Modeling the protective effect of aw and fat content on the high pressure resistance of Listeria monocytogenes in dry-cured ham

Food Research International 75 (2015) 194–199

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Modeling the protective effect of aw and fat content on the high pressure resistance of Listeria monocytogenes in dry-cured ham S. Bover-Cid ⁎, N. Belletti, T. Aymerich, M. Garriga IRTA, Food Safety Programme — Finca Camps i Armet, E-17121 Monells, Spain

a r t i c l e

i n f o

Article history: Received 24 February 2015 Received in revised form 19 May 2015 Accepted 27 May 2015 Available online 31 May 2015 Keywords: Inactivation High pressure processing Ready-to-eat meat products Piezoprotection Food safety

a b s t r a c t High pressure processing (HPP) is a promising food preservation technology as an alternative to thermal processing for microbial inactivation. The technological parameters, the type of microorganism, and the food composition can greatly affect the microbicidal potential of HPP against spoilage and pathogenic microorganisms. Presently, the number of available models quantifying the influence of food characteristics on the pathogen inactivation is scarce. The aim of this study was to model the inactivation of Listeria monocytogenes CTC1034 in drycured ham, as a function of pressure (347–852 MPa, 5 min/15 °C), water activity (aw, 0.86–0.96) and fat content (10–50%) according to a Central Composite Design. The response surface methodology, based on the equation obtained with a stepwise multivariate linear regression, was used to describe the relationship between bacterial inactivation and the studied variables. According to the best fitting polynomial equation, besides pressure intensity, both aw and fat content exerted a significant influence on HP-inactivation of L. monocytogenes. A clear linear piezoprotection trend was found lowering the aw of the substrate within the whole range of tested pressure. Fat content was included in the model through the quadratic term and as interaction term with pressure, resulting in a particular behavior. A protective effect due to the presence of high fat content was identified for pressure treatments above ca. 700 MPa. At lower pressure, higher inactivation of L. monocytogenes occurred by increasing the fat content above 30%. The results emphasize the relevant influence of intrinsic factors on the L. monocytogenes inactivation by HPP, making necessary to assess and validate the effectiveness of HPP on specific food products and consequently set process criteria adjusted to each particular food product. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Since 1980's Listeria monocytogenes emerged in many countries as one of the major safety concern for the food industry (Warriner & Namvar, 2009). Particularly, ready-to-eat (RTE) meat products have been identified as a potential vehicle for this pathogen. The risk of contamination by L. monocytogenes is particularly relevant during post-pasteurization/packaging operations, i.e. post-lethally exposure (Gilbert, Lake, Hudson, & Cressey, 2009). Among alternative food preservation technologies, high pressure processing (HPP) has attracted significant interest as a final decontamination step for RTE foods. As an inpackage cold pasteurization process, HPP can extend the shelf-life and enhance the microbiological safety providing an additional lethal treatment able to inactivate bacteria at ambient or refrigeration temperatures. Therefore, the effects that compromise food flavor, color, texture and appearance are minimized besides allowing a good retention of nutritional attributes of food (Garriga & Aymerich, 2009; Rastogi, Raghavarao, Balasubramaniam, Niranjan, & Knorr, 2007). HPP is internationally recognized by several organizations and institutions ⁎ Corresponding author at: IRTA — Finca Camps i Armet s/n, Monells E-17121 Spain. E-mail address: [email protected] (S. Bover-Cid).

http://dx.doi.org/10.1016/j.foodres.2015.05.052 0963-9969/© 2015 Elsevier Ltd. All rights reserved.

(CAC, 2007; HHS, 2008) and it is well accepted by the consumer (Olsen, Grunert, & Sonne, 2010). During the last decade, the HPP use in food processing has steadily increased, nowadays being commercially used in a high number of food processors in Europe, USA, Australia, Japan, etc. (Tonello, 2011). For RTE dry-cured meat products (such as Serrano ham, Parma ham, Iberian ham) HPP is particularly interesting for products indented to markets under the zero tolerance policy in relation to the presence of L. monocytogenes, e.g. USA and Japan (BoverCid, Belletti, Garriga, & Aymerich, 2011). However, the massive implementation of such emerging technology at an industrial level still requires a better understanding of the impact that different factors play in the degree of microbial inactivation, which should to be taken into account in the validation procedures of HPP treatments. In addition to the processing conditions (pressure, time and temperature of the treatment) additional factors can alter the performance of HPP in pasteurizing food products. For instance, cell piezoresistance is highly dependent on the type of microorganism, species and strain (Alpas et al., 1999). The complexity of foods, in terms of structure and composition can account for the piezoprotection reported in several studies. For instance, while acidity usually favor HPinactivation of microorganisms, the presence of proteins and sugars has been reported to act as protective agents on microbial cells (Black,

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Huppertz, Fitzgerald, & Kelly, 2007; Gao, Ju, & Wu, 2007; García-Graells, Masschalck, & Michiels, 1999). Similarly, high fat and/or low water activity (aw) values have been shown to decrease the antimicrobial effectiveness of HP treatments (Erkmen & Dogan, 2004b; Hayman, Kouassi, Anantheswaran, Floros, & Knabel, 2008; Hereu, Bover-Cid, Garriga, & Aymerich, 2012; Hereu, Dalgaard, Garriga, Aymerich, & Bover-Cid, 2012). A substantial number of studies dealing with the inactivation of microorganisms by HPP are available in the literature, though those applying a modeling approach are scarce and mainly developed in culture media and liquid foods (e.g. milk, juices) rather than in solid food matrixes (Serment-Moreno, Barbosa-Cánovas, Torres, & Welti-Chanes, 2014). The quantification of the influence of the food intrinsic factors with a piezoprotective effect on foodborne microorganisms like L. monocytogenes is presently one of the main gaps in the field of HPP (Georget et al., 2015; Rendueles et al., 2011). Therefore, there is the need for inactivation predictive models developed with a product oriented approach, especially when HPP is designed in the frame of hurdle technology. The present work was undertaken with the aim to model the HPP inactivation of L. monocytogenes in dry-cured ham as a function of two important intrinsic factors: aw and fat content, including pressure as the most important process parameter leading the lethality of the treatment. 2. Materials and methods 2.1. Bacterial strain and culture preparation L. monocytogenes CTC1034 was originally isolated from dry-cured ham. This strain was previously selected and used for the study of HPP inactivation for its higher resistance in comparison with other tested strains (Bover-Cid et al., 2011). To prepare the inoculum culture, 100 μl of a stock culture (stored in 20% glycerol at −80 °C) were transferred to 10 ml Brain Heart Infusion (BHI, from DB, NJ, USA) broth and incubated for 7 h at 37 °C. One hundred μl were transferred to a second tube of 10 ml BHI and incubated overnight for 18 h at 37 °C, resulting in an early stationary phase culture. An appropriate volume of this overnight culture was added to different aliquots of sterile water used for sample preparation (Section 2.2) in order to obtain a level of inoculum of about 106–107 cfu/g in dry-cured ham. 2.2. Experimental design, sample preparation and high pressure processing A Central Composite Design (CCD) involving three variables (aw, fat content and pressure) and five levels was followed, the experimental layout of which is shown in Table 1 (NIST/SEMATECH). Dry-cured hams were obtained directly from the producer and aseptically deboned in the laboratory. To adjust the aw and fat content in accordance with the CCD trials, different batches of dry-cured ham samples were prepared as follows. The lean section was aseptically Table 1 Selected factors (variables) and their values, corresponding to each level used in the Central Composite Design (CCD) of 3 factors and 5 levels. Levelsa

−1.68 −1 0 1 1.68

Factors aw

Fat (%)

Pressure (MPa)

0.86 0.88 0.92 0.94 0.96

10.00 18.18 30.18 42.18 50.36

347 450 600 750 852

a Levels were set for a circumscribed Central Composite Design to maintain rotability and orthogonality; i.e. for 3 factors, the scaled value for α relative to ±1 is 22/3 = 1.68 (NIST/SEMATECH).

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separated from the fat section of the deboned dry-cured ham. Each section was separately minced under aseptic conditions. For each trial of the CCD, the appropriate volume of the water containing the L. monocytogenes strain (see Section 2.1) was added to different portions of minced lean dry-cured ham (aw = 0.85; 6.73% fat) and mixed until homogenization to attain the target aw (i.e. in the range of 0.860– 0.960). Then, to reach the target concentration of fat (10%–50%), an appropriate quantity of minced fat was added to each portion of inoculated lean dry-cured ham. To ensure the homogeneous distribution when mixing two components of different size, the geometrical dilution approach was followed. The actual aw of the aliquots was verified by measuring it with an Aqualab™ equipment (Series 3, Decagon Devices Inc., Pullman, WA, USA). The aliquots with the desired aw, fat content (according to the CCD design shown in Table 1) and containing the inoculated L. monocytogenes strain at (ca. 107 cfu/g) were divided in ca. 15 g samples, each one vacuum packaged in plastic bags PET/PE (with oxygen permeability b50 cm3/m2/24 h and water vapor permeability b15 mg/m2/24 h; Sacoliva S.L., Barcelona, Spain). For each combination of conditions of the CCD, the actual concentration of L. monocytogenes in the packaged samples was measured in duplicate (Section 2.3) to determine the initial inoculum level (N0). Packaged samples were submitted to HPP according to the CCD, i.e. in the range of 347–852 MPa (for 5 min at an initial fluid temperature of 15 °C). The Wave 6000 Hiperbaric (Burgos, Spain) and the Thiot ingenierie — Hiperbaric (Bretenoux, France — Burgos, Spain) equipments were used for pressures up to and above 600 MPa, respectively. Pressure come up rate was on average 220 MPa/min. Pressure release was almost immediate. Samples for each combination of factors were prepared and sampled at least in duplicate to measure the L. monocytogenes concentration after the HPP (N). 2.3. Microbiological determinations Each dry-cured ham sample was homogenized (1/10 dilution) with tryptic soy broth with 0.6% yeast extract, TSBYE (DB, NJ, USA) in a Masticator Classic (IUL S.A., Barcelona, Spain). The homogenate was serially diluted in 0.1% Bacto Peptone (Difco Laboratories, Detroit, MI, USA) with 0.85% NaCl (Merck, Darmstadt, Germany) and plated onto the selective media Chromogenic Listeria Agar (CLA; Oxoid Basingstoke, UK), incubated at 37 °C for 48 h. For expected counts below the quantification limit (b4 cfu/g), the presence or absence of L. monocytogenes was determined after the TSBYE homogenate enrichment (at 37 °C for 48 h). The enriched homogenate was streaked onto a CLA plate; when necessary, colonies were confirmed by PCR (Aymerich, Jofré, Garriga, & Hugas, 2005). For modeling purposes, positive results below the quantification limit were recorded as 0 Log cfu/g, while absence in 15 g was computed as −1.18 Log cfu/g. The inactivation of L. monocytogenes was registered in terms of logarithmic reductions as Log (N / N0), i.e. difference between counts after the treatments (N) and the initial inoculum (N0). 2.4. Mathematical modeling The response surface methodology (RSM) was the empirical procedure used to study the relationship between the selected variables (aw, fat content and pressure) and the inactivation of L. monocytogenes in dry-cured ham. To uniform the order of magnitude of the experimental variables, their values were rescaled (i.e. aw ∗ 10, fat content / 10, pressure / 100) before the statistical modeling. The statistical package Statistica for Windows (v.8, Statsoft Inc., Tulsa, OK, USA) was used. The backward stepwise linear regression was the procedure chosen to generate the second order polynomial equation that best fitted our experimental inactivation data. The statistical significance and the goodness of fit of the model containing only statistical significant terms (p b 0.05) was evaluated using the determination coefficients (R2adj), the significance p-values derived from the F-test. Response surfaces were drawn by keeping constant at the central value of the

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experimental design the independent variables not shown in the graphs (i.e. fat at 30.18% and aw at 0.92). 3. Results

out a clear effect of the fat content on the L. monocytogenes inactivation, as it seems to depend on the pressure level applied. In dry-cured ham samples with 30.18% of fat, aw 0.92 and pressurized at 600 MPa (i.e. constituting the central points of the CCD, trial 9 to 14), the inactivation of L. monocytogenes was 4.5 Log units on average.

3.1. L. monocytogenes HP-inactivation 3.2. Modeling of inactivation results Table 2 shows the HP-inactivation of L. monocytogenes CTC1034 in the dry-cured ham, expressed as logarithmic reduction (Log N / N0) of viable cells, achieved for each one of the 20 combinations of aw, fat content and pressure tested in accordance with the CCD. The measured aw values are reported in brackets and were used in the mathematical modeling. All the experiments were conducted at constant holding time of 5 min and initial fluid temperature of 15 °C. All the trials conducted with pressure levels up to 450 MPa, resulted in a relative low to moderate microbial inactivation (runs 1, 3, 5, 7, and 19), with Log reductions varying from 0.92 to 3.92 depending on the fat content and aw value. The lethality of the process against L. monocytogenes CTC1034 increased with the rise of the pressure applied. However, the pressure alone did not guarantee the success of the pressurization in the range of the conditions tested. In fact, despite a pressure treatment of 600 MPa, a relatively low microbial inactivation (−2.24 Logs) was registered for the lowest aw value tested (0.857, trial 15). The complete inactivation of L. monocytogenes (absence in the 15 gsample), was achieved only in two conditions of the experimental design: in run 20 corresponding to the highest level of pressure (852 MPa) used, and in run 6 combining a quite high pressure level (750 MPa) with moderate aw (0.948). A high inactivation of L. monocytogenes of about 7 Logs was registered also in run 16, corresponding to the highest aw (0.961) tested; though some surviving cells (presence in 15 g) were detected after 48 h of sample enrichment. As expected, the aw of dry-cured ham affected the efficacy of the HPP in reducing L. monocytogenes counts, as evidenced by the comparison of the results obtained in trials performed at the same pressure and drycured ham samples with equal fat content and different aw (e.g. trials 15 and 16). An inactivation slightly exceeding 6 Logs was registered in dry-cured ham formulated with the lowest (10%, trial 17) and highest (50%, trial 18) fat content. However, from raw data it is difficult to find

Table 2 Central Composite Design (CCD) arrangement and results obtained in each trial about logarithmic reductions of Listeria monocytogenes CTC1034 in dry-cured ham after high hydrostatic processing. Trial

awa

Fat (%)

Pressure (MPa)

Inactivation log (N / N0)b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

0.880 (0.883) 0.880 (0.883) 0.880 (0.890) 0.880 (0.890) 0.940 (0.945) 0.940 (0.948) 0.940 (0.939) 0.940 (0.939) 0.920 (0.919) 0.920 (0.919) 0.920 (0.915) 0.920 (0.915) 0.920 (0.922) 0.920 (0.922) 0.860 (0.857) 0.960 (0.961) 0.920 (0.920) 0.920 (0.911) 0.920 (0.919) 0.920 (0.922)

18.18 18.18 42.18 42.18 18.18 18.18 42.18 42.18 30.18 30.18 30.18 30.18 30.18 30.18 30.18 30.18 10.00 50.36 30.18 30.18

450 750 450 750 450 750 450 750 600 600 600 600 600 600 600 600 600 600 347 852

−0.92 −5.26 −1.05 −5.75 −1.16 −7.96 −3.92 −6.07 −4.38 −4.12 −4.88 −4.64 −4.18 −4.71 −2.24 −6.82 −6.58 −6.28 −0.99 −7.04

a The column of aw reports target theoretical values according to the Central Composite Design; the actual measured values are reported in parenthesis, which were used for the model development. b Mean of the duplicate sample analysis.

To quantify the relationship between the HP-inactivation of L. monocytogenes, expressed as Log survivor data, and the studied factors (aw, fat content and pressure), a multivariate regression analysis was carried out. To obtain the equation best fitting to our data without compromising the parsimony, a backward stepwise regression method was applied, thus only the statistically significant (p b 0.05) terms deriving from each studied factor was retained in the final equation. The statistical approach used resulted in the quadratic polynomial expression reported as Eq. (1), in which only the significant terms are included. LogðN=N0 Þ ¼ 38:653−34:29  aw −0:0237  P−0:00349  F 2 þ 0:000334  P  F

ð1Þ

where Log (N / N0) represents the logarithmic reduction of L. monocytogenes CTC1034; aw, the measured water activity values of dry-cured ham; F, the fat content (%) of the dry-cured ham; and P, the pressure level (MPa) of the HPP (for 5 min at 15 °C). All the three factors considered in this study (aw, fat, pressure) significantly contributed to the HPP inactivation of L. monocytogenes CTC1034. The influence of pressure and aw was described by the corresponding linear terms. Fat content was statistically significant; it is present as second order term, and also as interactive factor with pressure. According to the ANOVA, the polynomial model obtained had an adjusted R2 of 0.842 for the dependent variable, indicating a satisfactory degree of correlation between the experimental inactivation data and the fitted ones; the significance of the model was high, as expressed by the F-value (26.40) and the associated low probability (p b 0.0001). 3.3. Response surface plots The response surface plots presented in Fig. 1 provide an overview of the influence of all the three independent variables on the inactivation of L. monocytogenes by HPP. Those surfaces were drawn on the basis of the empirical equation obtained (Eq. (1)); the two-way interactions between factors on L. monocytogenes inactivation are plotted keeping the variable not represented at the central value of the CCD (i.e. aw at 0.92, fat content at 30.18%). In Fig. 1A the inactivation is described as a function of pressure and aw. The increase of the L. monocytogenes HPinactivation was linked to the increase of both the pressure treatment and aw values of the dry-cured ham. At the range tested, the increase of pressure provided a more marked contribution, with respect to aw, as shown by the steepest slope of pressure effects. This fact is also supported by the higher standardized coefficient of the regression for pressure (beta = − 1.39) in comparison with that of aw (beta = − 0.40). The reduction of aw of samples exerted a protective effect on L. monocytogenes CTC1034 during the HPP, which followed a linear trend, irrespective of the pressure level applied. In Eq. (1), the fat content (%) appears as second order term accounting for the curvature of the surface reported in Fig. 1B. According to our results, the role of fat as piezoprotective agent seems to depend on the pressure level, owing to the interactive term present in the model (Eq. (1)). As a consequence, the influence of fat content on the lethality of HPP against L. monocytogenes is more complicated to explain with respect to aw. In the range of pressure treatments above ca. 700 MPa, a slight increase of the inactivation extent of L. monocytogenes CTC1034 was registered for decreasing levels of fat (below 30%). However, in the same pressure interval, increasing the fat content above 30% had

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B

Pressure (MPa)

aw

0 -1 -2 -3 -4 -5 -6 -7 -8 -9 -10

10

350 400 450 500 550 600 650 700 750 800 850

Inactivation (Log N/N0)

1 0 -1 -2 -3 -4 -5 -6 -7 -8 -9

350 400 450 500 550 600 650 700 750 800 850 0.86 0.87 0.88 0.89 0.90 0.91 0.92 0.93 0.94 0.95 0.96

Inactivation (Log N/N0)

A

197

Pressure (MPa)

15 20 25 30 35 40

Fat (%)

45 50

Fig. 1. Response surface plots describing the effect of aw and pressure (A) as well as fat content and pressure (B) on the inactivation of L. monocytogenes CTC1034 in the dry-cured ham according to the developed model (see Eq. (1)). In each plot, the factor not included is kept at the central value of the Central Composite Design, i.e. fat content at 30.18% (A), water activity at 0.92 (B).

practically no relevant effect. When pressure values below 650 MPa were applied, an increasing inactivation of L. monocytogenes was registered by increasing fat contents above 30%, and this increase was even more evident at the lowest pressure. 4. Discussion 4.1. L. monocytogenes inactivation Besides other factors, the compositional characteristics of the pressurized food have been proven to affect the degree of HPP inactivation of spoilage and pathogenic microorganism. The food system used in this work was designed with controlled aw and fat content with the aim to elucidate and quantify their possible protective role on L. monocytogenes behavior facing pressure treatments of different intensities. The experimental immediate inactivation results revealed that the pressure resistance of L. monocytogenes CTC1034 in the dry-cured ham was significantly higher than that usually reported in laboratory media or food products, including other RTE (cooked) meat products (Considine, Kelly, Fitzgerald, Hill, & Sleator, 2008; García-Graells et al., 1999; Gervilla, Ferragut, & Guamis, 2000; Hereu, Dalgaard, et al., 2012; Patterson, 2005; Smelt & Hellemons, 1998). The inactivation of L. monocytogenes in liquid laboratory media treated at 600 MPa for 3–10 min was reported to be of at least 7 Log units, as demonstrated in phosphate buffer (pH 7) and citrate buffer (pH 5.7) (Ritz et al., 2000). A similar inactivation extent (above 7 Logs) was reported for whole milk (Chen, 2007) and orange juice (pH 3.6) (Erkmen & Dogan, 2004a) treated at 600 MPa for 1-6 min and 4.5 min, respectively. In solid foods in general, the inactivation efficacy is reported to be lower than in liquid media. For instance, a treatment at 600 MPa for 5 min caused more than 5 Logs inactivation of the strain L. monocytogenes CTC1034 inoculated in cooked ham (aw ≥ 0.98) (Hereu, Bover-Cid, et al., 2012). A decrease of the process lethality against microorganisms was expected when lowering the aw of dry-cured ham. In fact, a difference of more than 4 Logs of inactivation was registered between trials 15 (− 2.24 Logs) and 16 (− 6.82 Logs) characterized by the lowest and highest aw respectively. Similarly to run 15, an average of −2.9 Log inactivation for L. monocytogenes CTC1034 (the same strain used in the present work) inoculated on sliced Iberian dry-cured ham (aw = 0.88; 33.3% fat) treated at 600 MPa was previously reported (Hereu, BoverCid, et al., 2012). An extreme demonstration of the protective effect of low aw values occurred with the pressurization of lyophilized cells of

L. monocytogenes, for which no inactivation at all was observed (Hayman et al., 2008). The piezoprotection given by low aw can be related to the stabilization of proteins (particularly enzymes), reducing its pressure-induced denaturation in biological systems (Georget et al., 2015). However, in the light of the application of HPP to real food matrices, it is important to highlight that this undesirable protective effect will be compensated by the inhibition of the recovery and potential growth of the cells during storage time (Hereu, Bover-Cid, et al., 2012; Jofré, Aymerich, Grèbol, & Garriga, 2009). So far, the influence of fat content on the HP-inactivation of bacteria has received less attention and the available published results are controversial. Based on the results obtained in the present work, a certain relationship with pressure can be hypothesized: the level of fat affects the lethality of the process depending on the intensity of the pressurization. In particular, piezoprotection occurred at the highest pressure intensities (above 650 MPa), while at lower pressure the higher fat content the higher inactivation was recorded. At 500 MPa a progressive protection against L. innocua inactivation was recorded in ovine milk with increasing fat contents from 0% to 50% (Gervilla et al., 2000), though the particular mechanisms have not been established. In meat products, the differential inactivation extent observed by mesophilic microorganisms in dry sausages (fatty product) and dry-cured beef (lean product) submitted to HPP (500 MPa for 5 min at 18 °C) was hypothesized to be due to the protective effect of the higher fat content present in dry sausages (Rubio, Martínez, García-Cachán, Rovira, & Jaime, 2007a,b). However, no clear relationship between the type fatty acid composition of the meat product and the effectiveness of highpressure treatment was observed (Rubio et al., 2007a). Conversely, no significant influence of fat on the HP-inactivation of L. monocytogenes and Salmonella in minced chicken treated at 400 MPa for 2 min was observed (Escriu & Mor-Mur, 2009). It is known that fat experiences higher compression heating (up to 8 °C/100 MPa) than aqueous media (about 3 °C/ 100 MPa) (Balasubramaniam, Martínez-Monteagudo, & Gupta, 2015). Consequently, a slight thermal inactivation effect synergistically with the non-thermal HP lethal effects could also be hypothesized at high fat contents and/or high pressure levels (Rasanayagam et al., 2003). On the basis of the results reviewed, it appears that the effect of fat is not simple and may be related to several influencing factors not yet elucidated, including its interaction with the other components of the product. 4.2. Model application The modeling approach can offer interesting benefits and provide reliable empirical equations for the quantification of the influence of

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key parameters on the fate of pathogenic microorganisms. These empirical equations can be used as a support tool for food operators in the management and optimization of the processes/product design as well as the validation of the combination of factors that ensures the microbiological safety and/or the quality of the product (CAC, 2008; FSIS, 2012). However, for a reliable application of mathematical models, they should be better developed with a product-oriented approach in order to implicitly take into account the specific characteristics of the commodity studied (Georget et al., 2015). Available studies for the quantification of the role of intrinsic parameters on the HPP inactivation of L. monocytogenes generally include experiments in controlled conditions but usually lack of the productoriented approach, failing to include food matrix complexity. The present study used a dry-cured ham and developed an empirical equation for the quantification of the role of the product aw and its fat content on the HP-inactivation of L. monocytogenes under specific and controlled conditions. As food composition can enhance or minimize the lethality of HPP, it is important to assess in specific food products whether the HPP achieves the required Performance Criteria (PC) and assure the product safety according to the established Food Safety Objectives (FSO). Some general and specific PC has been suggested for L. monocytogenes in RTE food products. A reduction of the number of viable cells of L. monocytogenes of six orders (Logs units) of magnitude is recommended by the U.S. administration for refrigerated RTE products in general

(HHS, 2008). According to the U.S. Food Safety Inspection Service guidelines for controlling L. monocytogenes in post-lethally exposed RTE meat products, a post-lethally treatment achieving at least 5 Logs inactivation would be suitable for reprocessing contaminated products (FSIS, 2012). The safety criteria of the Spanish Agency of Food Safety for RTE meat products was set at 4 Log reductions (AESAN, 2005). In products like dry-cured ham, according to the results presented in this work, both aw and fat are relevant factors affecting the efficacy of HPP. With the purpose of highlighting the significant influence of food characteristics on the accomplishment of this safety standard, the two dimensional isoreduction plots were drawn on the basis of the developed model (Eq. (1)). Two types of RTE dry-cured ham were considered, characterized by a considerable difference in aw and fat content: (a) Serrano-type ham (Jamón Serrano), which can show an aw of 0.92 and a fat content around 15%, and (b) Iberian-type ham (Paleta Iberica), with higher fat content (33%) and lower value of aw (0.88). Physicochemical characteristics of both types dry-cured ham are representative of commercial products as reported in a previous study (Hereu, BoverCid, et al., 2012). The contour plots representing the isoreduction diagrams were drawn and are presented in Fig. 2 for the two different types of drycured ham. Each line corresponds to the combination of factors (i.e. aw and pressure, fat and pressure) yielding the same inactivation extent. Thus, it is possible to locate the combination of HPP conditions providing each target isoreduction.

Serrano ham (a w 0.92 & 14% fat)

Iberian ham (aw 0.88 & 33% fat)

0.96

0.96

A

0.94

0.94

0.93

0.93

0.92

0.92

0.91

0.91

0.90

0.90

0.89

0.89

0.88

0.88

0.87

0.87

0.86 350

400

450

500

550

600

650

700

750

800

B

0.95

aw

aw

0.95

0.86 350

850

400

450

500

Pressure (MPa)

550

600

650

700

750

C

D

45

45

40

40

Fat content (%)

Fat content (%)

850

50

50

35 30

35 30

25

25

20

20

15

15

10 350

800

Pressure (MPa)

400

450

500

550

600

650

Pressure (MPa)

700

750

800

850

10 350

400

450

500

550

600

650

700

750

800

850

Pressure (MPa)

Fig. 2. Contour plots describing the Listeria monocytogenes isoreduction lines (from −1 to −7 Log units) achieved by HPP as a function of the pressure intensity as well as the combination of aw value and fat content of dry-cured ham. In each plot, the factor not included is kept to the value of the type of dry-cured ham considered in the example: Serrano ham (0.92 aw and 14% fat) and Iberian ham (0.88 aw and 33% fat).

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To achieve a PC of 6 Logs reduction, at least 655 MPa and 850 MPa (for 5 min at 15 °C), would be necessary for Serrano and Iberian ham, respectively (Fig. 2). According to the prediction of the model, the PC of 4 Log reduction will be achieved by processing Serrano ham at 550 MPa and Iberian ham at 685 MPa. Nowadays, industrial HPP equipments achieve maximum working pressures of 600 MPa (Serment-Moreno et al., 2014; Tonello, 2011), not allowing the attainment of the process criteria needed to accomplish with the above mentioned PC. Nevertheless, taking into consideration that L. monocytogenes is not able to grow in RTE dry-cured ham, no increase in the health hazard can be foreseen during product storage. Therefore, a milder PC could be tolerated for a listericidal treatment for dry-cured ham in order to meet the “zero tolerance” policy for L. monocytogenes. In this frame, a performance criterion of 2.4 Log reductions has been proposed as a reference (Hoz, Cambero, Cabeza, Herrero, & Ordóñez, 2008). According to the present study, a HP treatment up to 600 MPa would enable the achievement of 2.4 Logs inactivation, with intensity depending upon aw and, to a certain extent, also upon fat content. In particular, the application of our equation indicates that this target inactivation was achieved with pressures of 465 MPa for Serrano ham and 560 MPa for Iberian ham. 5. Conclusions The HP-inactivation of L. monocytogenes is strongly dependent on the physico-chemical characteristics of food matrix, which make necessary to assess and validate the effectiveness of HP on specific food products, in order to design pressure treatments achieving the required performance criteria. The product-oriented modeling approach presented here allowed the characterization of the piezoprotective effect of aw against HP treatments, which follows a linear trend irrespectively of the pressure level within the assessed range. The role of fat and its contribution as piezoprotective agent seems dependent upon the pressure level, though the mechanisms of its influence are still uncertain and would need further study. The developed model represents a useful tool in the design proper HPP of RTE dry-cured ham. Acknowledgments This work has been funded by the Spanish Ministerio de Ciencia e Innovación (INIA, ref. RTA2007-00032). Nicoletta Belletti acknowledges the fellowship of the “Subprograma DOC-INIA 2010”. References AESAN (2005). Opinión del Comité científico de la AESA sobre una cuestión presentada por la Dirección Ejecutiva, en relación con la aplicación de altas presiones en carne y productos cárnicos (Ref. AESA-2003-007). Revista del Comité Científico de la AESAN, 1, 36–71. Alpas, H., Kalchayanand, N., Bozoglu, F., Sikes, A., Dunne, C. P., & Ray, B. (1999). Variation in resistance to hydrostatic pressure among strains of food-borne pathogens. Applied and Environmental Microbiology, 65(9), 4248–4251. Aymerich, T., Jofré, A., Garriga, M., & Hugas, M. (2005). Inhibition of Listeria monocytogenes and Salmonella by natural antimicrobials and high hydrostatic pressure in sliced cooked ham. Journal of Food Protection, 68(1), 173–177. Balasubramaniam, V. M., Martínez-Monteagudo, S. I., & Gupta, R. (2015). Principles and application of high pressure-based technologies in the food industry. Annual Review of Food Science and Technology, 6, 435–462. Black, E. P., Huppertz, T., Fitzgerald, G. F., & Kelly, A. L. (2007). Baroprotection of vegetative bacteria by milk constituents: A study of Listeria innocua. International Dairy Journal, 17(2), 104–110. Bover-Cid, S., Belletti, N., Garriga, M., & Aymerich, T. (2011). Model for Listeria monocytogenes inactivation on dry-cured ham by high hydrostatic pressure processing. Food Microbiology, 28(4), 804–809. CAC (2007). Guidelines on the application of general principles of food hygiene to the control of Listeria monocytogenes in foods. CAC/GL 61–2007, 1–28. CAC (2008). Guidelines for the validation of food safety control measures. CAC/GL 69 — 2008, 1–28. Chen, H. (2007). Use of linear, Weibull, and log-logistic functions to model pressure inactivation of seven foodborne pathogens in milk. Food Microbiology, 24(3), 197–204. Considine, K. M., Kelly, A. L., Fitzgerald, G. F., Hill, C., & Sleator, R. D. (2008). High-pressure processing — effects on microbial food safety and food quality. FEMS Microbiology Letters, 281(1), 1–9.

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