Inactivation kinetics of a piezotolerant Staphylococcus aureus isolated from high-pressure-treated sliced ham by high pressure in buffer and in a ham model system: Evaluation in selective and non-selective medium

Available online at www.sciencedirect.com

Innovative Food Science and Emerging Technologies 8 (2007) 478 – 484 www.elsevier.com/locate/ifset

Inactivation kinetics of a piezotolerant Staphylococcus aureus isolated from high-pressure-treated sliced ham by high pressure in buffer and in a ham model system: Evaluation in selective and non-selective medium Chrysoula C. Tassou ⁎, Polymnia Galiatsatou, Fotis J. Samaras, Constantinos G. Mallidis National Agricultural Research Foundation, Institute of Technology of Agricultural Products, 1 Sof. Venizelou str., GR-141 23 Lykovrissi Attiki, Greece Received 22 February 2007; accepted 6 April 2007

Abstract The kinetics of inactivation by high pressure of a pressure-resistant strain of Staphylococcus aureus isolated from pressure-treated packaged sliced ham, in buffer and in a ham model system was studied. Selective (BP agar) and enrichment media (BHI agar) were used for enumeration in order to count healthy and sublethally injured cells of the pathogen. A first-order kinetic inactivation was observed in both suspension media, and a very significant increase in D values was apparent when the microorganism was suspended and pressurized in the model food system compared to buffer. In the case of phosphate buffer as suspension medium, the zp values obtained were 107.5 and 113.6 MPa for the two recovery media, i.e. BP and BHI agars, respectively. In contrast, in the case of the food model system, a two-phase linear relation was apparent and the PDT (Pressure Death Time) curve can be divided into two linear sections, so that two zp values could be defined, one for each section. Zp values of 100 and 79.4 MPa correspond to pressures b 500 MPa for the BP and BHI counts, respectively, while zp values of 416.7 and 333.3 MPa correspond to higher pressures N 500 MPa for the selective and non-selective medium, respectively. When S. aureus had been pressurized in phosphate buffer, the BHI agar was slightly better in cell recovery, while in the case of the ham model system, the BP agar proved superior and gave significantly higher colony counts. © 2007 Elsevier Ltd. All rights reserved. Keywords: High pressure; Staphylococcus aureus; Ham; Meat products; Inactivation; Sublethal injury; Selective media Industrial relevance: The paper provides significant information for the food processing industry as it deals with the effect of high-pressure technology on a piezotolerant pathogen that may survive in sliced ham. This technology is already applied in ham products and this paper supports the need for the use of real food in pressure studies in order to avoid underestimation of the effect and hence the processing times. It is also shown that different recovery media, i.e. selective and non-selective, should be used to avoid underestimation of the surviving cells.

1. Introduction High hydrostatic pressure (HHP), as an alternative nonthermal technology for processing of foods, is an interesting option to meet today's consumer requirements because it can achieve increased shelf life and stability whilst preserving natural taste, texture, colour and vitamin content (Knorr, 1993; Matser, Krebbers, van de Berg, & Bartels, 2004; Smelt, 1998). Although the idea of pressure-treated foods is over 100 years old, it is only in the last 25 years that it has become a commercial reality. Nowadays, HHP is being successfully ⁎ Corresponding author. Tel.: +30 210 2845940; fax: +30 210 2840740. E-mail address: [email protected] (C.C. Tassou). 1466-8564/$ - see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ifset.2007.04.002

applied to a variety of products including fruit juices, purées, sauces, desserts, rice dishes, oysters and sliced cooked and ready-to-eat meat products. Most applications are for pasteurization of foods that are distributed chilled. Sliced vacuum-packaged cooked ham is a highly perishable product due to its composition, pH and water activity and the lack of background flora competing with spoilage or pathogenic microorganisms (Hugas, Garriga, & Monfort, 2002). Its shelf life (usually 4 weeks) depends on the hygienic characteristics of the final product after post-processing as well as on the packaging methods where cross-contamination is more likely to occur. There are studies proving that HHP technology can successfully find application in sliced cooked ham and other

C.C. Tassou et al. / Innovative Food Science and Emerging Technologies 8 (2007) 478–484

delicatessen meat products sold in flexible pouches. It has been shown that high-pressure processing can cause a significant delay in the growth of spoilage microorganisms, avoiding the potential for off-flavors and gas to be produced (Garriga, Grebol, Aymerich, Monfort, & Hugas, 2004; Morales, Calzada, & Nunez, 2006; Taoukis, Katsaros, Galiatsatou, & Mallidis, 2003). Accordingly it can contribute to the shelf life extension and maintenance of sensory properties and organoleptic freshness for at least 60 days after treatment, under chilled storage (Patterson, 2005; Serra et al., 2007). However, HHP processing conditions necessary to diminish spoilage microorganisms should be sufficient to destroy pathogens, which can grow in the product, otherwise they should become the target microorganisms. This is the case in our study where a Staphylococcus aureus isolated from sliced ham in pouches after being subjected to a pressure of 450 MPa for 5 min – a pressure able to inactivate most spoilage microorganisms of ham such as lactic acid bacteria (Taoukis et al., 2003). S. aureus is a pathogen capable of producing enterotoxins responsible for staphylococcal food poisoning, one of the most prevalent causes of gastroenteritis worldwide. There is information about the occurrence of pathogens in cooked meat products like Listeria monocytogenes, Salmonella spp, Escherichia coli, Campylobacter spp. and S. aureus due mainly to post contamination during slicing and packaging (Grau & Vanderlinden, 1992; Jofre et al., 2005; Sheridan, Duffy, McDowell, & Blair, 1994; Uyttendaele, Troy, & Debevere, 1999; Wong, Carey-Smith, Hollis, & Hudson, 2005). S. aureus is referred to as one of the most resistant non-sporulated Gram-positive bacteria to HHP treatment (Alpas et al., 1999; Patterson, Quinn, Simpson, & Gilmour, 1995; Shigehisa, Ohmori, Saito, Taji, & Hayashi, 1991; Wuytack, Diels, & Michiels, 2002). Investigating the resistance of pathogens is important in recovering stressed cells, which, under some conditions, can grow and cause problems. It is known that selective media allow for differentiation and enumeration of specific target microorganisms, but these media also contain agents which may inhibit repair of sublethally injured cells (Wuytack et al., 2002; Yuste, Capellas, Fung, & Mor-Mur, 2004). Sublethally injured cells are therefore not detected on selective media and this increases the risk of overestimating the efficacy of the pressure treatment. In the present study, we investigated the kinetics of inactivation by high pressure of a pressure-resistant strain of S. aureus isolated from pressurized packaged sliced ham, in buffer and in a ham model system. Selective and enrichment media were used for enumeration in order to count healthy and sublethally injured cells of the pathogen. 2. Materials and methods 2.1. Cultures and preparation of inocula S. aureus, isolated from packed sliced ham after being subjected to a pressure of 450 MPa for 5 min, was used as test microorganism. Stock culture was kept in vials of treated beads

479

in a cryoprotective fluid (Protect bacterial preservers, Technical Service, Lancashire, UK) at − 80 °C until use. The microorganism was revived by inoculation in 100 ml BHI broth (1.10493, Merck, Darmstadt, Germany) followed by incubation at 37 °C for 24 h. The culture was kept at 4 °C and renewed to ensure viability. For experiments, culture was grown in 100 ml BHI broth for 18–20 h at 37 °C. The cells were harvested by centrifugation at 7000 rpm for 10 min, washed twice with sterile phosphate buffer, re-centrifuged and finally suspended in appropriate medium (sterile phosphate buffer pH 6.8 or model food system) to give a final concentration of about 1 × 108 cells/ ml. The model food system was a well-homogenized mixture of ham and water (4.4:1 or 52.36 g ham + 12 ml H2O). 2.2. High-pressure equipment The high-pressure system (Resato International B.V., Roden, The Netherlands) consists of: (i) A high-pressure intensifier unit responsible for the building up of pressure in the system. An electric motor drives a hydraulic pump. The oil of the hydraulic pump is used to propel the oil-driven double-acting intensifier, which is a hydraulically driven reciprocating pump. In the intensifier, the pressure of the pressure fluid (Resato International B.V.; high-pressure fluid – glycol emulsion) is transformed up to 1000 MPa. The pressure is adjustable in steps of approximately 25 MPa. (ii) High-pressure vessels. The system consists of a block of six small (42 ml) high-pressure (HP) vessels and one large vessel (1.5 l). The dimensions of the vessels are 2.5 cm in diameter and 10 cm in length for the small ones and 7 cm in diameter and 40 cm in length for the large one. The vessels are closed using a unique Resato thread connection on the top of the vessel. The pressure is transmitted from the intensifier to vessels by the pressure fluid through high-pressure stainless steel tubing. Air-operated high-pressure needle valves are used for the control of circulation of pressure fluid, so it is possible for the vessels to work in an independent way. Each vessel is provided with a cooling/heating jacket giving the ability to set the initial experimental temperature in a range from − 40 to + 100 °C. Temperature transmitters are mounted in each vessel in order to monitor the temperature. Two pressure transducers are used in order to control and measure the pressure in the system. 2.3. High-pressure tests An aliquot of 3 ml of the cell suspension was transferred to polyethylene bags (2 × 8 cm). The bags were heat-sealed taking care to expel most of the air in the bag. This bag was placed in a second slightly bigger bag in order to prevent accidental leakage of cell suspension and contamination of the pressurizing liquid. Two plastic bags were placed in each of the six small vessels. The vessels were filled up with pressurizing liquid and their caps were screwed tightly. In order to study the kinetics of cell death under the same experimental conditions with only one variable, i.e. time, the desired pressure was applied and at predetermined time intervals, different for each experiment, the pressure was released from each vessel so a replication of data

480

C.C. Tassou et al. / Innovative Food Science and Emerging Technologies 8 (2007) 478–484

Fig. 1. Inactivation kinetics of S. aureus by HHP in phosphate buffer (pH 6.7) with enumeration in Baird Parker (BP) agar (♦: 350 MPa, ■: 400 MPa, ▴: 450 MPa, ×: 475 MPa, –: 500 MPa, ●: 550 MPa).

for each time-pressure set were obtained. Each experiment was repeated twice with the same sets of pressure and time. The initial temperature of the vessel jacket was adjusted to 23 °C by circulating a water–glycerol solution from a water bath. The pressure in the system and the temperature in each vessel were monitored and recorded through the PLC system. The rate of pressure increase was about 100 MPa per 7 s and pressure release time was less than 3 s. Pressurization time, in this study, did not include the pressure increase and release times. 2.4. Enumeration of survivors 0.1 ml of at least three subsequent dilutions from the processed aliquot were transferred and spread-plated on to the non selective Brain Heart Infusion agar (BHI, 1.13825, Merck, Darmstadt, Germany) and the selective Baird Parker agar (BP, 1.05406, Merck, Darmstadt, Germany) for the growth of S. aureus. Colonies were counted initially after 24 h while all the plates were then re-incubated for 2 days more at 37 °C thus allowing damaged cells to form colonies. 2.5. Estimation of kinetic parameters

shown in Figs. 1 and 2. Fig. 1 presents the number of survivors on the selective medium (BP agar) and Fig. 2 on the nonselective (BHI agar). An apparent first-order kinetic behavior was observed and a first-order kinetic model was used to estimate the rate of destruction in these experiments. Higher pressures were found to be more effective in the destruction of microbial cells as shown by the increased steepness of survivor curves. The pressure death time (PDT) curves for both recovery media are presented in Fig. 3. The D and zp values of all tests are shown in Table 1. The pressure survivor curves for S. aureus at pressures ranging from 450 to 660 MPa in ham model system and enumerated on BP and BHI agars are shown in Figs. 4 and 5, respectively. Although higher pressures were used, the number of survivors was also higher compared to the case of buffer. A very significant increase in D values, as a result of the suspension of microorganisms in the model food system, was observed (Table 1). The same first order kinetics of destruction was applied in this case. The assumption that HHP inactivation of S. aureus follows the log-linear model is supported also by other authors (Butz & Ludwig, 1986; Gervilla, Sendra, Ferragut, & Guamis, 1999; O'Reilly, O'Connor, Kelly, Beresford, & Murphy, 2000). First-order inactivation kinetics by HHP have been proposed also for other vegetative bacteria in a number of food matrices, like Listeria innocua in dairy cream (Raffali et al., 1994), E. coli, L. innocua and Salmonella enteritidis in liquid whole egg (Ponce, Pla, Capellas, Guamis, & Mor-Mur, 1998; Ponce, Pla, Mor-Mur, Gervilla, & Guamis, 1998), E. coli in saline (Smelt & Rijke, 1992) or in carrot juice (Van Opstal, Vanmuysen, Wuytack, Masschalck, & Michiels, 2004) and L. monocytogenes in raw milk (Mussa, Ramaswamy, & Smith, 1999). Analysis of variance (Table 2) showed that pressure and type of substrate as well as their interaction were highly significant on D values of S. aureus. Moreover, the outputs of the kinetic characteristics of the Baranyi model for all cases examined are presented in Table 3. The experimental data fitted well with the model to generate survival curves as judged by the small standard error (S.E.) of fit and high correlation coefficients (r2). Inactivation rates presented in Table 3 regarding the suspension

The pressure survivor curves were plotted as log10(N/N0) against time. The D (decimal reduction time) values were estimated as the negative reciprocal of the slope of the survivor curves. From the pressure death time (PDT) curve, the zp value (e.g. the pressure increase required to accomplish an l-log cycle reduction in the D value), can be obtained as the negative reciprocal of the slope. The Excel ad-in programme DMFit (IFR, Norwich, UK) was used and the Baranyi's primary predictive model (Baranyi & Roberts, 1994; Baranyi, Roberts, & McClure, 1993) was fitted to the ratio log10(N/N0) for the estimation of the following kinetic parameters: the death rate constant k (log10(N/ N0) min− 1), the standard error of fit and the r2. 3. Results and discussion The pressure survivor curves at pressures ranging from 350 to 550 MPa for S. aureus in phosphate buffer (pH 7.0) are

Fig. 2. Inactivation kinetics of S. aureus by HHP in phosphate buffer (pH 6.7) with enumeration in brain heart infusion (BHI) agar (♦: 350 MPa, ■: 400 MPa, ▴: 450 MPa, ×: 475 MPa, –: 500 MPa, ●: 550 MPa).

C.C. Tassou et al. / Innovative Food Science and Emerging Technologies 8 (2007) 478–484

Fig. 3. Log D values of inactivation of S. aureus by HHP at the pressure range of 350–550 MPa in phosphate buffer after enumeration in BHI agar (■) or in BP agar (▴).

medium (buffer or ham model system) give similar assumptions to the ones elaborated from D values: the fact that resistance of the pathogen increases dramatically from buffer to food model system. Generally, the pressure resistance of microorganisms can be affected by many intrinsic and environmental parameters, with very important the nature of the suspension medium. Their ability to survive high pressures, can be greatly increased when they are treated in nutritionally rich media, containing substances that may provide protection against damage or nutrients essential for repair (Hoover, Metrick, Papineau, Farkas, & Knorr, 1989; Simpson & Gilmour, 1997). Cheftel and Culioli (1997), reported that resistance of microorganisms to pressure is generally higher in foods than in buffer solutions and increases with decreasing water activity. Recently, Panagou, Tassou, Manitsa, and Mallidis (in press) have demonstrated the protective effect of the food matrix (fish) compared to phosphate buffer on the resistance of a fish spoilage microorganism at pressures lower than 550 MPa, Table 1 Decimal reduction times (min) of S. aureus at the pressure range of 350– 660 MPa suspended in phosphate buffer or in a ham model system (ham/water, 4.4:1) and enumerated in BP agar or in BHI agar Pressure (MPa)

350 400 450 460 475 480 500 550 600 660 zp

481

Fig. 4. Inactivation kinetics of S. aureus by HHP in ham model system (ham/ water, 4.4:1) with enumeration in Baird Parker (BP) agar (▴: 450 MPa, ●: 460 MPa, ■: 480 MPa, ♦: 500 MPa, ▵: 550 MPa, ×: 600 MPa, ○: 660 MPa).

which was overcome by higher pressures of 600 and 650 MPa. Patterson (2005) in a review paper reports that certain food constituents such as proteins, carbohydrates and lipids can have a protective effect. This is evident in this study where ham model system rich in proteins and lipids has a baroprotective effect on S. aureus. Hugas et al. (2002) have also reported 1.12– 3.46 log units less inactivation of various lactic acid bacteria and Salmonella carnosus in cooked ham homogenized with water (3:1) than in phosphate buffer after treatment at 500 MPa/ 40 °C for 10 min. There are studies dealing with the inactivation of S. aureus either in buffer or in food systems after HHP treatment (Gervilla et al., 1999; O'Reilly et al., 2000; Patterson & Kilpatrick, 1998; Takahashi, 1992). However, in most of them, the pressure resistance of S. aureus has not been elaborated at combinations of different pressures and times which can lead to kinetic data (D and zp values). Usually the pathogen has been subjected in one set of pressure/time and the reductions in log10 counts are given. For comparison reasons of the present work's results to the previously published data, the log10 reductions given by other researchers has been expressed arbitrary as approximate D values using the equation of the classic log-linear survival model: log(Nt) = log(N0) − (1/D)t (Table 4). Indeed, the significant increase in pressure resistance of the pathogen in model food system compared to buffer solution observed in this work

D values (min) Phosphate buffer

Ham model system

BP

BP

BHI

26.4 16.0

18.6 11.9

13.8 7.3 5.6 3.8 3.1 (P b 500 MPa) 100 (P N 500 MPa) 416.7

7.4 4.1 2.2 1.7 1.3 (P b 500 MPa) 79.4 (P N 500 MPa) 333.3

BHI

20.2 11.6 6.7

21.1 17.3 7.0

2.7

2.5

1.6 0.5

1.6 0.6

107.5

113.6

Fig. 5. Inactivation kinetics of S. aureus by HHP in ham model system (ham/ water, 4.4:1) with enumeration in brain heart infusion (BHI) agar (▴: 450 MPa, ●: 460 MPa, ■: 480 MPa, ♦: 500 MPa, ▵: 550 MPa, ×: 600 MPa, ○: 660 MPa).

482

C.C. Tassou et al. / Innovative Food Science and Emerging Technologies 8 (2007) 478–484

Table 2 Analysis of variance of the effect of pressure and type of substrate on D values of S. aureus

Corrected model Intercept Pressure Type of substrate Pressure × Substrate Error Total Corrected total a

Type III sum of squares

df Mean square

F

Significance

1496.142 a 1169.010 836.686 347.320 312.136 95.258 2760.410 1591.400

5 299.228 56.543 0.000 1 1169.010 220.898 0.000 2 418.343 79.051 0.000 1 347.320 65.630 0.000 2 156.068 29.491 0.000 18 5.292 24 23

Table 4 Decimal reduction times a (min) of S. aureus at pressure range 300–600 MPa suspended in phosphate buffer, peptone water or in different food systems Substrate

Pressures 300

R2 = 0.940.

is in agreement with the results of other studies as shown in Table 4. Specifically, the D and z parameters estimated in the present study are similar to those recorded by other researchers for S. aureus either in buffer solution or in model food systems and real food (Table 4), with a few exceptions like those of Gao, Wang, and Jiang (2005) and Erkmen and Karatas (1997) who have reported much lower resistance for S. aureus. It is worth mentioned that despite the different strains used in various studies, the similarity of results verifies the validity of kinetic data obtained in this work. The pressure death time (PDT) curves from which the zp values could be obtained are shown in Figs. 3 and 6. In the case of phosphate buffer as suspension medium a linear relation is observed and the zp values obtained are 107.5 and 113.6 MPa

Buffer Phosphate buffer Phosphate buffer Phosphate buffer Phosphate buffer Peptone water

Reference

350

400

450

500 550 600

21.1 17.3

7.0

1.6

0.6

Present work

10

Alpas et al., 2000 1.5

Yuste et al., 2004 3.5

21

Food Ham slurry Poultry slurry Pork slurry Pork marengo Cheese slurry Milk Milk

1.2

Milk

2.5

Kalchayanand, 1998

26.4

38

33

Ogihara et al., 1998

7.3

5.6

3.8

Present work

5

Patterson et al., 1995

1.5

Shigehisa et al., 1991

20

Moerman, 2005

20

O'Reilly et al., 2000 7.5

Patterson et al., 1995 Erkmen & Karatas, 1997 Gao et al., 2005

a

Approximate D values calculated from log reduction data given by the corresponding references. Table 3 Outputs of the Baranyi model for the HHP inactivation of S. aureus at the pressure range of 350–660 MPa suspended in phosphate buffer or in a ham model system (ham/water, 4.4:1) and enumerated in BP agar or in BHI agar Pressure (MPa)

350 400 450 460 475 480 500 550 600 660

Phosphate buffer

Ham model system

BP

BP 2

k (log10N/ N0 min− 1)

S.E. of fit

r

−0.045 −0.087 −0.143

0.446 0.254 0.355

0.933 0.983 0.962

−0.416

0.217

0.988

−0.616 −2.502

0.295 0.351

0.968 0.944

BHI 350 400 450 460 475 480 500 550 600 660

k (log10N/ N0 day− 1)

S.E. of fit

r2

− 0.033 − 0.052

0.233 0.296

0.932 0.921

− 0.066 − 0.131 − 0.182 − 0.325 − 0.288

0.169 0.258 0.169 0.182 0.373

0.965 0.921 0.957 0.991 0.916

− 0.057 − 0.078

0.295 0.274

0.933 0.948

− 0.114 − 0.236 − 0.495 − 0.748 − 0.762

0.401 0.226 0.451 0.111 0.404

0.898 0.978 0.911 0.995 0.911

for the two recovery media, i.e. BP and BHI agars, respectively. On the contrary, in the case of the food model system, a twophase linear relation is apparent (Fig. 6). PDT curve can be divided in two linear sections, so that two zp values could be defined, one for each section. Zp values of 100 and 79.4 MPa

BHI

−0.049 −0.048 −0.130

0.519 0.307 0.333

0.946 0.962 0.961

−0.324

0.524

0.934

−0.713 −1.953

0.097 0.310

0.996 0.947

Fig. 6. Log D values of inactivation of S. aureus by HHP at the pressure range of 450–660 MPa in ham model system (ham/water, 4.4:1) after enumeration in BHI agar (■) or in BP agar (▴).

C.C. Tassou et al. / Innovative Food Science and Emerging Technologies 8 (2007) 478–484

correspond to pressures b 500 MPa for the BP and BHI counts, respectively, while zp values of 416.7 and 333.3 MPa correspond to higher pressures N 500 MPa for the selective and non-selective medium, respectively. Data on zp values are very limited and vary among different microorganisms and media. For instance, Zook, Parish, Braddock, and Balaban (1999) has reported zp values of 115–121 MPa for the ascospores of Saccharomyces cerevisiae in orange and apple juices. Mallidis, Galiatsatou, Taoukis, and Tassou (2003) reported zp values of 71–83 MPa for Lactobacillus brevis and 77–100 MPa for Lactobacillus plantarum when suspended in buffers of different pH. Mussa et al. (1999) observed zp values of 240–260 MPa for L. monocytogenes in milk, while O'Reilly et al. (2000) reported zp value of 350 MPa for S. aureus suspended in a cheese slurry. The zp values for S. aureus when suspended in phosphate buffer estimated in this study are similar to the above mentioned. Additionally, the extremely high zp values observed in the model food system for pressures N500 (420 MPa) are in agreement with those given by others for S. aureus and L. monocytogenes (Mussa et al., 1999; O'Reilly et al., 2000). Using different recovery media it is possible to reveal on some of them the presence of a population of stressed cells. Generally, it has been assumed that selective media result in a lower recovery rate than the non-selective ones (Bozoglou, Alpas, & Kaletunc, 2004; Bull, Hayman, Stewart, Szabo, & Knabel, 2005; Sherry, Patterson, & Madden, 2004). In the present study, two different recovery media were used, i.e., BP and BHI agars. When S. aureus pressurized in a phosphate buffer and only for pressures up to 450 MPa, slightly higher D values were observed for the BHI counts, which means higher number of survivors indicating that a small number of sublethally injured cells was recovered better on this medium (Table 1; Fig. 3). This observation can be attributed to the selective agents (lithium chloride and tellurite) present in BP agar that do not allow the damaged cells to recover. The differences in counts between the two recovery media, when buffer was used as suspension medium, are minimizing by increasing the pressure more than 450 MPa, thus assuming complete inactivation of cells and no existence of sublethally damaged subpopulation. On the contrary, when S. aureus was pressurized suspended in the ham model system, the D values and therefore the number of survivors were substantially higher on the selective BP agar in the whole range of pressures applied (Table 1; Fig. 6). Probably the selective stimulatory action of glycine and pyruvate present in BP agar on S. aureus has overcome the destructive effect of HHP, considering also that less detrimental effects have been occurred in the cell membranes in the ham model system. Indeed, Park, Sohn, Shin, and Lee (2001) have stated that, in a solid matrix such as ham, the microbial cells are more protected against pressure due either to lower water activity or to protein content. The fat and salt content of ham also may play a baroprotective effect (Garcia-Graells, Masschalck, & Michiels, 1999; Oxen & Knorr, 1993; Palou, Lopez-Malo, Barbosa-Canovas, Welti-Chanes, & Swanson, 1997; Styles, Hoover, & Farkas, 1991) preserving the membrane integrity (Molina-Hoppner, Doster, Vogel, & Ganzle, 2004).

483

Our observations show that the relation between the food matrix and/or the food ingredients on resistance of microorganisms towards pressure treatment is very complex. As low water activity protects the cells more from high-pressure lethal effect because the pressure is not transferred freely in the solid matrix (Park et al., 2001), the increased resistance from S. aureus in real ham is expected. According to our results in the ham model system, the pressure has to be increased by almost 420 MPa in order to reduce the D value by 1 log10. In practice, it means that for processing pressures above 600 MPa, a holding time of more than 20 min has to be used, in order to achieve a 6D inactivation. In conclusion, our results support the need to use real food in pressure studies in order to avoid underestimation of the effect and hence the processing times. It has also been shown that different recovery media, i.e. selective and nonselective, should be used to avoid underestimation of the surviving cells, as in our study the selective medium proved to be superior in cell recovery. Acknowledgements The present study was financially supported by the projects PAVE 2000 (400BE) and EPAN – Competitiveness 2003 (TR24). Technical work by Mrs. G. Charvourou is greatly appreciated. References Alpas, H., Kalchayanand, N., Bozoglu, F., & Ray, B. (2000). Interactions of high hydrostatic pressure, pressurization temperature and pH on death and injury of pressure-resistant and pressure-sensitive strains of foodborne pathogens. International Journal of Food Microbiology, 60, 33−42. Alpas, H., Kalchayanand, N., Bozoglu, F., Sikes, A., Dunne, C. P., & Ray, B. (1999). Variation in resistance to hydrostatic pressure among strains of foodborne pathogens. Applied and Environmental Microbiology, 65, 4248−4251. Baranyi, J., & Roberts, T. A. (1994). A dynamic approach to predicting bacterial growth in food. International Journal of Food Microbiology, 23, 277−294. Baranyi, J., Roberts, T. A., & McClure, P. (1993). A non-autonomous differential equation to model bacterial growth. Food Microbiology, 10, 43−59. Bozoglou, F., Alpas, H., & Kaletunc, 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. Butz, P., & Ludwig, H. (1986). Pressure inactivation of microorganisms at moderate temperatures. Physica B+C, 139–140, 875−877. Cheftel, J. C., & Culioli, J. (1997). Effects of high pressure on meat: A review. Meat Science, 46(3), 211−236. Erkmen, O., & Karatas, S. (1997). Effect of high hydrostaitic pressure on Staphylococcus aureus in milk. Journal of Food Engineering, 33, 257−262. Gao, Y. L., Wang, Y. X., & Jiang, H. H. (2005). Effect of high pressure and mild heat on Staphylococcus aureus in milk using response surface methodology. Process Biochemistry, 40, 1849−1854. Garcia-Graells, C., Masschalck, B., & Michiels, C. W. (1999). Inactivation of Escherichia coli in milk by high hydrostatic pressure treatment in combination with antimicrobial peptides. Journal of Food Protection, 62, 1248−1254.

484

C.C. Tassou et al. / Innovative Food Science and Emerging Technologies 8 (2007) 478–484

Garriga, M., Grebol, N., Aymerich, M. T., Monfort, J. M., & Hugas, M. (2004). Microbial inactivation after high pressure processing at 600 MPa in commercial meat products over its shelf life. Innovative Food Science & Emerging Technologies, 5, 451−457. Gervilla, R., Sendra, E., Ferragut, V., & Guamis, B. (1999). Sensitivity of Staphylococcus aureus and Lactobacillus helveticus in ovine milk subjected to high hydrostatic pressure. Journal of Dairy Science, 82, 1099−1107. Grau, F. H., & Vanderlinden, P. B. (1992). Occurrence, numbers and growth of Listeria monocytogenes on some vacuum-packaged processed meats. Journal of Food Protection, 55, 4−7. Hoover, D. G., Metrick, C., Papineau, A. M., Farkas, D. F., & Knorr, D. (1989). Biological effects of high hydrostatic pressure on food microorganisms. Food Technology, 43, 99−107. Hugas, M., Garriga, M., & Monfort, J. M. (2002). New mild technologies in meat processing: High pressure as a model technology. Meat Science, 62, 359−371. Jofre, A., Martin, B., Carriga, M., Hugas, M., Pla, M., Rodriguez-Lazaro, D., et al. (2005). Simultaneous detection of Listeria monocytogenes and Salmonella by multiplex PCR in cooked ham. Food Microbiology, 22(1), 109−115. Kalchayanand, N., Sikes, A., Dunne, C. P., & Ray, B. (1998). Factors influencing death and injury of foodborne pathogens by hydrostatic pressure-pasteurization. Food Microbiology, 15, 207−214. Knorr, D. (1993). Effects of high-hydrostatic pressure processes on food safety and quality. Journal of Food Protection, 59, 146−150. Mallidis, C. G., Galiatsatou, P., Taoukis, P., & Tassou, C. C. (2003). Highpressure destruction kinetics of L. plantarum and L. brevis isolated from traditional Greek salads. Journal of the Science of Food and Agriculture, 38, 579−585. Matser, A. M., Krebbers, B., van de Berg, R. W., & Bartels, P. V. (2004). Advantages of high pressure sterilization of food products. Trends in Food Science and Technology, 15, 79−85. Moerman, F. (2005). High hydrostatic pressure inactivation of vegetative microorganisms, aerobic and anaerobic spores in pork Marengo, a low acidic particulate food product. Meat Science, 69, 225−232. Molina-Hoppner, A., Doster, W., Vogel, R. F., & Ganzle, M. G. (2004). Protective effect of sucrose and sodium chloride for Lactococcus lactis during sublethal and lethal high-pressure treatments. Applied and Environmental Microbiology, 70(4), 2013−2020. Morales, P., Calzada, J., & Nunez, M. (2006). Effect of high-pressure treatment on the survival of Listeria monocytogenes Scott A in sliced vacuumpackaged Iberian and Serano hams. Journal of Food Protection, 69(10), 2539−2543. Mussa, D. M., Ramaswamy, H. S., & Smith, J. P. (1999). High pressure (HP) destruction kinetics of Listeria monocytogenes Scott A in raw milk. Food Research International, 31, 343−350. Ogihara, H., Hitomi, T., & Yano, N. (1998). Inactivation of some foodborne pathogens and indicator bacteria by high hydrostatic pressure. Journal of Food Hygiene Society of Japan, 39(6), 436−439. O'Reilly, C. E., O'Connor, P. M., Kelly, A. L., Beresford, T. P., & Murphy, P. M. (2000). Use of hydrostatic pressure for inactivation of microbial contaminants in cheese. Applied and Environmental Microbiology, 66(11), 4890−4896. Oxen, P., & Knorr, D. (1993). Baroprotective effects of high solute concentrations against inactivation of Rhodotorula rubra. LebensmittelWissenschaft+Technologie, 26, 220−223. Palou, E., Lopez-Malo, A., Barbosa-Canovas, G. V., Welti-Chanes, J., & Swanson, B. G. (1997). High hydrostatic pressure as a hurdle for Zygosaccharomyces bailii inactivation. Journal of Food Science, 62(4), 855−857. Panagou, E. Z., Tassou, C. C., Manitsa, C., & Mallidis, G. (in press). Modelling the effect of high pressure on the inactivation kinetics of a pressure-resistant strain of Pediococcus damnosus in phosphate buffer and gilt-head seabream (Sparus aurata). Journal of Applied Microbiology. doi:10.1111/j.13652672.2006.03201.x Park, S. W., Sohn, K. H., Shin, J. H., & Lee, H. J. (2001). High hydrostatic pressure inactivation of Lactobacillus viridescens and its effects on ultrastructure of cells. International Journal of Food Science & Technology, 36, 775−781. Patterson, M. F. (2005). Microbiology of pressure treated foods. Journal of Applied Microbiology, 98(6), 1400−1409.

Patterson, M. F., & Kilpatrick, D. J. (1998). The combined effect of high hydrostatic pressure and mild heat on inactivation of pathogens in milk and poultry. Journal of Food Protection, 61, 432−436. 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(5), 524−529. Ponce, E., Pla, R., Capellas, M., Guamis, B., & Mor-Mur, M. (1998). Inactivation of Escherichia coli inoculated in liquid whole egg by high hydrostatic pressure. Food Microbiology, 15, 265−272. Ponce, E., Pla, R., Mor-Mur, M., Gervilla, R., & Guamis, B. (1998). Inactivation of Listeria innocua inoculated in liquid whole egg by high hydrostatic pressure. Food Microbiology, 61, 119−122. Raffali, J., Rosec, J. P., Carlez, A., Dumay, E., Richard, N., & Cheftel, J. C. (1994). High pressure stress and inactivation of Listeria innocua in inoculated dairy cream. Science Alimentaries, 14, 349−358. Serra, X., Grebol, N., Guardia, M. D., Guerrero, L., Gou, P., Masoliver, P., et al. (2007). High pressure applied to frozen ham at different process stages: 2. Effect on the sensory attributes and on colour characteristics of dry-cured ham. Meat Science, 75, 21−28. Sheridan, J. J., Duffy, G., McDowell, D. A., & Blair, I. S. (1994). The occurrence and initial numbers of Listeria in Irish meat and fish products and the recovery of injured cells from frozen products. International Journal of Food Microbiology, 22, 105−113. Sherry, A. E., Patterson, M. F., & Madden, R. H. (2004). Comparison of 40 Salmonella enterica serovars injured by thermal, high-pressure and irradiation stress. Journal of Applied Microbiology, 96, 887−893. Shigehisa, T., Ohmori, T., Saito, A., Taji, S., & Hayashi, R. (1991). Effect of high pressure on characteristics of pork slurries and inactivation of microorganisms associated with meat and meat products. International Journal of Food Microbiology, 12, 207−216. Simpson, R. K., & Gilmour, A. (1997). The effect of high hydrostatic pressure on Listeria monocytogenes in phosphate-buffered saline and model food systems. Journal of Applied Microbiology, 83, 181−188. Smelt, J. P. P. M. (1998). Recent advances in the microbiology of high pressure processing. Trends in Food Science & Technology, 9, 152−158. Smelt, J. P. P. M., & Rijke, G. G. F. (1992). High pressure treatment as a tool for pasteurization of foods. In C. Balny, R. Hayashi, K. Heremans, & P. Masson (Eds.), High pressure and biotechnology, Vol. 224 (pp. 361−364). London: INSERM/John Libbey Eurotext. Styles, M. F., Hoover, D. G., & Farkas, D. F. (1991). Response of Listeria monocytogenes and Vibrio parahaemolyticus to high hydrostatic pressure. Journal of Food Science, 56, 1404−1407. Takahashi, K. (1992). Sterilization of microorganisms by high hydrostatic pressure at low temperature. High Pressure Biotechnology, 224, 303−307. Taoukis, P., Katsaros, G., Galiatsatou, P., & Mallidis, K. (2003). Shelf life study of high hydrostatic pressure processed packed ham. IFT Ann. Meeting, Chicago, USA. Uyttendaele, M., Troy, P. D., & Debevere, J. (1999). Incidence of Listeria monocytogenes in different types of meat products on the Belgian retail market. International Journal of Food Microbiology, 53, 75−80. Van Opstal, I., Vanmuysen, S. C. M., Wuytack, E. Y., Masschalck, B., & Michiels, C. W. (2004). Inactivation of Escherichia coli by high hydrostatic pressure at different temperatures in buffer and carrot juice. International Journal of Food Microbiology, 98(2), 179−191. Wong, T. L., Carey-Smith, G. V., Hollis, L., & Hudson, J. A. (2005). Microbiological survey of prepackaged pate and ham in New Zealand. Letters in Applied Microbiology, 41, 106−111. Wuytack, E. Y., Diels, A. M. J., & Michiels, C. W. (2002). Bacterial inactivation by high-pressure homogenisation and high hydrostatic pressure. International Journal of Food Microbiology, 77, 205−212. Yuste, J., Capellas, M., Fung, D. Y. C., & Mor-Mur, M. (2004). Inactivation and sublethal injury of foodborne pathogens by high pressure processing: Evaluation with conventional media and thin agar layer method. Food Research International, 37(9), 861−866. Zook, C. D., Parish, M. E., Braddock, R. J., & Balaban, M. O. (1999). High pressure inactivation kinetics of Saccharomyces cerevisiae ascospores in orange and apple juices. Journal of Food Science, 64(3), 533−535.