Synergistic combination of heat and ultrasonic waves under pressure for Cronobacter sakazakii inactivation in apple juice C. Arroyo, G. Cebrián, R. Pagán, S. Condón * Tecnología de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, C/ Miguel Servet, 177, 50013 Zaragoza, Spain article info Article history: Received 27 July 2011 Received in revised form 19 October 2011 Accepted 26 October 2011 Keywords: Hurdle technology Nonthermal technologies Ultrasound Food pasteurization Food preservation abstract The combined effect of the simultaneous application of heat and ultrasonic waves under pressure (manothermosonication, MTS) on the survival of a strain of Cronobacter sakazakii was studie d in apple juice. Below 45  C, the inactivation by ultrasound under pressure was independent of temperature. Above 64  C, the lethal effect of ultrasound under pressure was negligible when compared to the lethality of the heat treatment at the same temperature. Between 45  C and 64  C, the lethality of the combined process (MTS) was higher than expected if heat and ultrasound under pressure processes acted simul- taneously but independently, that is, a synergistic effect was observed. The maximum synergistic effect (38.2%) was found at 54  C. Recovery on selective media e with sodium chloride or bile salts e revealed that a certain proportion of the survivors after MTS treatments were sublethally injured. It was also observed that survivors after MTS treatments progressively died during refrigerated storage (up to 96 h at 4  C) in the apple juice. The practical implication of these findings is discussed. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Ultrasound treatment for food preservation is receiving a great deal of attention as an appealing alternative to the traditional heat processing offoods, which often may have negative side effectssuch as changes on the sensorial and nutritional properties of food (FDA, 20 00). Research into the application of ultrasound processing for food preservation began when Chambers and Gaines (1932) managed to inactivate 80% of the bacterial flora of raw milk, making feasible ultrasonic pasteurization treatments. Nonetheless, ultrasound lacks the power and versatility to inactivate a sufficient number of microorganisms reliably for purposes of food preserva- tion. Its low lethality on microorganisms, especially spore-formers, the reduced information related to microbial inactivation in foods, and the unavailability of suitable equipment hampered early applications of ultrasound for sanitation purposes. However, a number of combinations have been proposed to increase its lethality and, thus, enable the transfer of this technology to the industry for the development of minimally processed foods. Among them, probably the most promising ones are the combination of ultrasound with pressure (referred to as manosonication, MS), with temperature (thermosonication) or with both simultaneously (manothermosonication, MTS) (Chemat, Huma, & Khan, 2011; Condón, Raso, & Pagán, 2005; Sala, Burgos, Condón, López, & Raso, 1995). The combination of ultrasound and heat to achieve a high degree of bacterial inactivation was first reported by Ordóñez, Aguilera, García, and Sanz (1987) and since then, it has been studied by several authors (Adekunte et al., 2010; Álvarez, Mañas, Sala, & Condón, 2003; Baumann, Martin, & Feng, 2005; Ciccolini, Taillandier, Wilhem, Delmas, & Strehaiano, 1997; D’Amico, Silk, Wu, & Guo, 2006; Guerrero, López-Malo, & Alzamora, 2001; Lee, Zhou, Liang, Feng, & Martin, 2009; Pagán, Mañas, Palop, & Sala, 1999; Pagán, Mañas, Raso, & Condón, 1999; Raso, Pagán, Condón, & Sala, 1998; Raso, Palop, Pagán, & Condón, 1998; Zenker, Heinz, & Knorr, 2003). In these works, researchers demonstrated that when ultrasound was employed, both at lethal and sublethal tempera- tures, an increase in the inactivation rate occurred; and some of them reported an effect much greater than the additive effect of the two treatments considered independently. Nevertheless, there are still many aspects that are not fully known, including the resistance of many pathogenic microorganisms, the influence of environ- mental factors on the lethality of the process, the mechanisms leading to microbial inactivation and the effect of this process on enzymes and nutritive and sensorial properties of foods. Further work should be carried out in order to fully elucidate these points, which will leadto an efficientdesign of theprocesses and willenable the definitive transfer of this technology to the industry. Cronobacter sakazakii is an emerging foodborne pathogen that has increasingly gained the interest and concern of regulatory agencies, health care providers, the scientific community, and the * Corresponding author. Tel.: þ34 976 761581; fax: þ34 976 761590. E-mail address: scondon@unizar.es (S. Condón). Contents lists available at SciVerse ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.10.056 Food Control 25 (2012) 342e348 food industry because of its potential impact on human health (Chang, Chiang, & Chou, 2009). While infections caused by this species have predominantly involved neonates and infants less than one year of age, C. sakazakii has caused diseases in all age groups (FAO/WHO, 2004). Although most of the outbreaks caused by this species have been reported as being associated with powdered infant formula, C. sakazakii has been isolated in food or food products other than powdered infant formula (Baumgartner, Grand, Liniger, & Iversen, 2009; Friedemann, 2007; Turcovský, Kuniková, Drahovská, & Kaclíková, 2011). Therefore, its presence on or in foods poses some level of safety risk not only to neonates and infants but also to immunocompromised consumers (Beuchat et al., 2009). A wide range of environmental sources, beverages, and several foods e many of which are not subjected to processes that will inactivate the pathogen e have been found to be contaminated by C. sakazakii. According to Iversen and Forsythe (2003), soil, water, and vegetables may be the principal sources of C. sakazakii contamination. To the knowledge of the authors, the inactivation of C. sakazakii by ultrasound at different temperatures has only been studied in one food product: reconstituted powdered infant formula (Adekunte et al., 2010; Arroyo, Cebrián, Pagán, & Condón, 2011a). Information related to the combined effect of heat and ultrasound on the inactivation of C. sakazakii in other food products has not been reported. Vegetable acidic products, such as juices, are among the prod- ucts for which ultrasound processing has been proposed as an alternative to heat. Although ultrasound alone would hardly be capable of inactivating bacterial spores, the acidic pH of these products would hamper their germination, thus extending their shelf lives. In this study, we have examined the efficacy of ultra- sound under pressure treatments combined with heat for the inactivation of C. sakazakii, a microorganism which seems to be more acid-tolerant than most closely related enteric pathogens (Dancer, Mah, Rhee, Hwang, & Kang, 2009), inoculated into apple juice. The occurrence of sublethal damage and the possibility of its exploitation have also been explored. 2. Materials and methods 2.1. Microorganism, growth conditions, and treatment media C. sakazakii CECT 858 (ATCC Type strain 29544) was supplied by the Spanish Type Culture Collection (CECT, Valencia, Spain). During this investigation, the culture was maintained at À80  C in cryo- vials. Frozen stock cultures were activated by surface spreading onto Oh & Kang (OK) agar plates (Vitaltech Ibérica S.L., Spain) and incubated for 24 h at 37  C(Oh & Kang, 2004). A broth subculture was prepared by inoculating a flask containing 10 mL of fresh Tryptone Soya Broth (Biolife, Milan, Italy), supplemented with 0.6% yeast extract (w/v) (Biolife) (TSBYE), with one of the colonies iso- lated as described above. After inoculation, the flask was incubated overnight at 30  C in a rotary shaker at 150 rpm. Flasks containing 50 mL of fresh TSBYE were inoculated with the overnight subcul- ture to a concentration of 5  10 4 CFU/mL, and then incubated under agitation for 24 h at 30  C to reach the stationary growth phase with a final concentration of approximately 5  10 9 CFU/mL. C. sakazakii resistance to ultrasound under pressure in combi- nation with heat was studied in commercially sterilized apple juice (pH 3.4, a w > 0.99) (Alcampo, S.A., Spain), which was purchased from a local market in Zaragoza, Spain. 2.2. MS/MT S treatments MS/MTS treatments were carried out in a specially designed resistometer previously described (Raso, Pagán et al., 1998). In this investigation, a 450 W Branson Digital Sonifier Ò ultrasonic gener- ator (Branson Ultrasonics Corporation, Danbury, Connecticut, USA) with a constant frequency of 20 kHz was used. Survival curves to ultrasound treatments were obtained at different temperatures ranging from 35  Cto64  C, at constant peak-to-peak amplitude (117 m m) and constant gauge pressure (200 kPa). The power input (W) into the treatment medium was 5 W/mL. Temperature control during the experiments was achieved by dissipating excess heat evolved during sonication by circulating cool water through the cooling coil. The temperature of treatment medium was continu- ously monitored by a thermocouple (NiCreNi sensor class 1, ref. FTA05L0100, ALMEMO Ò , Ahlborn, Germany), which was insulated with heat-resistant silicone to ensure a constant target temperature value (Æ0.2  C). Once temperature, pressure, and amplitude had attained stability, 0.2 mL of an adequately diluted cell suspension were injected into the 23-mL treatment chamber containing the apple juice to a final concentration of approx. 3  10 5 CFU/mL. During treatment, 0.1 mL samples were collected at preset intervals and immediately pour-plated and incubated. 2.3. Heat treatments Heat treatments were carried out in a thermoresistometer TR- SC, as previously described by Condón, Arrizubieta, and Sala (1993). Survival curves to ultrasound treatments were obtained at different temperatures ranging from 45  Cto64  C. Once the preset temperature had attained stability (Æ0.05  C), 0.2 mL of an adequately diluted cell suspension were inoculated into the treat- ment chamber containing the apple juice (300 mL) to a final concentration of approx. 2  10 5 CFU/mL. After inoculation, 0.1 mL samples were collected at different times and immediately pour plated and incubated. Heat resistance displayed by bacteria was the same when using either the MTS or TR-SC equipment (data not shown). Considering ease of handling, thermal treatments were carried out in the TR-SC. 2.4. Incubation of treated samples and colony counting Tryptone Soya Agar (Biolife) supplemented with 0.6% yeast extract (TSAYE) used as a non-selective medium was added to the treated samples placed onto Petri dishes, and then incubated at 35  C for 24 h. Previous experiments demonstrated that longer incubation times did not change the viable counts (data not shown). The sublethal damage of C. sakazakii cells after the treat- ments was evaluated by comparing the counts grown on TSAYE with the counts grown on TSAYE supplemented with 5% (w/v) sodium chloride (Probus, Barcelona, Spain) (TSAYE-SC) and on TSAYE supplemented with 0.3% (w/v) bile salts (Biolife) (TSAYE-BS). These percentages of sodium chloride and bile salts were the maximum concentrations that did not affect the growth of healthy cells (data not shown). The loss of tolerance to the presence of sodium chloride is attributed to loss of osmotic functionality and/or integrity of the cytoplasmic membrane, whereas cells become sensitized and thus, unable to grow on selective media containing bile salts if the outer membrane is damaged (Mackey, 2000; Thanassi, Cheng, & Nikaido, 1997). The physiological basis of increased sensitivity to sodium chloride or bile salts in sublethally injured cells is thus complex but is used here as an indication of cytoplasmic and outer membrane “damage”, respectively. Samples recovered in the selective media TSAYE-SC or TSAYE-BS were incubated for 48 h. Longer incubation times did not influence the viable counts (data not shown). After incubation, viable colonies were enumerated with an Image Analyzer Automatic Colony Counter (Protos, Synoptics, Cambridge, UK) as described elsewhere (Condón, Palop, Raso, & Sala, 1996). C. Arroyo et al. / Food Control 25 (2012) 342e348 343 2.5. Curve fitting, resistance parameters, and statistical analyses Survival curves were obtained by plotting the log 10 number of survivors versus the treatment time (min). Under heat treatments, curves showing a concave downward profile (presence of a shoulder) were observed. Therefore, a mathematical model based on the Weibull distribution was used to fit the survival curves. This model is described by the following equation (Mafart, Couvert, Gaillard, & Leguerinel, 2002): Log 10 SðtÞ¼ðÀt= d Þ r (1) where S(t) is the survival fraction, t is the treatment time (min), d value is the scale parameter or the time for the first decimal reduction, and r value is the shape parameter, which indicates the profile of the survival curve ( r < 1 for concave upward curves, r ¼ 1 for linear curves, and r > 1 for concave downward curves). Decimal reduction time (DRT) curves were obtained by plotting the log 10 time to inactivate the 1 st ( d values), 2 nd ,3 rd ,and4 th log cycle of inactivation versus the treatment temperature. z 1 ,z 2 , z 3 ,andz 4 values (  C) represent the temperature increase required for a 1Àlog 10 decrease in the time to inactivate the 1 st ,2 nd ,3 rd ,and4 th log cycle of inactivation,respectively;andarededuced fromtheregression line of their corresponding DRT curves. To fit the model to the experimental data and to calculate d and r values, GraphPad PRISM Ò 4.1 software (GraphPad Software, Inc., San Diego, CA, USA) was used. Experiments were conducted in triplicate on independent working days, and the standard deviations are given in the figures as error bars. Regarding statistical analyses, t-tests were performed with the same software and differences were considered significant for a p 0.05. 3. Results 3.1. Kinetics of inactivation Table 1 includes the values for the scale and shape parameters from the fitting of the Mafart equation to the survival curves to heat and ultrasound (MS and MTS) obtained in this study. Root mean square error (RMSE) and determination coefficient (R 2 ) values are also included to show the fitting’s accuracy. As can be observed, the survival curves of C. sakazakii cells to heat in apple juice showed a downward concavity ( r > 1). By contrast, all the survival curves to MS/MTS treatments showed a linear profile ( r z 1). 3.2. C. sakazakii resistance to heat, MS, and MTS in apple juice Fig.1 shows the C. sakazakii inactivation rates by heat ( d T values) and ultrasound under pressure at non-lethal ( d MS values) and lethal temperatures ( d MTS values) in apple juice. As can be seen, the resistance of C. sakazakii cells to heat decreased with temperature. An exponential relationship between d values and temperature (T) was found, and a z 1 value of 6.6  C (standard error ¼ 0.14) was deduced. Therefore, an increase in temperature of 6.6  Cwas necessary to reduce the d value by ten-fold when C. sakazakii was heat treated in apple juice. As concave downward profiles are found for survival curves to heat, representing the d values (time for the first decimal reduction) against temperature might not be repre- sentative for the following log cycles of inactivation. Therefore, the influence of temperature on the time for the 2 nd ,3 rd and 4 th log cycle of inactivation was also studied (data not shown). A similar exponential relationship between the variables was found, with z 2 , z 3 , and z 4 values of 6.6  C, 6.5  C, and 6.5  C, respectively (p > 0.05). Regarding the MS/MTS processes, the lethality of ultrasound treatments remained near constant below 45  C(p > 0.05). Above this temperature, the MS process would become a MTS process. In other words, below this temperature, the lethality of the process would only be caused by the effect of ultrasound, and above 45  C, the lethality of the process would result from the combination of the lethality of both technologies. Hence, over 45  C, the lethality of MTS quickly increased with temperature. For instance, raising the treatment temperature from 35  Cto60  C caused an 8.5-fold decrease in the d value (Fig. 1, Table 1). If we compare the DRT curve of heat with the DRT curve of ultrasound treatments (Fig. 1), it can be seen that the combined process (MTS) is more efficient on reducing microbial population than heat acting alone. For instance, whereas 0.86 min are needed under a heat treatment at 56  C for inactivating 90% of the C. sakazakii population, the same level of inactivation can be ach- ieved after 0.28 min of MTS treatments at the same temperature. Therefore, a 3-fold reduction of treatment time can be obtained (Fig. 1, Table 1). In order to determine whether this increase in lethality by MTS processes over heat processes was due to an additive effect (the lethality of the combined process is the sum of the inactivation rates of heat and ultrasound treatments acting simultaneously but individually) or to a synergistic effect (the lethality of the combined process is higher than the expected for heat and ultrasound treat- ments acting simultaneously but individually), the experimental MTS-DRT curve (Fig. 1) was compared with the corresponding Table 1 Heat and MS/MTS resistance parameters ( d and r values) from the fitting of the Weibull equation to the survival curves of C. sakazakii cells treated in apple juice. In all cases, determination coefficient R 2 > 0.99. The asterisk ( * ) indicates the temperature at which the MS process becomes a MTS process (p 0.05). T (  C) Heat MS/MTS d value (min) mean (SD) r value mean (SD) RMSE d value (min) mean (SD) r value mean (SD) RMSE 35 nd ee0.940 (0.020) 0.94 (0.11) 0.070 45 43.57 (4.721) 1.45 (0.07) 0.053 0.782 (0.003) * 1.00 (0.03) 0.024 50 5.959 (1.149) 1.30 (0.08) 0.051 0.684 (0.090) 1.00 (0.21) 0.044 54 1.626 (0.085) 1.50 (0.04) 0.102 0.368 (0.016) 1.06 (0.07) 0.117 56 0.862 (0.080) 1.61 (0.10) 0.073 0.278 (0.046) 1.01 (0.18) 0.139 60 0.203 (0.073) 1.51 (0.52) 0.201 0.111 (0.032) 1.03 (0.10) 0.140 62 0.123 (0.047) 1.80 (0.63) 0.128 nd ee 64 0.050 (0.002) 1.76 (0.08) 0.188 0.036 (0.006) 1.04 (0.14) 0.093 T, temperature (  C), d , scale parameter (min), r , shape parameter (dimensionless), SD, standard deviation, nd, non determined, RMSE, root mean square error. Fig. 1. Influence of temperature on C. sakazakii inactivation by heat (-) and ultra- sound (C) treatments in apple juice. Data points represent the mean values of at least three independent replicates, and the error bars show the standard deviations. C. Arroyo et al. / Food Control 25 (2012) 342e348344 theoretical MTS-DRT curve. This theoretical MTS-DRT curve repre- sents the additive effect, and was obtained representing the theo- retical d MTS values against temperature. The theoretical d MTS values were calculated with the equation proposed by Raso, Pagán et al. (1998) and adapted to our resistance parameters: Theorethical d MTS value ¼ ð d T  d MS Þ ð d T þ d MS Þ (2) Since, as described before, survival curves to heat and MTS showed different profiles and, therefore, conclusions drawn from the comparison of the d values might not be applicable for the following log cycles of inactivation, the theoretical times for the 2 nd , 3 rd and 4 th log cycles of inactivation by MTS at different tempera- tures were also calculated. For this purpose, the d values to heat ( d T ) and MS ( d MS ) appearing in Eq. (2) were replaced for the times for the 2 nd ,3 rd and 4 th log cycle of inactivation e calculated with the parameters obtained from curve fitting shown in Table 1.These theoretical values were also compared to the experimental results. For each level of inactivation, the comparison of the experi- mental and theoretical MTS-DRT curves demonstrates that a synergistic effect occurs in a certain range of temperatures. Synergism for each temperature and level of inactivation was calculated as follows: % Synergism ¼ Theoretical value À Experimental value Theoretical value  100 (3) where value refers to the time to inactivate the 1 st ,2 nd ,3 rd ,or4 th log cycle of inactivation. The magnitude of the synergism observed for the different levels of inactivation and at each treatment temperature is represented in Fig. 2. As can be seen, for all levels of inactivation, in the range of temperatures from 45  Cto64  C, the lethal effect of MTS was higher than the expected for if heat and ultrasound would occur simultaneously but independently, which in turn is translated into a synergistic effect. At temperatures higher than 64  C, no advan- tages were observed by adding sonication to the heat treatment, thus the inactivating effect would be solely due to heat. The maximum synergistic effect was obtained at 54  C(Fig. 2). It is also observed that the maximum synergistic effect (38.2%) occurs for the first log cycle of inactivation and decreases with the inactivation (maximum synergistic effect for the 4 th cycle of inactivation ¼ 34%). 3.3. Occurrence of sublethal damages after heat and MTS treatments in apple juice and counts evolution during storage under refrigeration In order to explore the possibility of exploiting sublethal damages as a mean to increase the lethality of MTS treatments in apple juice, we studied the presence of sublethally damaged cells and the evolution of microbial counts during storage under refrigeration (4  C) in apple juice after MTS treatments at 54  C, the temperature at which the maximum synergism was observed. For comparison purposes, the presence of sublethally damaged cells and the evolution of counts during refrigerated storage was also studied for heat-treated cells at the same temperature (54  C) and unprocessed cells. As can be observed in Fig. 3A, MTS treatments caused sublethal damages in the cytoplasmic and outer membranes of C. sakazakii cells. Thus, recovery in the medium with sodium chloride (TSAYE- SC) and medium with bile salts (TSAYE-BS) resulted in a decrease in the d value from 0.38 min (recovery in the non-selective medium) Fig. 2. Occurrence and magnitude of the synergistic effect (%) after ultrasound treat- ments at different temperatures in apple juice. 0 1 2 3 4 -5 -4 -3 -2 -1 0 Time ( min ) Log N t /N 0 0.00 0.25 0.50 0.75 1.00 1.25 -5 -4 -3 -2 -1 0 Time (min) Log N t /N 0 A B Fig. 3. Survival curves of C. sakazakii cells to a MTS treatment (54  C, 117 m m, 200 kPa) (A), and to a heat treatment (54  C) (B). Cells were treated in apple juice and recovered in the non-selective medium TSAYE (:) and in the selective media TSAYE-SC ( D ) and TSAYE-BS ( 7 ). Data points represent the mean values of at least three independent replicates, and the error bars show the standard deviations. C. Arroyo et al. / Food Control 25 (2012) 342e348 345 to 0.21 min, a 1.8-fold decrease, and to 0.12 min, a 3.1-fold decrease, respectively. Similarly, a certain proportion of C. sakazakii cells also were sublethally damaged in their cytoplasmic and outer membranes after a heat treatment at the same temperature (Fig. 3B). A 1.7-fold and a 5.4-fold decrease in d values were found when heat-treated cells were recovered in TSAYE-SC and TSAYE-BS, respectively, when compared with those cells recovered in TSAYE. Survival counts immediately after 1 min-MTS treatment at 54  C in apple juice showed 2.7 log cycles of inactivated cells, as well as, 1 log cycle of survivors with damaged cytoplasmic membranes and more than 3 log cycles of survivors with damaged outer membranes as revealed by the survival counts in TSAYE, TSAYE-SC, and TSAYE-SB, respectively (time 0, Fig. 4A). Immediately after the treatment, MTS-treated cells were kept under refrigeration (4  C) in the apple juice for up to 96 h. This subsequent storage revealed that survivors À recovered in TSAYE À remaining after the MTS treat- ment progressively died. Thus, after 96 h of incubation in apple juice, more than 5 log cycles of C. sakazakii cells had lost their viability. Furthermore, the number of cells sensitized to sodium chloride also increased with incubation time (Fig. 4A). Heat-treated (1 min; 54  C) and unprocessed controls were also stored under the same conditions. Results indicated that the evolution of the counts e in both non-selective and the two selective media e during refrigerated storage of heat-treated cells showed the same trend that described for MTS-treated cells. Thus, up to 1.8, 3.5, and 4.5 log cycles of C. sakazakii cells were inactivated after a heat treatment followed by 96 h of incubation under refrigeration when recovered in TSAYE, TSAYE-SC, and TSAYE-SB, respectively (Fig. 4B). By contrast, when a non-treated population e control cells e was exposed to the same storage (in apple juice at 4  C for 96 h), neither inactivation nor sublethal damage was observed (data not shown). As an example, 0.48 log cycles were inactivated in apple juice by heat (1 min, 54  C), 1.1 log cycles by MS (1 min, 35  C), and 2.7 log cycles by MTS (1 min, 54  C), which implies a 71% of additional inactivation over heat and ultrasound acting independently but simultaneously. After the MTS treatment, the inactivation increased up to 5.3 log cycles upon subsequent storage under refrigeration (96 h, 4  C), whereas only 1.8 log cycles were achieved after a 1 min- heat treatment followed by the same refrigerated storage. 4. Discussion The development of combined processes with ultrasound is encouraged by the low lethality of ultrasound treatments applied alone and by economical reasons since the energetic cost is high and combinations, for instance, with heat, would significantly reduce the costs (Chemat et al., 2011; Knorr, Zenker, Heinz, & Lee, 2004). On the other hand, if heat and ultrasound are applied simultaneously, process times and temperatures can be reduced to achieve the same lethality values (Mason, Paniwnyk, & Lorimer, 1996; Villamiel, van Hamersveld, & De Jong, 1999), which would result in an extended sensory and quality shelf life (Piyasena, Mohareb, & McKellar, 2003; Zenker et al., 2003). Synergies between heat and ultrasound have been reported for microbial inactivation in neutral pH products such as milk (Arroyo et al., 2011a) and buffer of low water activity (Álvarez et al., 2003), but not for low pH media. We therefore studied the possible development of synergies in apple juice as a model of acidic pH food product, which has been proposed to be processed by ultra- sound, in C. sakazakii, a microorganism which seems to be more acid-tolerant than most closely related enteric pathogens (Dancer et al., 2009). Results here reported indicated that the combination of ultrasound under pressure with heat is synergistic for the inac- tivation of C. sakazakii cells in apple juice. The occurrence of sublethally injured cells after MTS treatments was also explored, with special emphasis on its potential exploitation for increasing the lethality of the treatments. All the survivalcurvesto MS/MTS obtained were linear, as already described, for this species when exposed to MS (Arroyo, Cebrián, Pagán, & Condón, 2011b), to MTS in buffer and milk (Arroyo et al., 2011a), and for the survival curves to MTS of other species (Álvarez et al., 2003; López-Malo, Guerrero, & Alzamora, 1999; Pagán, Mañas, Raso et al., 1999). This linear shape in MTS survival curves was also found when C. sakazakii was treated at temperatures at which survival curves to heat showed shoulders. Similar results have been observed for the same microorganism when treated in milk (Arroyo et al., 2011a) and for heat-shocked Listeria monocytogenes cells(Pagán,Mañas, Palop et al.,1999).It can be speculatedthat these differences would arise asa consequence of thedifferent mechanism of inactivation of heat and ultrasound, but further studies would be required in order to elucidate this point. Results obtained demonstrate that the resistance of C. sakazakii to ultrasound would vary as a function of the treatment tempera- ture. There are few data available in the literature concerning the influence of treatment temperature on microbial ultrasound resistance in food products of acidic pH. Moreover, of those studies in which ultrasound is applied in combination with heat, the A time 0 0,5 5 24 48 72 96 0 1 2 3 4 5 6 ** ** * ** Incubation time (h) Log 10 cycles of inactivation B time 0 24 96 0 1 2 3 4 5 6 Incubation time ( h ) Log 10 cycles of inactivation Fig. 4. (A) Log 10 cycles of C. sakazakii inactivated cells after a MTS treatment in apple juice (1 min at 54  C, 117 m m, 200 kPa; time 0) and after subsequent incubation at 4  C for up to 96 h in apple juice. Asterisks indicate more than 6 log 10 cycles of cell inac- tivation. (B) Log 10 cycles of C. sakazakii inactivated cells after a heat treatment in apple juice (1 min at 54  C; time 0) and after subsequent incubation at 4  C for up to 96 h in apple juice. Cells were recovered in the non-selective medium TSAYE (white bars) and in the selective media TSAYE-SC (gray bars) and TSAYE-BS (black bars). Error bars show the standard deviations of the mean value. C. Arroyo et al. / Food Control 25 (2012) 342e348346 number of temperatures tested is scarce and do not verify whether the effect obtained is additive or synergistic. Data accumulated over the last 15 years indicated that, in most cases, the combinationof heatand ultrasound underpressurewould have an additive effect as it has been described for L. monocytogenes in apple cider (Baumann et al., 2005), Yersinia enterocolitica (Raso, Pagán et al., 1998), Salmonella Enteritidis and Aeromonas hydro- phila (Pagán, Mañas, Rasoet al.,1999) inpH7.0 buffer, althoughsome exceptions have been reported for Bacillus subtilis (Raso, Palop et al., 1998) and Enterococcus faecium (Pagán, Mañas, Raso et al., 1999)in pH 7.0 buffer. The occurrence of an additive effect has been attrib- uted tothe different mechanism of inactivation of both technologies (Raso, Pagán etal.,1998) whereas thesynergies havebeen attributed to a sensitizing phenomena caused by heat that would render cells more sensitive to ultrasound (Álvarez et al., 2003; Condón, Mañas, & Cebrián, 2011; Pagán, Mañas, Palop et al., 1999). The occurrence of these effects would depend on the microorganism investigated, the range of temperatures, and the treatment media tested. In fact, the temperature atwhich additive orsynergistic effects wouldappear in MTS treatments would be determined by the microbial heat resis- tance. Thus, it might be expected that in media in which the heat resistance is lower, the temperatures at which these phenomena would occur would be lower, the opposite also being true. Further- more, it should be remarked that, up to date, all the conditions leading to the occurrence of synergies were coincident with condi- tions leading to an increase in heat resistance, which suggests that those factors leading to an increased heat resistance would not protect cells against ultrasound. By contrast, our results demon- strate that in acidicconditions (apple juice, pH 3.4)e where the heat resistance of C. sakazakii is reduced (Arroyo, Condón, & Pagán, 2009) e a synergistic effect can also be found. This could be due to the acidic pH or to the composition of the apple juice. In order to check whether thesynergism between ultrasound andheat for C. sakazakii inactivation does occur both at neutral and acidic pH, the heat and MTS resistance in citrate-phosphate buffers of different pH was studied and the synergism of the combination was calculated. Results obtained demonstrated that not only a synergistic effect can be found when cells are MTS-treated in acid pH media, but also that this synergism is higher in acid than in neutral pH media (see Supplementary data). The second part of this investigation was designed to explore the occurrence of sublethally injured cells after MTS treatments. Results here reported demonstrate that after MTS treatments in apple juice, a certain proportion of the C. sakazakii population were sublethally injured in their cytoplasmic and outer membranes. This finding provides an opportunity to develop other combined processes to take advantage of the sensitivity of the damaged cells to increase the lethality of treatments without raising the treat- ment intensity. Our results also show that the decrease in the d values calculated upon recovery in medium with added sodium chloride e when compared to those calculated in the non-selective medium e was similar for heat and MTS treatments, and that the decrease upon recovery in medium with added bile salts was 1.7- fold higher for MTS-treated cells than for heat-treated ones. Two relevant conclusions can be inferred from these results. On one hand, as already pointed out in Arroyo et al. (2011a), the synergistic effect obtained after combining ultrasound and heat would not be due to the lethal effect of ultrasound on cells with damaged cyto- plasmic membranes caused by heat. Similarly, the synergism observed cannot be attributed, at least solely, to the lethal effect of ultrasound on cells with damaged outer membranes caused by heat. On the other hand, these results show that MTS treatments would remain advantageous e when compared to heat e in an eventual combined process in which these damages to the inner and/or outer membranes are exploited. The study of the evolution of survival counts in refrigerated apple juice after MTS treatments was encouraged, among other reasons, because we supposed that its acidic pH would lead to the death of sublethally damaged cells caused by MTS as already observed with Escherichia coli for others technologies such as high pressure (García-Graells, Hauben, & Michiels, 1998) or pulsed electric fields (García, Hassani, Mañas, Condón, & Pagán, 2005), which would provide an additional advantage for acidic products. Results obtained indicate that C. sakazakii cells progressively died during refrigerated storage, but even upon 96 h, a certain propor- tion of cells still remained damaged in their cytoplasmic and outer membranes. Furthermore, the number of cells recovered in media with added sodium chloride also decreased with incubation time, and the number of MTS-treated cells and recovered in TSAYE after 96 h was lower than the number of cells recovered in TSAYE-SC just after the MTS treatment (time 0). All these findings indicate that, at least for C. sakazakii MTS-treated cells, damages detected by the recovery in media with added sodium chloride would not be directly related to the ability of these cells to maintain their pH homeostasis during refrigerated storage. Besides, the counts in media with added sodium chloride immediately after the treat- ment might underestimate the number of cells that would be inactivated by an adequately designed combined process. On the other hand, given the important role of the outer membrane in pH homeostasis (Booth, Cash, & O’Byrne, 2002), it can be hypothesized that the progressive inactivation of cells e both when the recovery was carried out in TSAYE and TSAYE-SC e might be due to the inability of cells with injured outer membranes to maintain pH homeostasis. Finally, from a practical point of view, our results indicate that MTS treatments might constitute an alternative to conventional thermal pasteurization treatments also in thermo-sensitive prod- ucts such as fruit juices (Char, Mitilinaki, Guerrero, & Alzamora, 2010; Ugarte-Romero, Feng, Martin, Cadwallader, & Robinson, 20 06; Valero et al., 2007; Zenker et al., 2003). Furthermore, since the acidic pH of these products would hamper the germination of spores, adequately designed MTS treatments would guarantee their safety and would extend their shelf lives, in spite of the fact that ultrasound, when applied at these temperatures, requires high amounts of energy for bacterial spore inactivation. It should also be noted that, apart from the increase in the lethality of the process, another advantage of the combined use of ultrasound and heat is that it would reduce the treatment costs when compared to ultrasound applied at non-lethal temperatures, not only because the increase in temperature would reduce the treatment time, but also because the heat dissipated by the ultrasound waves might be used to achieve the final process temperature. Further work is required in order to validate the results obtained here in other species and also to study the infl uence of MTS treatments on the organolep tic and nutritive attributes of food products. 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Synergistic combination of heat and ultrasonic waves under pressure for Cronobacter sakazakii inactivation in apple juice C. Arroyo, G. Cebrián, R. Pagán,. combined effect of the simultaneous application of heat and ultrasonic waves under pressure (manothermosonication, MTS) on the survival of a strain of Cronobacter sakazakii was studie d in apple juice. . of inactivation and decreases with the inactivation (maximum synergistic effect for the 4 th cycle of inactivation ¼ 34%). 3.3. Occurrence of sublethal damages after heat and MTS treatments in