ARTICLE IN PRESS

FOODMICROBIOLOGY

0740-0020/$ - se

doi:10.1016/j.fm

�CorrespondE-mail addr

Food Microbiology 24 (2007) 492–499

www.elsevier.com/locate/fm

Elimination by ozone of Shigella sonnei in shredded lettuce and water

Marıa Victoria Selmaa, David Beltrana, Ana Allendea,Eliseo Chacon-Verab, Marıa Isabel Gila,�

aResearch Group on Quality, Safety and Bioactivity of Plant Foods, Food Science and Technology Department, CEBAS-CSIC, P.O. Box 164,

Espinardo, Murcia, E-30100, SpainbDifferential Equations and Numerical Analysis Department, Mathematical Building, University of Seville, C/Tarfia, s/n, Sevilla, E-41080 Spain

Received 31 May 2006; received in revised form 4 September 2006; accepted 12 September 2006

Available online 13 November 2006

Abstract

Several outbreaks of shigellosis have been attributed to the consumption of contaminated fresh-cut vegetables. The minimal processing

of these products make it difficult to ensure that fresh produce is safe for consumer. Chlorine-based agents have been often used to

sanitize produce and reduce microbial populations in water applied during processing operations. However, the limited efficacy of

chlorine-based agents and the production of chlorinated organic compounds with potential carcinogenic action have created the need to

investigate the effectiveness of new decontamination techniques. In this study, the ability of ozone to inactivate S. sonnei inoculated on

shredded lettuce and in water was evaluated. Furthermore, several disinfection kinetic models were considered to predict S. sonnei

inactivation with ozone. Treatments with ozone (1.6 and 2.2 ppm) for 1min decreased S. sonnei population in water by 3.7 and

5.6 log cfumL�1, respectively. Additionally, it was found that S. sonnei growth in nutrient broth was affected by ozone treatments. After

5.4 ppm s ozone dose, lag-phases were longer for injured cells recovered at 10 1C than 37 1C. Furthermore, treated cells recovered in

nutrient broth at 10 1C were unable to grow after 16.5 ppm s ozone dose. Finally, after 5min, S. sonnei counts were reduced by 0.9 and 1.4

log units in those shredded lettuce samples washed with 2 ppm of ozonated water with or without UV-C activation, respectively. In

addition, S. sonnei counts were reduced by 1.8 log units in lettuce treated with 5 ppm for 5min. Therefore, ozone can be an alternative

treatment to chlorine for disinfection of wash water and for reduction of microbial population on fresh produce due to it decomposes to

nontoxic products.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Disinfection; Fresh-cut; Microbial inactivation; Pathogen; Sanitation

1. Introduction

Fresh and fresh-cut produce is responsible for a growingnumber of foodborne disease outbreaks each year (Beu-chat, 2002). Sources of microbial contamination on fruitsand vegetables during production include animal andhuman feces, contaminated manure, inorganic amend-ments, irrigation water, water used for pesticide applicationor other agricultural purposes and contaminated dust(Beuchat and Ryu, 1997). The minimal processing of theseproducts make it difficult to ensure that fresh produce issafe for consumer. Moreover, tissue damage during

e front matter r 2006 Elsevier Ltd. All rights reserved.

.2006.09.005

ing author. Tel.: +34968 396 315; fax: +34 968 396 213.

ess: migil@cebas.csic.es (M.I. Gil).

processing and subsequent release of nutrients in fresh-cut produce enhance microbial development (Harris et al.,2003).Although the reported incidence of foodborne shigellosis

is lower than that of salmonellosis or other entericpathogens, each year there are a significant number ofoutbreaks, mainly associated with consumption of freshproduce (Smith, 1987; Harris et al., 2003). Consumption ofcontaminated foods results in gastrointestinal illness, sincethe infective dose of Shigella may be as low as 10–500organisms (DuPont et al., 1989). Several outbreaks ofshigellosis have been attributed to the consumption ofcontaminated raw vegetables such as lettuce salads,shredded cabbage, green onions and parsley (Beuchat,1996; Long et al., 2002; Naimi et al., 2003). Although

ARTICLE IN PRESSM.V. Selma et al. / Food Microbiology 24 (2007) 492–499 493

relatively fragile, some strains of Shigella are able totolerate adverse conditions such as acidic environment.Shigella species normally grow over the range of pH 4.5–9but can remain viable following several hours of exposureat pH 2–3 (Bagamboula et al., 2002). Therefore, saladswith an acidic pH (commercial carrot salad pH 2.7–2.9,potato salad pH 3.3–4.4, coleslaw pH 4.1–4.2, and crabsalad pH 4.4–4.5) can also be implicated in shigellosisoutbreaks (Rafii and Lunsford, 1997). Furthermore,Shigella can survive at refrigeration temperatures underlow O2 packaging conditions (Gil and Selma, 2006).Shigella survives at 4 1C in commercial salads for at least11 days and up to 20 days in crab salad (Rafii andLunsford, 1997).

Extensive research has been carried out to determine theefficacy of different sanitizers and modified atmospherepackaging (MAP) to inhibit the growth of foodbornepathogens on fresh and fresh-cut fruits and vegetables(Sapers and Miller, 1998; Park and Beuchat, 1999; Kimand Yousef, 2000; Bai et al., 2001; Emmambux andMinnaar, 2003). Chlorine-based agents have beenoften used to sanitize produce and reduce microbialpopulations in water applied during processing operations(Delaquis et al., 2004). However, various disadvantagesare associated with these agents, such as the productionof chlorinated organic compounds with potential carcino-genic action and the limited antimicrobial efficacy outof pH range 6–7.5 (Fawell, 2000). Although US govern-ment regulations consider that benefits of proper chlorina-tion compensate the presence of potential carcinogenicby-products, chlorine is banned in an extended numberof European countries (Baur et al., 2004; CouncilDirective 98/83/EC, 1998). It has created the need toinvestigate the effectiveness of new decontaminationtechniques.

Ozone has been evidenced as an effective treatment fordisinfection of drinking water where ozone decomposesspontaneously to nontoxic products (Korich et al., 1990;Restaino et al., 1995). Furthermore, ozonated water hasbeen applied to fresh-cut vegetables for sanitation purposesreducing microbial populations and extending the shelf-lifeof some of these products (Beltran et al., 2005a, b).However, scarce information is currently available aboutinactivation of foodborne pathogens such as S. sonnei byozone. An often-cited disadvantage of using ozone as adisinfectant is its high instability, making difficult topredict how ozone reacts in the presence of organic matter(Cho et al., 2003). However, disinfection kinetic models arequick and economical ways to assess food safety objec-tively. Moreover, these kinetic models could also be used topredict the influence of important parameters on thedisinfection processes.

The objective of this study was to investigate the effect ofozone for inactivating S. sonnei inoculated on shreddedlettuce and in water. Also, the validity of four disinfectionkinetic models to predict S. sonnei inactivation by ozonewas assessed.

2. Materials and methods

2.1. Culture preparation

A five-strain cocktail of S. sonnei (CECT 413, 457, 542,4631, and 4887T) was used in this study. Strains wereobtained from the Spanish-type culture collection (Valen-cia, Spain). Cultures were rehydrated in nutrient broth(NB) and then inoculated onto separate nutrient agar (NA)plates and incubated for 24 h at 37 1C. After incubation,stock bacterial cultures were kept at �70 1C in NBcontaining 10% (vol/vol) glycerol (Sigma Chemical, St.Louis, MO). All growing media were purchased fromScharlau Chemie S.A. (Barcelona, Spain). Shigella cultureswere made resistant to ampicillin (Merck, Darmstadt,Germany) by consecutive 24 h transfers of isolated coloniesto NA with increasing concentration of ampicillin untilcolonies were resistant to 80mg of ampicillin per liter.Additionally, growth curves at 37 1C were achieved in NBsupplemented with ampicillin (NB-Amp) for selection ofampicillin-resistant (Amp+) bacterial colonies. After selec-tion, Amp+ colonies were inoculated onto separate NAplates supplemented with ampicillin (8� 10�2 g L�1) (NA-Amp) and incubated for 24 h at 37 1C. Stock bacterialcultures were kept at �70 1C in NB- Amp containing 10%(vol/vol) glycerol (Sigma Chemical, St. Louis, MO) andsubcultured twice in NB-Amp at 37 1C for 24 h before use.

2.2. Lettuce

Iceberg lettuces (L. sativa L.) were obtained from a localsupplier in Murcia (Spain). Lettuce heads were transportedto the laboratory in a cooler (15 1C) and kept at 4 1C and70% relative humidity (RH) in darkness until processingthe next day. Wrapper leaves were hand-removed andheads were then shredded in 3� 3 cm pieces using a sharpstainless steel knife. All the processing operations wereconducted at 8 1C under sanitary conditions. Immediatelyafter cutting, lettuce pieces were maintained at 4 1C/30minbefore the inoculation with S. sonnei.

2.3. Inoculation of water and lettuce with S. sonnei

For water inoculation, each culture of non-ampicillin-resistant S. sonnei was subcultured from the stock in 10mLof sterile NB tubes and incubated at 37 1C until reached thestationary phase. From each culture, 0.1mL was subcul-tured in 50mL NB flasks followed by incubation for a 24 hinterval. The final bacterial strain concentration of about109 cfumL�1 was determined by plating onto NA platesand incubating for 24 h at 37 1C. Fifty milliliters of eachstrain were mixed together and washed three times with0.05M sterile phosphate buffer pH 7 by centrifugation(centrifuge model Centronic-BL, JP, Selecta, Barcelona,Spain) at 1800g for 10min at 20 1C. Cell pellet wasresuspended in 20mL of 0.05M phosphate buffer pH 7(final cell concentration of 1010 cfumL�1). One hundred

ARTICLE IN PRESSM.V. Selma et al. / Food Microbiology 24 (2007) 492–499494

milliliter glass bottles with 50mL of ozonated deionizedwater were inoculated with 0.5mL cell cocktail suspensionprepared in phosphate buffer pH 7 to attain a count ofabout 108 cfumL�1.

For the inoculation of lettuce, each 50 g of shreddedlettuce were placed in a plastic bag and 1mL cocktail ofAmp+ bacteria overnight cultures (approximately109 cfumL�1) was added. Then, bags were manuallyshaken for 5min to ensure a homogeneous distribution ofthe microorganism in the product. A total of 6 kgof inoculated shredded lettuce was air-dried to allowthe cell attachment, under a biohazard safety cabinet for1 h at 20 1C. To obtain homogeneous samples, shreddedlettuce was well mixed and divided into batches of 200 g,five (1 kg) for each washing treatment and stored at 4 1Cduring 2 h.

2.4. Ozone production and measurement

Extra-dry compressed air (0.7 Pa) was passed through awater-cooled corona discharge generator (model 1A,Steriline, Ozono Electronica Iberica, Granada, Spain) toproduce ozone. Gaseous ozone production (3 g h�1) wasmeasured with an ozone gas analyzer (model H1-SPT, INUSA Inc., Needham, MA). An ozone flow of 150L h�1 wasdissolved in deionized water by an inverse mixer. Theexcess gas was neutralized by a thermal destroyer (modelDOT 1.1, Ozono Electronica Iberica) at 550 1C. Thedissolution tank had a volume of 100L. Ozonated waterwas impelled out by a pump at a flow rate of 1m3 h�1, andconduced through a stainless-steel plate heat exchanger(model UFX 6-11, Barriquand, Roanne Cedex, France),coupled to a water cooling unit of 1.98 kW capacity (modelTAE 015 PO, MTA Srl, Conselve, Italy). Finally, theozonated water arrived to the 50L treatment tank andreturned to the dissolution tank by a second pump closingthe circuit. The applied ozone dose was controlled with anintegration system of concentration by temperature im-plemented in a programmable automaton (model SiemensS7+TD200, Ingenieria y Control Remoto S.L., Granada,Spain). An ultraviolet C (UV-C) lamp is coupled to theozone equipment in order to achieve photolysis of ozone byUV-C radiation.

An amperometric selective probe equipped with atemperature compensation sensor was used to monitordissolved ozone. The probe was connected to a dissolvedozone analyzer (B&C Electronics Srl, Carnate, Milano,Italy), which measures ozone concentration in tworanges (0–2 and 2–20mgL�1). The indigo trisulfonatespectrophotometric method was used to calibrate theanalyzer, and also to check the applied ozone concentra-tion (APHA, 1998; Bader and Hoigne, 1981). The decreasein absorbance was measured at 2570.1 1C using aspectrophotometer (UV-1603 Shimadzu, Tokyo, Japan)equipped with a temperature controller (CPS 240,Shimadzu).

2.5. Inactivation of S. sonnei inoculated in ozonated water

Fifty milliliters water with different ozone concentra-tions were transferred from the ozone treatment tank to a100mL ozone demand-free glass bottles. The bottles wereinoculated with 0.5mL microbial suspension and immedi-ately stirred using a magnetic bar at 100 rpm. Samples(1mL each) of the reaction mixture were pipetted atintervals, and each one was instantly mixed with 0.5mLneutralizing solution of 0.005M sodium thiosulfate. Ozoneconcentrations at different times were determined by theindigo trisulfonate method (APHA, 1998; Bader andHoigne 1981).

2.6. Inactivation kinetic models by ozone

A disinfection kinetic model needs to be determined forpredicting the influence of important parameters on thedisinfection processes such as ozone concentration, reac-tion time and ozone demand. The experimental data ofS. sonnei inoculated in ozonated water were analysed usingnonlinear regression kinetic models (Modified Chick,Modified Chick–Watson and Modified Multiple Target)(Table 1). These models were considered due to theirexponential decay behavior similar to the experimentalresults. Furthermore, Chick–Watson model was used tovalidate the effectiveness of a unique CT concept forS. sonnei inactivation, independently of the ozone con-centration and when all other water quality parameters arekept constant (Table 1). The CT value is concentra-tion� time and allows comparison of the efficacy ofdifferent ozone treatments where ozone concentrationand contact time are different. During analysis usingChick–Watson model, CT values were calculated with theintegrated expression:

CT ¼C0 �D

K�1� exp �K�tð Þ½ �. (2)

Nonlinear regression analyses were used to predict thesurvival of microorganisms in terms of time (t) and applieddisinfectant doses (C0) (Table 1). Some of these modelsincluding the Modified Chick and Modified Chick–Watsoncome from assuming that the remaining population Nt isrelated to the changes in residual ozone at time t through adifferential rate law with several parameters (Eq. (3)).

dNtdt¼ �kC

qt N

pt t40;

Nt ¼ N0; t ¼ 0:

((3)

In this equation q is termed the dilution coefficient, p isthe reaction order and k is the independent of theconcentrations. The different values of the parameters q

and p allow the mixing of linear (cases q ¼ 1 and/or p ¼ 1)and nonlinear effects (cases q and/or p different from 1) onthe ozone dilution and reaction order, respectively. Thesedifferent situations yield the two models mentioned above.On the other hand, modified multiple target model is based

ARTICLE IN PRESS

Table 1

Disinfection kinetic models with instantaneous ozone demand

Model name Model Kinetic parameters References

Chick–Watson log10ðStÞ ¼ �kCT k Haas et al. (1995)

Modified Chick log10ðStÞ ¼ �kðC0�DÞ

k�½1� expð�k�tÞ� k, k*,D Kaymak, 2003

Modified Chick–Watson log10ðStÞ ¼ �kðC0�DÞq

qk�½1� expð�qk�tÞ� k, k*,q, D Cho et al. 2003

Modified multiple target log10 Stð Þ ¼ log10 1� 1� exp kðC0�DÞðexp ð�k�tÞ�1Þk�

� �� �ncn o

k, k*,nc , D Kaymak, 2003

St: survival ratio.

CT: Ozone dose.

C0: initial applied ozone dose.

D: Instantaneous ozone demand.

k*: Ozone decay rate.

t: time.

k, p, q: rate parameters.

nc: number of critical targets.

M.V. Selma et al. / Food Microbiology 24 (2007) 492–499 495

on the assumption that a particle (in our case microorgan-isms) contains a finite number (nc) of discrete criticaltargets, all of which must be hit for complete destruction ofthe particle (Haas and Karra, 1984).

2.7. Inactivation of S. sonnei inoculated on shredded lettuce

For the inactivation of Amp+ S. sonnei inoculated onshredded lettuce, ozone was dissolved until different ozoneconcentrations were reached in the 50L treatment tank.One kilogram of inoculated shredded lettuce was sub-merged without agitation in 5 different solutions in a ratioof lettuce (kg): solution (l) 1:50: (A) sterile deionized water,(B) 1 ppm ozone concentration, (C) 2 ppm ozone concen-tration, (D) 5 ppm ozone concentration and (E) 2 ppm ofozone activated by UV-C. All the ozonated solutions had apH ¼ 6.68. The washing time was until a maximum of5min. Samples (30 g each) were taken at appropriate timeintervals. Ozone concentration at different times wasdetermined by dissolved ozone analyzer connected to theozone equipment as well as by the indigo method.

2.8. Recovery of inoculated S. sonnei in water

Samples (1mL) neutralized with 0.5mL neutralizingsolution (0.005M sodium thiosulfate) were added to8.5mL of 1% buffered peptone water (AES Laboratoire,Combourg, France) to recover S. sonnei from ozonatedwater. Dilutions were made if necessary in 1% bufferedpeptone water, and plate counts in NA were performedafter incubation for 24 h at 37 1C.

Alternatively, inoculum before and after treatment withozonated water during 5 and 20 s (initial concentration of2.2 ppm) were serially diluted if necessary in 1% bufferedpeptone water, and 1mL was inoculated in 50mL NBflasks to give an initial concentration of 10 cfumL�1.Inoculated flasks with S. sonnei were stored at 4, 10 and37 1C. Samples were taken at appropriate time intervals,dilutions were made if necessary in 1% buffered peptone

water and plate counts in NA were performed by pourplate technique. Flasks were monitored for at least 60 daysif growth was not detected.

2.9. Enumeration of surviving S. sonnei in lettuce

Twenty-five grams of unwashed and washed lettucesamples inoculated with S. sonnei were transferred intostomacher bags (Seeward Medical, London, UK) in a 1:10dilution with sterile 1% buffered peptone water andhomogenized with a stomacher (IUL Instrument, Barcelo-na, Spain) during 90 s. Aliquots were taken from each bag,serially diluted in 1% buffered peptone water, and plated induplicate on NA-Amp. Plates were counted after incuba-tion at 37 1C for 48 h.

2.10. Statistical analysis and data modeling

Nonlinear disinfection kinetic models were considered tofit the experimental results concerning the inactivation ofS. sonnei inoculated in ozonated water (Table 1). Therewere three replications per treatment and reaction time.Inactivation data of S. sonnei were fitted to the nonlinearmodels by using the curve fitting toolbox provided by thesoftware MATLABs 6.5 (Table 1). Finally, a cross valida-tion technique to predict the properties of the models at eachexperimental condition was carried out three times. For eachmodel, two sets of experimental data out of the three wereused to estimate the model parameters. Then, the third onewas used to estimate, via the SSE, how well the modelpredicts this experimental data (Selma et al., 2006).Inactivation by ozone of S. sonnei inoculated on

shredded lettuce was performed by triplicate. Analysis ofvariance (ANOVA), followed by Tukey’s method with asignificant level of Pp0.05 was carried out on these datausing SPSS (Windows 2000, Statistical Analysis).Experimental growth data of S. sonnei were fitted to the

Baranyi et al. (1993) function using a complementary toolof Microsoft Excel (D-model, J. Baranyi, Institute of Food

ARTICLE IN PRESS

Time (seconds)

(B)

Time (seconds)

0 50 100 150 200 250 300

Ozo

ne c

once

ntra

tion

(ppm

)

0

1

2

3

4

52ppm+UV 5ppm2ppm1ppm

(A)

0 10 20 30 40 50 60

Ozo

ne c

once

ntra

tion

(ppm

)

-0.5

0.0

0.5

1.0

1.5

2.0

2.5 1.6 ppm2.2 ppm

0.5 ppm

Fig. 1. Disappearance kinetic of residual ozone concentration Ct ¼ ðC0 �DÞ expð�ktÞ. In: (A) 100mL glass bottles after inoculation of Shigella sonnei.

(B) 50L tank during washing of lettuce inoculated with Shigella sonnei. Ct: residual ozone at time t. D: instantaneous demand of ozone.

Table 2

Effect of ozone concentration on the inactivation of Shigella sonnei in

water

Initial ozone

concentration (ppm)

Exposure time (s) S. sonnei reduction

log10 (N0/N)

0.5 0 0.170.1 a

10 0.270.2 a

15 0.270.2 a

20 0.270.2 a

30 0.270.2 a

60 0.370.2 a

M.V. Selma et al. / Food Microbiology 24 (2007) 492–499496

Research, Norwich, UK). Kinetic parameters, includinglag time and exponential growth rate for each growth curvewere calculated from the generated Baranyi values. Onlygrowth curves with at least 10 data points were used formodelling, as suggested by the authors. The coefficient ofdetermination (R2) indicating the goodness of fit was alsocalculated. Triplicate growth curves for each conditionwere obtained. Finally, an ANOVA analysis followed byby Tukey’s method were applied to determine whichtreatments and recovery conditions had mean parameterssignificantly different from each others.

1.6 0 0.170.1 a

10 2.870.3 b

15 3.170.1 b

20 3.570.5 b

30 3.670.5 b

60 3.770.5 b

2.2 0 0.170.2 a

5 1.970.1 c

10 3.470.1 b

15 4.970.1 d

20 5.570.1 e

30 5.470.2 e

60 5.670.3 e

Values are means7standard deviations of three replicates which were

plated in duplicate. Means followed by different letters are significantly

different (Pp0.05).

3. Results and discussion

3.1. Effective ozone concentration

An instantaneous demand of ozone, denoted by D

(mgL�1), followed by the gradual disappearance of ozonewas observed in the ozonated water inoculated withS. sonnei (Fig. 1A). When the initial ozone concentrationwas 0.5 ppm in 100mL glass bottles, no residual ozone wasfound after 5 s from S. sonnei inoculation (Fig. 1A).However, for 1.6 and 2.2 ppm ozone, the residual ozoneconcentrations in the glass bottles after 5 s were 0.5 and 1,respectively (Fig. 1A). The rate of ozone disappearance hasbeen modeled by a first-order kinetic (Haas and Karra,1984; Kaymak, 2003):

Ct ¼ ðC0 �DÞ exp ð�k�tÞ, (1)

where C0 is the initial applied ozone concentration(mgL�1), Ct is the residual ozone at time t (mgL�1), D isthe instantaneous demand of ozone (mgL�1) and is afunction of the inoculum cell density, and k* is the first-order disinfectant decay rate (time�1). The instantaneousozone demand of the Shigella inoculum (108 cfumL�1) wascalculated experimentally by Eq. (1). D value was 1.1 ppm(Fig. 1A).

On the other hand, when using the 50L ozone tank forinoculated lettuce treatment, ozone concentration did notdecrease after 300 s (Fig. 1B). These results showed that theintegration system of the ozone concentration coupled to theozone equipment was able to maintain the desired concen-tration in the water tank controlling the ozone demand.

3.2. Inactivation by ozone of S. sonnei inoculated in water

The effects of the different ozone concentrations (0.5, 1.6and 2.2 ppm) on the inactivation of S. sonnei inoculated in

ARTICLE IN PRESSM.V. Selma et al. / Food Microbiology 24 (2007) 492–499 497

ozonated water are shown in Table 2. Water and 0.5 ppmozonated water did not reduce S. sonnei populationbecause the initial ozone concentration of 0.5 ppm wasnot enough to satisfy the initial ozone demand (1.1 ppm).When initial ozone concentrations were 1.6 and 2.2 ppm, S.

sonnei population (108 cfumL�1) decreased by 3.7 and 5.6log units, respectively, after 60 s. Therefore, microbialinactivation rate was significantly higher at the highestozone concentration (Table 2). The CT values (concen-tration� time) for achieving 3.7 and 5.6-log reductions ofS. sonnei were 12 and 17 ppm s independently of the initialozone concentration (Fig. 2).

Regarding nonlinear regression analysis, the fittingprocess was performed by minimizing the sum of squaresof the errors (SSE) and root mean square error (RMSE)(Ratkowsky, 1983; Ratkowsky, 2002) where a RMSEcloser to 0 indicates a better fit. The statistics SSE andRMSE all with a significant level of Po0.05, have beenused to determine the best model for fitting the experi-mental reductions of S. sonnei in ozonated water withinitial ozone concentrations of 1.6 and 2.2 ppm. (Table 3).Although all the models behaved well, the model whichbest fitted the experimental results was found to be themodified multiple target model. This model had thesmallest RMSE among the four models.

Table 3

Comparison of SSE, RMSE, and average SSE for the nonlinear proposed mo

SSE

Initial ozone concentration (ppm) 1.6 2.2

Modified Chick 8.34 10.55

Modified Chick–Watson 8.34 10.55

Modified multiple target 16.89 165.14

SSE: Sum of squares of the errors.RMSE: Root mean square error.�Average SSE in predicting, computed according with the cross validation

-8

-7

-6

-5

-4

-3

-2

-1

0

0 10 20 30 40 50

CT (ppm s)

Log

10 (

N/N

0)

0.5 ppm

1.4 ppm

2 ppm

Fig. 2. Effect of ozone doses on the inactivation kinetics of Shigella sonnei

inoculated in water.

On the other hand, the cross validation technique waseffective to predict the properties of our models. Weconsidered two repetitions to fit each model and the oneleft was used to validate the fitting (Selma et al., 2006).Therefore, for each model, three SSE values were obtained,according to the three different choices of the key process‘‘two data set as estimating group and one data set asvalidation group’’ and finally, the average value of each ofthe three SSE was calculated (Table 3).

3.3. Inactivation by ozone of S. sonnei inoculated on lettuce

Approximately 5.670.4 log cfu of S. sonnei per gram wasrecovered from shredded lettuce after inoculation ofbacteria and drying. Therefore, there was a reduction ofabout 1.7 log cfumL�1 from the inoculum level. In thisstudy, the effect of ozone and ozone activated with UV-Con S. sonnei inoculated on lettuce is shown in Fig. 3. Thewashes with sterile deionized water did not significantlyreduce microbial population after 5min (Fig. 3). One ppmof ozonated water reduced the microbial population by 0.7log units after 3min (Fig. 3). Cell inactivation was notincreased when prolonging this ozone treatment andcounts remained unchanged for up to 5min of treatment.After 5min, S. sonnei counts were reduced by 0.9, 1.4 and1.8 log units in those samples washed with 2 ppm ofozonated water with or without UV-C activation and

dels on the inactivation of S. sonnei in water by ozone

RMSE Average SSE�

1.6 2.2 1.6 2.2

0.80 0.78 5.43 3.59

0.80 0.81 5.42 3.60

1.14 3.21 5.92 3.67

process, significant level Po0.05.

Time (seconds)

0 50 100 150 200 250 300

Log

10 (

N/N

0)

-2.0

-1.5

-1.0

-0.5

0.0

Water1 ppm2 ppm

5 ppm2 ppm + UV

Fig. 3. Reduction kinetics by ozone of Shigella sonnei inoculated on

shredded lettuce.

ARTICLE IN PRESS

Table 4

Growth characteristics of S. sonnei suspended in water exposed or not to different ozone doses

Ozone doses (ppm s) Temperature (1C) Lag time (h) Exponential growth

rate Log10 cfumL�1 h�1R2

0 37 1.6370.24 a 0.6970.03 x 0.99

10 62.7276.79 b 0.0270.01 y 0.99

4 No Growth No Growth —

5.4 37 2.2170.40 a 0.8570.02 x 0.99

10 165.2871.70 c 0.0470.01 y 0.99

4 No Growth No Growth —

16.5 37 2.1370.82 a 0.9770.21 x 0.99

10 No Growth No Growth —

4 No Growth No Growth —

*Standard deviation (s.d). Numbers followed by the same letter are not significantly different (Pp0.05) for the same incubation temperature within

columns.

M.V. Selma et al. / Food Microbiology 24 (2007) 492–499498

5 ppm, respectively. Therefore, significant differences werefound between the numbers of S. sonnei in shredded lettucewashed with different ozone concentrations. Microbialinactivation rate was significantly higher at the highestozone concentration. On the other hand, 2 ppm ozonetreatment was not more effective when it was combinedwith UV-C. Therefore, in this study UV-C activation didnot improve the efficacy of this ozone treatment in spite ofsimultaneous application of these sanitizing factors couldcause photolysis of ozone by UV radiation to generatehydroxyl radicals, which are highly reactive with organicmatter and microbial cells.

3.4. Sublethal damage of S. sonnei by ozone

The effect of two different ozone doses on the ability ofS. sonnei to grow at different temperatures was evaluated(Table 4). Both treated and untreated cells were unable togrow at 4 1C (Table 4). S. sonnei growth in NB at 10 1C wasaffected by 5.4 ppm s ozone treatment. The specific growthrate was not modified, but the lag period was prolongedfrom 62 to 165 h (Table 3). It indicated that ozonetreatment produced damage of the cell surface architectureas previously shown (Diao et al., 2004). Furthermore,ozone treatment produced sub-lethal injury since lag-phaseelongation has been related to the repair and adaptationperiod (Shin and Pyun, 1997). The subsequent growth ratedid not vary because once cells were recovered they couldgrow normally. When S. sonnei treated with the sameozone dose was incubated at 37 1C in NB, the lag phase wasnot prolonged. Similar results have been shown both for E.

aerogenes (Selma et al., 2003) and L. monocytogenes (Shinand Pyun, 1997) where lag-phases were longer for injuredcells recovered under stress conditions such as lowtemperatures.

When ozone dose was increased to 16.5 ppm s, survivalcells were able to grow in NB at 37 1C. No significantdifferences were found among the two different ozonetreatments on the sub-sequent growth at incubationtemperature of 37 1C (Table 4). However, treated cells

recovered in NB at 10 1C were unable to grow. Therefore,parameters such as ozone doses and growth temperaturehave a relevant role in the ability of bacteria to recoverafter exposure to this technological treatment.

Acknowledgements

The authors are grateful to Spanish CICYT (ComisionInterministerial de Ciencia y Tecnologıa) projectAGL2004-03060 for financial support. D. Beltran is holderof a grant from Ministerio de Ciencia y Tecnologıa (Spain)reference BES-2002-0216. Thanks are also due to OzonoElectronica Iberica for helping with the ozone facility.

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