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

Synergistic combination of heat and ultrasonic waves under pressurefor Cronobacter sakazakii inactivation in apple juice Tecnología de los Alimentos, Facultad de Veterinaria, Universidad

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Synergistic combination of heat and ultrasonic waves under pressure

for Cronobacter sakazakii inactivation in apple juice

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

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

a b s t r a c t

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 studied in apple juice Below 45C, the inactivation by ultrasound under pressure was independent of temperature Above 64C, the lethal effect of ultrasound under pressure was negligible when compared to the lethality

of the heat treatment at the same temperature Between 45C and 64C, 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 54C Recovery on selective mediae 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 4C) in the apple juice The practical implication of theseﬁndings is discussed

1 Introduction

Ultrasound treatment for food preservation is receiving a great

deal of attention as an appealing alternative to the traditional heat

processing of foods, which often may have negative side effects such

as changes on the sensorial and nutritional properties of food (FDA,

2000) 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 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 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).

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Food Control

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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 foodse many of which are not subjected to processes

that will inactivate the pathogen e have been found to be

contaminated by C sakazakii According toIversen 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 at80C 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 37C (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, theﬂask was incubated

overnight at 30C 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 5 104 CFU/mL, and then incubated

under agitation for 24 h at 30C to reach the stationary growth

phase with aﬁnal concentration of approximately 5 109CFU/mL

C sakazakii resistance to ultrasound under pressure in

combi-nation with heat was studied in commercially sterilized apple juice

(pH 3.4, aw> 0.99) (Alcampo, S.A., Spain), which was purchased

from a local market in Zaragoza, Spain

2.2 MS/MTS 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 SoniﬁerÒultrasonic 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 35C to 64C, at constant peak-to-peak amplitude (117mm) 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, ALMEMOÒ, Ahlborn, Germany), which was insulated with heat-resistant silicone to ensure a constant target temperature value (0.2C) 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 3 105 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 45C to 64C Once the preset temperature had attained stability (0.05 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 105CFU/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)

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2.5 Curveﬁtting, resistance parameters, and statistical analyses

Survival curves were obtained by plotting the log10number 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 toﬁt the survival curves This

model is described by the following equation (Mafart, Couvert,

Gaillard, & Leguerinel, 2002):

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, andrvalue is the shape parameter, which indicates the

proﬁle of the survival curve (r< 1 for concave upward curves,r¼ 1

for linear curves, andr> 1 for concave downward curves)

Decimal reduction time (DRT) curves were obtained by plotting

the log10time to inactivate the 1st(dvalues), 2nd, 3rd, and 4thlog cycle

of inactivation versus the treatment temperature z1, z2, z3, and z4

values (C) represent the temperature increase required for a 1log10

decrease in the time to inactivate the 1st, 2nd, 3rd, and 4thlog cycle of

inactivation, respectively; and are deduced from the regression line of

their corresponding DRT curves Toﬁt the model to the experimental

data and to calculatedandrvalues, GraphPad PRISMÒ4.1 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 theﬁgures 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 1includes the values for the scale and shape parameters

from theﬁtting 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 (R2) values are

also included to show theﬁtting’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 (rz 1)

3.2 C sakazakii resistance to heat, MS, and MTS in apple juice

Fig 1shows the C sakazakii inactivation rates by heat (dTvalues)

and ultrasound under pressure at non-lethal (dMSvalues) and lethal

temperatures (dMTS values) in apple juice As can be seen, the resistance of C sakazakii cells to heat decreased with temperature

An exponential relationship betweendvalues and temperature (T) was found, and a z1value of 6.6C (standard error¼ 0.14) was deduced Therefore, an increase in temperature of 6.6 C was necessary to reduce thedvalue by ten-fold when C sakazakii was heat treated in apple juice As concave downward proﬁles are found for survival curves to heat, representing thedvalues (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 2nd, 3rdand 4thlog cycle of inactivation was also studied (data not shown) A similar exponential relationship between the variables was found, with z2,

z3, and z4values of 6.6C, 6.5C, and 6.5C, respectively (p> 0.05) Regarding the MS/MTS processes, the lethality of ultrasound treatments remained near constant below 45C (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 45C, the lethality of the process would result from the combination of the lethality of both technologies Hence, over 45C, the lethality of MTS quickly increased with temperature For instance, raising the treatment temperature from 35C to 60 C caused an 8.5-fold decrease in thedvalue (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 (dandrvalues) 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).

dvalue (min)

mean (SD)

rvalue mean (SD)

RMSE dvalue (min) mean (SD)

rvalue mean (SD)

RMSE

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

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),

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.

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theoretical MTS-DRT curve This theoretical MTS-DRT curve

repre-sents the additive effect, and was obtained representing the

theo-reticaldMTSvalues against temperature The theoreticaldMTSvalues

were calculated with the equation proposed byRaso, Pagán et al

(1998)and adapted to our resistance parameters:

Theorethical dMTS value ¼ ðdTdMSÞ

ðdTþdMSÞ (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 2nd,

3rdand 4thlog cycles of inactivation by MTS at different

tempera-tures were also calculated For this purpose, thedvalues to heat (dT)

and MS (dMS) appearing in Eq.(2)were replaced for the times for the

2nd, 3rd and 4th log cycle of inactivation e calculated with the

parameters obtained from curve ﬁtting shown inTable 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:

% Synergism¼ Theoretical value Experimental value

Theoretical value 100

(3)

where value refers to the time to inactivate the 1st, 2nd, 3rd, or 4thlog

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 45C to 64C, 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 64C, 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 54C (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 4thcycle of inactivation¼ 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 (4C) in apple juice after MTS treatments at 54C, 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 (54C) and unprocessed cells

As can be observed inFig 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 thedvalue from 0.38 min (recovery in the non-selective medium)

Fig 2 Occurrence and magnitude of the synergistic effect (%) after ultrasound

-5 -4 -3 -2 -1

0

Time (min)

/N 0

-5 -4 -3 -2 -1

0

Time (min)

/N 0

A

B

Fig 3 Survival curves of C sakazakii cells to a MTS treatment (54C, 117mm, 200 kPa) (A), and to a heat treatment (54C) (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

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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 indvalues 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 54C

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 (4C) in

the apple juice for up to 96 h This subsequent storage revealed that

survivors recovered in TSAYE 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; 54C) 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 mediae 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, 54C), 1.1 log cycles by MS (1 min, 35C), and 2.7 log cycles by MTS (1 min, 54C), 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, 4C), 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 survival curves to 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 speculated that these differences would arise as a consequence of the different 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

0

1

2

3

4

5

Incubation time (h)

B

0

1

2

3

4

5

6

Incubation time (h)

g 10

Fig 4 (A) Log 10 cycles of C sakazakii inactivated cells after a MTS treatment in apple

juice (1 min at 54 C, 117mm, 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 54C; time 0) and after subsequent incubation at 4C 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

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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 combination of heat and ultrasound under pressure would

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, Raso et al.,1999) in pH 7.0 buffer, although some

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 to the different mechanism of inactivation of both technologies

(Raso, Pagán et al.,1998) whereas the synergies have been 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 at which additive or synergistic effects would appear 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 acidic conditions (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 the synergism between ultrasound and heat 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

dvalues calculated upon recovery in medium with added sodium

chloridee when compared to those calculated in the non-selective

mediume 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 inArroyo 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 advantageouse 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 theseﬁndings 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 cellse both when the recovery was carried out in TSAYE and TSAYE-SCe 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, 2006; 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 influence of MTS treatments on the organoleptic and nutritive attributes of food products Finally, the finding that MTS treatments lead to the occurrence of sublethally damaged cells opens the possibility for the development of more-complex combined processes including MTS

Acknowledgments This work was supported by Universidad de Zaragoza (UZ2007-CIE-12) The authors further extend thanks to Gobierno de Aragón (Spain) for the fellowship for C Arroyo PhD thesis

Appendix Supplementary data Supplementary data related to this article can be found online at

doi:10.1016/j.foodcont.2011.10.056

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