ORIGINAL PAPER

Inactivation of Listeria monocytogenes and Escherichia coliby Ultrasonic Waves Under Pressure at Nonlethal(Manosonication) and Lethal Temperatures(Manothermosonication) in Acidic Fruit Juices

Burcin Hulya Guzel & Cristina Arroyo & Santiago Condón & Rafael Pagán &

Alev Bayindirli & Hami Alpas

Received: 16 April 2013 /Accepted: 26 September 2013 /Published online: 11 October 2013# Springer Science+Business Media New York 2013

Abstract The inactivation of Listeria monocytogenes andEscherichia coli suspended in apple and orange juices byultrasound under pressure at nonlethal (manosonication, MS)and lethal temperatures (manothermosonication, MTS) wasevaluated. Significant differences were found in the MS resis-tance (35 °C, 110 μm, 200 kPa) of three strains of L.monocytogenes and three of E. coli in pH 3.5 buffer, L.monocytogenes STCC 5672 and E. coli O157:H7 being themost resistant strains. Regarding the interspecific differences,L. monocytogenes showed higher MS resistance than E. coli .Although the pH and treatment medium composition did notsignificantly change the bacterial MS resistance, the effective-ness of ultrasound increased by both raising the amplitude ofultrasonic waves and the pressure. The energy transmitted tothe fruit juices by ultrasonic waves at different combinationsof amplitudes (46.5, 90, 110, and 130.5 μm) and pressures (0,100, and 200 kPa) was also studied, obtaining an exponentialrelationship between the DMS values and power input: anincrease of 116W increased the inactivation rate approximate-ly 10-fold in both juices. The MS resistance of both speciesdecreased when heat was applied jointly with ultrasound(MTS), which was more effective in inactivating L.monocytogenes and E. coli than the sum of MS and

heat acting simultaneously but independently. Therefore,MTS showed a synergistic lethal effect in acidic juices,whose magnitude was dependent on the treatmentconditions.

Keywords Listeria monocytogenes .Escherichia coli .

Ultrasound .Manosonication .Manothermosonication .

Acidic fruit juices

AbbreviationsMS ManosonicationTS ThermosonicationMTS ManothermosonicationTT Thermal treatmentTSAYE Tryptone soy agar supplemented with 0.6 %

of yeast extractTSBYE Tryptone soy broth supplemented with 0.6 %

of yeast extractTR-SC Thermo-resistometer Sala-CondónCFU Colony forming unitD Decimal reduction timezTT Increase in temperature (in degree Celsius) for

the DTT value to drop 1 log cyclezMS Increase in power (in watts) for the DMS value

to drop 1 log cycle

Introduction

Thermal treatment is the most common processing method forfood preservation due to its capacity to inactivate microorgan-isms and enzymes responsible for human diseases and fooddeterioration. However, this treatment can cause undesirablealterations of food. Therefore, the use of non-thermal

B. H. Guzel :A. Bayindirli :H. Alpas (*)Department of Food Engineering, Middle East Technical University,Üniversiteler Mah, 06800 Çankaya, Ankara, Turkeye-mail: imah@metu.edu.tr

B. H. GuzelDepartment of Food Engineering, Inonu University, 44280 Malatya,Turkey

C. Arroyo : S. Condón : R. PagánTecnología de los Alimentos, Facultad deVeterinaria, Universidad deZaragoza, C/ Miguel Servet, 177, 50013 Zaragoza, Spain

Food Bioprocess Technol (2014) 7:1701–1712DOI 10.1007/s11947-013-1205-6

treatments or the combination of these methods with otherpreservation technologies is gaining importance as they pro-vide the opportunity to introduce safe and less processedproducts into the food market (Condón et al. 2011; Raso andBarbosa-Cánovas 2003). Among them, ultrasound is one ofthe non-thermal treatments proposed as an alternative to cur-rent thermal treatments in order to inactivate microorganismsin food products (US FDA 2000).

Ultrasound is the acoustic energy generated by soundwaves with frequencies above the human hearing range(>16 kHz). From the beginning of the twentieth century upto this date, ultrasound has been used as a food processingtechnology in multiple fields such as defoaming, emulsifica-tion, and degassing, among others (Villamiel and De Jong2000). The destruction of bacterial cells by ultrasound as apreservation technique has been studied by a number ofdifferent researchers to ensure food safety (Piyasena et al.2003; Sagong et al. 2011). Recent studies have shown thatultrasound treatment alone had low lethal effect on variousbacterial strains to provide adequate food safety or stability(Condón et al. 2011; Entezari et al. 2004; Lee et al. 2013). Forthat reason, combining ultrasound with heat and pressure hasbeen proposed to inactivate microorganisms and enzymes(Álvarez et al. 2006; Kuldiloke 2002; López-Malo et al.2005; Ordóñez et al. 1984; Sala et al. 1995). The high micro-bial inactivation of ultrasound with moderate heat treatment(thermosonication, TS) was firstly mentioned by Ordóñezet al. (1984). In addition, Sala et al. (1995) described greatmicrobial inactivation by ultrasound when combined withpressure at both nonlethal (manosonication, MS) and lethaltemperatures (manothermosonication, MTS).

The production of fresh-like and microbiologically safefruit juices is a great challenge for the food industry. To ensurethe microbiological safety of juice products, the US Food andDrug Administration’s Guidance for Industry included a rulerequiring all juice producers to follow a 5-log10 reduction rule(99.999 % reduction) in the pertinent pathogens under theirHazard Analysis and Critical Control Points systems (USFDA 2001a). Listeria monocytogenes is a psychrotrophicbacteria widely recognized as an important hazard in the foodindustry as being responsible for a number of cases offoodborne diseases (US FDA 2003). According to Kellerand Miller (2005), the lowest pH value for the growth of L.monocytogenes in apple juice varies from 4.4 to 4.6. AlthoughL. monocytogenes cannot grow at pH values lower than 4.4(US FDA 2001b), the survival of Escherichia coli O157:H7and Salmonella spp. has been reported (Beales 2004; Isomet al. 1995). The presence of E. coli serotypes O157:H7 infresh juices is believed to be due to fecal contamination, whichmay cause diarrhea and the hemolytic–uremic syndrome(Erkmen and Bozoglu 2008a, b; Feng 2012). From epidemi-ological data, it has been demonstrated thatE. coli O157:H7 iscapable of surviving in low-pH juices (Keller andMiller 2005;

US FDA 2001b), and although the pH of most apple andorange juices is low enough to either significantly slow orinhibit the growth of E. coli , enterohemorrhagic strains(EHEC—especially O157:H7) have shown tolerance to highacid levels, causing an extended survival time (Glass et al.1992; Keller and Miller 2005; Zhao and Doyle 1994).Different studies are available in the literature studying thelethal effect of ultrasound combined with heat in differentmedia such as buffer (Arroyo et al. 2011b; Lee et al. 2009;Mañas et al. 2000b; Pagán et al. 1999a; Zenker et al. 2003),milk (Arroyo et al. 2011b; Pagán et al. 1999a; Villamiel andDe Jong 2000; Zenker et al. 2003), orange juice (Zenker et al.2003), carrot juice (Zenker et al. 2003), apple juice (Arroyoet al. 2012), distilled water (Cabeza et al. 2004), broth(Guerrero et al. 2001; López-Malo et al. 2005), and liquidwhole egg (Mañas et al. 2000b). The influence of the combi-nation of ultrasound under pressure and heat at differenttreatment temperatures on L. monocytogenes and E. coliO157:H7 in acidic food juices is still unknown. Moreover,in some of these studies, researchers reported a synergisticeffect between heat and ultrasound treatments for the inacti-vation of different microorganisms, such as Cronobactersakazakii (Arroyo et al. 2011a), Salmonella enterica serovarEnteritidis (Álvarez et al. 2003), Streptococcus faecium(Pagán et al. 1999b), and Bacillus subtilis (Raso et al.1998b). Nevertheless, there are not enough researches relatedto the synergistic effect of heat and ultrasound under pressureon the inactivation of L. monocytogenes and E. coli O157:H7by MTS in acidic fruit juices. Therefore, in this study, weevaluated the inactivation of L. monocytogenes and E. colisuspended in apple and orange juices by high-power ultra-sound treatments under pressure at nonlethal (MS) and lethal(MTS) temperatures. As a reference, the experiments werealso carried out in citrate phosphate buffer (pH 3.5 and 7.0).

Materials and Methods

Microorganisms, Growth Conditions, and Media

L. monocytogenes STCC 4031 (ATCC 15313), STCC 7467(ATCC 19111), and STCC 5672 and E. coli STCC 4201(ATCC 11303) were obtained from the Spanish TypeCulture Collection (STCC, Burjassot, Valencia, Spain). Thestrains of E. coli O157:H7 VTEC-Phage type 34 (Chapmanet al. 1993) and E. coli W3110 (ATCC 27325) were kindlyprovided by Dr. B. M. Mackey from the University ofReading (UK). During this investigation, the cultures weremaintained frozen at −80 °C in cryovials.

A plate of tryptone soy agar (Biolife, Milan, Italy) supple-mented with 0.6 % of yeast extract (TSAYE; Biolife) wasstreaked with a loopful of microorganisms from each cryovial.The plates were incubated for 48 h at 30 °C for L.

1702 Food Bioprocess Technol (2014) 7:1701–1712

monocytogenes and at 37 °C for E. coli . A single colony fromthe stock plate was transferred to a 10-mL flask of steriletryptone soy broth (Biolife) supplemented with 0.6 % of yeastextract (TSBYE). The inoculated broth was held overnight at30 °C for L. monocytogenes and at 37 °C for E. coli in a rotaryshaker (Selecta, mod. Rotabit, Barcelona, Spain) at 150 rpm.Flasks containing 50 mL of fresh TSBYE were inoculatedwith the overnight subculture up to a concentration of approx-imately 5×104 CFU/mL and then incubated in the rotaryshaker for 24 h at 30 °C for L. monocytogenes and at 37 °Cfor E. coli to reach the stationary phase of growth (109 CFU/mL, approximately).

McIlvaine citrate phosphate buffer (Dawson et al. 1974) ofpH 3.5 and 7.0, and commercially sterilized apple (pH 3.4) andorange (pH 3.7) juices were used as treatment media. The apple(Alcampo S.A., Spain) and orange (García Carrión S.A., Spain)juices were purchased from a local market in Zaragoza, Spain.

MS/MTS Treatments

MS and MTS treatments were carried out in a speciallydesigned resistometer previously described by Raso et al.(1998a). A 450-W Digital Sonifier ultrasonic generator witha constant frequency of 20 kHz (Branson UltrasonicsCorporation, Danbury, CT, USA) was used. Four differentwave amplitudes (46.5, 90, 110, and 130.5 μm) and threedifferent pressures (0, 100, and 200 kPa) were applied duringthe experiments. All MS treatments were carried out at con-stant temperature (35±0.2 °C). For the MTS experiments, aconstant ultrasonic wave amplitude (110 μm) and a constantpressure (200 kPa) were applied at 50, 55, and 60 °C(±0.2 °C). These process parameters were selected so as tocompare the microbial resistance to MS and MTS in fruitjuices with that in laboratory media—in order to describe theinfluence of the treatment medium composition—and in viewof further comparison with other studies found in the literatureunder similar experimental conditions (Arroyo et al. 2011a, b,2012; Pagán et al. 1999a, b, c; Raso et al. 1998a, b)

Temperature control during the experiments was achievedby dissipating excess heat evolved during sonication by cir-culating cool water through the cooling coil. The temperatureof the treatment medium was continuously monitored using athermocouple (NiCr-Ni sensor class 1, ref. FTA05L0100,ALMEMO, Ahlborn, Germany), which was insulated with aheat-resistant silicone to ensure a constant target temperaturevalue (±0.2 °C).

After the stabilization of the temperature, pressure, andamplitude of ultrasonic waves, 0.2 mL of an appropriatedilution of the cell suspension was injected into the treatmentvessel containing the treatment medium (23 mL) to obtain aconcentration of approximately 3×105 CFU/mL. After injec-tion, samples of 0.1 mL were collected for definite timeintervals, directly pour-plated, and incubated.

Thermal Treatments

Thermal treatments were performed in a specially designedthermo-resistometer (TR-SC; Condón et al. 1993). To com-pare the microbial resistance to thermal treatments (TT) andMTS in fruit juices, three different temperatures (50, 55, and60±0.2 °C) at constant pressure (200 kPa) were selected. The350-mL treatment vessel was filled with the treatment medi-um and, once the temperature and pressure were stabilized,0.2 mL of an appropriate dilution of the cell suspension wasinjected into the treatment vessel to obtain a concentration ofapproximately 3×105 CFU/mL. After injection, samples of0.1 mL were collected for definite time intervals, directlypour-plated, and incubated.

Incubation of Treated Samples and Colony Counting

The collected samples were pour-plated in TSAYE and incu-bated for 24 h at 30 °C for L. monocytogenes and at 37 °C forE. coli . After incubation, CFUs were counted with anImproved Image Analyzer Automatic Colony Counter(Protos, Synoptics, Cambridge, UK), as described inCondón et al. (1987).

Resistance Parameters and Statistical Analysis

Survival curves for heat, MS, and MTS treatments wereobtained by plotting the log10 number of survivors vs. thetreatment time (in minutes). A mathematical model based onthe Weibull distribution proposed by Mafart et al. (2002) wasused for modeling these curves.

log10S tð Þ ¼ −t=δð Þρ ð1Þ

where S (t ) is the survival fraction; t is the treatment time (inminutes); δ value is the scale factor or the time for the firstdecimal reduction; and ρ value is the shape factor, whichindicates the profile of the survival curve (ρ <1 for concaveupward curves, ρ =1 for linear curves or a first order kinetics,and ρ >1 for concave downward curves). For comparisonpurposes, microbial resistance was expressed as 1D or 4Dvalues, i.e., the time necessary to inactivate 90 % (1-log10cycle) or 99.99 % (4-log10 cycles) population, respectively.

To fit the mathematical model to the experimental data, theGraphPad PRISM® software (GraphPad Software Inc., SanDiego, CA, USA) was used. For statistical analysis, ANOVAtest followed by Tukey’s test (p =0.05) was used with theSPSS software (SPSS Inc., Chicago, IL, USA); differenceswere considered significant if p ≤0.05. All experiments wereperformed in triplicate on independent days, and the error barsin the figures indicate the standard deviations.

Food Bioprocess Technol (2014) 7:1701–1712 1703

Power Measurement

The power input (in watts) into the treatment medium by theultrasonic waves at different combinations of amplitudes(46.5, 90, 110, and 130.5) at a constant pressure (200 kPa)or of different pressures (0, 100, and 200 kPa) at a constantamplitude (110μm)was calculated bymeans of a calorimetricmethod. This method is based on the temperature change withtime of a mass of liquid absorbing the acoustic power (Berlanand Mason 1992; Margulis and Margulis 2003).

In each experiment, the temperature rise was estimatedfrom the slope of the straight line obtained during the firstseconds of the experiment. Ultrasonic power delivered to thetreatment mediumwas calculated with the following equation:

P ¼ Cp� m� dT

dtð2Þ

where Cp is the heat capacity of the treatment medium (injoules per kilogram Kelvin), m is the mass of the treatmentmedium (in kilograms), and dT /dt is the temperature rise persecond (Kelvin per second). The Cp values used for orangeand apple juices were 4.82 kJ/kg °C (Tiwari et al. 2009) and3.89 kJ/kg °C, respectively (Lozano 2006). A thermocoupleconnected to a data logger (ref. OA2390-5S, ALMEMO®,Ahlborn, Holzkirchen, Germany) was used to measure the

temperature of the treatment medium. Before measuring thetemperature increase, the initial temperature was set at 30 °Cin all the experiments.

Synergistic or Additive Effect Calculation

To determine whether an additive or a synergistic effect occurswhen combining heat and ultrasound (MTS treatments), foreach temperature, theoretical 4DMTS values were calculatedand compared with the experimentally obtained 4DMTS

values. Theoretical 4DMTS values were calculated with theequation proposed by Raso et al. (1998a), which represents anadditive effect (the lethality of the combined treatment equalsthe lethality of heat and ultrasound treatments acting simulta-neously but independently).

Theoretical 4DMTS ¼ 4DMS � 4DTTð Þ4DMS þ 4DTTð Þ ð3Þ

where 4DMS and 4DTT values were obtained from the fit ofthe inactivation curves for the MS and thermal treatments,respectively. Afterwards, for each treatment temperature, themagnitude of the synergistic effect was calculated with thefollowing equation:

% Synergism ¼ Theoretical 4DMTSvalue−Experimental 4DMTSvalue

Theoretical 4DMTSvalue� 100 ð4Þ

Results and Discussion

Ultrasound treatment applied alone may not be adequate forthe food industry because of its low lethal effect and longtreatment time (Lee et al. 2013; Sagong et al. 2011). However,the greater intensity reached when combining high-powerultrasound with pressure and mild heat significantly increasesthe number of ultrasound applications in the food industry(Condón et al. 2011). In this study, the inactivation of micro-bial populations suspended in acidic fruit juices by MTS hasbeen explored. Previously, the study examines the variation inMS resistance among bacterial strains and compares MSmicrobial resistance obtained in laboratory media with dataobtained in acidic fruit juices.

Variation in MS Resistance Among L. monocytogenes and E.coli Strains

The lethal effect of MS (35 °C, 110 μm, 200 kPa) in citratephosphate buffer (pH 3.5) was studied in three strains of L.monocytogenes and E. coli (Table 1). The survival curves ofthe six strains under MS treatments followed first-order

kinetics (R2≥0.97). Equation 1 (ρ value fixed at 1) was usedto estimate the DMS values.

As can be observed in Table 1, statistically significantdifferences were observed in the DMS values of strains ofthe same species, L. monocytogenes STCC 5672 and E. coliO157:H7 (Table 1) being the most resistant strains to MS foreach species (p ≤0.05), showing DMS values of 1.74 and0.76 min, respectively. Although no statistically significantdifferences in MS resistance were observed for Salmonellaspp. (Mañas et al. 2000b) and C. sakazakii strains (Arroyoet al. 2011a), Rodriguez-Calleja et al. (2006) also foundsignificant differences in MS resistance of variousStaphylococcus aureus strains. For these authors, the maxi-mum difference in resistance was four times, whereas in ourcase, the MS resistance varied approx. two times between themost and the least resistant strains for both L. monocytogenesand E. coli species.

Regarding the interspecific differences, all strains of theGram-positive L. monocytogenes displayed a higher resistantto ultrasound than all strains of the Gram-negative E. coli(Table 1). As an example, theDMS value of L. monocytogenesSTCC 5672 was twofold higher than that of E. coli O157:H7

1704 Food Bioprocess Technol (2014) 7:1701–1712

Table 1 Resistance parameters (DMS/MTS) of L. monocytogenes and E. coli to ultrasound treatments

Strain Treatment conditions Fit parameters

pH Treatment media Amplitude(μm)

Pressure(kPa)

T (°C) DMS/MTS

(min)SD Significance

levelR2

Listeria monocytogenes

STCC 4031 3.5 Buffer 110 200 35 0.96a 0.02 p ≤0.05 0.99

STCC 7467 3.5 Buffer 110 200 35 1.05a 0.05 0.99

STCC 5672 3.5 Buffer 110 200 35 1.74b 0.15 0.97

STCC 5672 3.5 Buffer 110 200 35 1.74 0.15 Ns 0.97

7.0 Buffer 110 200 35 1.75 0.04 0.98

3.4 Apple juice 110 200 35 1.81 0.08 0.98

3.7 Orange juice 110 200 35 1.87 0.08 0.99

3.4 Apple juice 46.5 200 35 3.51d 0.23 p ≤0.05 0.99

3.4 90 200 35 2.19c 0.08 0.98

3.4 110 200 35 1.81b 0.08 0.98

3.4 130.5 200 35 1.32a 0.05 0.99

3.7 Orange juice 46.5 200 35 4.41c 0.71 p ≤0.05 0.99

3.7 90 200 35 3.40b 0.16 0.99

3.7 110 200 35 1.87a 0.08 0.99

3.7 130.5 200 35 1.05a 0.04 0.99

3.4 Apple juice 110 0 35 5.83b 0.48 p ≤0.05 0.98

3.4 110 100 35 2.51a 0.08 0.99

3.4 110 200 35 1.81a 0.08 0.98

3.7 Orange juice 110 0 35 4.25c 0.03 p ≤0.05 0.98

3.7 110 100 35 3.05b 0.18 0.99

3.7 110 200 35 1.87a 0.08 0.99

3.4 Apple juice 110 200 35 1.81d 0.08 p ≤0.05 0.98

3.4 110 200 50 1.05c 0.00 0.99

3.4 110 200 55 0.77b 0.05 0.97

3.4 110 200 60 0.23a 0.01 0.97

3.7 Orange juice 110 200 35 1.87d 0.08 p ≤0.05 0.99

3.7 110 200 50 1.20c 0.00 0.99

3.7 110 200 55 0.76b 0.12 0.96

3.7 110 200 60 0.31a 0.01 0.95

Escherichia coli

STCC 4201 3.5 Buffer 110 200 35 0.45a 0.02 p ≤0.05 0.99

W 3110 3.5 Buffer 110 200 35 0.45a 0.01 0.99

O157:H7 3.5 Buffer 110 200 35 0.76b 0.04 0.98

O157:H7 3.5 Buffer 110 200 35 0.76 0.04 Ns 0.98

7.0 Buffer 110 200 35 0.80 0.04 0.95

3.4 Apple juice 110 200 35 0.92 0.03 0.98

3.7 Orange juice 110 200 35 0.93 0.00 0.99

3.4 Apple juice 46.5 200 35 3.59b 0.10 p ≤0.05 0.96

3.4 90 200 35 1.09a 0.11 0.99

3.4 110 200 35 0.92a 0.03 0.98

3.4 130.5 200 35 0.85a 0.12 0.98

3.7 Orange juice 46.5 200 35 3.07c 0.06 p ≤0.05 0.99

3.7 90 200 35 1.50b 0.04 0.99

3.7 110 200 35 0.93a 0.00 0.99

3.7 130.5 200 35 0.82a 0.08 0.99

3.4 Apple juice 110 0 35 2.41c 0.33 p ≤0.05 0.99

Food Bioprocess Technol (2014) 7:1701–1712 1705

under similar conditions. Our results also demonstrate that L.monocytogenes STCC 5672 would display a MS resistancesimilar to that of other Gram-positive species (Pagán et al.1999a, b; Villamiel and De Jong 2000) and higher than that ofother Gram-negative species studied in the literature (Arroyoet al. 2011a; Mañas et al. 2000b; Pagán et al. 1999b; Rasoet al. 1998a; Villamiel and De Jong 2000). The higher resis-tance to ultrasound of the Gram-positive in comparisonwith theGram-negative bacteria has been previously reported (Pagánet al. 1999b; Villamiel and De Jong 2000) and has beenassociated with specific morphological features (Ahmed andRussell 1975; Alliger 1975). In this case, the higher MS resis-tance of L. monocytogenes might be related to its smaller size(Ahmed and Russell 1975) and coccus shape (Alliger 1975).

The most MS-resistant strains for each species, L.monocytogenes STCC 5672 and E. coli O157:H7, were cho-sen to carry out the evaluation of the effect of the processparameters.

Effect of the Treatment Media on MS Resistance

The influence of pH on ultrasonic effectiveness is not clear.Although some authors reported higher ultrasound sensitivityat acidic pH values (Sala et al. 1995; Salleh-Mack and Roberts2007) or at neutral pH values (Pagán et al. 1999a), othersreported no influence of pH on microbial MS resistance(Arroyo et al. 2011a; Guerrero et al. 2001). Table 1 showsthe inactivation by MS (35 °C, 110 μm, 200 kPa) of L.monocytogenes STCC 5672 and E. coli O157:H7 suspended

Table 1 (continued)

Strain Treatment conditions Fit parameters

pH Treatment media Amplitude(μm)

Pressure(kPa)

T (°C) DMS/MTS

(min)SD Significance

levelR2

3.4 110 100 35 1.46b 0.10 0.99

3.4 110 200 35 0.92a 0.03 0.98

3.7 Orange juice 110 0 35 2.79c 0.45 p ≤0.05 0.98

3.7 110 100 35 1.71b 0.02 0.98

3.7 110 200 35 0.93a 0.00 0.99

3.4 Apple juice 110 200 35 0.92c 0.03 p ≤0.05 0.98

3.4 110 200 50 0.89c 0.08 0.99

3.4 110 200 55 0.52b 0.07 0.98

3.4 110 200 60 0.27a 0.02 0.96

3.7 Orange juice 110 200 35 0.93d 0.00 p ≤0.05 0.99

3.7 110 200 50 0.77c 0.06 0.99

3.7 110 200 55 0.59b 0.01 0.99

3.7 110 200 60 0.29a 0.01 0.92

ANOVA test (p ≤0.05) was conducted within each group, as shown in the significance level column. To identify intergroup differences after significantdifferences in the ANOVA test, multiple comparisons were performed using Tukey’s test. Values with the same lowercase letters did not showstatistically significant differences (p>0.05)

SD standard deviation, Ns, not significant (p>0.05), R2 determination coefficient

0 50 100 150-0.5

0.0

0.5

1.0

A

Amplitude ( m)

Log

DM

S

0 50 100 150-0.5

0.0

0.5

1.0

B

Amplitude ( m)

Log

DMS

µ

µ

Fig. 1 Influence of amplitude of ultrasonic waves on the resistance of L.monocytogenes STCC 5672 (circle) andE. coli O157:H7 (square) in apple(a) and orange (b) juices by ultrasound under pressure (MS treatment;35 °C, 200 kPa). Data are the means±standard deviation (error bars)

1706 Food Bioprocess Technol (2014) 7:1701–1712

in buffer (pH 3.5 and 7.0) and apple (pH 3.4) and orange(pH 3.7) juices. As is shown, there were no statisticallysignificant differences (p >0.05) among the DMS values ob-tained in these four media of different pH values for both L.monocytogenes STCC 5672 and E. coli O157:H7 (Table 1).Therefore, the MS resistance of L. monocytogenes and E. coliwas not affected by the pH values tested regardless of the foodmatrix, buffer vs. juices.

The effect of the treatment medium composition on themicrobial inactivation by ultrasound has also been exploredby other authors. For instance, Arroyo et al. (2011a) men-tioned that the resistance of C. sakazakii to MS increasedwhen liquid food products were used as the treatment mediain comparison to laboratory media of the same pH. Similarly,Wang et al. (2010) found that the ultrasonic resistance ofAlicylobacilli in apple juice was higher than in buffer.Moreover, the DMS values of L. monocytogenes in milk(pH 6.7) were slightly higher (<50 %) than those obtained inMcIlvaine buffer of the same pH (Pagán et al. 1999a). Thedifferent ultrasonic resistance of bacteria in food matrices and

buffers has been related to their different composition. On thecontrary, some other authors have found no influence oftreatment medium compositions on bacterial resistance toultrasound (Mañas et al. 2000b; Zenker et al. 2003). Mañaset al. (2000b) mentioned that three different serotypes ofSalmonella had the same MS resistance in liquid whole egg(pH 7.7) and citrate phosphate buffer (pH 7.0). Besides, E.coli K12DH 5α treated with ultrasound had the sameD valuein carrot juice (pH 5.9), UHTmilk (pH 6.7), and pH 7.0 buffer(Zenker et al. 2003). As indicated above, in our study, the MSresistance of L. monocytogenes and E. coli was similar whensuspended in apple juice (pH 3.4), orange juice (pH 3.7), andMcIlvaine buffer at a similar pH (3.5, p >0.05).

Effect of Amplitude of Ultrasonic Waves and Pressure on MSLethal Effect

The influence of the amplitude of ultrasonic waves and pres-sure on the resistance to MS of the selected strains suspendedin acidic fruit juices was studied. As can be seen in Table 1, an

0 100 200-0.5

0.0

0.5

1.0A

Pressure (kPa)

Lo

gD

MS

0 100 200-0.5

0.0

0.5

1.0B

Pressure (kPa)

Lo

gD

MS

Fig. 2 Influence of static pressure on the resistance of L. monocytogenesSTCC 5672 (circle) and E. coli O157:H7 (square) in apple (a) andorange (b ) juices by MS treatment (35 °C) at constant amplitude(110 μm). Data are the means±standard deviation (error bars)

0 30 60 90 120 150-0.5

0.0

0.5

1.0

1.5A

Power (W)

Lo

gD

MS

0 30 60 90 120 150-0.5

0.0

0.5

1.0

1.5B

Power (W)

Lo

gDMS

Fig. 3 Influence of power input (in watts) on the inactivation rate (LogDMS) of L. monocytogenes STCC 5672 (circle) and E. coli O157:H7(square) by ultrasound treated in apple (a) and orange (b) juices atdifferent combinations of amplitudes (46.5, 90, 110, and 130.5 μm) andpressures (0, 100, and 200 kPa). Data are the means±standard deviation(error bars)

Food Bioprocess Technol (2014) 7:1701–1712 1707

increase in the lethality of MS treatment in both fruit juiceswas caused by both increasing the amplitude and the pressure.

Figure 1 shows the relationship between the amplitude ofultrasonic waves and the lethality of MS treatment on L.monocytogenes STCC 5672 and E. coli O157:H7 at a con-stant pressure (200 kPa) in apple and orange juices. For bothspecies, the DMS values decreased exponentially when theamplitude of ultrasonic waves increased in the range of 46.5–130.5 μm at a constant pressure. For instance, theMS lethalityon L. monocytogenes STCC 5672 increased by 62 and 76 %in apple and orange juices, respectively, when the amplitudeincreased from 46.5 to 130.5 μm. Similarly, the same ampli-tude variation increased the MS lethality on E. coli by 76 and73% in apple and orange juices, respectively. Previous reportshave also shown that the logarithm of the DMS values de-creases linearly with the ultrasonic wave amplitudes in labo-ratory media (Arroyo et al. 2011a; Pagán et al. 1999a, b, c;Raso et al. 1998a, b; López-Malo et al. 2005). Moreover, nostatistically significant differences (p >0.05) were foundamong the slopes of the regression lines for both species inboth juices (data not shown), which indicates that the magni-tude of the influence of ultrasonic waves amplitude on themicrobial resistance is similar for L. monocytogenes and E.coli in both fruit juices. Thus, the influence of the amplitudeon MS resistance would be the same regardless of the treat-ment media and the species investigated. On the other hand, itcan also be seen that, as stated before, L. monocytogenesSTCC 5672 was always more resistant than E. coli O157:H7.

The influence of the static pressure on the MS lethality onL. monocytogenes STCC 5672 and E. coli O157:H7 at con-stant amplitude (110 μm) in apple and orange juices ispresented in Fig. 2. The inactivation of both microorganismsby MS treatments increased when raising the pressure. Forinstance, as the pressure was increased from 0 to 100 kPa, theDMS values of L. monocytogenes STCC 5672 and E. coliO157:H7 in apple juice dropped to 57 and 39 % of theiroriginal values, respectively. In the same way, when pressurewas increased from 100 to 200 kPa, theDMS values decreasedan extra 28 and 37 %. Similar results were observed in orangejuice (Table 1 and Fig. 2b). Other authors have proposed thatthe relationship between pressure and MS inactivation can bedescribed using a quadratic equation (Arroyo et al. 2011a;Pagán et al. 1999a, b; Raso et al. 1998b); however, in therange of pressures investigated here, an exponential functionadequately described the relationship.

Relationship Between Power Measurements and MSInactivation

As previously stated by Mañas et al. (2000a), the lethality ofultrasound would rely on the amount of energy delivered intothe treatment medium. Thus, the rate of microbial inactivationbyMSwould be determined by the energy transferred into themedium regardless of the combination of pressure and ampli-tude necessary to transfer that energy. In this study, the rela-tionship between the energy transmitted to the treatment

0 1 2 3 4-4

-3

-2

-1

0

A

Time (min)

Log

(N

t/N

0)

0 1 2 3 4-4

-3

-2

-1

0B

Time (min)

Log

(N

t/

N0)

0 1 2 3 4-4

-3

-2

-1

0C

Time (min)

Log

(N

t/ N

0)

0 1 2 3 4-4

-3

-2

-1

0D

Time (min)

Log

(N

t/

N0)

Fig. 4 Survival curves of L.monocytogenes STCC 5672subjected to heat (60 °C, filledsquare), MS (35 °C, 200 kPa,110μm, filled triangle), andMTS(60 °C, 200 kPa, 110 μm, filledcircle) treatments in apple(a) and orange (b) juices and ofE. coli O157:H7 subjected to heat(60 °C, empty square), MS(35 °C, 200 kPa, 110 μm, emptytriangle), and MTS (60 °C,200 kPa, 110 μm, empty circle)treatments in apple (c) andin orange (d) juices. Data are themeans±standard deviation(error bars)

1708 Food Bioprocess Technol (2014) 7:1701–1712

medium and the MS resistance of L. monocytogenes STCC5672 and E. coli O157:H7 is shown in Fig. 3. The existence ofan exponential relationship between the log10DMS values andthe energy transmitted by ultrasound waves to the treatmentmedium has been reported, defining a zMS value similar to thattraditionally used when describing the kinetics of microbialinactivation by heat (Condón et al. 2011). In Fig. 3, thisexponential relationship between the DMS values and thepower delivered can be seen, with zMS mean values of 107and 121 W for L. monocytogenes STCC 5672 and 107 and116 W for E. coli O157:H7 in apple and orange juices,respectively. No significant differences (p >0.05) were foundamong the slopes of the regression lines for the two speciesstudied in both fruit juices and, consequently, between the zMS

values, obtaining a zMS mean value of 116 W. This indicatesthat an increase in 116 W in the energy transferred into theapple and orange juices by ultrasound will make the inactiva-tion rate of L. monocytogenes and E. coli increase by 10times. Other authors have reported values in the same rangefor C. sakazakii suspended in pH 7.0 buffer (zMS=134 W;Arroyo et al. 2011a).

Inactivation of L. monocytogenes STCC 5672 and E. coliO157:H7 by Manothermosonication (MTS Treatments)

Before the valuation of the lethal effect of the combinedtreatment based on the simultaneous application of ul-trasound, pressure, and heat (MTS treatment), the resis-tance of the selected microorganism to each hurdleacting alone was explored. Figure 4 shows the survivalcurves of the selected microorganisms to heat (TT), MS,and to the simultaneous application of both hurdles(MTS) in apple and orange juices.

Regarding the kinetics of inactivation, while the survivalcurves obtained after MS treatment were linear (R2≥0.97), aspreviously stated, those obtained after TT showed a down-ward concavity. Therefore, the survival curves were fitted toMafart’s equation (Eq. 1) to obtain the resistance parameters(δ and ρ values) and, thus, to estimate the time to achieve acertain degree of inactivation (1D or 4D ). Moreover, it isnoteworthy to mention that the concave downward profilesonly appeared when apple and orange juices were used as thetreatment media, but not when buffer of the same pHwas usedas the treatment medium (data not shown). The occurrence ofa downward concavity in the survival curves to heat treat-ments has been reported by some researchers (Arroyo et al.2009; Mafart et al. 2002; Peleg 2000) and the possible expla-nation of its occurrence extensively reviewed (Geeraerd et al.2000). The practical conclusion is the fact that these devia-tions from linearity disappear when applying ultrasound, asstated above, which thereby simplifies the kinetics of inacti-vation and eases the calculation of the treatment time requiredfor a specific level of inactivation. On the other hand, in

contrast to the interspecific strain behavior shown under MStreatments, it should be noticed that the Gram-negative E. coliO157:H7 displayed a higher heat resistance than the Gram-positive L. monocytogenes STCC 5672 in both fruit juices(Figs. 4 and 5 and Table 2). These results would point out E.coli O157:H7 instead of L. monocytogenes as a target micro-organism when processing fruit juices by heat.

Although different studies are available in the literaturestudying the lethal effect of ultrasound combined with heatin different media, as indicated in “Introduction,” the influ-ence of the combination of ultrasound under pressure and heatat different treatment temperatures on L. monocytogenes andE. coli O157:H7 in acidic food juices is still unknown.Therefore, in this work, the inactivation of L. monocytogenesand E. coli O157:H7 by MS in apple and orange juices wasstudied at different temperatures (35, 50, 55, and 60 °C;Fig. 5a, b). Figure 5a, b also includes the heat resistance ofboth species in both juices at 50, 55, and 60 °C.

30 40 50 60-2

-1

0

1

2A

Temperature (oC)

Log

D

30 40 50 60-2

-1

0

1

2B

Temperature (oC)

Log

D

Fig. 5 Influence of temperature on L. monocytogenes STCC 5672inactivation by heat (empty circle) and MS/MTS (filled circle) and onE. coli O157:H7 inactivation by heat (empty square) andMS/MTS (filledsquare) treatments in apple (a) and orange (b) juices. Data are the means±standard deviation (error bars)

Food Bioprocess Technol (2014) 7:1701–1712 1709

As expected, the DTT values decreased with temperature.From the exponential relationship between the Log DTT

values and temperature, zTT values of 9.5, 5.8, and 8.8,5.9 °C could be deduced for L. monocytogenes and E. coliin apple and orange juices, respectively (Fig. 5). In contrast,the inactivation byMSwas independent of temperature up to athreshold temperature, above which the rate of inactivationquickly increased with temperature. This limiting temperaturedistinguishes between MS and MTS treatments and is depen-dent on the microorganism and the treatment media (Arroyoet al. 2011b, 2012). Thus, raising the treatment temperature ofthe combined treatment from 35 to 60 °C caused an eightfolddecrease in the D value of L. monocytogenes in apple juiceand a threefold decrease in the D value of E. coli in orangejuice (Fig. 5a).

The comparison of the inactivation by heat with the inac-tivation by ultrasound treatments shows that both the MS andthe combined process (MTS) were more efficient in reducingthe microbial population than heat acting alone (Fig. 5a, b).For establishing comparisons, the 4D parameter was used. Forinstance, whereas 2.20 and 3.12 min are needed under heattreatment at 60 °C in apple juice for inactivating 99.99 % ofthe L. monocytogenes STCC 5672 and E. coli O157:H7 cellpopulations, respectively, the same level of inactivation mightbe achieved after 0.92 and 1.08 min of MTS treatments at thesame temperature.

Synergies between heat and ultrasound have been reportedfor microbial inactivation of different microorganisms (Arroyoet al. 2011a; Álvarez et al. 2003; Pagán et al. 1999b; Raso et al.

1998b), but not for L. monocytogenes and E. coli in apple andorange juices.

Therefore, and in order to determine whether this increasein lethality by MTS processes over heat processes was due toan additive effect (the lethality of the combined process is thesum of the inactivation rates of heat and ultrasound treatmentsacting simultaneously but individually) or to a synergisticeffect (the lethality of the combined process is higher thanthe expected for heat and ultrasound treatments acting simul-taneously but individually), the experimental 4DMTS valueswere compared with their corresponding theoretical 4DMTS

values (Table 2). According to the results reported here, thecombination of heat and ultrasound under pressure was syn-ergistic for the inactivation of L. monocytogenes and E. colicells byMTS at all treatment temperatures in apple and orangejuices. Maximum synergism was observed when inactivatingL. monocytogenes in apple juice by MTS at 60 °C: theexperimental 4DMTS value was 45 % lower than the theoret-ical 4DMTS corresponding to the sum of the effectiveness ofheat and MS treatment acting simultaneously butindependently.

On the other hand, it is noteworthy to point out that despiteL. monocytogenes STCC 5672 being considered as the targetmicroorganism under MS treatments and the same for E. coliO157:H7 under heat treatments, no significant differenceswere found between the DMTS values for both strains at60 °C in both juices (p >0.05). The lower treatment tempera-ture to achieve a required level of inactivation and this reduc-tion in the interspecific differences in resistance are the two

Table 2 Relationship between temperature and the experimental andtheoretical 4DMTS values (calculated with Eq. 4) of L. monocytogenesSTCC 5672 and E. coli O157:H7 in apple and orange juices and the

synergistic effect of the combined MTS treatment at different tempera-tures (calculated with Eq. 5)

Treatment media Microorganism T ª (°C) 4DMS

(min)4DTT

(min)Theoretical4DMTS (min)

Experimental4DMTS (min)

% synergism

Apple juice L. monocytogenesSTCC 5672

35 7.24 – – – –

50 – 24.64 5.60 4.20 24.94

55 – 9.16 4.04 3.08 23.83

60 – 2.20 1.69 0.92 45.47

E. coli O157:H7 35 3.68 – – – –

50 – 162.48 3.60 3.56 1.07

55 – 26.52 3.23 2.04 36.87

60 – 3.12 1.69 1.08 36.04

Orange juice L. monocytogenesSTCC 5672

35 7.48 – – – –

50 – 26.64 5.84 4.80 17.81

55 – 10.36 4.34 3.04 30.01

60 – 1.92 1.53 1.24 18.84

E. coli O157:H7 35 3.72 – – – –

50 – 157.52 3.63 3.08 15.25

55 – 38.48 3.39 2.36 30.43

60 – 3.24 1.73 1.16 33.01

1710 Food Bioprocess Technol (2014) 7:1701–1712

main advantages of using MTS treatments to process acidicfruit juices.

Conclusions

In this paper, the inactivation of L. monocytogenes STCC 5672and E. coli O157:H7 suspended in apple and orange juices byMS andMTS treatments has been studied for the first time. Thefacts that (1) pH is a factor of minor relevance in this kind ofprocess as well as the interspecific differences in resistancebecoming smaller; (2) the bacterial cell inactivation rates of L.monocytogenes STCC 5672 and E. coli O157:H7 by MSincreased with increases in ultrasonic wave amplitude andpressure; (3) MTS treatments at a specific temperature are moreeffective on inactivating microbial loads than TS, MS, or heat;and (4) the combination of ultrasound and heat treatments hasbeen demonstrated to be synergistic for the inactivation of L.monocytogenes STCC 5672 and E. coli O157:H7 are of greatadvantage for the processing of acidic fruit juices by MTS.Therefore, our data suggest that MTS could be a plausiblealternative to the current pasteurization treatments for fruitjuices. The optimum conditions for treatment will mainly relyon the thermotolerance of the target microorganism and wouldcorrespond to those conditions in which the synergism betweenthe hurdles (ultrasound, pressure, and heat) is maximum.

Acknowledgment This study was financially supported by BAP-08-11-DTP.2002K120510 (METU, Ankara, Turkey).

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