J. Dairy Sci. 90:99-109
© American Dairy Science Association, 2007.
Response of Two Salmonella enterica Strains Inoculated in Model Cheese Treated with High Hydrostatic Pressure
S. De Lamo-Castellví,
A. X. Roig-Sagués1,
T. López-Pedemonte,
M. M. Hernández-Herrero,
B. Guamis and
M. Capellas
Centre Especial de Recerca Planta de Tecnologia dels Aliments, CeRTA, XIT, Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
1 Corresponding author: ArturXavier.Roig{at}uab.es
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ABSTRACT
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The aim of this work was to determine the response to high hydrostatic pressure and the ability for survival, recovery, and growth of 2 strains of Salmonella enterica (Salmonella enteritidis and Salmonella typhimurium) inoculated in a washed-curd model cheese produced with and without starter culture. Inoculated samples were treated at 300 and 400 MPa for 10 min at room temperature and analyzed after treatment and after 1, 7, and 15 d of storage at 12 ° C to study the behavior of the Salmonella population. Cheese samples produced with starter culture and treated at 300 and 400 MPa showed maximum lethality; no significant differences in the baroresistant behavior of both strains were detected. Nevertheless, when starter culture was not present, the maximum lethality was only observed in cheese samples treated at 400 MPa, in the case of S. enteritidis. Ability to repair and grow was not observed in model cheese produced with starter culture and cell counts of treated samples decreased after 15 d of storage at 12 ° C. In cheese produced without starter culture, Salmonella cells showed the ability to repair and grow during the storage period, reaching counts over 3 log10 (cfu/mL) in both applied treatments and serotypes. These results suggest that high hydrostatic pressure treatments are effective to reduce Salmonella population in this type of cheese, but the presence of the starter culture affects the ability of this microorganism to repair and grow during the storage period.
Key Words: high hydrostatic pressure • Salmonella • cheese • starter culture
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INTRODUCTION
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Salmonella is ubiquitous in nature and the serotypes that can cause human infections occur in all types of animals, including a significant but unknown number of domestic animals. Infections exhibit a number of clinical manifestations, but gastrointestinal disorders are the most common. The severity and duration of symptoms depend on the serotype of Salmonella present, the amount of food eaten, and the susceptibility of the person involved (El-Gazzar and Marth, 1992). The 2 most prevalent serotypes of Salmonella currently isolated from foodborne outbreaks in the United States and Europe are Salmonella enteritidis and Salmonella typhimurium (Mattick et al., 2001; Anonymous, 2003). Moreover, S. enteritidis is one of the Salmonella serotypes most commonly associated with morbidity and mortality in humans (Ahmed et al., 2000).
Raw and pasteurized milk and different types of cheese have been involved in several outbreaks (Altekruse et al., 1998; Haeghebaert et al., 2003). Some studies have shown that when milk becomes contaminated with Salmonella spp. after pasteurization, the pathogen could survive the cheese-making process and persist for several months in the cheese (Leyer and Johnson, 1992). In ripened Cheddar cheese stored at 7 ° C, Salmonella was detected for up to 7 mo. In cold-packed cheeses, Salmonella was found depending on the pH value and preservative used (El-Gazzar and Marth, 1992). Moreover, fat and proteins in cheese can protect foodborne pathogens from gastric acidity, reducing the number of organisms necessary to cause clinical infections (Altekruse et al., 1998). These trends reassert the importance of detecting low numbers of Salmonella cells in cheese.
High hydrostatic pressure (HHP) has been proposed as a viable alternative to conventional heat treatment for preserving food. In contrast to thermal processing, the application of HHP to foods causes negligible impairment of nutritional value, taste, color, or flavor (Smelt, 1998). Several factors are known to affect the resistance of bacteria to HHP: temperature, magnitude and duration of pressure treatment, stage of growth, and composition of the medium (McClements et al., 2001). Moreover, in food, 2 effects determine microbiological safety and stability: the effect during treatment (Patterson and Kilpatrick, 1998), and the effect after treatment during the repair phase of the microorganism (Smelt, 1998).
In cheese, differences in the degree of microbial inactivation obtained by applying HHP may be due to the species and the quantity of starter cultures used as well as cheese acidity and composition (O’Reilly et al., 2001). Several authors have studied the effect of high hydrostatic pressure on microorganisms and spores in different types of cheese (O’Reilly et al., 2001; López-Pedemonte et al., 2003; De Lamo-Castellví et al., 2005) but it is also necessary to evaluate the effect on Salmonella. For this purpose, we selected a washed-curd model cheese following the protocol proposed by López-Pedemonte et al. (2003), which allowed us to work under controlled microbiological conditions.
The aim of this study was to evaluate the effect of HHP on S. enteritidis and S. typhimurium inoculated in washed-curd model cheese produced with or without starter culture and to analyze the behavior of both pathogens during a storage period of 15 d at 12 ° C.
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MATERIALS AND METHODS
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Bacterial Culture Preparation
Salmonella enteritidis (CECT 4300) and S. typhimurium (CECT 443) were obtained from Spanish Type Culture Collection (CECT, Universidad de Valencia, Valencia, Spain) and were kept in cryobeads (Nalgene System 100, Microkit Iberica S.L., Madrid, Spain) at – 20 ° C. The first bacterial culture was obtained by inoculating a cryobead in 10 mL of brain heart infusion broth (Oxoid, Basingstoke, UK) containing 0.6% yeast extract (BHIYE, Oxoid) and incubated at 37 ± 1 ° C for 24 ± 2 h. One milliliter of this first culture was transferred to 10 mL of BHIYE and incubated for an additional 18 h at 37 ± 1 ° C to allow the culture to reach the stationary phase. Cells were centrifuged at 1,250 x g for 15 min at room temperature, washed once in 10 mM PBS (Oxoid), and the pellets were resuspended in PBS to a final concentration of 8 to 9 log10 (cfu/mL).
Starter Culture Preparation
A mixture of commercial lyophilized strains of Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris (Ezal MAO 11, Rhodia Iberia S.A., Madrid, Spain), which is known as a nonbacteriocin producer, was used as a starter culture for the washed-curd model cheese manufacture. The culture was revived by inoculating 0.015 g of the mixture in 1,000 mL of commercial sterilized skimmed milk and by incubating at 30 ± 1 ° C for 24 ± 2 h. A volume of 50 mL was used to prepare a subculture in 200 mL of sterilized skimmed milk, which was incubated under the same temperature and time conditions reaching a final concentration of about 9 log10 (cfu/mL).
Model Cheese Manufacture
Raw cow’s milk obtained from a local farm was transported and stored at 4 ° C. Before inoculation, milk was pasteurized at 65 ° C for 30 min and then cooled to 32 ° C in an ice and water bath. Two percent (vol/vol) of starter culture and 0.01% (vol/vol) of a 35% CaCl2 dilution (Arroyo, Santander, Spain) were added. Milk was kept in a water bath at 32 ° C for 20 min and then inoculated with 1% (vol/vol) of S. enteritidis or S. typhimurium inoculum (except blanks). We added 0.02% (vol/vol) of liquid rennet extract of bovine origin (520 mg/L of active chymosin, Arroyo, Santander, Spain) as coagulating agent. Centrifugation bottles (Nalgene, Nalge Nunc International, Rochester, NY) were filled with 225 mL of inoculated milk and placed in a bath at 32 ° C for 40 min until the curd was formed. Curd was cut into small pieces and kept in the water bath while the temperature was increased to 37 ° C for more than 5 min. The bottles were kept in the bath at 37 ° C for a further 15 min. Curds were washed to avoid excessive acidification substituting 40% (vol/vol) of whey with sterile water. Bottles were then centrifuged at 7,000 x g for 40 min at 20 ° C and kept in their containers in a water bath at 37 ° C until pH 5.5 was reached. When the pH of the resulting cheeses dropped to 5.5 (only in the case of cheese made with starter), whey was completely removed from the bottles by decantation and cheeses were salted by adding 100 mL of 20% (wt/vol) NaCl sterile brine into each bottle for 15 min. After this, cheeses were removed from the centrifugation bottles using sterile pincers and dried on sterile paper. They had a final weight of approximately 23 g with 55% DM, 1.5% salt, and 25.16% fat in moisture content; the water activity was 0.99. Cheeses were vacuum packed (EVT-7-CD model, 97% vacuum, Tecnotrip S.A., Terrassa, Spain) twice in plastic bags (Cryovac Packaging, Sant Boi de Llobregat, Spain), and stored overnight at 12 ° C before HHP treatment.
In the case of model cheeses produced without starter culture, the same protocol without adding starter culture was followed.
High Pressure Treatment
Cheese samples were pressurized in a discontinuous isostatic press (Pilote HP, ACB, Nantes, France) with a pressure chamber measuring 30 cm in diameter and 70 cm in length. The temperature of the pressurization fluid (water) was measured by a thermocouple. The pressurization and depressurization times and initial, final, and maximum temperatures during the treatments are shown in Table 1
. Samples (initially at 12 ° C) were placed in a water bath for 10 min to reach room temperature. Cheese samples (8 for each treatment) were pressurized at 300 and 400 MPa for 10 min. In previous studies performed with model cheeses inoculated with Yersinia enterocolitica or Escherichia coli strains (De Lamo-Castellví et al., accepted), 500 MPa was also applied but the cheese texture and the starter culture were affected too much by this treatment.
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Table 1. Pressurization and depressurization times and initial, final, and maximum temperatures during the high hydrostatic pressure (HHP) treatments
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Sampling Protocol
Pasteurized milk samples inoculated with S. enteritidis or S. typhimurium were analyzed to count the number of these microorganisms before cheese making. Three groups of cheese samples were analyzed: noninoculated (blank cheese samples), inoculated with S. enteritidis or S. typhimurium but not treated by HHP (control cheese samples), and inoculated and treated (pressurized cheese samples). All samples were stored at 12 ° C. At the end of cheese production, control samples were analyzed to assess the population of S. enteritidis and S. typhimurium. Microbiological analyses were performed at 0 d (immediately after HHP treatment), and at 1, 7, and 15 d after treatment of all samples. Experiments were run 3 times with duplicate analyses each time.
Microbiological Analyses
Ten grams of each sample was homogenized in 90 mL of maximum recovery diluent (MRD, Oxoid) for 1 min using an electromechanical blender (BagMixer, Interscience, St-Nom, France) at room temperature. Ten-fold serial dilutions were prepared in MRD and 1 mL of the appropriate dilution was plated into Salmonella chromogenic agar base (SC, Oxoid) containing Salmonella selective supplement (Oxoid) to count non-injured cells of S. enteritidis and S. typhimurium (Cassar and Cuschieri, 2003). Kang and Fung (1999) proposed the thin agar layer (TAL) method to recover injured microorganisms. A modification of this method (TALm), tested in previous studies (De Lamo Castellví et al., 2005), was used to count both injured and noninjured cells to a detection level of 10 cfu/g. After a selective medium layer (SC, 20 mL), the first layer of nutritive medium (7 mL of BHIYE) was added. Then, 1 mL of the appropriate dilution of cheese sample and a second layer (7 mL) of nutritive medium was added. During the first few hours of incubation of TAL plates, injured cells recover and start to grow on the nonselective medium top layer, whereas the agents of the selective medium gradually diffuse to the top layer. Then, the target microorganism performs most reactions that it typically does on selective medium, and growth of most other microorganisms is inhibited by the now lower concentration of selective agents. All plates were incubated at 37 ± 1 ° C for 24 ± 2 h. Representative colonies were picked and inoculated onto BHIYE plates and incubated at 37 ° C for 24 h before identification using a biochemical test (API 20E, BioMérieux, Marcy-L’Etoile, France).
Besides this, 1 mL of appropriate dilution was plated into M17 agar (Oxoid) supplemented with 5% (vol/vol) of lactose (Oxoid) at 20% (wt/vol) to evaluate the count of Lactococcus in model cheese produced with starter culture. All plates were incubated at 30 ± 1 ° C for 48 ± 2 h.
Results are expressed as the logarithm of the colony-forming units per gram. Lethality was calculated as the difference between the logarithms of the colony counts of the control (N0) and treated samples at d 0 (log10 N0 – log10 N).
An enrichment procedure was used to check the presence of injured S. enteritidis and S. typhimurium cells in cheese samples that did not show colonies growing in either culture media. For this purpose, the first dilution of each sample obtained after HHP treatment and after 1, 7, and 15 d of storage, consisting of 10 g of cheese diluted in 90 mL of MRD, was stored at 37 ± 1 ° C for 24 ± 2 h. A loopful of this culture was streaked onto a plate of SC and incubated at 37 ± 1 ° C for 24 ± 2 h. Representative colonies were identified using biochemical tests (API 20E),
pH Measurement
Model cheese pH was determined using a penetration electrode (Crison, Crison Instruments S.A., Alella, Spain); pH values were based on the mean of 9 measurements.
Statistical Analyses
Analysis of variance was performed using the GLM procedure of SAS software (SAS System for Windows, 8.02, 1999; SAS Institute, Inc., Cary, NC). The Student-Newman-Keuls tests were used to obtain paired comparisons among sample means. Level of significance was set at P < 0.05.
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RESULTS
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Behavior of Initial Inocula During Model Cheese Production
Cell counts of S. enteritidis and S. typhimurium inoculated in milk and in model cheese produced with and without starter culture are shown in Table 2. It is clear from the results obtained that S. enteritidis and S. typhimurium increased in numbers during the cheese-making process in both types of cheese. Moreover, it is important to note that no significant differences were detected between the increased values of different strains and culture media obtained during the production of cheese made with starter culture (data not shown). However, in cheese produced without starter culture, the increased value of S. enteritidis and S. typhimurium observed during the cheese manufacture was highly significant (P < 0.001) in TALm, but not in selective medium. No differences were detected between serotypes (data not shown).
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Table 2. Cell counts of Salmonella enteritidis and Salmonella typhimurium inoculated in milk (initial inoculum) and in model cheese produced with and without starter culture (at the end of cheese production)
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At 300 MPa, the effect of HHP treatment on starter population was minimum, whereas at 400 MPa, it was close to 3 log10 (cfu/g) (Table 3). Moreover, no significant difference in pressure resistance behavior of starter was detected between cheese samples inoculated with S. enteritidis and S. typhimurium.
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Table 3. Lethality values1 of starter culture inoculated in model cheese and pressurized at 300 and 400 MPa for 10 min at d 0
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High Pressure Effect at Day 0 in Model Cheese
Lethality results obtained immediately after HHP treatments in washed-curd model cheese inoculated with and without starter culture and with S. enteritidis or S. typhimurium are shown in Table 4. In the case of cheese produced with starter culture, 300 and 400 MPa treatments produced the same lethality values in both strains and culture media. In cheese made without starter and inoculated with S. enteritidis, the treatment that caused the highest lethality was 400 MPa, and it is worth mentioning that this value was more significant (P < 0.001) in TALm than in SC. No significant differences were detected in the lethality values obtained after applying 300 and 400 MPa in model cheese made without starter culture and inoculated with S. typhimurium except in the case of 400 MPa in TALm which was highly significant (P < 0.001).
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Table 4. Lethality values1 of Salmonella enteritidis and Salmonella typhimurium inoculated in model cheese produced with and without starter culture and pressurized at 300 and 400 MPa for 10 min at d 0
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Behavior of S. enteritidis and S. typhimurium After HHP Treatment in Model Cheese Produced With Starter Culture
Significant differences between cell counts of S. enteritidis and S. typhimurium were only observed in cheese samples inoculated with S. enteritidis and S. typhimurium and treated at 300 MPa (P < 0.05). Salmonella strains inoculated in model cheese treated at 400 MPa were not able to repair after 15 d of storage at 12 ° C (Figure 1, a and b) and kept their counts below the detection level (10 cfu/g) in both culture media. Moreover, after the enrichment process (24 h at 37 ° C), cells were only detected at d 1 of storage at 12 ° C (data not shown). At 300 MPa, cheese samples inoculated with S. enteritidis decreased their counts during the time of storage (Figure 1a) without reaching the level of detection and samples inoculated with S. typhimurium decreased their cell counts below the detection level after 7 d of storage (Figure 1b) in both culture media. Cell counts of S. typhimurium were found after the enrichment process from 0 to 15 d of storage.
None of the S. typhimurium or S. enteritidis cells inoculated in control samples had the ability to grow after 15 d of ripening at 12 ° C (Figure 1, a and b
) and their counts showed a tendency to decrease in after 1 and 7 d of storage, respectively. This tendency was more accentuated in the case of S. typhimurium (Figure 1b), which showed counts close to the detection level of 10 cfu/g after 15 d of storage at 12 ° C.
Behavior of S. enteritidis and S. typhimurium After HHP Treatment in Model Cheeses Produced Without Starter Culture
Salmonella enteritidis and S. typhimurium inoculated in model cheese and treated at 300 and 400 MPa had the ability to repair and grow during the storage period at 12 ° C (Figure 2, b and c). Nevertheless, depending on the HHP treatment, the time necessary to detect this behavior and the level of Salmonella population after the storage period were different. At 300 MPa, both strains began to grow after 1 d of storage (Figure 2b) reaching counts close to 4 and 8 log10 (cfu/g) in selective medium and TALm, respectively, at the end of storage. At 400 MPa, S. enteritidis and S. typhimurium increased their cell counts after 1 and 7 d of storage in TALm and selective medium (Figure 2c) respectively. At the end of storage period, both strains showed cell counts over 3 and 7 log10 (cfu/g) in selective medium and TALm, respectively.
Control samples inoculated with S. enteritidis and S. typhimurium did not show significant differences in their cell counts during the time of storage in any culture media (Figure 2a), except for S. typhimurium in TALm (P < 0.05).
Behavior of Starter Culture After HHP Treatments in Model Cheeses
Cell counts in the starter population treated by HHP showed a tendency to decrease during the storage time (Figure 3, a and b). At 300 MPa, cells counts began to decrease at the same day of treatment in the case of S. enteritidis, and after 1 d of storage at 12 ° C, in the case of S. typhimurium. At 400 MPa, this tendency to decrease was observed after 1 d of storing cheeses under refrigerated conditions. In control and blank samples, this behavior was observed after 7 d.
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DISCUSSION
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The increase in Salmonella counts during cheese manufacture observed in this study has been reported previously by several authors (Wood et al., 1984; Modi et al., 2001). This increase, in regard to inoculated milk, could be considered a consequence of physical concentration and growth of the inocula during cheese production. Spahr and Url (1994) found that the increase of 1 log10 (cfu/g) in bacterial counts during the first steps of the cheese-making process is due to the physical concentration phenomenon caused by syneresis of the curd rather than bacterial growth. Also, some studies have demonstrated the ability of different species of pathogenic microorganisms to grow during the cheese-making process when they have been added to pasteurized milk after being grown in favorable conditions (Johnson et al., 1990).
A population of bacteria after physical preservation treatment may contain 3 physiological types of cells: noninjured cells that are capable of growth and multiplication both in selective and nonselective culture medium; injured cells that are capable of multiplication in a nonselective medium but not in a selective medium; and dead cells, which are incapable of multiplication under any conditions (Wuytack et al., 2003). In this research, a selective culture medium (SC) and a modification of TALm were used to detect the injured Salmonella population produced by the cheese-making process, HHP treatments, and low pH of cheese. Wu et al. (2001) reported that TAL was an adequate method to enumerate acid-injured S. typhimurium cells. In previous experiments carried out on the same washed-curd model cheese, we found that the combination of both culture media was an appropriate method for detecting injured cells of Y. enterocolitica (De Lamo-Castellví et al., 2005). In the present study, the population of injured cells of S. enteritidis and S. typhimurium induced by cheese making and low pH of cheese was not able to recover and grow in TALm medium. Other authors have reported similar behavior; Brashears et al. (2001) found that Salmonella cells subjected to stress by lactic acid (pH 3.5 after 18 h of incubation at 4 ° C) were not able to repair themselves using another recovery media method: stressed cells were put on a 5-mL thin layer of tryptic soy agar, incubated for 2 h at room temperature, and then overlaid with xylose lysine tergitol4 agar.
The HHP treatments used in this work were effective in reducing the initial counts on selective medium and TALm of S. enteritidis and S. typhimurium inoculated in cheese produced with and without starter culture. Other studies have evaluated the effect of HHP on inoculated microorganisms in cheese. Capellas et al. (1996) made fresh cheese inoculated with Escherichia coli CECT 405. Samples were treated using different combinations of pressure (400 to 500 MPa), temperature (2, 10, or 25 ° C), and time (5, 10, or 15 min) and subsequently stored at 2 to 4 ° C. No survival of E. coli cells was detected 1 d after pressurization, except in samples treated for 5 min and 25 ° C at pressures of 400 to 450 MPa. O’Reilly et al. (2000) did not detect cell counts of Staphylococcus aureus and E. coli inoculated in cheese slurries after applying HHP treatments higher than 600 MPa at 20 ° C and 400 MPa at 30 ° C, respectively. In a previous study (De Lamo-Castellví et al., 2005), we reported that HHP treatments at 400 and 500 MPa for 10 min at 20 ° C were effective in reducing the initial population of Y. enterocolitica inoculated in model cheese produced with starter culture. Besides this, it is important to note that in cheese samples produced without starter culture, it was necessary to apply more pressure to obtain the same reduction than in cheese produced with starter culture. Differences in pH between the 2 types of cheese (Table 5) and the presence of starter could be the main reasons for this behavior. In fact, several studies have demonstrated that bacteria can become much more pressure-sensitive at low pH and that efficient inactivation of even the most pressure-resistant vegetative bacteria could be produced at relatively mild pressure in acidic foods (García-Graells et al., 1998; Alpas et al., 2000; Jordan et al., 2001).
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Table 5. Behavior of pH in blank cheese samples (noninoculated with Salmonella) produced with and without starter culture and stored at 12 ° C
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High hydrostatic pressure treatments produce sublethally injured cells (McClements et al., 2001; Bozoglu et al., 2004), which are not able to repair in a stressful environment. Analysis of the behavior of cell counts after treatment indicates that the cheese matrix did not allow recovery and growth of injured cells. The main contributing factor to this effect seems to be low pH, which is mainly caused by the conversion of lactose in lactic acid and by the presence of the starter culture. When pH is low, most microbes become more susceptible to HHP inactivation and recovery of sublethally injured cells is reduced. Some authors suggest that HHP could restrict the pH range that bacteria can tolerate, therefore reducing recovery of sublethally injured cells. Factors that have been proposed to explain this effect are the inhibition of ATPase-dependent transfer of protons and cations, ATPase denaturation, or the dislocation of bound ATPase in the membrane (Wouters et al., 1998; Pagán et el., 2001). In preliminary research (De Lamo-Castellví et al., 2005), we made washed-curd model cheeses inoculated with 3 strains of Y. enterocolitica (serotypes O:1, O:3, and O:8) and starter culture, and treated them with HHP at 300, 400, and 500 MPa for 10 min at 20 ° C. None of the Y. enterocolitica strains inoculated in samples treated at 400 and 500 MPa showed repair ability after 15 d of storage at 8 ° C, and their counts remained below the level of detection [1 log10 (cfu/g)]. Serotype O:1 treated at 300 MPa was not able to repair, and serotypes O:3 and O:8 treated at 300 MPa showed a decrease in their cell counts during storage.
Moreover, the decreasing trend detected in Salmonella cell counts of control samples indicates that both strains are affected by low pH or starter culture, and that this effect is more intense for S. typhimurium.
Molina-Höppner (2002) reported that HHP treatment of Lactococcus lactis spp. cremoris MG1363 inoculated in milk buffer initially affected metabolic activity and subsequently damaged membrane integrity. After treatment at 300 MPa for 5 min, metabolic activity was 10 to 12% of the activity of untreated microorganisms; after 12 min of treatment, the cells did not show any metabolic activity. During the treatment at 300 MPa, cell death was closely followed by the loss of metabolic activity, but cultures retained about 25% of metabolic activity, even after a 3 log10 (cfu/mL) reduction in cell count. Nevertheless, the addition of a high concentration of salts and solutes showed a protective effect during HHP treatment. Moreover, Molina-Höppner (2002) found that L. lactis cell counts on GM17 supplemented with 3% of NaCl were generally lower than cell counts on GM17, indicating formation of a sublethally injured population during the application of HHP treatments.
We conclude that HHP treatments can be useful to accelerate the reduction of initial levels of Salmonella in cheese, but it is necessary to combine this technology with the low pH and the presence of the starter culture to inhibit recovery and growth and to increase the death rate of the injured population.
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ACKNOWLEDGEMENTS
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This study was supported financially by CAL-00-005-C2-1 project (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria) and by the Departament d’Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya, which provided S. De Lamo-Castellvía fellowship to carry out this investigation. We would like to thank the Spanish Type Culture Collection for providing S. enteritidis and S. typhimurium strains.
Received for publication June 8, 2006.
Accepted for publication August 7, 2006.
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ABSTRACT
INTRODUCTION
= TALm control (inoculated with Salmonella but not treated by HHP); 
= SC control; • = TALm 300 MPa; 
= SC 300 MPa; 
= TALm 400 MPa and SC 400 MPa. The presented data are the mean values of 3 experiments ± SE; detection level = 1 log10 (cfu/g).
= Blank (noninoculated with Salmonella);