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

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: (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 Â 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 Â 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 Â 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 Â 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
Â
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. 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.
C. Arroyo et al. / Food Control 25 (2012) 342e348 347
References
Adekunte,A.,Valdramidis, V. P.,Tiwari,B.K.,Slone, N.,Cullen, P.J., Donnell,C.P. O., et al.
(2010). Resistance of Cronobacter sakazakii in reconstituted powdered infant
formuladuring ultrasoundatcontrolled temperatures: a quantitative approachon
microbial responses. International Journal of Food Microbiology, 142,53e59.
Álvarez, I., Mañas, P., Sala, F. J., & Condón, S. (2003). Inactivation of Salmonella
enterica Serovar Enteritidis by ultrasonic waves under pressure at different
water activities. Applied and Environmental Microbiology, 69,668e672.
Arroyo, C., Cebrián, G., Pagán, R., & Condón, S. (2011a). Inactivation of Cronobacter
sakazakii by manothermosonication in buffer and milk. International Journal of
Food Microbiology, 151,21e28.
Arroyo, C., Cebrián, G., Pagán, R., & Condón, S. (2011b). Inactivation of Cronobacter
sakazakii by ultrasonic waves under pressure in buffer and foods. International
Journal of Food Microbiology, 144,446e454.
Arroyo, C., Condón, S., & Pagán, R. (2009). Thermobacteriological characterization of
Enterobacter sakazakii. International Journal of Food Microbiology, 136,110e118.
Baumann, A. R., Martin, S. E., & Feng, H. (2005). Power ultrasound treatment of
Listeria monocytogenes in apple cider. Journal of Food Protection, 68,2333e2340.

Baumgartner, A., Grand, M., Liniger, M., & Iversen, C. (2009). Detection and
frequency of Cronobacter spp. (Enterobacter sakazakii) in different categories of
ready-to-eat foods other than infant formula. International Journal of Food
Microbiology, 136,189e192.
Beuchat, L. R., Kim, H., Gurtler, J. B., Lin, L., Ryu, J., & Richards, G. M. (2009). Cro-
nobacter sakazakii in foods and factors affecting its survival, growth, and inac-
tivation. International Journal of Food Microbiology, 136, 204e21 3.
Booth, I. R., Cash, P., & OByrne, C. (2002). Sensing and adapting to acid stress.
Antonie van Leeuwenhoek, 81,33e42.
Chambers, L. A., & Gaines, N. (1932). Some effects of intense audible sound on living
organisms and cells. Journal of Cellular and Comparative Physiology, 1,451e473.
Chang, C., Chiang, M., & Chou, C. (20 09). The effect of temperature and length of
heat shock treatment on the thermal tolerance and cell leakage of Cronobacter
sakazakii BCRC 13988. International Journal of Food Microbiology, 134,184e189.
Char, C. D., Mitilinaki, E., Guerrero, S. N., & Alzamora, S. M. (2010). Use of high-
intensity ultrasound and UV-C light to inactivate some microorganisms in
fruit juices. Food and Bioprocess Technology, 3,797e803.
Chemat, F., Huma, Z., & Khan, M. K. (2011). Applications of ultrasound in food
technology: processing, preservation and extraction. Ultr
asonics Sonochemistry,
18,813e835.
Ciccolini, L., Taillandier, P., Wilhem, A. M., Delmas, H., & Strehaiano, P. (1997). Low
frequency thermo-ultrasonication of Saccharomyces cerevisiae suspensions:
effect of temperature and of ultrasonic power. Chemical Engineering Journal, 65,
145e149.
Condón, S., Arrizubieta, M. J., & Sala, F. J. (1993). Microbial heat resistance deter-
minations by the multipoint system with the thermoresistometer TR-SC. Journal
of Microbiological Methods, 18,357e366.
Condón, S., Mañas, P., & Cebrián, G. (2011). Manothermosonication for microbial
inactivation. In H. Feng, G. V. Barbosa-Cánovas, & J. Weiss (Eds.), Ultrasound tech-

nologies for food and bioprocessing (pp. 287e320). New York: Springer Science.
Condón, S., Palop, A., Raso, J., & Sala, F. J. (1996). Influence of the incubation
temperature after heat treatment upon the estimated heat resistance values of
spores of Bacillus subtilis. Letters in Applied Microbiology, 22,149e152.
Condón, S., Raso, J., & Pagán, R. (2005). Microbial inactivation by ultrasound. In
G. V. Barbosa-Cánovas, M. S. Tapia, & M. P. Cano (Eds.), Novel food processing
technologies (pp. 423e442). Boca Ratón: CRC Press.
D’Amico, D. J., Silk, T. M., Wu, J., & Guo, M. (2006). Inactivation of microorganisms in
milk and apple cider treated with ultrasound. Journal of Food Protection, 69,
556e563.
Dancer, G. I., Mah, J. H., Rhee, M. S., Hwang, I. G., & Kang, D. H. (2009). Resistance of
Enterobacter sakazakii (Cronobacter spp.) to environmental stresses. Journal of
Applied Microbiology, 107, 1606e1614.
Food and Agriculture Organization/World Health Organization (FAO/WHO). (2004).
Cronobacter sakazakii and other microorganisms in powdered infant formula;
meeting report. In Microbiological risk assessment series 6. World Health Orga-
nization/Food and Agriculture Organization of the United Nations, Geneva and
Rome. Geneva, Switzerland: WHO Press, Available at: />foodsafety/publications/micro/mra6/en/.
Friedemann,M.(2007).Enterobacter sakazakii in foodandbeverages (other than infant
formula and milk powder). International Journal of Food Microbiology, 116,1e10.
García, D., Hassani, M., Mañas, P., Condón, S., & Pagán, R. (2005). Inactivation of
Escherichia coli O157:H7 during the storage under refrigeration of apple juice
treated by pulsed electric fields. Journal of Food Safety, 25,30e42.
García-Graells, G., Hauben, K. J. A., & Michiels, C. W. (1998). High-Pressure inacti-
vation and sublethal injury of pressure-resistant Escherichia coli mutants in fruit
juices.
Applied and Environmental Microbiology, 64,
1566e1568.
Guerrero, S., López-Malo, A., & Alzamora, S. M. (2001). Effect of ultrasound on the
survival of Saccharomyces cerevisiae:influence of temperature, pH and ampli-

tude. Innovative Food Science and Emerging Technologies, 2,31e39.
Iversen, C., & Forsythe, S. (2003). Risk profile of Enterobacter sakazakii, an emergent
pathogen associated with infant milk formula. Trends in Food Science and
Technology, 14,443e454.
Knorr, D., Zenker, M., Heinz, V., & Lee, D. U. (2004). Applications and potential of
ultrasonics in food processing. Trends in Food Science and Technology, 15,
261e266.
López-Malo, A., Guerrero, S., & Alzamora, S. M. (1999). Saccharomyces cerevisiae
thermal inactivation kinetics combined with ultrasound. Journal of Food
Protection, 62, 1215e121 7.
Lee, H., Zhou, B., Liang, W., Feng, H., & Martin, S. E. (2009). Inactivation of Escherichia
coli cells with sonication, manosonication, thermosonication, and man-
othermosonication: microbial responses and kinetics modeling. Journal of Food
Engineering, 93, 354e364.
Mackey, B. M. (2000). Injured bacteria. In M. Lund, T. C. Baird-Parker, & G. W. Gould
(Eds.), The microbiological safety and quality of food, Vol. I (pp. 315e341). Gai-
thersburg, Maryland: Aspen Publisher.
Mafart, P., Couvert, O., Gaillard, S., & Leguerinel, I. (2002). On calculating sterility in
thermal preservation methods: application of the Weibull frequency distribu-
tion model. International Journal of Food Microbiology, 72,107e113.
Mason, T. J., Paniwnyk, L., & Lorimer, J. P. (1996). The uses of ultrasound in food
technology. Ultrasonics Sonochemistry, 3, S253eS260.
Oh, S., & Kang, D. (2004). Fluorogenic selective and differential medium for isolation
of Enterobacter sakazakii. Applied and Environmental Microbiology, 70,
5692e5694.
Ordóñez, J. A., Aguilera, M. A., García, M. L., & Sanz, B. (1987). Effect of combined
ultrasonic and heat treatment (thermoultrasonication) on the survival of
a strain of Staphylococcus aureus. Journal of Dairy Research, 54,61e67.
Pagán, R., Mañas, P., Palop, A., & Sala, F. J. (1999). Resistance of heat-shocked cells of
Listeria monocytogenes to manosonication and manothermosonication. Letters

in Applied Microbiology, 28,71e75.
Pagán, R., Mañas, P., Raso, J., & Condón, S. (1999). Bacterial resistance to ultrasonic
waves under pressure at nonlethal (Manosonication) and lethal (Man-
othermosonication) temperatures. Applied and Environmental Microbiology, 65
,
29
7e300.
Piyasena, P., Mohareb, E., & McKellar, R. C. (2003). Inactivation of microbes using
ultrasound: a review. International Journal of Food Microbiology, 87,207e216.
Raso, J., Pagán, R., Condón, S., & Sala, F. J. (1998). Influence of temperature and
pressure on the lethality of ultrasound. Applied and Environmental Microbiology,
64, 465e471 .
Raso, J., Palop, A., Pagán, R., & Condón, S. (1998). Inactivation of Bacillus subtilis
spores by combining ultrasonic waves under pressure and mild heat treatment.
Journal of Applied Microbiology, 85, 849e854.
Sala, F. J., Burgos, J., Condón, S., López, P., & Raso, J. (1995). Effect of heat and
ultrasound on micro-organisms and enzymes. In G. W. Gould (Ed.), New
methods of food preservation (pp. 176e204). London: Blackie Academic &
Professional.
Thanassi, D. G., Cheng, L. W., & Nikaido, H. (1997). Active efflux of bile salts by
Escherichia coli. Journal of Bacteriology, 179,2512e2518.
Turcovský, I., Kuniková, K., Drahovská, H., & Kaclíková, E. (2011). Biochemical and
molecular characterization of Cronobacter spp. (formerly Enterobacter sakazakii)
isolated from foods. Antonie van Leeuwenhoek, 99,257e269.
Ugarte-Romero, E., Feng, H., Martin, S. E., Cadwallader, K. R., & Robinson, S. J. (2006).
Inactivation of Escherichia coli with power ultrasound in apple cider. Journal of
Food Science, 71,E102eE108.
U. S. Food and Drug Administration, Center for Food Safety and Applied Nutrition.
(June 2, 2000). Kinetics of microbial inactivation for alternative food processing
technologies. Ultrasound. Available at: />ScienceResearch/ResearchAreas/.

Valero, M., Recrosio, N., Saura, D., Muñoz, N., Martí, N., & Lizama, V. (2007). Effects
of ultrasonic treatments in orange juice processing. Journal of Food Engineering,
80, 509e516.
Villamiel, M., van Hamersveld, E. H., & De Jong, P. (1999). Review: effect of ultra-
sound processing on the quality of dairy products. Milchwissenschaft, 54,69e73.
Zenker, M., Heinz, V., & Knorr, D. (2003). Application of ultrasound-assisted thermal
processing for preservation and quality retention of liquid foods. Journal of Food
Protection, 66, 1642e1649.
C. Arroyo et al. / Food Control 25 (2012) 342e348348