High pressure carbon dioxide combined with high power ultrasound processing of dry cured ham spiked with Listeria monocytogenes

Food Research International 66 (2014) 264–273

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High pressure carbon dioxide combined with high power ultrasound processing of dry cured ham spiked with Listeria monocytogenes Spilimbergo Sara, Cappelletti Martina, Ferrentino Giovanna ⁎ Department of Industrial Engineering, University of Trento, via Sommarive 9, 38123 Povo, Trento, Italy

a r t i c l e

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Article history: Received 19 June 2014 Accepted 26 September 2014 Available online 5 October 2014 Keywords: Listeria monocytogenes Pasteurization Dry cured ham High pressure carbon dioxide High power ultrasound Quality

a b s t r a c t Traditionally, thermal treatments for the inactivation of Listeria monocytogenes in meat products involve undesirable changes of the product quality. In recent years, efforts have been carried out to develop alternative methods to inactivate L. monocytogenes without affecting the product quality attributes. In this context, the feasibility of combined high pressure carbon dioxide and high power ultrasound (HPCO2 + HPU) treatment to inactivate L. monocytogenes inoculated on the surface of dry cured ham was investigated. Inactivation data were determined at 6, 8 and 12 MPa, as a function of temperature (22, 35, 45 °C) and treatment time (0.5 to 30 min), and compared to those obtained after thermal and HPCO2 treatments. Color, pH and acidity changes of the samples after both thermal and HPCO2 + HPU treatments were measured and compared, sensorial profile of the treated samples was evaluated by a sensory panel and shelf-life was determined by a storage study for 4 weeks at 4 °C. The results clearly revealed that HPU alone was not able to induce any microbial inactivation while HPCO2 + HPU treatment always assured a certain level of inactivation, variable with the process temperature used: the inactivation efficiency was demonstrated higher at 35 °C rather than 22 °C and no enhancement was observed at 45 °C compared to 35 °C. Process conditions of 12 MPa, 35 °C, at 10 W for 5 min assured inactivation to undetectable level of L. monocytogenes spiked on the surface of the product with an initial concentration of about 109 CFU/g. No differences were detected between acidity, pH, color and sensory attributes of the untreated and HPCO2 + HPU treated dry cured ham surface, while slight differences were measured between the values obtained for the untreated and thermally treated samples. Additionally, the storage study demonstrated that a full microbial and quality shelf-life was assured for 4 weeks at 4 °C. The results obtained may open the doors to the application of such an innovative process at industrial level, in particular to treat ham-type or meat products. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Cured ham is one of the most popular “Ready-To-Eat” (RTE) foods around the world (Toldrá, 2004). Although its physical–chemical parameters do not let the growth of the bacteria, microbial contamination risks could occur during slicing and packaging operations of the product and Listeria monocytogenes is considered as the major pathogen affecting its safety. Difficult to eradicate from the production environment, it can survive during refrigeration and storage, causing severe infections in humans (McLauchlin, 1997), such as listeriosis, which is commonly regarded as an invasive disease (Franciosa, Tartaro, Wedell-Neergaard, & Aureli, 2001). Considering the severity and the high mortality rate, about 20% for listeriosis (European Food Safety Authority, 2014), the elimination or reduction of L. monocytogenes is a compulsory step before marketing the product. The USA (CFSAN/ FSIS, 2003) adopted a zero-tolerance policy for L. monocytogenes in RTE meat products where this bacterium can grow, which means a ⁎ Corresponding author. Tel.: +39 0461 282 485. E-mail address: [email protected] (F. Giovanna).

http://dx.doi.org/10.1016/j.foodres.2014.09.024 0963-9969/© 2014 Elsevier Ltd. All rights reserved.

Food Safety Objective (FSO) of 0.04 CFU/g at the retailer stage. The European Union established an FSO value of 100 CFU/g for these products, and if the manufacturer cannot demonstrate the achievement of this objective, the criterion “not detected in 25 g” is applied before the product leaves the production plant (Commission, 2005). It is well-known that traditional heat treatment (85 °C for 10 s for the inactivation of L. monocytogenes) involves undesirable changes of the quality characteristics of the product. In recent years, efforts have been carried out to develop alternative methods to eliminate, or at least reduce, the presence of L. monocytogenes or other pathogens in RTE foods, among others: chemical and physical methods as mixtures of organic or inorganic salts and organic acids (Shabala, Lee, Cannesson, & Ross, 2008); mixtures of essential oils (Turgis, Han, Bursa & Lacroix, 2008); combination of peroxyacetic acid/nisin and protective starter cultures (Minei, Gomes, Ratti, D'Angelis, & De Martinis, 2008), new preservation processes, i.e. high hydrostatic pressure (Gola, Frustoli, Rovere, & Miglioli, 2003) and ozone (Kim, Yousef, & Dave, 1999), innovative packaging methods as active antimicrobial food packaging (Quintavalla & Vicini, 2002); combination of modified atmosphere packaging or vacuum packaging and low storage temperature (Grisenti et al., 2004).

S. Sara et al. / Food Research International 66 (2014) 264–273

Nowadays, high hydrostatic pressure (HHP) is probably the most attractive new preservation technology: it has been already applied at industrial scale due to its promising potentials as listericidal treatment for dry cured ham products and to its capability to meet the consumer demand for high quality foods. Hugas, Garriga, and Monfort (2002) stated that from both physical– chemical and microbiological points of view, dry cured ham high pressure treated at 600 MPa for 6 min at 31 °C was substantially equivalent to the untreated products, and that the decontamination efficacy of HHP depended on many other factors such as treatment temperature, microbial strain, exposure time, pH, water activity and food composition. Rubio, Martìnez, Garcìa-Cachàn, Rovira, and Jaime (2007) demonstrated that HHP at 500 MPa for 5 min avoided the growth of enterobacteria, enterococci and Pseudomonas in sliced dry cured ham and delayed the growth of lactic acid bacteria, Micrococcaceae and yeasts and molds. Besides, no changes in physical–chemical and sensory parameters were found after the treatment and during a refrigerated storage. Hereu, Bover-Cid, Garriga, and Aymerich (2012) indicated that HHP, as post-processing listericidal treatment, was more effective (both immediately and long term) than the use of nisin. Biopreservation and natural antimicrobials have been also extensively used in the meat industry to increase flavor and extend shelflife of the products (Aymerich, Picouet, & Monfort, 2008). In Europe, their use is regulated by the European directive 95/2/CE (European Parliament and of the Council, 1995). In USA, they are recognized as antilisterial agents and the Federal Register (Food Safety and Inspection Service, U, 2000) regulates their use. It has been demonstrated that combined hurdles, i. e. biopreservation and HHP, provided a wider margin of safety in the control of L monocytogenes during the storage of RTE cured meat products (Hereu et al., 2012). Among the innovative non-thermal technologies, high pressure carbon dioxide (HPCO2) has been increasingly investigated as a technique able to induce the inactivation of not only the natural microbial flora but also pathogens occurring in solid and liquid matrices (Arreola, Balaban, Marshall, et al., 1991; Zhou, Wang, Hu, Wu, & Liao, 2009; Spilimbergo & Ciola, 2010; Ferrentino & Spilimbergo, 2011). CO2 is considered a GRAS (Generally Recognized as Safe) substance, which means that it can be used for food products. The critical temperature (31.1 °C) is compatible with the thermal stability of most materials, and the critical pressure (7.3 MPa) is easily reached in industrial processes (Spilimbergo, Komes, Vojvodic, Levaj, & Ferrentino, 2013). Theories explaining the inactivation mechanism of HPCO2 involve the diffusion and solubility of CO2 in the culture medium, the decrease of the medium pH, the increase of the membrane fluidity and permeability, the diffusion of CO2 into the cells, the cell membrane rupture caused by the increase of the internal pressure, and the resultant changes in the cellular environment, such as a decrease in pH, the inactivation of key enzymes, and the extraction of critical intracellular materials (Garcìa-Gonzalez, Geeraerd, Spilimbergo, et al., 2007; Pataro, Ferrentino, Ricciardi, & Ferrari, 2010). Ferrentino, Plaza, Ramirez-Rodrigues, Ferrari, and Balaban (2009) studied the effects of HPCO2 pasteurization on the physical and quality attributes of red grapefruit juice. A 5 Log(CFU/ml) reduction of yeasts and molds and total aerobic microorganisms occurred at 40 °C, 34.5 MPa and 7 min of treatment and during the storage study the HPCO2-treated juice showed no growth of these microorganisms. The experimental results evidenced that the treatment could maintain the physical and quality attributes of the product. Spilimbergo and Ciola (2010) investigated the microbial inactivation and quality parameters (pH, sugar content, acidity, turbidity) of peach and kiwi juices treated with HPCO2 at 35 °C and 10 MPa: inactivation to undetectable level of natural microbial flora occurred after 15 min of treatment for both juices. No significant changes in chemical–physical or sensorial characteristics between untreated and treated samples were detected. Cappelletti, Ferrentino, Endrizzi, et al. (2014) recently applied HPCO 2 treatment to the pasteurization of coconut water: 120 bar, 40 °C, and 30 min were the optimal process conditions to

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induce 5 Log(CFU/ml) reduction of mesophilic microorganisms, lactic acid bacteria, yeasts and molds and 7 Log(CFU/ml) reduction of total coliforms. No differences in chemical composition, vitamins and amino acids were detected between HPCO2 and untreated samples; however, differences in the volatile compounds of the three products were clearly distinguishable. Ferrentino, Balzan, and Spilimbergo (2012b) investigated the feasibility of HPCO2 treatment to inactivate L. monocytogenes inoculated on the surface of dry cured ham. Treatment at 50 °C, 12 MPa for 15 min resulted in inactivation to undetectable level of L. monocytogenes with an initial microbial load of 107 CFU/g. Less severe conditions (45 °C, 12 MPa, 5 min) were sufficient to reach inactivation to undetectable level if the initial microbial load was 103 CFU/g. The process slightly influenced the color and sensory attributes of the sample. Ferrentino, Balzan, Dorigato, Pegoretti, and Spilimbergo (2012) evaluated the effectiveness of HPCO2 as non-thermal technology for the pasteurization of fresh-cut coconut. A treatment of 15 min at 45 °C and 12 MPa induced 4 Log(CFU/g) reduction of mesophilic microorganisms, lactic acid bacteria, total coliforms and yeasts and molds. The hardness of coconut was not affected by the treatment, but the samples developed an irregular and disorderly microstructure. Although several experimental results demonstrated the efficiency of the process, long treatment times and temperatures were often needed to guarantee the safety and stability of some food products, limiting the efficiency of HPCO2 inactivation processes (Garcìa-Gonzalez, Geeraerd, Elst, et al., 2009; Liu, Hu, Zhao, & Song, 2012). Accordingly, the scientific interest towards combining HPCO2 with other lowtemperature techniques is increasing (Ortuño, Martínez-Pastor, Mulet, & Benedito, 2012a). High power ultrasound (HPU) at low frequencies (20 to 100 W) has the potential to be used for the inactivation of bacterial populations. Ultrasound is known to have a significant effect on the velocity of food industry processes involving heat and mass transfer. There are many potential applications in food processing, such as extraction, filtration, extrusion, freezing or crystallization (Piyasena, Mohareb, & McKellar, 2003). The application of ultrasound in food preservation processing is relatively recent. Cameron, McMaster, and Britz (2009) proved the effectiveness of the ultrasonication for the destruction of Escherichia coli, Pseudomonas fluorescens and L. monocytogenes with no detrimental effect on the total protein or casein content of pasteurized milk. It was demonstrated that high-intensity ultrasonic waves could cause cell rupture and enzyme denaturalization, although using only ultrasound the effects were not sufficiently severe to reduce the product microbial content (Butz & Tauscher, 2002). An effective microbial inactivation can be achieved by combining ultrasound with either heat, or pressure or both. The combination of heat or pressure and ultrasound increases the efficiency with respect to the treatment time and energy consumption, compared to each individual treatment (Chemat, Zill-e-Huma, & Khan, 2011). Ordonez, Sanz, Hernandez, and Lopez-Lorenzo (1984) investigated the combined destructive effect of ultrasonic waves and heating on microorganisms (thermosonication, TS), using as a model of two species of thermoduric streptococci: the combination of ultrasound of 20 kHz, 160 W with temperatures ranging from 5 to 62 °C was much more efficient with respect to treatment time and energy consumption compared to the single treatments. Raso, Palo, Pagan, and Condon (1998) studied the inactivation of Bacillus subtilis spores by ultrasonic treatments under static pressure (manosonication, MS) and combined pressure/heat (mano-thermosonication, MTS), revealing that the sporicidal effect of these processes depended on the static pressure, amplitude of ultrasonic waves and treatment temperature: a MS treatment at 500 kPa and 117 μm of amplitude for 12 min at 70 °C inactivated approximately 99% of the B. subtilis spore population. Furthermore, MS treatments at temperatures higher than 70 °C (MTS) led to a synergistic effect on spore inactivation. HPU inactivation mechanism concerns cavitation phenomena: the molecules of the liquid subjected to the ultrasonic waves vibrate and the vibration amplitude increases with the increase of the loaded

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power. Over a certain amplitude of vibration, the intermolecular forces are no more able to hold together the molecules, thus leading to the formation of bubbles, which during the ultrasound cycles, decrease and increase their size until reaching a critical size and collapsing. The pressure changes resulting from these implosions are the main bactericidal effect in ultrasound. The hot zones can kill some bacteria, but they are very localized and do not affect a large enough area (Chemat et al., 2011; Piyasena et al., 2003; Yun, Kedie, Shulai, Adshiri, & Arai, 1997). The main drawback of the HPU application is the transmission of the acoustic wave from the emitter's surface to the sample. The air, in fact, is a high attenuating medium that absorbs the acoustic energy preventing its transfer to the solids to be treated. In addition, the high impedance difference between the solid surface of the emitters and the air, and between the air and the solid samples, produces the reflection of a high proportion of the generated acoustic signal (García-Pérez, Cárcel, De la Fuente, & Riera, 2006). The use of a “dense fluid”, such as CO2, as a medium can overcome this problem, allowing the application of the technology to solid products. The simultaneous application of CO2 in supercritical phase and HPU has been shown to improve the extraction processes (Gao, Nagy, Liu, Simandi, & Wang, 2009; Riera, Blanco, García, et al., 2010) or to accelerate chemical reactions (Trofimov, Samsonov, Lee, Smart, & Wai, 2001). In the ultrasound assisted supercritical extraction processes, the ultrasound induced micro-stirring and solvent cavitation had some physical consequences, including cracked or damaged plant cell walls, increased solvent diffusion, interfacial turbulence and reduction of the external resistance to mass transfer (Gao et al., 2009). Just a pair of references has been found in the literature covering the simultaneous application of HPCO2 and HPU for microbial inactivation in liquid media. Ortuño, Martínez-Pastor, Mulet, and Benedito (2012b) demonstrated that when HPCO2 and HPU were coupled, the time needed to reach 8 Log(CFU/ml) reductions was shortened, on average by 95%, compared to HPCO2 alone. Cappelletti, Ferrentino, and Spilimbergo (2014) recently compared the effect of HPCO2 alone or in combination with HPU on both the natural microbial flora of coconut water and the pathogenic Gram-negative bacteria Salmonella enterica inoculated in the product. The synergistic effect of HPCO2 + HPU was evident: at 12 MPa and 40 °C about 5 Log(CFU/ml) reductions were achieved for the natural microbial flora in about 15 min, while about 30 min was needed for HPCO2 treatment. The storage study highlighted that HPCO2 treated coconut water resulted microbiologically unstable and showed heavy regrowth phenomena during the storage, while, a full shelf life of 4 weeks was assured for HPCO2+ HPU treated samples. With regard to the simultaneous application of HPCO2 and HPU for preservation of solid foods, no references have been found in the literature. The present work focused on the feasibility of combined HPCO2 + HPU treatment for the inactivation of L. monocytogenes spiked on dry cured ham surface, considering both microbial and quality aspects. Accordingly, our work studied the optimization of the process parameters (temperature, pressure, and time) to achieve inactivation to undetectable level of L. monocytogenes, thus obtaining a very complete comparison between thermal, HPCO2 and HPCO2 + HPU treatments. Secondly, the study investigated the changes of color, acidity and pH of dry cured ham, after both thermal and HPCO2 + HPU and the effect of these treatments on the appearance, texture, and aroma, by a sensory panel. Finally, in order to evaluate if the product was stable, from both microbial and quality points of view, a storage study at refrigerated conditions (4 °C) for 4 weeks was performed. 2. Materials and methods 2.1. Culture and cell suspension L. monocytogenes ATCC 19115 (DSMZ, Braunschweig, Germany) strain was used. The microbial culture was grown on solid Luria Bertani (LB) agar medium at 37 °C for 16 h. One colony was picked up and

inoculated into 10 ml of the medium. Bacterial culture was incubated at 37 °C with constant shaking (200 rpm) to stationary phase (16 h), with a final concentration of about 107 CFU/ml. Cells were collected by centrifugation at 6000 rpm for 10 min and then re-suspended in 5 ml of sterile phosphate buffered solution (PBS; 0.01 M, pH 7.4) to a final concentration of about 109 CFU/ml.

2.2. Sample preparation and inoculation Slices of dry cured ham surface were purchased from a local market, cut in 2 g with a rectangular shape (surface area of about 2 cm2) and spiked with 50 μl of L. monocytogenes suspension, obtaining a concentration of about 107 CFU/g. The samples were left 1 h in a sterile chamber at room temperature to let the microbial suspension absorb on dry cured ham, and subsequently treated with HPU, HPCO2, thermal treatment and combined HPCO2 + HPU treatments.

2.3. HPU combined HPCO2 apparatus The HPCO2 apparatus consisted in a sapphire high pressure visualization cell (Separex S.A.S., France) with an internal volume of 50 ml, designed to withstand up to 40 MPa and 100 °C. The plant includes a CO2 tank, kept at room temperature, a chiller reservoir, an HPLC pump, and a thermostatic bath to keep the inactivation vessel at the desired temperature. The cell is equipped with a safety device which consists of a rupture disc calibrated for operations up to 40 MPa, a thermocouple to measure the inside temperature of the cell and a manometer which shows the pressure inside the cell. The system was equipped with an ultrasound system (Aktive Arc Sarl, Switzerland) designed on purpose and embedded in the HPCO2 plant (Fig. 1). The HPU system consists of a transducer, a buster, a special retainer (M36 × 1.5), a sonotrode and a power generator unit (40 W and 30 kHz). For the experiments with HPU, the ultrasound unit was turned on (time zero) when the desired pressure and temperature were reached in the vessel. HPU conditions were chosen in order to preserve the quality attributes (color and texture) of the products. As the power output was found to be a strong function of the loaded pressure, the amplitude of the generator was modulated for each condition of pressure and temperature to produce an applied power of about 10 W ± 2 W. With this set up, it was verified that the temperature of the samples did not increase too much in 2 min of HPCO2 + HPU treatment. After constant intervals of 2 min, the sonotrode was switch off to let the samples cooled down and avoid the deleterious effect of the temperature. In other words, HPCO2 + HPU treatments were performed based on cycles of 2 min of HPCO2 + HPU and 2 min of HPCO2 alone, that were necessary to cool down the sample to the process temperature. Temperature profiles were evaluated for 2 min of HPCO2 + HPU and 2 min of HPCO2 at each pressure and temperature conditions tested. For the treatment performed at 12 MPa and 35 °C, HPCO2 + HPU started when the temperature inside the vessel reached the value of 35 °C. Switching on the sonotrode, a temperature profile was generated with an increase from 35 °C to a maximum value of 43 °C while the mean temperature, evaluated over the 2 min of treatment, was equal to 37.2 °C. Switching off the sonotrode, the sample cooled down and 2 min of HPCO2 was enough to reach again the initial temperature of 35 °C. During these cycles, the pressure was kept constant using the pump and the valve on the decompression line. After the treatments, the samples were collected in individual sterile tubes for microbial analyses. The vessel was cleaned and disinfected with ethanol (96% v/v) after each sampling. In order to investigate the synergistic effect of HPCO2 + HPU combined treatment in terms of lower treatment time/lower temperature or pressure assuring the same inactivation level, or a higher inactivation at the same operative conditions compared to HPCO2 alone, the following conditions were tested: 6, 8 and 12 MPa, at 22, 35 and 45 °C, for 0.5–30 min.

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Fig. 1. Combined HPCO2 + HPU apparatus.CO2: tank; V1, V2, V3: valves; PI: pressure manometers; TI: temperature probe; P1: volumetric pump; R1: sonotrode.

2.4. Thermal treatment Sample were put in plastic falcon tubes and placed into a preheated water bath. Experiments were performed changing the temperature from 80 to 90 °C and the treatment times from 1 to 10 min. The process conditions were chosen testing the resistance of L. monocytogenes to heat.

2.5. Microbial analysis The standard plate count method (ISO 6887-1, 1999; ISO 6887-2, 2003; ISO 7218, 2007) was used to determine the initial microbial load and the effectiveness of the treatments in reducing the number of L. monocytogenes spread on the surface of the sample (ISO 11290-2, 1998). After each treatment, dry cured ham samples were prepared and mixed with 4 ml of PBS in a sterile vial, which was stomached at 230 rpm for 2 min (Stomacher 400; International P.B.I., Milan, Italy). The homogenate was serially diluted in PBS and plated in duplicate onto the selective media O.A. Listeria agar (Ottaviani and Agosti, Liofilchem). The incubation temperature and time were 37 °C and 48 h. The plates were incubated for more than 48 h at 37 °C to assure the complete microbial death and to observe no recovery of injured cells at the process conditions inducing inactivation to undetectable level, The degree of inactivation was determined by evaluating the Log(N/N0) versus time, where N0 (in CFU/g) was the number of microorganisms initially present in the untreated sample and N (in CFU/g) was the number of survivors after the treatment. Three independent experiments were carried out for each experimental condition and the results were calculated as the mean value of 3 replications. Standard deviations were shown in the graphs by error bars.

2.6. Quality analyses An analysis of the quality attributes (pH, total acidity (TA), color and sensory) was performed on dry cured ham surface treated with thermal and HPCO2 + HPU combined treatment at the optimal process conditions in order to evaluate the features of the product treated with the new process and compare it with the thermal conventional one.

2.6.1. TA and pH determination The sample was homogenized with 2 ml of distilled water and the pH was measured using a digital pH meter (Eutech Instruments, Nijkerk, The Netherlands), after calibration with commercial buffer solutions at pH 7.0 and 4.0 (Crison, Vetrotecnica s.r.l., PD, Italy). The measurements were performed in triplicate, and mean values and standard deviations were evaluated. TA measurements were performed by titrating 2 ml of the homogenized sample with standardized NaOH (0.05 N) to the phenolphthalein end point (pH = 8.2 ± 0.1). The volume of NaOH was converted to grams of lactic acid per milliliters of the homogenized sample and TA calculated based on the following formula: Total acidityðlactic acid g=lÞ ðml NaOH usedÞ  ðnormality of NaOHÞ  ðLactic acid molecular weightÞ : ml homogenized sample ð1Þ 2.6.2. Color measurements The color of the samples was measured with a new spectroscopic apparatus designed to reduce expensive rework or out-of-specification product disposal after HPCO2 pasteurization (Ferrentino, Balzan, & Spilimbergo, 2012b). The system consisted of a high-resolution miniature spectrometer (HR2000+; Ocean Optics Inc., Dunedin, FL, USA) to which a fiber optic reflection probe (Ocean Optics Inc., Dunedin, FL, USA) was connected. The probe transmitted the light from a halogen lamp to the sample by the illuminating fibers while the reflected light from the sample was acquired by the reading fiber and measured by the spectrometer (Apruzzese, Balke, & Diosady, 2000). After the calibration of the signal, the reflectance spectrum of the treated samples was acquired by a specific software (Spectra Suite®, Ocean Optics Inc., Dunedin, FL, USA) providing L* (lightness), a* (redness), and b* (yellowness) parameters. Color measurements were performed in triplicate, and mean values and standard deviations were evaluated. Chroma (C) and total color difference (ΔE) were calculated from the numerical values of L* (lightness), a* (redness) and b* (yellowness) according to Eqs. (1) and (2), respectively: ¼

C¼

pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi a2 þ b2

ΔE ¼

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi        L1 −L2 2 þ a1 −a2 2 þ b1 −b2 :

ð1Þ ð2Þ

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Total color difference (ΔE) was calculated between untreated and HPCO2 + HPU treated samples. 2.6.3. Sensory analysis The sample quality was evaluated by a panel of 10 untrained judges, 5 males and 5 females between 21 and 50 years old, by a descriptive analysis, using a modified version of the procedure described by Komes, Belščak-Cvitanović, Horžić, et al. (2011). The evaluations were conducted the day after the preparation of the samples, in a quiet room with sufficient space between the testers, adequate light and ventilation, and at midmorning (because it was considered the best time before extraneous aromas and odors fill the air). A rank order test was performed and the panelists were asked to independently evaluate 3 sensory characteristics: appearance, texture, and aroma of each sample presented in plastic white cups at ambient temperature. The volunteers were asked to judge the samples using a ten point scale: 1 corresponded to the lowest preference and 10 corresponded to the highest preference; untreated and treated samples with thermal and HPCO2 + HPU combined treatments at optimal conditions were evaluated. The results were expressed as the average for each sensory attribute, and the standard deviations were calculated. 2.7. Storage study Dry cured ham surface samples spiked with L. monocytogenes and processed with HPCO2 + HPU combined treatment at the optimal conditions (12 MPa, 35 °C, 5 min with 10 W of HPU, delivered every 2 min) were stored at 4 °C for 4 weeks. During the storage microbial load, pH, TA and color were monitored. Microbial and quality parameters of untreated sample, stored at 4 °C for 4 weeks, were also monitored as a control. 2.8. Statistical analysis Differences between mean values were tested using the analysis of variance followed by multiple comparisons between means with the Duncan test. The general procedure of Statistica 7.0 software (StatSoft Inc., Tulsa, OK, U.S.A.) was used. All the data were analyzed at a significance level of p N 0.05. 3. Results and discussion 3.1. Thermal treatment The results obtained after a thermal treatment at 85 °C indicate that 1 min was sufficient to induce inactivation to undetectable levels of L. monocytogenes (detection limit of 30 CFU/g) spiked on dry cured ham surface. 3.2. HPCO2, HPU and HPCO2 + HPU combined treatments Fig. 2 shows the inactivation data of L. monocytogenes spiked on dry cured ham surface obtained after HPCO2, HPU and HPCO2 + HPU combined treatment at 22 °C and 6 (a), 8 (b), and 12 MPa (c). The results clearly demonstrated that HPU treatment was not able to induce any microbial inactivation at 22 °C, even after 30 min of treatment and that HPCO 2 and HPU treatments applied simultaneously did not show any synergistic effect compared to HPCO2 alone: the inactivation values of HPCO 2 and HPCO 2 + HPU were almost overlapped, for all the pressures tested. This result could be probably due to the low temperature: at 22 °C the bacterial cell membrane is particularly rigid and not sensitive to CO2 action, despite the simultaneous application of HPU which increases the solubility rate of CO2 inside the liquid phase, thus, theoretically, the ability of the gas to interfere with the cell membrane.

Conversely, the increase of the process temperature up to 35 °C highlights the synergistic effect of HPCO2 + HPU combined treatment, compared to HPCO2 alone, as shown in Fig. 3: HPCO2 induced in 30 min 3.4, 4.2 and 7.0 Log reductions at 6, 8 and 12 MPa respectively, while HPCO2 + HPU about 7.5 Log reductions after just 8, 6 and 5 min at 6, 8 and 12 MPa respectively. Indeed, from Fig. 3 it can be observed that the inactivation ratio of HPU applied to the sample was negligible even at 35 °C and for long treatment times. Comparing Fig. 2 with Fig. 3, it can be pointing out that HPCO2 + HPU is much more efficient at 35 °C rather than at 22 °C: probably at 35 °C cell membrane mobility is higher, the phospholipid-bilayer is more fluid and, additionally, CO2 is in supercritical phase, characterized by a high diffusivity into the liquid phase and high solvent power, which allows faster penetration into the cell membrane and an easy extraction of material from the cytoplasm (Garcìa-Gonzalez et al., 2009). Furthermore, ultrasound, other than causing cavitation phenomena, highly affects the mixing at microscale thus inducing a better contact of the pressurized CO2 with the surface of the cells, consequently increasing the inactivation rates. The enhanced contact between the microorganisms and the pressurized CO2 accelerates the diffusion of CO2 through the cell membrane, the decrease of the intracellular pH and all the phenomena that contribute to the microorganism death. In other words, the application of HPU accelerates both the solubilization of CO2 and the cell-medium mass transfer, causing a drastic drop in intracellular pH and an extraction of vital constituents (Piyasena et al., 2003). To investigate further the effect of the temperature, experiments were also performed at 45 °C (data not shown). No enhancement of HPCO2 + HPU inactivation efficiency was observed compared to 35 °C, especially at higher pressures. For this reason and in order to perform the combined process at temperature as low as possible, avoiding the deleterious effect of temperature on the product quality, process conditions of 35 °C, 12 MPa, for 5 min with 10 W ultrasound, delivered power every 2 min were used to perform the chemical–physical and sensorial characterizations of the product. It could be meaningful to compare our present results to those obtained with other promising non-thermal technologies, in particular the inactivation of Listeria spp. with HHP. For instance, Lucore and co-workers found that HHP pressurization at 700 MPa for 9 min assured 6 Log reduction of L. monocytogenes on frankfurters (Lucore, Shellhammer, & Yousef, 2000), while Bover-Cid, Belletti, Garriga, and Aymerich (2011) investigated the inactivation of L. monocytogenes on dry-cured ham at different HHP processing conditions. They achieved 7 and 8 Log reductions after 13 min at 11 °C and 750 MPa and after 9 min at 16 °C and 850 MPa, respectively. In general, pressure was found to be the most important factor determining the inactivation extent. HHP at 250 MPa did not inactivate L. monocytogenes in smoked salmon, but significant lag phases of 17 and 10 days were observed during the storage at 5 °C and 10 °C, respectively (Lakshmanan & Dalgaard, 2004). Ponce, Pla, Mor-Mur, Gervilla, and Guamis (1998) obtained over 5 Log reductions of L. innocua in liquid whole egg at 450 MPa, 20 °C for 15 min: the results indicated that microbial inactivation was enhanced if the exposure to pressure was prolonged. The present work demonstrated that HPCO2 + HPU combined technology allowed reducing significantly the pressure values of the pasteurization process, compared to HHP technology, almost halving the treatment times and thus potentially lowering the costs of an industrial application. 3.3. Quality analyses Quality analysis (pH, TA, color and sensory evaluation) performed on dry cured ham surface subjected to thermal (85 °C, 1 min) and to HPCO2 + HPU combined treatment (12 MPa, 35 °C, 5 min with 10 W of HPU delivered every 2 min) is reported.

0

0

-2

-2 Log(N/No)

Log(N/No)

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-4

-6

269

-4

-6

HPU HPCO2

HPU HPCO2

(a)

HPCO2+HPU

(b)

HPCO2+HPU

-8

-8

0

5

10

15

20

25

30

35

0

5

10

15

20

25

30

35

Treatment time (min)

Treatment time (min)

0

Log(N/No)

-2

-4

-6 HPU HPCO 2

(c)

HPCO 2+HPU

-8 0

5

10

15

20

25

30

35

Treatment time (min) Fig. 2. Inactivation of L. monocytogenes spiked on dry cured ham surface treated by HPCO2, HPU and HPCO2 + HPU at 22 °C and 6 MPa (a), 8 MPa (b), and 12 MPa (c).

3.3.1. TA, pH and color measurements Fig. 4 shows TA and pH of dry cured ham surface samples after both thermal and HPCO2 + HPU treatments. The results were compared to the one obtained for the untreated sample. No significant differences were detected between TA of the untreated and HPCO2 + HPU treated samples, while significant differences were measured between the values obtained for the untreated and thermally treated dry cured ham surface. These differences could be probably attributed to the high temperature used for the thermal process that induced the melting of the fatty part of the sample thus influencing TA values. As regard pH, no significant differences were detected between the treated and untreated samples. Fig. 5 shows the results of the color parameters (L*, a*, and b*) for the untreated, thermal and HPCO2 + HPU treated samples. No significant differences were detected between the 3 samples. Probably the process conditions were not too severe to influence the color attributes. As concerns the effect of HHP treatment on dry cured ham quality attributes, it has been demonstrated that the process causes minimal changes in “fresh” characteristics of food because it can be conducted at ambient or refrigerated temperatures. However, there is no doubt that HHP causes quality changes of meat. Some of the changes such as color and lipid oxidation are detrimental, whereas other changes such as pressure tenderization and pressure-assisted gelation are beneficial (Cheftel & Culioli, 1997).

samples. The results, reported in Fig. 6, demonstrated that the panelists could perceive significant differences between the 3 samples regarding the appearance and texture attributes. No significant differences were detected for the aroma attributes. Both treated samples were judged less bright compared to the untreated, probably the high temperature of thermal treatment and the extraction properties of CO2 in supercritical phase could affect appearance attributes. As concern the texture, treated samples were stickier, hard and pasty compared to the untreated. No significant differences could be detected between the dry cured ham treated with thermal treatment and the dry cured ham treated with the HPCO2 + HPU combined treatment. The results were in agreement with the ones obtained by Ferrentino, Balzan, and Spilimbergo (2012b).

3.3.2. Sensory analysis Untrained panelists were asked to judge 3 samples: the control untreated dry cured ham surface, the thermally and HPCO2 + HPU treated

3.4.2. TA, pH and color measurements In Table 2, the results of TA, pH, Chroma and ΔE of untreated and treated dry cured ham surface during the refrigerated storage are

3.4. Storage study 3.4.1. Microbial analysis Table 1 shows the results of the microbial storage: L. monocytogenes was never detected in the treated samples during the whole storage period while in the untreated samples its concentration tended to decrease after 2 weeks of storage, reaching 5.91 Log(CFU/g) after 4 weeks of storage: probably after a first period of sustenance, Listeria cells started dying, because of the lack of nutrients and the formation of inhibitory products on dry cured ham surface.

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0

0

(b)

(a) -2

Log(N/No)

Log(N/No)

-2

-4

-4

-6

-6

HPU HPCO 2

HPU HPCO2

HPCO 2+HPU

HPCO2+HPU

-8

-8 0

5

10

15

20

25

30

0

35

5

10

15

20

25

30

35

Treatment time (min)

Treatment time (min)

0

(c)

Log(N/No)

-2

-4

-6 HPU HPCO 2 HPCO 2+HPU

-8 0

10

20

30

40

Treatment time (min) Fig. 3. Inactivation of L. monocytogenes spiked on dry cured ham surface treated by HPCO2, HPU and HPCO2 + HPU at 35 °C and 6 MPa (a), 8 MPa (b), and 12 MPa (c).

reported. As noticed, pH and TA of treated samples did not change significantly. For the untreated samples, the behavior was similar; however, after 4 weeks of storage a significant pH decrease could be noticed probably associated to the microbial contamination. With regard to the results of the color attributes (L*, a*, and b*), Chroma and ΔE values were evaluated. No significant differences of Chroma values were detected for the untreated and treated samples during 4 weeks of refrigerated storage. The color changes between untreated and HPCO2 + HPU treated samples were evaluated in terms of ΔE resulting equal to 4.4 after the treatment and not significantly

different during the storage. An absolute threshold value for human color discrimination has only been determined for few specific products (Martínez, Melgosa, Pérez, Hita, & Negueruela, 2001); nevertheless, ΔE N 4 is usually considered a clearly distinguishable color difference to the average person. 4. Conclusions The results above assess the feasibility of the combined treatment for the inactivation of L. monocytogenes spiked on dry cured ham surface 7 6

0.5

5 0.4

4

pH

Total acidity (mg of lactic acid/L)

0.6

0.3

3 0.2

2 0.1

1

0.0

0 Untreated

Thermally treated

HPCO2+HPU

Untreated

Thermally treated

Fig. 4. TA and pH of dry cured ham surface thermally and HPCO2 + HPU treated compared with the untreated.

HPCO2+HPU

S. Sara et al. / Food Research International 66 (2014) 264–273

100

Color parameters

Table 1 Behavior of L. monocytogenes on untreated and treated samples during 4 weeks of storage.

L* a* b*

80

271

60

40

Storage time (week)

Untreated sample Log(CFU/g)

HPCO2 + HPU treated sample Log(CFU/g)

0 1 2 3 4

7.20 7.22 7.66 5.92 5.91

n.d. n.d. n.d. n.d. n.d.

± ± ± ± ±

0.04 0.06 0.05 0.11 0.19

n.d. = not detected.

20

differences just in few quality attributes, as appearance and texture, of HPCO2 + HPU treated products compared to the untreated, but the overall quality of the products was judged good. The storage study confirmed the positive results obtained from the microbial study. No differences were observed in color, pH and TA between the products after the combined treatment after 4 weeks of refrigerated storage. In conclusion, the results demonstrated the feasibility and the potential of HPCO2 + HPU as an innovative non-thermal pasteurization technology of food surface, in particular ham-type meat products. Further studies are needed, anyhow, before the application of such an innovative technique at industrial level, both to deep further the inactivation mechanism and to perform an economic analysis of a hypothetical HPCO2 + HPU plant.

0 Untreated

Thermally treated

HPCO2+HPU

Fig. 5. Color parameters of dry cured ham surface thermally and HPCO2 + HPU treated compared with the untreated.

highlighting the synergistic effect of the two techniques operating in parallel. As concerns the microbial study, the combined treatment showed a faster inactivation rate, thus achieving the FSO value required for human safety: 5 min at 12 MPa, 35 °C with 10 W delivered every 2 min of treatment was enough to achieve about 7 Log reductions, while HPCO2 alone needed 30 min to obtain the same inactivation level. The quality analyses revealed no differences in pH, TA and color of the samples treated with the combined process. The panelists perceived

(a)

overall colour homogenicity 10

(b) adhesiviness 10

8

8

6 4

6

hollow incidence

brightness

hardness

4

2

2

colour intensity

0

0

stringiness

crunbly

marbling

pasty

metallic 10

(c)

8 off-odour

6

cooked ham

4 2 0

aged

fishy

cured Fig. 6. Net chart of the characteristic (axis) of the three sensory attributes (a, appearance; b, texture; c, aroma) of dry cured ham surface. Different samples are represented by different lines on the chart: dashed, untreated sample; continuous, HPCO2 + HPU treated samples (12 MPa, 35 °C, 5 min); dotted, thermally treated samples (85 °C, 1 min).

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Table 2 TA, pH, Chroma and ΔE values of untreated and HPCO2 + HPU treated dry cured ham surface during refrigerated storage. Storage time (week)

Untreated sample pH

0 1 2 3 4

6.2 5.7 5.9 5.7 5.7

± ± ± ± ±

0.1a 0.6b 0.1b 0.5b 0.2b

ΔE

HPCO2 + HPU treated sample TA (mg of lactic acid/l) 0.28 0.28 0.28 0.27 0.28

± ± ± ± ±

0.1a 0.1a 0.1a 0.2a 0.1a

Chroma 19.9 21.8 24.2 22.2 20.2

± ± ± ± ±

3.8a 2.7a 0.5a 3.6a 3.3a

pH 6.3 6.3 6.1 6.2 6.3

TA (mg of lactic acid/l) ± ± ± ± ±

0.1a 0.3a 0.2a 0.1a 0.1a

0.23 0.23 0.23 0.17 0.17

± ± ± ± ±

0.1a 0.1a 0.2a 0.1a 0.1a

Chroma 21.8 21.4 22.2 20.5 24.0

± ± ± ± ±

0.7a 2.0a 1.9a 3.0a 3.2a

4.4 5.2 4.1 6.1 4.7

± ± ± ± ±

1.1a 1.4a 1.5a 0.5a 0.3a

Data are mean values ± standard deviations. Values with similar letters within rows are not significantly different (Duncan's test, p N 0.05).

Acknowledgments The research leading to these results received funding from the European Community Seventh Framework Program (FP7/2007–2013) under grant agreement no. 245280, also known under the acronym PRESERF. References Apruzzese, F., Balke, S.T., & Diosady, L.L. (2000). On-line colour and composition monitoring in the extrusion cooking process. Food Research International, 33, 621–628. Arreola, A.G., Balaban, M.O., Marshall, M.R., et al. (1991). Supercritical carbon dioxide effects on some quality attributes of single strength orange juice. Journal of Food Science, 56, 1030–1033. Aymerich, T., Picouet, P.A., & Monfort, J.M. (2008). Decontamination technologies for meat products. Meat Science, 78, 114–129. 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, 804–809. Butz, P., & Tauscher, B. (2002). Emerging technologies: Chemical aspects. Food Research International, 35, 279–284. Cameron, M., McMaster, L.D., & Britz, T.J. (2009). Impact of ultrasound on dairy spoilage microbes and milk components. Dairy Science & Technology, 89, 83–98. Cappelletti, M., Ferrentino, G., Endrizzi, I., et al. (2014). High pressure carbon dioxide pasteurization of coconut water: A sport drink with high nutritional and sensory quality. Journal of Food Engineering, http://dx.doi.org/10.1016/j.jfoodeng.2014.08.012. Cappelletti, M., Ferrentino, G., & Spilimbergo, S. (2014). Supercritical carbon dioxide combined with high power ultrasound: An effective method for the pasteurization of coconut water. Journal of Supercritical Fluids, 92(2014), 257–263. CFSAN/FSIS (2003). Quantitative assessment of relative risk to public health from foodborne Listeria monocytogenes among selected categories of ready-to-eat foods. Silver Spring: Food and Drug Administration. Cheftel, J.C., & Culioli, J. (1997). Effects of high pressure on meat: A review. Meat Science, 46, 211–236. Chemat, F., Zill-e-Huma, S., & Khan, M.K. (2011). Applications of ultrasound in food technology: Processing, preservation and extraction. Ultrasonics Sonochemistry, 18, 813–835. Commission, E. (2005). Regulation (EC) no 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs. Official Journal of the European Union, 338, 1–26. European Food Safety Authority (2014). The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2012. EFSA Journal, 12, 3547–3859. European Parliament and of the Council (1995). European Directive 95/2/CE relative to food additives other than colors and sweeteners. Official Journal, L61, 1–40. Ferrentino, G., Balzan, S., Dorigato, A., Pegoretti, A., & Spilimbergo, S. (2012). Effect of supercritical carbon dioxide pasteurization on natural microbiota, texture, and microstructure of fresh-cut coconut. Journal of Food Science, 7(55), E137–E143. Ferrentino, G., Balzan, S., & Spilimbergo, S. (2012a). On-line color monitoring of solid foods during supercritical CO2 pasteurization. Journal of Food Engineering, 110, 80–85. Ferrentino, G., Balzan, S., & Spilimbergo, S. (2012b). Supercritical carbon dioxide processing of dry cured ham spiked with Listeria monocytogenes: Inactivation kinetics, color, and sensory evaluation. Food and Bioprocess Technology, 6, 1164–1174. Ferrentino, G., Plaza, M.L., Ramirez-Rodrigues, M., Ferrari, G., & Balaban, M.O. (2009). Effects of dense phase carbon dioxide pasteurization on the physical and quality attributes of a red grapefruit juice. Journal of Food Science, 74, E333–E341. Ferrentino, G., & Spilimbergo, S. (2011). High pressure carbon dioxide pasteurization of solid foods: current knowledge and future outlooks. Trends in Food Science & Technology, 22, 427–441. Food Safety and Inspection Service, U (2000). Food additives for use in meat and poultry products: Sodium diactetate, sodium acetate, sodium lactate and potassium lactate. Federal Register, 65, 3121–3123. Franciosa, G., Tartaro, S., Wedell-Neergaard, C., & Aureli, P. (2001). Characterization of L. monocytogenes strains involved in invasive and non-invasive listeriosis outbreaks by PCR-based fingerprinting techniques. Applied and Environmental Microbiology, 67, 1793–1799. Gao, Y., Nagy, B., Liu, X., Simandi, B., & Wang, Q. (2009). Supercritical CO2 extraction of lutein esters from marigold (Tagetes erecta L.) enhanced by ultrasound. Journal of Supercritical Fluids, 49, 345–350.

Garcìa-Gonzalez, L., Geeraerd, A.H., Elst, K., et al. (2009). Inactivation of naturally occurring microorganisms in liquid whole egg using high pressure carbon dioxide processing as an alternative to heat pasteurization. Journal of Supercritical Fluids, 51, 74–82. Garcìa-Gonzalez, L., Geeraerd, A.H., Spilimbergo, S., et al. (2007). High pressure carbon dioxide inactivation of microorganisms in foods: The past, the present and the future. International Journal of Food Microbiology, 117, 1–28. García-Pérez, J.V., Cárcel, J.A., De la Fuente, S., & Riera, E. (2006). Ultrasonic drying of foodstuff in a fluidized bed. Parametric study. Ultrasonics, 44, e539–e543. Gola, S., Frustoli, M., Rovere, P., & Miglioli, L. (2003). Inattivazione di Listeria monocytogenes in prosciutto crudo trattato con la pressione idrostatica [Inactivation of Listeria monocytogenes in dry cured ham by hydrostatic pressure treatment]. Industria Conserve, 78, 441–449. Grisenti, M.S., Lori, D., Vicini, L., et al. (2004). Comportamento di Listeria monocytogenes in prosciutto crudo stagionato in rapporto all'atmosfera di confezionamento e alla temperatura di conservazione [Behavior of Listeria monocytogenes in dry cured ham depending on the packaging atmosphere and storage temperature]. Industria Conserve, 79, 3–12. Hereu, A., Bover-Cid, S., Garriga, M., & Aymerich, T. (2012). High hydrostatic pressure and biopreservation of dry-cured ham to meet the food safety objectives for Listeria monocytogenes. International Journal of Food Microbiology, 154, 107–112. 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. ISO 11290-2 (1998). Microbiology of food and animal feeding stuffs—Horizontal methods for the detection and enumeration of Listeria monocytogenes. Part 2: Enumeration method. ISO 6887-1 (1999). Microbiology of food and animal feeding stuffs—Preparation of test samples, initial suspension and decimal dilutions for microbiological examination—Part 1: General rules for the preparation of the initial suspension and decimal dilutions. ISO 6887-2 (2003). Microbiology of food and animal feeding stuffs—Preparation of test samples, initial suspension and decimal dilutions for microbiological examination—Part 2: Specific rules for the preparation of meat and meat products. ISO 7218 (2007). Microbiology of food and animal feeding stuffs — General requirements and guidance for microbiological examinations. Kim, J.G., Yousef, A.E., & Dave, S. (1999). Application of ozone for enhancing the microbiological safety and quality of foods: A review. Journal of Food Protection, 62, 1071–1087. Komes, D., Belščak-Cvitanović, A., Horžić, D., et al. (2011). Bioactive and sensory properties of herbal spirit enriched with cocoa (Theobroma cacao L.) polyphenolics. Food Bioprocess Technology, http://dx.doi.org/10.1007/s11947-011-0630-7. Lakshmanan, R., & Dalgaard, P. (2004). Effects of high-pressure processing on Listeria monocytogenes, spoilage microflora and multiple compound quality indices in chilled cold-smoked salmon. Journal of Applied Microbiology, 96, 398–408. Liu, Y., Hu, X., Zhao, X., & Song, H. (2012). Combined effect of high pressure carbon dioxide and mild heat treatment on overall quality parameters of watermelon juice. Innovative Food Science and Emerging Technologies, 13, 112–119. Lucore, L.A., Shellhammer, T.H., & Yousef, A.E. (2000). Inactivation of Listeria monocytogenes Scott A on artificially contaminated frankfurters by highpressure processing. Journal of Food Protection, 63, 662–664. Martínez, J.A., Melgosa, M., Pérez, M.M., Hita, E., & Negueruela, A.I. Note (2001). Visual and instrumental color evaluation in red wines. Food Science and Technology International, 7, 439–444. McLauchlin, J. (1997). The pathogenicity of Listeria monocytogenes. A public health perspective. Reviews in Medical Microbiology, 8, 1–14. Minei, C.C., Gomes, B.C., Ratti, R.P., D'Angelis, C.E.M., & De Martinis, E.C.P. (2008). Influence of peroxyacetic acid and nisin and coculture with Enterococcus faecium on Listeria monocytogenes biofilm formation. Journal of Food Protection, 71, 634–638. Ordonez, J.A., Sanz, B., Hernandez, P.E., & Lopez-Lorenzo, P. (1984). A note on the effect of combined ultrasonic and heat treatments on the survival of thermoduric streptococci. Journal of Applied Bacteriology, 56, 175–177. Ortuño, C., Martínez-Pastor, M.T., Mulet, A., & Benedito, J. (2012a). Supercritical carbon dioxide inactivation of Escherichia coli and Saccharomyces cerevisiae in different growth stages. Journal of Supercritical Fluids, 63, 8–15. Ortuño, C., Martínez-Pastor, M.T., Mulet, A., & Benedito, J. (2012b). An ultrasoundenhanced system for microbial inactivation using supercritical carbon dioxide. Innovative Food Science and Emerging Technologies, 15, 31–37. Pataro, G., Ferrentino, G., Ricciardi, C., & Ferrari, G. (2010). Pulsed electric fields assisted microbial inactivation of S. cerevisiae cells by high pressure carbon dioxide. Journal of Supercritical Fluids, 54, 120–128. Piyasena, P., Mohareb, E., & McKellar, R.C. (2003). Inactivation of microbes using ultrasound: A review. International Journal of Food Microbiology, 87, 207–216.

S. Sara et al. / Food Research International 66 (2014) 264–273 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. Journal of Food Protection, 61, 119–122. Quintavalla, S., & Vicini, L. (2002). Antimicrobial food packaging in meat industry. Meat Science, 62, 373–380. Raso, J., Palo, A., Pagan, R., & Condon, S. (1998). Inactivation of Bacillus subtilis spores by combining ultrasonic waves under pressure and mild heat treatment. Journal of Applied Microbiology, 85, 849–854. Riera, E., Blanco, A., García, J., et al. (2010). High power ultrasonic system for the enhancement of mass transfer in supercritical CO2 extraction processes. Ultrasonics, 50, 306–309. Rubio, B., Martìnez, B., Garcìa-Cachàn, M.D., Rovira, J., & Jaime, I. (2007). Effect of high pressure preservation on the quality of dry cured beef “Cecina de León”. Innovative Food Science and Emerging Technologies, 8, 102–110. Shabala, L., Lee, S.H., Cannesson, P., & Ross, T. (2008). Acid and NaCl limits to growth of Listeria monocytogenes and influence of sequence of inimical acid and NaCl levels on inactivation kinetics. Journal of Food Protection, 71, 1169–1177. Spilimbergo, S., & Ciola, L. (2010). Supercritical CO2 and N2O pasteurisation of peach and kiwi juice. International Journal of Food Science and Technology, 45, 1619–1625.

273

Spilimbergo, S., Komes, D., Vojvodic, A., Levaj, B., & Ferrentino, G. (2013). High pressure carbon dioxide pasteurization of fresh-cut carrot. Journal of Supercritical Fluids, 79, 92–100. Toldrá, F. (2004). Dry-cured ham meat products. New York: Wiley-Blackwell. Trofimov, T.I., Samsonov, M.D., Lee, S.C., Smart, N.G., & Wai, C.M. (2001). Ultrasound enhancement of dissolution kinetics of uranium oxides in supercritical carbon dioxide. Journal of Chemical Technology and Biotechnology, 76, 1223–1226. Turgis, M., Han, J., Borsa, J., & Lacroix, M. (2008). Combined effect of natural essential oils, modified atmosphere packaging and gamma radiation on the microbial growth on ground beef. Journal of Food Protection, 71, 1237–1243. Yun, C., Kedie, C., Shulai, C., Adshiri, T., & Arai, K. (1997). Effects of ultrasound on mass transfer in supercritical extraction. 4th International Symposium on Supercritical Fluids, 11–14 May, Sendai, Japan (pp. 707–710). Zhou, L., Wang, Y., Hu, X., Wu, J., & Liao, X. (2009). Effect of high pressure carbon dioxide on the quality of carrot juice. Innovative Food Science and Emerging Technologies, 10, 321–327.