure to low temperature and high hydrostatic pressure. Applied and Environmental Microbiology 68 (2), 456–463. Wen, J., Anantheswaran, R.C., Knabel, S.J., 2009. Changes in barotolerance, thermotolerance, and cellular morphology throughout the life cycle of Listeria monocytogenes. Applied and Environmental Microbiology 75 (6), 1581–1588.