Synergistic combinations of high hydrostatic pressure and essential oils or their constituents and their use in preservation of fruit juices

International Journal of Food Microbiology 161 (2013) 23–30

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Synergistic combinations of high hydrostatic pressure and essential oils or their constituents and their use in preservation of fruit juices Laura Espina a, Diego García-Gonzalo a, Amin Laglaoui b, Bernard M. Mackey c, Rafael Pagán a,⁎ a b c

Departamento de Producción Animal y Ciencia de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain Université Abdelmalek Essaâdi, Faculté des Sciences et Techniques. Equipe de Recherche en Biotechnologies et Génie des Biomolécules (ERBGB), B.P. 416, Tanger, Morocco Department of Food and Nutrition Sciences, The University of Reading, PO Box 226, Whiteknights, Reading RG6 6AP, United Kingdom

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Article history: Received 31 August 2012 Received in revised form 2 November 2012 Accepted 11 November 2012 Available online 28 November 2012 Keywords: High hydrostatic pressure Essential oils Sublethal injury Synergistic effect Listeria monocytogenes Escherichia coli O157:H7

a b s t r a c t This work addresses the inactivation achieved with Escherichia coli O157:H7 and Listeria monocytogenes EGD-e by combined processes of high hydrostatic pressure (HHP) and essential oils (EOs) or their chemical constituents (CCs). HHP treatments (175–400 MPa for 20 min) were combined with 200 μL/L of each EO (Citrus sinensis L., Citrus lemon L., Citrus reticulata L., Thymus algeriensis L., Eucalyptus globulus L., Rosmarinus officinalis L., Mentha pulegium L., Juniperus phoenicea L., and Cyperus longus L.) or each CC ((+)-limonene, α-pinene, β-pinene, p-cymene, thymol, carvacrol, borneol, linalool, terpinen-4-ol, 1,8-cineole, α-terpinyl acetate, camphor, and (+)-pulegone) in buffer of pH 4.0 or 7.0. The tested combinations achieved different degrees of inactivation, the most effective being (+)-limonene, carvacrol, C. reticulata L. EO, T. algeriensis L. EO and C. sinensis L. EO which were capable of inactivating about 4–5 log10 cycles of the initial cell populations in combination with HHP, and therefore showed outstanding synergistic effects. (+)-Limonene was also capable of inactivating 5 log10 cycles of the initial E. coli O157:H7 population in combination with HHP (300 MPa for 20 min) in orange and apple juices, and a direct relationship was established between the inactivation degree caused by the combined process with (+)-limonene and the occurrence of sublethal injury after the HHP treatment. This work shows the potential of EOs and CCs in the inactivation of foodborne pathogens in combined treatments with HHP, and proposes their possible use in liquid food such as fruit juices. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Essential oils (EOs) are aromatic and volatile oily liquids obtained from plant materials (Burt, 2004). Their biological properties are determined by their composition, which is characterized by two or three major components and others present in trace amounts (Bakkali et al., 2008). These components include terpenes and terpenoids, as well as aromatic and aliphatic constituents (Bakkali et al., 2008). Although the antibacterial properties of EOs have been widely studied, the interest to use them as natural preservatives is a growing trend, in accordance with consumers' increasing health consciousness (Lopez et al., 2005; Marino et al., 2001). However, their actual use in food has been limited because high concentrations are needed to achieve sufficient antimicrobial activity (Hyldgaard et al., 2012), which results in undesirable flavor changes in the food product (Yamazaki et al., 2004). Following the hurdle theory proposed by Leistner and Gorris (1995), EOs or their constituents (chemical constituents, CCs) have been studied

⁎ Corresponding author at: Dpto. PACA. Facultad de Veterinaria, Universidad de Zaragoza, C/Miguel Servet, 177, 50013, Zaragoza, Spain. Tel.: +34 976 761581; fax: +34 976 761590. E-mail address: [email protected] (R. Pagán). 0168-1605/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2012.11.015

in combination with other preservation methods to inactivate foodborne microorganisms. This way, synergistic combinations can permit the decrease of the intensity of each hurdle, therefore resulting in a better maintenance of the nutritional and sensorial properties of fresh food. For example, preservatives such as nisin (Yamazaki et al., 2004) and sodium citrate (Blaszyk and Holley, 1998) have been successfully combined with EOs or their constituents. Mild heat has also been proven to act synergistically in combination with CCs like S-carvone (Karatzas et al., 2000) or EOs such as citrus fruit EOs (Espina et al., 2011), cinnamon and clove (Knight and McKellar, 2007). In recent studies, the outstanding synergistic effect between mild heat and certain compounds has been attributed to the occurrence of sublethal injury to the bacterial envelopes, which facilitated the access of the antimicrobial compound to the cellular target (Ait-Ouazzou et al., 2011a; Espina et al., 2010; Somolinos et al., 2010). Sublethal injury is a consequence of exposure to a chemical or physical process that damages but does not kill a microorganism (Wesche et al., 2009), and its detection could indicate those circumstances in which two technologies may act synergistically in a combined process for food preservation (Mackey, 2000). Sublethal injury in bacteria is also known to be caused by other preservation technologies, including high hydrostatic pressure (HHP) (Wesche et al., 2009). The cell membrane is a primary site of pressure

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damage (Yuste et al., 2004) and membrane — damaged cells may show enhanced sensitivity to antimicrobials (Hauben et al., 1997). For example, synergistic effects have been shown with HHP combined with nisin (Lee and Kaletunç, 2010) or bacteriocins (Alpas and Bozoglu, 2000). Little research has been done regarding combinations of HHP with EOs or CCs. Somolinos et al. (2008) studied the synergistic inactivation of E. coli by HHP and citral, and Karatzas et al. (2011) studied the combination with carvacrol to inactivate Listeria monocytogenes. These promising results open up the possibility of exploring the combination of other EOs and CCs with HHP in the inactivation of target pathogens. On the other hand, HHP is known to cause minimal changes to the organoleptic and nutritional properties of food (Rendueles et al., 2011), and has emerged as a viable alternative to thermal processes (Esteve and Frigola, 2007). Pressure treatments have been applied to fruit juices to inactivate pathogens such as E. coli (Garcia-Graells et al., 1998; Linton et al., 1999). Furthermore, a processing protocol based on hurdle technology combining HHP with an EO or CC for the preservation of fruit juices could result in the reduction of the intensity of the HHP treatment applied, and therefore decrease initial and maintenance costs and lengthen the life of the equipment. The objectives of this work were (i) to determine the inactivation of E. coli O157:H7 and L. monocytogenes EGD-e by combined treatments of HHP and EOs or CCs in buffer of pH 4.0 and 7.0, and (ii) to study the combination of HHP with selected compounds for the preservation of apple and orange juices. To accomplish these objectives, nine EOs with previously studied chemical composition and antibacterial properties (Ait-Ouazzou et al., 2011b, 2012; Espina et al., 2011) and 13 of the main CCs constituting those EOs were used. 2. Material and methods 2.1. Micro-organisms and growth conditions The strains used were E. coli O157:H7 VTEC (Phage type 34) (Chapman et al., 1993) and L. monocytogenes EGD-e (Chatterjee et al., 2006). The cultures were maintained in cryovials at −80 °C. Broth subcultures were prepared by inoculating, with one single colony from a plate, a test tube containing 10 mL of sterile tryptic soy broth (Biolife, Milan, Italy) with 0.6% yeast extract added (Biolife) (TSBYE). After inoculation, the tubes were incubated overnight at 37 °C (E. coli O157:H7) or 30 °C (L. monocytogenes). With these subcultures, 250 mL Erlenmeyer flasks containing 50 mL of TSBYE were inoculated to a final concentration of 10 4 colony-forming units (CFU)/mL. These flasks were incubated under agitation (160 rpm) at the appropriate temperature (see above) until the stationary growth phase was reached. Stationary phase was chosen because cells show higher resistance to HHP at this stage than at exponential phase (Pagan and Mackey, 2000), and to match previously published data (Ait-Ouazzou et al., 2011a; Espina et al., 2010, 2011; Somolinos et al., 2010). 2.2. Essential oils Citrus fruit (orange, (Citrus sinensis L.); lemon (Citrus lemon L.) and mandarin (Citrus reticulata L.)) EOs were obtained as described by Espina et al. (Espina et al., 2011). Thymus algeriensis L., Eucalyptus globulus L., Rosmarinus officinalis L., Mentha pulegium L., Juniperus phoenicea L. and Cyperus longus L. EOs were obtained as described by Ait-Ouazzou et al. (Ait-Ouazzou et al., 2011b, 2012). The composition of all these EOs was analyzed and reported in the cited papers.

hydrocarbon monoterpenes ((+)-limonene (97%), α-pinene (98%), β-pinene (99%), and p-cymene (99%)), and 9 oxygenated monoterpenes (thymol (99%), carvacrol (98%), borneol (97%), linalool (≥95%), terpinen-4-ol (≥95%), 1,8-cineole (99%), α-terpinyl acetate (≥90%), camphor (96%), and (+)-pulegone (98%)). As thymol, borneol and camphor were solid at the experimental temperature, they were dissolved in 95% ethanol to obtain a stock solution of 100 mg/mL. 2.4. HHP treatments Cells were centrifuged at 6000 ×g for 5 min at 20 °C, and pellets were resuspended in the same medium that of the treatment medium. Treatment media were: citrate–phosphate buffer of pH 4.0 or 7.0, and shelf-stable apple juice (pH 3.6) or orange juice (pH 3.8) (purchased from a local supermarket). Where indicated, each EO or CC was added to a final concentration of 200 μL/L (corresponding to 200 mg/L of solid compounds in case of thymol, borneol and camphor) to the treatment medium. This concentration was chosen from previous work (Ait-Ouazzou et al., 2011a; Espina et al., 2010, 2011; Somolinos et al., 2010). Following the procedure explained by Friedman et al. (2002), a vigorous shaking method through Vortex shaking was used to mix EOs and CCs with the treatment medium. Because solvents and detergents can decrease the antimicrobial effect of essential oils and damage cell envelopes, these substances were not used in these experiments. Afterwards, microorganisms (E. coli O157:H7 or L. monocytogenes EGD-e) were added at a concentration of 3· 107 CFU/mL and, additionally, some experiments were performed at an initial concentration of 3·104 CFU/mL. These concentrations were chosen according to previously published data (Ait-Ouazzou et al., 2011a; Espina et al., 2010, 2011). Cell suspensions (1 mL each) were placed in sterile plastic pouches that were heat sealed and kept on ice before pressurization. Samples were pressure-treated in a 300-mL pressure vessel (model S-FL-850-9-W; Stansted Fluid Power, Stansted, United Kingdom) at room temperature (20 ±2 °C). The pressure-transmitting fluid was monopropylene glycol-water (30:70). Cells were exposed to pressures from 175 to 400 MPa for different times (5 to 20 min). The maximum temperature reached during pressurization was 30 °C. The pressure come up and release times were about 3 and 0.5 min, respectively. After decompression, the pouches were removed from the unit and placed on ice until viable counts were evaluated. 2.5. Counts of viable cells Samples were adequately diluted in maximum recovery diluent (Oxoid, UK) (1:9 dilutions) and 0.02 mL volumes were spread on tryptic soy agar supplemented with 0.6% yeast extract (TSAYE) (Oxoid). For each dilution, 10–200 colonies were counted on the surface of the agar. The detection limit was of 5 log10 cycles when working at an initial concentration of 3 · 10 7 CFU/mL. When working at an initial concentration of 3 · 10 4 CFU/mL, an additional sample of 0.9 mL was spread from the treated sample, and the detection limit was of 3 log10 cycles. Plates were incubated at 37 °C for 24 h (E. coli O157:H7) or 30 °C for 48 h (L. monocytogenes). Previous experiments showed that longer incubation times did not influence the amount of surviving cells. Experimental data was obtained from at least three independent experiments performed in different days. ANOVA and t-tests were performed with GraphPad PRISM® (GraphPad Software, Inc., San Diego, CA, USA) and differences were considered significant if p ≤ 0.05. 2.6. Detection of sublethal injury

2.3. Chemical constituents Out of the major constituents of the tested EOs, those with known antibacterial properties were selected and purchased from SigmaAldrich (Sigma-Aldrich Chemie, Steinheim, Germany). They were 4

In order to determine microbial cell injury in the cytoplasmic membrane, treated samples were also plated onto TSAYE containing 3% NaCl (E. coli O157:H7) or 6% NaCl (L. monocytogenes) (TSAYE-SC). Treated E. coli O157:H7 cells were also plated onto TSAYE containing

L. Espina et al. / International Journal of Food Microbiology 161 (2013) 23–30 Table 1 Log10 cycles of inactivation (mean ± standard deviation) of E. coli O157:H7 and L. monocytogenes EGD-e after HHP treatments at different pressures for 20 min. Cells were treated in McIlvaine's buffer of pH 4.0 or pH 7.0 and recovered in tryptic soya agar with 0.6% yeast extract (TSAYE), TSAYE with 3% NaCl added (TSAYE-SC) and TSAYE with 0.35% bile salts added (TSAYE-BS). E. coli O157:H7

L. monocytogenes

pH 4 (300 MPa) pH 7 (400 MPa) pH 4 (175 MPa) pH 7 (325 MPa) TSAYE 0.50 ± 0.16 TSAYE-SC >5.0 TSAYE-BS 4.5 ± 0.40

0.30 ± 0.16 4.8 ± 0.75 4.7 ± 0.69

0.77 ± 0.10 >5.0

0.47 ± 0.21 >5.0

0.35% bile salts No. 3 (Oxoid) (TSAYE-BS) to determine microbial cell injury in the outer membrane. These were the maximum concentrations of sodium chloride and bile salts that caused no reduction in colony counts of untreated cells. The extent of sublethal injury in a population of pressure-treated cells was expressed as the difference between the log10 count (CFU) on non selective medium (TSAYE) and the log10 count on selective medium (TSAYE-SC or TSAYE-BS). According to this representation “two logs10 of injury” means a 2-log10 difference in the count on selective and non-selective medium or that 99% of survivors were sublethally injured. 3. Results Table 1 shows the number of decimal logarithmic (log10) cycles of inactivation after the HHP treatments acting alone, for each microorganism and pH. As can be seen, different pressures were applied for 20 min in order to get a similar degree of inactivation (b1 log10 cycle) and sublethal damage (about 4.5 log10 cycles) for each microorganism and pH. These treatment conditions were selected from

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preliminary work. According to the selected pressure values, both E. coli O157:H7 and L. monocytogenes showed the greatest sensitivity at pH 4.0, being E. coli O157:H7 more pressure resistant that L. monocytogenes. Fig. 1 shows the number of log10 cycles of inactivation after combined treatments of HHP and 200 μL/L of each CC (Fig. 1A) or EO (Fig. 1B). The CCs and the EOs are arranged in order of the average effectiveness in inactivating both microorganisms and at both pHs. All the tested compounds inactivated less than 1.0 log10 cycle of the initial bacterial populations after holding at 20 °C for 20 min (data not shown), and HHP acting alone reached no more than 0.77 log10 cycles of inactivation (Table 1). Combined processes achieving a greater log10 reduction than the sum of the separate processes were considered to be synergistic. Therefore a reduction of more than 1.3 (for E. coli O157:H7 at pH 7.0), 1.5 (for E. coli O157:H7 at pH 4.0 and L. monocytogenes at pH 7.0) or 1.8 (for L. monocytogenes at pH 4.0) log10 cycles of the initial population were considered synergistic. Fig. 1A shows that the hydrocarbon monoterpene limonene and the phenol carvacrol were the most effective CCs in combination with HHP and even surpassed the detection limit of inactivation for the four conditions assayed. On the contrary, combined processes with some CCs like the phenol thymol, the ether 1,8-cineole, the alcohol (−)-borneol, and the ketone camphor achieved less than 2 log10 cycles of inactivation. Combinations of HHP with the ester α-terpinyl acetate or the alcohol linalool were significantly more active against E. coli than against L. monocytogenes at pH 7.0, although the treatment with linalool was more active against L. monocytogenes than against E. coli at pH 4.0. The hydrocarbon monoterpene (−)-β-pinene, the oxygenated monoterpene (+)-pulegone, the hydrocarbon monoterpene p-cymene, the alcohol (−)-terpinen-4-ol and the hydrocarbon monoterpene α-pinene showed intermediate bactericidal action in

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Fig. 1. Inactivation by combined treatments of HHP plus pure compounds or essential oils. E. coli O157:H7 ( , ) or L. monocytogenes EGDe ( , ) cells (initial concentration of 3 · 107 CFU/mL) were exposed to HHP plus 200 μL/L of pure compound (A) or essential oil (B) in McIlvaine's buffer of pH 4.0 ( , ) or 7.0 ( , ). Pure compounds (A) were: (+)-limonene (1); carvacrol (2); α-terpinyl acetate (3); linalool (4); (−)-β-pinene (5); (+)-pulegone (6); p-cymene (7); (−)-terpinen-4-ol (8); α-pinene (9); thymol (10); 1,8-cineole (11); (−)-borneol (12); and camphor (13). Essential oils (B) were: C. reticulata L. (1); T. algeriensis L. (2); C. sinensis L. (3); C. lemon L. (4); M. pulegium L. (5); R. officinalis L. (6); J. phoenicea L. (7); E. globulus L. (8); and C. longus L. (9). High hydrostatic pressure treatments were 20 min long at 300 MPa (E. coli O157:H7 at pH 4.0), 400 MPa (E. coli O157:H7 at pH 7.0), 175 MPa (L. monocytogenes at pH 4.0), or 325 MPa (L. monocytogenes at pH 7.0). The error bars in the figures indicate the standard deviations of the means for data obtained from three independent experiments.

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combination with HHP (around 2–4 log10 cycles of inactivation at pH 4.0 and 1–3 log10 cycles at pH 7.0). Regarding the EOs (Fig. 1B), the citrus fruit EOs (C. sinensis L., C. reticulata L. and C. lemon L.) and T. algeriensis L. were the most effective ones in inactivating E. coli O157:H7 and L. monocytogenes at both pHs in the combined processes. On the other hand, the least effective EOs (J. phoenicea L., E. globulus L. and C. longus L.) inactivated about 1– 3 log10 cycles of both pathogens in combination with HHP. A similar level of inactivation of E. coli O157:H7 was achieved at both pHs by most CCs and EOs in combination with HHP. However, the effectiveness of the combined processes against L. monocytogenes was higher at pH 4.0 than at pH 7.0 at the assayed conditions. In fact, most of the combined processes with CCs or EOs showed a similar effectiveness in inactivating both E. coli O157:H7 and L. monocytogenes at pH 4.0, but at pH 7.0 the observed synergistic effects were similar or higher against E. coli O157:H7 than against L. monocytogenes. The ester α-terpinyl acetate showed the highest difference (about 2.5 log10 cycles) between the inactivation degrees achieved by E. coli O157:H7 and L. monocytogenes at pH 7.0. The inactivation of L. monocytogenes by the alcohol linalool was also highly influenced by the pH, being the major effect obtained at acid pH. Fig. 2 shows the effect of storage at 4 °C for 24 h after exposure to HHP alone and to the combined treatments. As a control, samples treated with HHP alone and stored at 4 °C for 24 h achieved 1.9 log10 cycles of inactivation of both microorganisms at pH 4.0, and 0.8 log10 cycles of E. coli O157:H7 cells and 1.4 log10 cycles of L. monocytogenes cells at pH 7.0. After storage under refrigeration, combined processes with most compounds achieved an inactivation of 5 log10 cycles with E. coli O157:H7 at both pHs and with L. monocytogenes at pH 4.0. The maximum extra inactivation when compared to sampling without subsequent storage under refrigeration was 3 log10 cycles with C. longus at pH 4.0.

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Limonene and citrus fruit EOs were selected to be further studied in apple and orange juices due to their citrus-like flavour and their high effectiveness in combination with HHP. Out of the two bacteria tested, E. coli O157:H7 was selected for the rest of the determinations due to its relevance in juice-associated outbreaks (Parish, 2009) and its higher pressure resistance in comparison with L. monocytogenes. Fig. 3 shows the survival curves for E. coli O157:H7 for 20 mintreatments at different pressure values in absence of EOs or CCs, or in presence of (+)-limonene, C. sinensis L. or C. reticulata L. EOs in orange and apple juices. In both juices, the HHP treatment at 300 MPa inactivated less than 0.5 log10 cycles of the initial population. At this same pressure, C. lemon L. EO was discarded due to its low effectiveness, inactivating less than 2 log10 cycles of the initial population in combination with HHP. The addition of 200 μL/L of C. sinensis L. or C. reticulata L. EOs resulted in the inactivation of about 3 extra log10 cycles in orange juice (Fig. 3A), and 1.5–2 extra log10 cycles in apple juice (Fig. 3B), while the addition of (+)-limonene was able to inactivate 5 or more log10 cycles of the initial cell population in apple juice or orange juice, respectively. Increasing the pressure intensity maintained or increased this synergistic effect: for example, in orange juice, increasing the pressure up to 400 MPa in combination with C. sinensis L. or C. reticulata L. EOs achieved the inactivation of about 5 log10 cycles of E. coli O157:H7 in comparison with 1.5 log10 cycles of inactivation with HHP alone. In apple juice, on the other hand, 2 extra log10 cycles of inactivation were achieved at 400 MPa when adding C. sinensis L. or C. reticulata L. EO. The inactivation of 5 log10 cycles of the population in absence of EOs was achieved at 550 MPa in both juices. Since (+)-limonene was the most effective compound in combination with HHP and it is the major CC present in citrus fruit EOs (Espina et al., 2011; Fisher and Phillips, 2008), it was selected to

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Fig. 2. Inactivation by combined treatments of HHP plus pure compounds or essential oils and after storage at 4 °C for 24 h. E. coli O157:H7 ( , ) or L. monocytogenes EGDe ( , ) cells (initial concentration of 3 · 107 CFU/mL) were exposed to HHP plus 200 μL/L of pure compound (A) or essential oil (B) in McIlvaine's buffer of pH 4.0 ( , ) or 7.0 ( , ). Pure compounds (A) were: (+)-limonene (1); carvacrol (2); α-terpinyl acetate (3); linalool (4); (−)-β-pinene (5); (+)-pulegone (6); p-cymene (7); (−)-terpinen-4-ol (8); α-pinene (9); thymol (10); 1,8-cineole (11); (−)-borneol (12); and camphor (13). Essential oils (B) were: C. reticulata L. (1); T. algeriensis L. (2); C. sinensis L. (3); C. lemon L. (4); M. pulegium L. (5); R. officinalis L. (6); J. phoenicea L. (7); E. globulus L. (8); and C. longus L. (9). High hydrostatic pressure treatments were 20 min long at 300 MPa (E. coli O157:H7 at pH 4.0), 400 MPa (E. coli O157:H7 at pH 7.0), 175 MPa (L. monocytogenes at pH 4.0), or 325 MPa (L. monocytogenes at pH 7.0). The error bars in the figures indicate the standard deviations of the means for data obtained from three independent experiments.

L. Espina et al. / International Journal of Food Microbiology 161 (2013) 23–30

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study the implication of the sublethal injury in the inactivation achieved by the combined process in each treatment medium. Fig. 4 shows the survival curves for E. coli O157:H7 at a constant pressure (300 MPa) in McIlvaine buffer of pH 4.0, orange juice and apple juice after recovery in non selective (TSAYE) or selective (TSAYE-SC and TSAYE-BS) media, as well as the survival curves of combined treatments with 200 μL/L of (+)-limonene and after recovery in TSAYE. In McIlvaine buffer, the inactivation of 5 log10 cycles of the initial population was achieved before 15 min of the combined treatment. On the other hand, in orange and apple juices, the inactivation rate after the combined process was similar to the inactivation rate after HHP and recovery in selective media (Fig. 4B and C): no statistically significant differences were found in the times to reach 5 log10 cycles of inactivation (p > 0.05). These figures show that this level of inactivation after the combined process in both juices was achieved after 20 min of treatment. Since common total contamination levels in fruit juices range from 10 3 to 10 5 CFU/mL (Stratford et al., 2000), the initial contamination of E. coli O157:H7 was decreased from 3 · 10 7 to 3 · 10 4 CFU/mL in an attempt to reproduce more realistic conditions. As the antimicrobial molecule concentration needed to inactivate bacterial cells is related to the microbial contamination level (Somolinos et al., 2010; Stratford et al., 2000), a decrease in the initial cell contamination would be expected to diminish the amount of (+)-limonene needed. Fig. 5 shows inactivation curves in orange and apple juices with different concentrations of (+)-limonene at an initial E. coli O157:H7 concentration of 3 · 104 CFU/mL. Survival was also determined with a higher initial cell concentration of 3 · 107 CFU/mL with 200 μL/L of (+)-limonene. Comparison of the survival curves shows that 150 μL/L

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Fig. 3. Log10 cycles of inactivation of E. coli O157:H7 (initial concentration: 3·107 CFU/mL) by HHP for 20 min at different intensities without (○) or with 200 μL/L of C. sinensis L. essential oil (■), C. reticulata L. essential oil (▼), C. lemon L. essential oil (♦) or (+)limonene ( ) in orange juice (A) or apple juice (B). The horizontal dotted lines indicate the detection limit. The error bars in the figures indicate the standard deviations of the means for data obtained from three independent experiments.

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Treatment time (min) Fig. 4. Survival curves for E. coli O157:H7 (initial concentration: 3 ·107 CFU/mL) after HHP (300 MPa for 20 min) acting alone (○) or with 200 μL/L of (+)-limonene ( ) recovered in TSAYE. Cells after HHP treatments alone were also recovered in TSAYE-SC (□) and TSAYE-BS (Δ). Treatments were applied in McIlvaine buffer of pH 4.0 (A), orange juice (B) and apple juice (C). The error bars in the figures indicate the standard deviations of the means for data obtained from three independent experiments.

of (+)-limonene gave the same inactivation rate at the lower E. coli O157:H7 concentration as 200 μL/L did at the higher E. coli O157:H7 concentration in both juices. For each juice, no significant differences were found between the slopes of the survival curve at an initial cell concentration of 3 · 107 CFU/mL and with the addition of 200 μL/L and the survival curve at 3 · 10 4 CFU/mL and 150 μL/L (p >0.05). 4. Discussion In synergistic combinations of two hurdles, the overall inactivating effect surpasses the sum of the inactivation achieved by each hurdle acting alone, as explained by the hurdle theory (Leistner and Gorris, 1995). In previously published studies, mild thermal treatments in

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Treatment time (min) Fig. 5. Inactivation of Escherichia coli O157:H7 cells at 300 MPa in orange juice (A) or apple juice (B) with (+)-limonene. Survival curves in grey: an initial population of 3 · 104 CFU/mL was treated with 0 ( ), 10 ( ), 20 ( ), 50 ( ), 100 ( ) or 200 ( ) μL/L. Survival curves in black (selected concentration): an initial population of 3 · 104 CFU/mL was treated with 150 μL/L of (+)-limonene (●), and an initial population of 3 · 107 CFU/mL was treated with 200 μL/L of (+)-limonene (Δ). The horizontal dotted lines indicate the detection limit for an initial concentration of 3 · 104 CFU/mL. The error bars in the figures indicate the standard deviations of the means for data obtained from three independent experiments.

presence of 200 μL/L of carvacrol, linalool, citral or citrus fruit EOs inactivated more than 5 log10 cycles of E. coli O157:H7 in buffers (Ait-Ouazzou et al., 2011a; Espina et al., 2010, 2011), and this outstanding synergistic effect was attributed to the inactivation by the chemical compounds of cells sublethally injured by the thermal treatment. In the present work, HHP treatments were established to inflict a minimal degree of inactivation but maximum sublethal damage to treated cells. Since E. coli O157:H7 and L. monocytogenes showed different sensitivity to HHP at both tested pHs, a different pressure level was selected for each microorganism and each pH (Table 1). The higher inactivation of cells by HHP treatments at acid pH is in concordance with previous studies showing that a reduction in the pH of the suspending medium causes a progressive increase in cell sensitivity to pressure (Mackey et al., 1995; Pagan et al., 2001). Although Gram-negative bacteria are generally more sensitive than Gram-positive bacteria to HHP (Alpas et al., 1999; Mackey et al., 1995), the high variance among strains (Alpas et al., 1999; Pagan and Mackey, 2000) and the intrinsic sensitivity of each species can account for the lower resistance of the tested L. monocytogenes strain compared with E. coli O157:H7. Synergism was observed in combinations of HHP with the majority of the tested CCs or EOs (Fig. 1). Furthermore, for most CCs and EOs, similar effects were achieved at each pH and against two different microorganisms. This may be related to the occurrence of a similar degree of sublethal damage for the four assayed conditions, while

specific mechanisms of inactivation of each compound would determine their global effectiveness. However, some compounds showed different inactivation degrees as a function of the pH or the microorganism investigated, so a deeper study on the mechanism of inactivation of each CC would help understand the relationship between occurrence of sublethal damage and the inactivation degree achieved at each treatment condition, as well as the influence of the type of microorganism and the treatment medium. No relationship was found between the chemical structure of CCs and the degree of inactivation achieved with the combined process (Fig. 1A). For example, there was great variation between the effect of the combined process with carvacrol (which surpassed the detection limit) and with thymol (one of the least effective CCs), when these compounds have shown similar antibacterial activity (Botelho et al., 2007; Xu et al., 2008) and only differ in the position of the hydroxyl group in their phenolic rings. A similar phenomenon was found when studying the combination of carvacrol or thymol with pulsed electric fields (PEF) where a synergistic effect was seen only with carvacrol (Ait-Ouazzou et al., 2011a). Concerning the hydrocarbon monoterpenes, the inactivating effect achieved with limonene in combination with HHP exceeded by more than 2 log10 cycles for each microorganism and pH that of p-cymene, the latter being a product of the dehydrogenation of limonene. Combinations of HHP with CCs containing an alcohol group reached different degrees of inactivation: for example, the inactivation of L. monocytogenes at pH 4.0 varied from about 1 log10 cycle with borneol to more than 5 log10 cycles with linalool. Although the antimicrobial properties of alcohols are known to increase with molecular weight (Morton, 1983), the molecular weight of the three alcohols was the same, so the presence and position of specific molecular groups is expected to dramatically influence the bactericidal action of each CC in combination with HHP. Regarding the combined processes with EOs, the observation that citrus fruit EOs were among the most effective ones was in concordance with limonene being the major CC in their composition (Espina et al., 2011). Concentrations of (+)-limonene under 200 μL/L did not achieve the inactivation of 5 log10 cycles of the initial E. coli and L. monocytogenes populations in combination with HHP (data not shown), so this would fit with C. lemon L. EO, the citric EO with the least amount of limonene (Espina et al., 2011), showing the smallest bactericidal effect in the combined processes at pH 7.0. However, C. reticulata L. EO surpassed C. sinensis L. EO's inactivating effect against L. monocytogenes at pH 7.0 despite having a minor amount of limonene. Since interactions between the components of an EO are known to play a role in the overall activity of an EO (Dorman and Deans, 2000), this result could be related to the presence of other compounds in these EOs, which would either enhance (+)-limonene's action in C. reticulata L. EO or diminish its activity in C. sinensis L. EO. Previous work in our research group had reported that oxygenated monoterpenes could be responsible for the greater antibacterial activity of C. reticulata L. EO when compared with that of C. sinensis L. and C. lemon L. EOs (Espina et al., 2011), so similar interactions between CCs could occur in the combination with HHP. More research is needed to determine the influence of the chemical composition of CCs, as well as the effect of varying proportions of different CCs, on the efficacy of CCs and EOs, respectively, in inactivating microorganisms in combination with technologies such as HHP. Storage of the treated samples under refrigeration resulted in inactivation of up to 3 extra log10 cycles when compared with the inactivation achieved right after the combined treatments (Fig. 2). Previous work with HHP and citral also resulted in an enhancement of the inactivation after holding the treated samples at refrigeration temperatures (Somolinos et al., 2008). Inactivation of sublethally injured cells could occur during this holding time, similarly to that observed by García et al. (2005) after PEF treatment and storage at acid conditions. Considering that all foods pasteurized by any technology require storage under refrigeration during their shelf life, the

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effect of this storage on the inactivation of treated cells might be taken into account when designing preservation processes. Studies of HHP treatments have usually shown that inactivation of bacteria is more difficult in a food than in a water or buffer system (Ogihara et al., 2009; Patterson et al., 1995), since nutritionally rich media contain substances which may provide protection against damage or nutrients essential for repair (Hoover et al., 1989). In the present study, the degree of inactivation and the occurrence of sublethal injury did not differ between the buffer system of pH 4.0 and both juices after HHP treatments at 300 MPa (Fig. 4): no statistically significant differences were found in the level of inactivation achieved after 20 min for each recovery medium when comparing different treatment media (p > 0.05). However, differences were found in the inactivation achieved after the combined processes as a function of the media used: a faster inactivation rate of E. coli O157:H7 in the combined process with (+)-limonene was observed in buffer when compared to both juices (Fig. 4). As with HHP, nutrients are also known to protect the bacteria from the action of the EOs (Burt, 2004), so greater concentrations of EOs are required to achieve the same effect in foods as in buffer systems. Since the inactivation achieved by the combined processes with citrus fruit EOs was greater in orange juice than in apple juice (Fig. 3), influence of the specific composition of each juice on the overall activity of each combined process is an important factor to consider when designing these preservation treatments. When treating with HHP alone, it was necessary to increase the pressure level up to 550 MPa to obtain the same inactivation degree as achieved at 300 MPa in presence of 200 μL/L of (+)-limonene in both juices (Fig. 3). As a consequence, a decrease of 250 MPa in the pressure intensity would result in lower operating costs when applying preservation treatments (Malinowska-PaDczyk and KoBodziejska, 2009). In orange juice, a decrease of 150 MPa would also be possible with the addition of 200 μL/L of Citrus sinensis L. or C. reticulata L. EOs (Fig. 3). Working with 3 · 10 7 CFU/mL allowed for the detection of a 5-fold decrease in the initial E. coli O157:H7 population, in order to meet FDA's recommendation that juices should be higienized by the inactivation of a theoretical 5-log10 in the population of the pertinent pathogen (FDA, 2001). However, when considering a more realistic initial contamination, the required concentration of (+)-limonene to achieve the desired 5-fold decrease of E. coli O157:H7 could be reduced to 150 μL/L (Fig. 5). The concentration of EOs or CCs tested in this study, 200 μL/L (0.020% v/v), was much lower than the concentrations used in other studies on the preservation of fruit juices, either acting alone or in combination with other hurdles. For example, exposure to 0.60% (w/v) of vanillin was applied to reduce L. monocytogenes and E. coli O157:H7 populations in apple juice, respectively (Corte et al., 2004; Moon et al., 2006). Such low concentrations are likely to be tolerated in fruit juices, as reported in other studies: concentrations ≤ 0.015% of carvacrol and ≤0.020% of citrus, mandarin or lemon oils did not affect the sensory attributes of tested fruit juices (Raybaudi-Massilia et al., 2009). Further research could be done on the possibility of increasing effectiveness by combining low concentrations of mixtures of the tested EOs or CCs with HHP in order to control foodborne pathogens in other fruit juices or liquid foods. Besides, most of the tested CCs and EOs are included in the FDA's GRAS (Generally Recognized as Safe) lists (FDA, Revised 2012), so their addition to food would be permitted if they are used in the minimum quantity required to produce their intended effect. In summary, combinations of 200 μL/L of several EOs and CCs with HHP were tested for their effectiveness in the inactivation of E. coli O157:H7 and L. monocytogenes (initial concentration: 3 · 10 7 CFU/mL) in buffers of pH 4.0 and 7.0. Some compounds, such as (+)-limonene, carvacrol, C. reticulata L. EO, T. algeriensis L. EO or C. sinensis L. EO inactivated about 4–5 log10 cycles of the initial population at all the assayed conditions, showing an important synergistic effect when

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compared to each hurdle acting alone. In apple and orange juices, combined treatments of HHP (300 MPa for 20 min) with C. sinensis L. or C. reticulata L. EOs reached 1.5–2 extra log10 cycles of E. coli O157:H7 inactivation in apple juice and 2.5 extra log10 cycles in orange juice, and increasing the pressure up to 400 MPa achieved the same extra number of inactivation log10 cycles in each juice. The combination of HHP (300 MPa for 20 min) with 200 μL/L of (+)-limonene in orange or apple juice achieved a 5-log10 reduction in the initial E. coli O157: H7 concentration, and this outstanding synergistic effect was related to the occurrence of sublethally injured cells after HHP processing. Furthermore, reduction of this (+)-limonene concentration to 150 μL/L for inactivation of E. coli O157:H7 was possible when considering an initial contamination level of E. coli O157:H7 of 3 · 104 CFU/mL. 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