Synergistic combination of heat and ultrasonic waves under pressure for Cronobacter sakazakii inactivation in apple juice

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Synergistic combination of heat and ultrasonic waves under pressure for Cronobacter sakazakii inactivation in apple juice has many contents: Microorganism, growth conditions, and treatment media, MS/MTS treatments, Heat treatments, Incubation of treated samples and colony counting, Kinetics of inactivation, Occurrence of sublethal damages after heat and MTStreatments in apple juice and counts evolution during storage underrefrigeration,...

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Nội dung Text: Synergistic combination of heat and ultrasonic waves under pressure for Cronobacter sakazakii inactivation in apple juice

Food Control 25 (2012) 342e348 Contents lists available at SciVerse ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont 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 a r t i c l e i n f o a b s t r a c t Article history: The combined effect of the simultaneous application of heat and ultrasonic waves under pressure Received 27 July 2011 (manothermosonication, MTS) on the survival of a strain of Cronobacter sakazakii was studied in apple Received in revised form juice. Below 45 C, the inactivation by ultrasound under pressure was independent of temperature. 19 October 2011 Above 64 C, the lethal effect of ultrasound under pressure was negligible when compared to the lethality Accepted 26 October 2011 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- Keywords: taneously but independently, that is, a synergistic effect was observed. The maximum synergistic effect Hurdle technology Nonthermal technologies (38.2%) was found at 54 C. Recovery on selective media e with sodium chloride or bile salts e revealed Ultrasound that a certain proportion of the survivors after MTS treatments were sublethally injured. It was also Food pasteurization observed that survivors after MTS treatments progressively died during refrigerated storage (up to 96 h Food preservation at 4 C) in the apple juice. The practical implication of these ﬁndings is discussed. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Condón, Raso, & Pagán, 2005; Sala, Burgos, Condón, López, & Raso, 1995). The combination of ultrasound and heat to achieve a high Ultrasound treatment for food preservation is receiving a great degree of bacterial inactivation was ﬁrst reported by Ordóñez, deal of attention as an appealing alternative to the traditional heat Aguilera, García, and Sanz (1987) and since then, it has been processing of foods, which often may have negative side effects such studied by several authors (Adekunte et al., 2010; Álvarez, Mañas, as changes on the sensorial and nutritional properties of food (FDA, Sala, & Condón, 2003; Baumann, Martin, & Feng, 2005; Ciccolini, 2000). Research into the application of ultrasound processing for Taillandier, Wilhem, Delmas, & Strehaiano, 1997; D’Amico, Silk, food preservation began when Chambers and Gaines (1932) Wu, & Guo, 2006; Guerrero, López-Malo, & Alzamora, 2001; Lee, managed to inactivate 80% of the bacterial ﬂora of raw milk, Zhou, Liang, Feng, & Martin, 2009; Pagán, Mañas, Palop, & Sala, making feasible ultrasonic pasteurization treatments. Nonetheless, 1999; Pagán, Mañas, Raso, & Condón, 1999; Raso, Pagán, Condón, & ultrasound lacks the power and versatility to inactivate a sufﬁcient Sala, 1998; Raso, Palop, Pagán, & Condón, 1998; Zenker, Heinz, & number of microorganisms reliably for purposes of food preserva- Knorr, 2003). In these works, researchers demonstrated that when tion. Its low lethality on microorganisms, especially spore-formers, ultrasound was employed, both at lethal and sublethal tempera- the reduced information related to microbial inactivation in foods, tures, an increase in the inactivation rate occurred; and some of and the unavailability of suitable equipment hampered early them reported an effect much greater than the additive effect of the applications of ultrasound for sanitation purposes. However, two treatments considered independently. Nevertheless, there are a number of combinations have been proposed to increase its still many aspects that are not fully known, including the resistance lethality and, thus, enable the transfer of this technology to the of many pathogenic microorganisms, the inﬂuence of environ- industry for the development of minimally processed foods. Among mental factors on the lethality of the process, the mechanisms them, probably the most promising ones are the combination of leading to microbial inactivation and the effect of this process on ultrasound with pressure (referred to as manosonication, MS), with enzymes and nutritive and sensorial properties of foods. Further temperature (thermosonication) or with both simultaneously work should be carried out in order to fully elucidate these points, (manothermosonication, MTS) (Chemat, Huma, & Khan, 2011; which will lead to an efﬁcient design of the processes and will enable 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 * Corresponding author. Tel.: þ34 976 761581; fax: þ34 976 761590. E-mail address: scondon@unizar.es (S. Condón). agencies, health care providers, the scientiﬁc community, and the 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.10.056
C. Arroyo et al. / Food Control 25 (2012) 342e348 343 food industry because of its potential impact on human health investigation, a 450 W Branson Digital SoniﬁerÒ ultrasonic gener- (Chang, Chiang, & Chou, 2009). While infections caused by this ator (Branson Ultrasonics Corporation, Danbury, Connecticut, USA) species have predominantly involved neonates and infants less with a constant frequency of 20 kHz was used. Survival curves to than one year of age, C. sakazakii has caused diseases in all age ultrasound treatments were obtained at different temperatures groups (FAO/WHO, 2004). Although most of the outbreaks caused ranging from 35 C to 64 C, at constant peak-to-peak amplitude by this species have been reported as being associated with (117 mm) and constant gauge pressure (200 kPa). The power input powdered infant formula, C. sakazakii has been isolated in food or (W) into the treatment medium was 5 W/mL. Temperature control food products other than powdered infant formula (Baumgartner, during the experiments was achieved by dissipating excess heat Grand, Liniger, & Iversen, 2009; Friedemann, 2007; Turcovský, evolved during sonication by circulating cool water through the Kuniková, Drahovská, & Kaclíková, 2011). Therefore, its presence cooling coil. The temperature of treatment medium was continu- on or in foods poses some level of safety risk not only to neonates ously monitored by a thermocouple (NiCreNi sensor class 1, ref. and infants but also to immunocompromised consumers (Beuchat FTA05L0100, ALMEMOÒ, Ahlborn, Germany), which was insulated et al., 2009). A wide range of environmental sources, beverages, with heat-resistant silicone to ensure a constant target temperature and several foods e many of which are not subjected to processes value (Æ0.2 C). Once temperature, pressure, and amplitude had that will inactivate the pathogen e have been found to be attained stability, 0.2 mL of an adequately diluted cell suspension contaminated by C. sakazakii. According to Iversen and Forsythe were injected into the 23-mL treatment chamber containing the (2003), soil, water, and vegetables may be the principal sources of apple juice to a ﬁnal concentration of approx. 3 Â 105 CFU/mL. C. sakazakii contamination. To the knowledge of the authors, the During treatment, 0.1 mL samples were collected at preset intervals inactivation of C. sakazakii by ultrasound at different temperatures and immediately pour-plated and incubated. has only been studied in one food product: reconstituted powdered infant formula (Adekunte et al., 2010; Arroyo, Cebrián, Pagán, & 2.3. Heat treatments Condón, 2011a). Information related to the combined effect of heat and ultrasound on the inactivation of C. sakazakii in other food Heat treatments were carried out in a thermoresistometer TR- products has not been reported. SC, as previously described by Condón, Arrizubieta, and Sala Vegetable acidic products, such as juices, are among the prod- (1993). Survival curves to ultrasound treatments were obtained at ucts for which ultrasound processing has been proposed as an different temperatures ranging from 45 C to 64 C. Once the preset alternative to heat. Although ultrasound alone would hardly be temperature had attained stability (Æ0.05 C), 0.2 mL of an capable of inactivating bacterial spores, the acidic pH of these adequately diluted cell suspension were inoculated into the treat- products would hamper their germination, thus extending their ment chamber containing the apple juice (300 mL) to a ﬁnal shelf lives. In this study, we have examined the efﬁcacy of ultra- concentration of approx. 2 Â 105 CFU/mL. After inoculation, 0.1 mL sound under pressure treatments combined with heat for the samples were collected at different times and immediately pour inactivation of C. sakazakii, a microorganism which seems to be plated and incubated. more acid-tolerant than most closely related enteric pathogens Heat resistance displayed by bacteria was the same when using (Dancer, Mah, Rhee, Hwang, & Kang, 2009), inoculated into apple either the MTS or TR-SC equipment (data not shown). Considering juice. The occurrence of sublethal damage and the possibility of its ease of handling, thermal treatments were carried out in the TR-SC. exploitation have also been explored. 2.4. Incubation of treated samples and colony counting 2. Materials and methods Tryptone Soya Agar (Biolife) supplemented with 0.6% yeast 2.1. Microorganism, growth conditions, and treatment media extract (TSAYE) used as a non-selective medium was added to the treated samples placed onto Petri dishes, and then incubated at C. sakazakii CECT 858 (ATCC Type strain 29544) was supplied by 35 C for 24 h. Previous experiments demonstrated that longer the Spanish Type Culture Collection (CECT, Valencia, Spain). During incubation times did not change the viable counts (data not this investigation, the culture was maintained at À80 C in cryo- shown). The sublethal damage of C. sakazakii cells after the treat- vials. Frozen stock cultures were activated by surface spreading ments was evaluated by comparing the counts grown on TSAYE onto Oh & Kang (OK) agar plates (Vitaltech Ibérica S.L., Spain) and with the counts grown on TSAYE supplemented with 5% (w/v) incubated for 24 h at 37 C (Oh & Kang, 2004). A broth subculture sodium chloride (Probus, Barcelona, Spain) (TSAYE-SC) and on was prepared by inoculating a ﬂask containing 10 mL of fresh TSAYE supplemented with 0.3% (w/v) bile salts (Biolife) (TSAYE-BS). Tryptone Soya Broth (Biolife, Milan, Italy), supplemented with 0.6% These percentages of sodium chloride and bile salts were the yeast extract (w/v) (Biolife) (TSBYE), with one of the colonies iso- maximum concentrations that did not affect the growth of healthy lated as described above. After inoculation, the ﬂask was incubated cells (data not shown). The loss of tolerance to the presence of overnight at 30 C in a rotary shaker at 150 rpm. Flasks containing sodium chloride is attributed to loss of osmotic functionality and/or 50 mL of fresh TSBYE were inoculated with the overnight subcul- integrity of the cytoplasmic membrane, whereas cells become ture to a concentration of 5 Â 104 CFU/mL, and then incubated sensitized and thus, unable to grow on selective media containing under agitation for 24 h at 30 C to reach the stationary growth bile salts if the outer membrane is damaged (Mackey, 2000; phase with a ﬁnal concentration of approximately 5 Â 109 CFU/mL. Thanassi, Cheng, & Nikaido, 1997). The physiological basis of C. sakazakii resistance to ultrasound under pressure in combi- increased sensitivity to sodium chloride or bile salts in sublethally nation with heat was studied in commercially sterilized apple juice injured cells is thus complex but is used here as an indication of (pH 3.4, aw > 0.99) (Alcampo, S.A., Spain), which was purchased cytoplasmic and outer membrane “damage”, respectively. Samples from a local market in Zaragoza, Spain. recovered in the selective media TSAYE-SC or TSAYE-BS were incubated for 48 h. Longer incubation times did not inﬂuence the 2.2. MS/MTS treatments viable counts (data not shown). After incubation, viable colonies were enumerated with an Image Analyzer Automatic Colony MS/MTS treatments were carried out in a specially designed Counter (Protos, Synoptics, Cambridge, UK) as described elsewhere resistometer previously described (Raso, Pagán et al., 1998). In this (Condón, Palop, Raso, & Sala, 1996).
344 C. Arroyo et al. / Food Control 25 (2012) 342e348 2.5. Curve ﬁtting, resistance parameters, and statistical analyses Survival curves were obtained by plotting the log10 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): r Log10 SðtÞ ¼ ðÀt=dÞ (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 log10 time to inactivate the 1st (d values), 2nd, 3rd, and 4th log cycle of inactivation versus the treatment temperature. z1, z2, z3, and z4 values ( C) represent the temperature increase required for a 1Àlog10 Fig. 1. Inﬂuence of temperature on C. sakazakii inactivation by heat (-) and ultra- decrease in the time to inactivate the 1st, 2nd, 3rd, and 4th log cycle of sound (C) treatments in apple juice. Data points represent the mean values of at least inactivation, respectively; and are deduced from the regression line of three independent replicates, and the error bars show the standard deviations. their corresponding DRT curves. To ﬁt the model to the experimental data and to calculate d and r values, GraphPad PRISMÒ 4.1 software temperatures (dMTS values) in apple juice. As can be seen, the (GraphPad Software, Inc., San Diego, CA, USA) was used. Experiments resistance of C. sakazakii cells to heat decreased with temperature. were conducted in triplicate on independent working days, and the An exponential relationship between d values and temperature (T) standard deviations are given in the ﬁgures as error bars. Regarding was found, and a z1 value of 6.6 C (standard error ¼ 0.14) was statistical analyses, t-tests were performed with the same software deduced. Therefore, an increase in temperature of 6.6 C was and differences were considered signiﬁcant for a p 0.05. necessary to reduce the d value by ten-fold when C. sakazakii was heat treated in apple juice. As concave downward proﬁles are found 3. Results for survival curves to heat, representing the d values (time for the ﬁrst decimal reduction) against temperature might not be repre- 3.1. Kinetics of inactivation sentative for the following log cycles of inactivation. Therefore, the inﬂuence of temperature on the time for the 2nd, 3rd and 4th log Table 1 includes the values for the scale and shape parameters cycle of inactivation was also studied (data not shown). A similar from the ﬁtting of the Mafart equation to the survival curves to heat exponential relationship between the variables was found, with z2, and ultrasound (MS and MTS) obtained in this study. Root mean z3, and z4 values of 6.6 C, 6.5 C, and 6.5 C, respectively (p > 0.05). square error (RMSE) and determination coefﬁcient (R2) values are Regarding the MS/MTS processes, the lethality of ultrasound also included to show the ﬁtting’s accuracy. As can be observed, the treatments remained near constant below 45 C (p > 0.05). Above survival curves of C. sakazakii cells to heat in apple juice showed this temperature, the MS process would become a MTS process. In a downward concavity (r > 1). By contrast, all the survival curves to other words, below this temperature, the lethality of the process MS/MTS treatments showed a linear proﬁle (r z 1). would only be caused by the effect of ultrasound, and above 45 C, the lethality of the process would result from the combination of 3.2. C. sakazakii resistance to heat, MS, and MTS in apple juice the lethality of both technologies. Hence, over 45 C, the lethality of MTS quickly increased with temperature. For instance, raising the Fig. 1 shows the C. sakazakii inactivation rates by heat (dT values) treatment temperature from 35 C to 60 C caused an 8.5-fold and ultrasound under pressure at non-lethal (dMS values) and lethal decrease in the d value (Fig. 1, Table 1). If we compare the DRT curve of heat with the DRT curve of Table 1 Heat and MS/MTS resistance parameters (d and r values) from the ﬁtting of the ultrasound treatments (Fig. 1), it can be seen that the combined Weibull equation to the survival curves of C. sakazakii cells treated in apple juice. In process (MTS) is more efﬁcient on reducing microbial population all cases, determination coefﬁcient R2 > 0.99. The asterisk (*) indicates the than heat acting alone. For instance, whereas 0.86 min are needed temperature at which the MS process becomes a MTS process (p 0.05). under a heat treatment at 56 C for inactivating 90% of the T ( C) Heat MS/MTS C. sakazakii population, the same level of inactivation can be ach- d value (min) r value RMSE d value (min) r value RMSE ieved after 0.28 min of MTS treatments at the same temperature. mean (SD) mean (SD) mean (SD) mean (SD) Therefore, a 3-fold reduction of treatment time can be obtained 35 nd e e 0.940 (0.020) 0.94 (0.11) 0.070 (Fig. 1, Table 1). 45 43.57 (4.721) 1.45 (0.07) 0.053 0.782 (0.003)* 1.00 (0.03) 0.024 In order to determine whether this increase in lethality by MTS 50 5.959 (1.149) 1.30 (0.08) 0.051 0.684 (0.090) 1.00 (0.21) 0.044 processes over heat processes was due to an additive effect (the 54 1.626 (0.085) 1.50 (0.04) 0.102 0.368 (0.016) 1.06 (0.07) 0.117 lethality of the combined process is the sum of the inactivation 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 rates of heat and ultrasound treatments acting simultaneously but 62 0.123 (0.047) 1.80 (0.63) 0.128 nd e e individually) or to a synergistic effect (the lethality of the combined 64 0.050 (0.002) 1.76 (0.08) 0.188 0.036 (0.006) 1.04 (0.14) 0.093 process is higher than the expected for heat and ultrasound treat- T, temperature ( C), d, scale parameter (min), r, shape parameter (dimensionless), ments acting simultaneously but individually), the experimental SD, standard deviation, nd, non determined, RMSE, root mean square error. MTS-DRT curve (Fig. 1) was compared with the corresponding
C. Arroyo et al. / Food Control 25 (2012) 342e348 345 theoretical MTS-DRT curve. This theoretical MTS-DRT curve repre- 3.3. Occurrence of sublethal damages after heat and MTS sents the additive effect, and was obtained representing the theo- treatments in apple juice and counts evolution during storage under retical dMTS values against temperature. The theoretical dMTS values refrigeration were calculated with the equation proposed by Raso, Pagán et al. (1998) and adapted to our resistance parameters: In order to explore the possibility of exploiting sublethal damages as a mean to increase the lethality of MTS treatments in ðdT Â dMS Þ apple juice, we studied the presence of sublethally damaged cells Theorethical dMTS value ¼ (2) ðdT þ dMS Þ and the evolution of microbial counts during storage under refrigeration (4 C) in apple juice after MTS treatments at 54 C, the Since, as described before, survival curves to heat and MTS temperature at which the maximum synergism was observed. For showed different proﬁles and, therefore, conclusions drawn from comparison purposes, the presence of sublethally damaged cells the comparison of the d values might not be applicable for the and the evolution of counts during refrigerated storage was also following log cycles of inactivation, the theoretical times for the 2nd, studied for heat-treated cells at the same temperature (54 C) and 3rd and 4th log cycles of inactivation by MTS at different tempera- unprocessed cells. tures were also calculated. For this purpose, the d values to heat (dT) As can be observed in Fig. 3A, MTS treatments caused sublethal and MS (dMS) appearing in Eq. (2) were replaced for the times for the damages in the cytoplasmic and outer membranes of C. sakazakii 2nd, 3rd and 4th log cycle of inactivation e calculated with the cells. Thus, recovery in the medium with sodium chloride (TSAYE- parameters obtained from curve ﬁtting shown in Table 1. These SC) and medium with bile salts (TSAYE-BS) resulted in a decrease in theoretical values were also compared to the experimental results. the d value from 0.38 min (recovery in the non-selective medium) 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. A 0 Synergism for each temperature and level of inactivation was calculated as follows: -1 Theoretical value À Experimental value % Synergism ¼ Â 100 Theoretical value -2 Log Nt /N0 (3) where value refers to the time to inactivate the 1st, 2nd, 3rd, or 4th log cycle of inactivation. -3 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 -4 temperatures from 45 C to 64 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 -5 a synergistic effect. At temperatures higher than 64 C, no advan- 0.00 0.25 0.50 0.75 1.00 1.25 tages were observed by adding sonication to the heat treatment, thus the inactivating effect would be solely due to heat. The Time (min) 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 4th cycle of inactivation ¼ 34%). B 0 -1 -2 Log Nt /N 0 -3 -4 -5 0 1 2 3 4 Time (min) Fig. 3. Survival curves of C. sakazakii cells to a MTS treatment (54 C, 117 mm, 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 Fig. 2. Occurrence and magnitude of the synergistic effect (%) after ultrasound treat- TSAYE-BS (7). Data points represent the mean values of at least three independent ments at different temperatures in apple juice. replicates, and the error bars show the standard deviations.
346 C. Arroyo et al. / Food Control 25 (2012) 342e348 to 0.21 min, a 1.8-fold decrease, and to 0.12 min, a 3.1-fold decrease, selective media e during refrigerated storage of heat-treated cells respectively. Similarly, a certain proportion of C. sakazakii cells also showed the same trend that described for MTS-treated cells. Thus, were sublethally damaged in their cytoplasmic and outer up to 1.8, 3.5, and 4.5 log cycles of C. sakazakii cells were inactivated membranes after a heat treatment at the same temperature after a heat treatment followed by 96 h of incubation under (Fig. 3B). A 1.7-fold and a 5.4-fold decrease in d values were found refrigeration when recovered in TSAYE, TSAYE-SC, and TSAYE-SB, when heat-treated cells were recovered in TSAYE-SC and TSAYE-BS, respectively (Fig. 4B). By contrast, when a non-treated population respectively, when compared with those cells recovered in TSAYE. e control cells e was exposed to the same storage (in apple juice at Survival counts immediately after 1 min-MTS treatment at 54 C 4 C for 96 h), neither inactivation nor sublethal damage was in apple juice showed 2.7 log cycles of inactivated cells, as well as, observed (data not shown). 1 log cycle of survivors with damaged cytoplasmic membranes and As an example, 0.48 log cycles were inactivated in apple juice by more than 3 log cycles of survivors with damaged outer heat (1 min, 54 C), 1.1 log cycles by MS (1 min, 35 C), and 2.7 log membranes as revealed by the survival counts in TSAYE, TSAYE-SC, cycles by MTS (1 min, 54 C), which implies a 71% of additional and TSAYE-SB, respectively (time 0, Fig. 4A). Immediately after the inactivation over heat and ultrasound acting independently but treatment, MTS-treated cells were kept under refrigeration (4 C) in simultaneously. After the MTS treatment, the inactivation increased the apple juice for up to 96 h. This subsequent storage revealed that up to 5.3 log cycles upon subsequent storage under refrigeration survivors À recovered in TSAYE À remaining after the MTS treat- (96 h, 4 C), whereas only 1.8 log cycles were achieved after a 1 min- ment progressively died. Thus, after 96 h of incubation in apple heat treatment followed by the same refrigerated storage. juice, more than 5 log cycles of C. sakazakii cells had lost their viability. Furthermore, the number of cells sensitized to sodium 4. Discussion chloride also increased with incubation time (Fig. 4A). Heat-treated (1 min; 54 C) and unprocessed controls were also The development of combined processes with ultrasound is stored under the same conditions. Results indicated that the encouraged by the low lethality of ultrasound treatments applied evolution of the counts e in both non-selective and the two 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, A * * * * * * * 2004). On the other hand, if heat and ultrasound are applied 6 simultaneously, process times and temperatures can be reduced to Log10 cycles of inactivation 5 achieve the same lethality values (Mason, Paniwnyk, & Lorimer, 1996; Villamiel, van Hamersveld, & De Jong, 1999), which would 4 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 3 microbial inactivation in neutral pH products such as milk (Arroyo et al., 2011a) and buffer of low water activity (Álvarez et al., 2003), 2 but not for low pH media. We therefore studied the possible development of synergies in apple juice as a model of acidic pH 1 food product, which has been proposed to be processed by ultra- sound, in C. sakazakii, a microorganism which seems to be more 0 time 0 0,5 5 24 48 72 96 acid-tolerant than most closely related enteric pathogens (Dancer et al., 2009). Results here reported indicated that the combination Incubation time (h) of ultrasound under pressure with heat is synergistic for the inac- tivation of C. sakazakii cells in apple juice. The occurrence of B sublethally injured cells after MTS treatments was also explored, 6 with special emphasis on its potential exploitation for increasing Log10 cycles of inactivation the lethality of the treatments. 5 All the survival curves to MS/MTS obtained were linear, as already described, for this species when exposed to MS (Arroyo, Cebrián, 4 Pagán, & Condón, 2011b), to MTS in buffer and milk (Arroyo et al., 2011a), and for the survival curves to MTS of other species (Álvarez 3 et al., 2003; López-Malo, Guerrero, & Alzamora, 1999; Pagán, Mañas, Raso et al., 1999). This linear shape in MTS survival curves 2 was also found when C. sakazakii was treated at temperatures at which survival curves to heat showed shoulders. Similar results have 1 been observed for the same microorganism when treated in milk (Arroyo et al., 2011a) and for heat-shocked Listeria monocytogenes 0 cells (Pagán, Mañas, Palop et al.,1999). It can be speculated that these time 0 24 96 differences would arise as a consequence of the different mechanism Incubation time (h) of inactivation of heat and ultrasound, but further studies would be Fig. 4. (A) Log10 cycles of C. sakazakii inactivated cells after a MTS treatment in apple required in order to elucidate this point. juice (1 min at 54 C, 117 mm, 200 kPa; time 0) and after subsequent incubation at 4 C Results obtained demonstrate that the resistance of C. sakazakii for up to 96 h in apple juice. Asterisks indicate more than 6 log10 cycles of cell inac- to ultrasound would vary as a function of the treatment tempera- tivation. (B) Log10 cycles of C. sakazakii inactivated cells after a heat treatment in apple ture. There are few data available in the literature concerning the 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 inﬂuence of treatment temperature on microbial ultrasound in the selective media TSAYE-SC (gray bars) and TSAYE-BS (black bars). Error bars show resistance in food products of acidic pH. Moreover, of those studies the standard deviations of the mean value. in which ultrasound is applied in combination with heat, the
C. Arroyo et al. / Food Control 25 (2012) 342e348 347 number of temperatures tested is scarce and do not verify whether The study of the evolution of survival counts in refrigerated the effect obtained is additive or synergistic. apple juice after MTS treatments was encouraged, among other Data accumulated over the last 15 years indicated that, in most reasons, because we supposed that its acidic pH would lead to the cases, the combination of heat and ultrasound under pressure would death of sublethally damaged cells caused by MTS as already have an additive effect as it has been described for L. monocytogenes observed with Escherichia coli for others technologies such as high in apple cider (Baumann et al., 2005), Yersinia enterocolitica (Raso, pressure (García-Graells, Hauben, & Michiels, 1998) or pulsed Pagán et al., 1998), Salmonella Enteritidis and Aeromonas hydro- electric ﬁelds (García, Hassani, Mañas, Condón, & Pagán, 2005), phila (Pagán, Mañas, Raso et al.,1999) in pH 7.0 buffer, although some which would provide an additional advantage for acidic products. exceptions have been reported for Bacillus subtilis (Raso, Palop et al., Results obtained indicate that C. sakazakii cells progressively died 1998) and Enterococcus faecium (Pagán, Mañas, Raso et al., 1999) in during refrigerated storage, but even upon 96 h, a certain propor- pH 7.0 buffer. The occurrence of an additive effect has been attrib- tion of cells still remained damaged in their cytoplasmic and outer uted to the different mechanism of inactivation of both technologies membranes. Furthermore, the number of cells recovered in media (Raso, Pagán et al.,1998) whereas the synergies have been attributed with added sodium chloride also decreased with incubation time, to a sensitizing phenomena caused by heat that would render cells and the number of MTS-treated cells and recovered in TSAYE after more sensitive to ultrasound (Álvarez et al., 2003; Condón, Mañas, & 96 h was lower than the number of cells recovered in TSAYE-SC just Cebrián, 2011; Pagán, Mañas, Palop et al., 1999). The occurrence of after the MTS treatment (time 0). All these ﬁndings indicate that, at these effects would depend on the microorganism investigated, the least for C. sakazakii MTS-treated cells, damages detected by the range of temperatures, and the treatment media tested. In fact, the recovery in media with added sodium chloride would not be temperature at which additive or synergistic effects would appear in directly related to the ability of these cells to maintain their pH MTS treatments would be determined by the microbial heat resis- homeostasis during refrigerated storage. Besides, the counts in tance. Thus, it might be expected that in media in which the heat media with added sodium chloride immediately after the treat- resistance is lower, the temperatures at which these phenomena ment might underestimate the number of cells that would be would occur would be lower, the opposite also being true. Further- inactivated by an adequately designed combined process. On the more, it should be remarked that, up to date, all the conditions other hand, given the important role of the outer membrane in pH leading to the occurrence of synergies were coincident with condi- homeostasis (Booth, Cash, & O’Byrne, 2002), it can be hypothesized tions leading to an increase in heat resistance, which suggests that that the progressive inactivation of cells e both when the recovery those factors leading to an increased heat resistance would not was carried out in TSAYE and TSAYE-SC e might be due to the protect cells against ultrasound. By contrast, our results demon- inability of cells with injured outer membranes to maintain pH strate that in acidic conditions (apple juice, pH 3.4) e where the heat homeostasis. resistance of C. sakazakii is reduced (Arroyo, Condón, & Pagán, 2009) Finally, from a practical point of view, our results indicate that e a synergistic effect can also be found. This could be due to the MTS treatments might constitute an alternative to conventional acidic pH or to the composition of the apple juice. In order to check thermal pasteurization treatments also in thermo-sensitive prod- whether the synergism between ultrasound and heat for C. sakazakii ucts such as fruit juices (Char, Mitilinaki, Guerrero, & Alzamora, inactivation does occur both at neutral and acidic pH, the heat and 2010; Ugarte-Romero, Feng, Martin, Cadwallader, & Robinson, MTS resistance in citrate-phosphate buffers of different pH was 2006; Valero et al., 2007; Zenker et al., 2003). Furthermore, since studied and the synergism of the combination was calculated. the acidic pH of these products would hamper the germination of Results obtained demonstrated that not only a synergistic effect can spores, adequately designed MTS treatments would guarantee their be found when cells are MTS-treated in acid pH media, but also that safety and would extend their shelf lives, in spite of the fact that this synergism is higher in acid than in neutral pH media (see ultrasound, when applied at these temperatures, requires high Supplementary data). amounts of energy for bacterial spore inactivation. It should also be The second part of this investigation was designed to explore noted that, apart from the increase in the lethality of the process, the occurrence of sublethally injured cells after MTS treatments. another advantage of the combined use of ultrasound and heat is Results here reported demonstrate that after MTS treatments in that it would reduce the treatment costs when compared to apple juice, a certain proportion of the C. sakazakii population were ultrasound applied at non-lethal temperatures, not only because sublethally injured in their cytoplasmic and outer membranes. This the increase in temperature would reduce the treatment time, but ﬁnding provides an opportunity to develop other combined also because the heat dissipated by the ultrasound waves might be processes to take advantage of the sensitivity of the damaged cells used to achieve the ﬁnal process temperature. Further work is to increase the lethality of treatments without raising the treat- required in order to validate the results obtained here in other ment intensity. Our results also show that the decrease in the species and also to study the inﬂuence of MTS treatments on the d values calculated upon recovery in medium with added sodium organoleptic and nutritive attributes of food products. Finally, the chloride e when compared to those calculated in the non-selective ﬁnding that MTS treatments lead to the occurrence of sublethally medium e was similar for heat and MTS treatments, and that the damaged cells opens the possibility for the development of more- decrease upon recovery in medium with added bile salts was 1.7- complex combined processes including MTS. fold higher for MTS-treated cells than for heat-treated ones. Two relevant conclusions can be inferred from these results. On one Acknowledgments hand, as already pointed out in Arroyo et al. (2011a), the synergistic effect obtained after combining ultrasound and heat would not be This work was supported by Universidad de Zaragoza (UZ2007- due to the lethal effect of ultrasound on cells with damaged cyto- CIE-12). The authors further extend thanks to Gobierno de Aragón plasmic membranes caused by heat. Similarly, the synergism (Spain) for the fellowship for C. Arroyo PhD thesis. 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 Appendix. Supplementary data would remain advantageous e when compared to heat e in an eventual combined process in which these damages to the inner Supplementary data related to this article can be found online at and/or outer membranes are exploited. doi:10.1016/j.foodcont.2011.10.056.
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