Original Article

Synergistic Effects of Ultrasound and Sodium Hypochlorite(NaOCl) on Reducing Listeria monocytogenes ATCC19118

in Broth, Stainless Steel, and Iceberg Lettuce

Na-Young Lee, Seok-Won Kim, and Sang-Do Ha

Abstract

This study was performed in order to determine whether a combined treatment of ultrasound and sodium hypo-chlorite (NaOCl) is more effective than individual treatment on reducing Listeria monocytogenes ATCC19118 onstainless steel and iceberg lettuce. The bactericidal effect of ultrasound and NaOCl was investigated in tryptic soybroth (TSB), on stainless steel and iceberg lettuce. Various concentrations of NaOCl (50, 100, 150, and 200 ppm)were tested along with various ultrasound treatment times (5, 20, 40, 60, 80, and 100 min). The combined treatmentof ultrasound and NaOCl resulted in greater bacterial reductions than either treatment alone, without causing anysignificant changes in lettuce texture. The synergistic values of combined ultrasound and NaOCl treatments in TSB,on stainless steel, and on iceberg lettuce were 0.01–0.99 log10 colony-forming units (CFU)/mL, 0.01–0.62 log10

CFU/g, and 0.12–1.66 log10 CFU/g, respectively. These results suggest that the combination of ultrasound andNaOCl was more effective than each treatment against Listeria monocytogenes, and that this combination caneffectively sanitize fresh products such as iceberg lettuce.

Introduction

The demand for minimally processed fresh producehas increased over recent years according to consumers

interest in healthy, fresh, and easy-to-prepare products (Umet al., 2005). However, minimally processed fresh produceposes serious food poisoning risks because it is eaten raw,unlike most meat and seafood, which are typically cooked(Cha et al., 2004). Leafy vegetables such as iceberg lettuce,Chinese chive, and cucumber are the products most widelyconsumed fresh. Managing hygiene for the food safety ofthese products is critical, as spoilage is likely initiated uponexposure to various bacteria through the soil and water dur-ing/after harvest (Hong et al., 2007; Kim et al., 2009).

Listeria (L.) monocytogenes, a psychotropic foodbornepathogen, commonly occurs in the environment (Shan et al.,2012; Botticella et al., 2013; Ding et al., 2013). In general,L. monocytogenes can be isolated from many types of foodsincluding beef (Kang et al., 1991), chicken (Bailey et al.,1989; Kang et al., 1991), milk (Kang et al., 1991; Marshallet al., 1999), vegetables (Heisick et al., 1989; Botticella et al.,2013) and fish (Farber and Daley, 1994). In addition,L. monocytogenes causes listeriosis, which can lead to sep-ticemia, meningitis, and spontaneous abortion (Gellin andBroome, 1989; Pinner et al., 1992; Shan et al., 2012). For this

reason, many researchers are focusing on controllingL. monocytogenes contamination in various foods, especiallyfresh produce.

In general, chlorine is the most widely used disinfectant infresh-cut food due to the low cost and convenience. It isavailable on the commercial market in the form of sodiumhypochlorite solution (NaOCl) (Kim et al., 2000; Niemira,2007; Allende et al., 2008). However, when it is used at highconcentration for a long time, NaOCl can cause deteriorationin organoleptic quality, an unpleasant odor, residual chlorine,and production of byproducts including trihalomethane,which is a carcinogenic substance (Chang et al., 2000; Kimet al., 2008). In addition, it is difficult to completely removebacteria from the lettuce leaf surface with a single treatmentof NaOCl. Lettuce leaves may have stomata and folds thatencourage attachment of bacteria (Babic et al., 1996) andallow microorganisms to form biofilms (Fett, 2000; Ryu andBeuchat, 2005). Thus, novel techniques to disinfect foodproducts need to be developed to reduce the carcinogeniceffects of sodium hypochlorite and address other issues as-sociated with the substance.

Recently, hurdle technology—combining chemical andphysical techniques—has been developed to reduce patho-genic bacteria in fresh-cut produce (Leistner, 2000; Lombardet al., 2000; Chawla and Chander, 2004). Physical treatment

School of Food Science and Technology, Chung-Ang University, Gyunggido, Korea.

FOODBORNE PATHOGENS AND DISEASEVolume 11, Number 7, 2014ª Mary Ann Liebert, Inc.DOI: 10.1089/fpd.2013.1722

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techniques used for applying hurdle technology include highhydrostatic pressure (Cecile et al., 2004), ultrasound (Bril-hante and Dantas, 2012; Demirdoven and Baysal, 2009; Sa-gong et al., 2011; Zhou et al., 2009), and ultraviolet radiation(Ukuku and Geveke, 2010; Ha et al., 2011). Among thesetechnologies, ultrasound has been identified as a potentialphysical technique with antimicrobial effects (Piyasena et al.,2003; Baumann et al., 2005; Arroyo et al., 2011).

Ultrasound in the food industry is generally used at fre-quencies from 20 kHz to 10 MHz, and it acts as an antimi-crobial by producing powerful cavitation bubbles that disruptthe lipid membrane of bacteria and detach bacteria attachedto the surfaces of fresh-cut produce (Scouten and Beuchat,2002; Seymour et al., 2002; Piyasena et al., 2003). Thus, thedisinfecting efficacy of NaOCl could be increased whencombined with ultrasound treatment, as ultrasound mighthelp NaOCl penetrate inaccessible sites such as stomata andfolds in the leaves of fresh-cut produce (Sagong et al., 2011).

Therefore, the objective of this study was to assess thesynergistic effect of combined NaOCl and ultrasound treat-ment on L. monocytogenes in tryptic soy broth (TSB) onstainless steel and iceberg lettuce, compared to treatments ofNaOCl or ultrasound alone.

Materials and Methods

Bacterial strains

L. monocytogenes ATCC19118 isolated from chicken wasused to evaluate the bactericidal effects of single or combinedtreatments of chemical disinfection and ultrasound. Stockculture was maintained at - 70�C in 0.1 mL of TSB (DifcoLaboratories, Detroit, MI) with the addition of 50% (vol/vol)glycerol (Fisher Scientific, Itasca, IL). Working cultures werecultured twice at 37�C for 24 h in TSB, streaked onto a trypticsoy agar (TSA; Difco, Becton Dickinson, Franklin Lakes, NJ)plate, incubated at 37�C for 24 h, and examined for typicaland homogeneous colony morphology.

Inoculation of TSB, stainless steel, and iceberg lettuce

Populations of L. monocytogenes ATCC19118 used for theinoculums comprised 7–8 log10 CFU/mL. The inoculum wasprepared in 10 mL TSB by incubation at 37�C for 24 h. Thecell suspension was centrifuged at 13,000 · g for 10 min at4�C and resuspended in 10 mL 0.1% peptone water (Oxoid,Basingstoke, Hampshire, England).

Iceberg lettuce was purchased from a local market in An-seong, Korea and stored at 4�C before the experiment. Iceberglettuce was washed with distilled water for 2 min, treated withthe sanitizer for 2 min to remove natural bacterial contamina-tion. Fresh iceberg lettuce was uniformly cut into 10-g pieces(– 0.5) using a sterile stainless-steel knife, and the sliced ice-berg lettuce was used at room temperature. The surfaces of thestainless steel chip (20 · 20 mm) and the sliced iceberg lettucewere inoculated with 0.1 mL of the strain suspension. The in-oculated samples were dried for 1 h on a clean bench at roomtemperature. The initial pathogen level inoculated on stainlesssteel and iceberg lettuce was about 6–7 log10 CFU/g.

Ultrasound

Ultrasound (P 300 H model, 230 V; Hucom System Co.,Elmasonic, Germany) was chosen as physical treatment to

inactivate or detach bacteria from TSB, stainless steel, oriceberg lettuce. For the ultrasound treatment, an ultra-sound tank was filled with 28 L of distilled water and usedat an operating frequency of 37 kHz and a power up to1200 W. A 500-mL sterile glass beaker was placed in theultrasound tank and filled with 90 mL of sterile water.Inoculated samples were immersed in the glass beaker andtreated with ultrasound alone for 5, 20, 40, 60, 80, and100 min.

Sanitizing solution and evaluationof chemical disinfectants

Sodium hypochlorite (NaOCl, 12%; Shimadzu Co., Kyoto,Japan) dissolved in distilled water was used as the chemicaldisinfectant at 50–200 ppm. All NaOCl solutions were pre-pared immediately before use. The efficacy of NaOCl wasestimated using the European CEN EN 1276 method (dilu-tion neutralization method) based on quantitative suspensiontesting (AOAC, 1995; BSI, 1997).

Eight milliliters of NaOCl solution was added to a mixturecontaining 1 mL of TSB or one piece of stainless steel orsliced iceberg lettuce (7–8 log10 CFU/g) and 1 mL of inter-fering substance. This mixture was treated at 20 – 1�C(mean – standard deviation) for 5 min and then agitated, and1 mL of the mixture was added to a different mixture con-taining 8 mL of neutralizing agent and 1 mL of distilled dis-infecting product. This mixture was maintained for 5 min at20 – 1�C to ensure complete neutralization, after which 1 mLof the mixture was immediately applied to a sterilized Petridish with an Oxford agar base with Bacto Oxford antimi-crobial supplement (MOX, Difco) to count the number ofsurviving bacteria.

The sterile interfering substance samples, designed tosimulate clean and dirty conditions, were prepared by melting0.3 g of bovine serum albumen (Sigma, St. Louis, MO) in100 mL of water, which was then filtered with a membranefiltration system prior to use (0.45-lm pore diameter; Sar-torius AG 3770770, Gottingen, Germany).

The neutralizing agent used to stop the chemical reactionwas created by mixing 3 g of lecithin (Fluka, Buchs, Swit-zerland), 30 g of polysorbate 80 (Fluka), 5 g of sodium thio-sulfate (Sigma), 1 g of l-histidine (Sigma), and 30 g saponin(Fluka) in a 1-L flask. The mixture was then diluted witha diluting agent to increase its mass, melted, and sterilizedprior to use.

Combined ultrasound treatmentand sodium hypochlorite

The method described by Koivunen and Heinonen-Tanski(2005) was used for the combined ultrasound and NaOCltreatments. Ultrasound treatments and NaOCl disinfectantwere performed at room temperature. Disinfectant efficacy ofthe combined treatment was compared with each treatment ofultrasound and NaOCl treatments to estimate any synergisticeffects. The combination was conducted by applying ultra-sound as a primary disinfectant and NaOCl as a secondarydisinfectant.

The efficacy of different treatment was assessed based onthe population reduction of Listeria. The synergistic effectvalues of combined ultrasound and NaOCl disinfection werecalculated using the following equation:

2 LEE ET AL.

Synergistic effect values¼A� (BþC)

A : log10 CFU=g reduction by combined

ultrasound and NaOCl disinfection

B : log10 CFU=g reduction by single

ultrasound detachment

C : log10 CFU=g reduction by single NaOCl disinfection

A synergistic effect was confirmed with any positive val-ues, while antagonistic and no effects were confirmed withnegative and zero value, respectively.

Bacterial enumeration

After combined ultrasound and NaOCl treatments,L. monocytogenes in TSB was enumerated by plating onTSA, which was incubated at 37�C for 24 h. L. mono-cytogenes on the stainless steel chips were detached from thechips by vortexing with glass beads and then cultured on TSAwith the pour-plate technique and incubated at 37�C for 24 h.Finally, the prepared lettuce-leaf samples were transferredinto a sterile stomacher bag (Nasco Whirl-Pak, Janesville,WI) containing 90 mL of sterile 0.1% peptone water and thenhomogenized for 1 min using a stomacher (Bag mixe� 400;Interscience Co., France). Media for enumeration ofL. monocytogenes was prepared using an Oxford agar basewith Bacto Oxford antimicrobial supplement (MOX, Difco).All plates were incubated at 37�C for 24 h.

Texture measurement

In order to identify the change in iceberg lettuce qualitytreated with combined ultrasound and NaOCl, the texture of alltreated samples was measured. The texture analysis was per-formed according to procedures described earlier (Sagong et al.,2011). Changes in iceberg lettuce–leaf texture were evaluatedwith a Texture analyzer (Model TAHDi, 500; Stable MicroSystems, Boochun, South Korea) with SMSP/2 probe. Samplesmeasuring about 5 cm · 5 cm · 1.5 cm (1 piece,*2.5 g of greentissue) was placed onto the press holder and penetrated by aprobe. Maximum force was recorded, and all experiments werereplicated three times with independently prepared samples.

Statistical analysis

Experiments were repeated three times, with duplicate andaverage of duplicate plate from three replications calculated.Data were analyzed by the analysis of variance procedureusing SAS software (Version 9.1; SAS Institute Inc., Cary,NC) for a completely randomized design. When the effect wassignificant ( p < 0.05), mean separation was accomplished withDuncan’s multiple-range test. The results were expressed aslog10 CFU/g, and the response surface was described using theSigmaPlot software system (SigmaPlot 7.0).

Results

Bactericidal effects of ultrasoundwith NaOCl treatments

Figure 1 shows the bactericidal effects on L. monocytogenesunder different ultrasound treatment times (0–100 min) and

FIG. 1. Reduction in Listeria monocytogenes number(log10 colony-forming units (CFU)/mL) from combined ul-trasound and NaOCl treatment in tryptic soy broth (a), onstainless steel (b), and on iceberg lettuce (c).

REDUCTION OF L. MONOCYTOGENES BY NAOCL AND ULTRASOUND 3

NaOCl concentrations (0–200 ppm). Reduction of L. mono-cytogenes by 5–100 min ultrasound treatment in TSB, stainlesssteel, and iceberg lettuce were between 0.03 and 0.27, 0.38 and1.09, and 0.13 and 0.68 log10 CFU/g depending on the treat-ment time, respectively. Reductions in the populations ofL. monocytogenes in TSB, on the surface of stainless steel, andiceberg lettuce were between 1.22 and 4.20, 1.48 and 3.79 and1.25 and 4.17 log10 CFU/mL, depending on the NaOCl con-centration (50–200 ppm), respectively.

Treatment with NaOCl alone had a significant impact onL. monocytogenes populations when compared to no treat-ment, while ultrasound alone did not have a significant effecton L. monocytogenes populations. However, when sampleswere treated with combined treatment using 200 ppm (max-imum concentration) of NaOCl and 100 min of ultrasound(maximum treatment time), L. monocytogenes populationswere reduced by 4.54, 5.50, and 6.51 log10 CFU/mL in TSB,on stainless steel, and on iceberg lettuce, respectively. Theresults of these experiments demonstrated that the com-bined ultrasound and NaOCl treatment resulted in a greaterreduction in L. monocytogenes than the treatments appliedindividually.

Synergistic effects of ultrasoundwith NaOCl treatments

Table 1 shows the synergistic effects of combined treat-ment of ultrasound and NaOCl against L. monocytogenes inTSB, on the surface of stainless steel or iceberg lettuce. Sy-nergistic effects were observed in all combined treatments,although most synergistic values were well below 1 log10

CFU/mL. The highest synergistic values in TSB, on thesurface of stainless or iceberg lettuce were 0.99 (50 ppmNaOCl/100 min ultrasound), 0.62 (200 ppm NaOCl/100 minultrasound), and 1.66 log10 CFU/g (50 ppm NaOCl/100 minultrasound). The results suggested that the synergistic effectsagainst L. monocytogenes were not dependent on NaOClconcentration or ultrasound treatment time, unlike reductionlevels of L. monocytogenes.

Texture measurement

Iceberg lettuce is primarily used for salads, and thus tex-ture property for crispness is measured using the loadingvalue (N) of a Texture analyzer. Table 2 shows the textureresults of iceberg lettuce treated with combined ultrasound

Table 1. Values (log10 Colony-Forming Units [CFU]/g) of Synergistic Effecta

of Combined

Ultrasound and NaOCl Treatment Against Listeria monocytogenes

in Tryptic Soy Broth (TSB), Stainless Steel, and Iceberg Lettuce

Ultrasound (min)

SubjectNaOCl(ppm) 5 20 40 60 80 100

TSB 50 0.23 – 0.01a,z 0.33 – 0.05a,yz 0.39 – 0.04b,yz 0.53 – 0.18a,y 0.97 – 0.08a,x 0.99 – 0.01a,x

100 0.14 – 0.01b,y 0.24 – 0.21a,x 0.69 – 0.04a,x 0.72 – 0.01a,x 0.67 – 0.02b,x 0.71 – 0.01b,x

150 0.01 – 0.01c,x 0.1 – 0.01a,x 0.07 – 0.01c,x 0.11 – 0.07b,x 0.11 – 0.15c,x 0.16 – 0.11c,x

200 0.01 – 0.01c,x 0.03 – 0.04a,x 0.02 – 0.04c,x 0.04 – 0.06b,x 0.08 – 0.08c,x 0.07 – 0.09c,x

Stainless steel 50 0.24 – 0.06a,x 0.44 – 0.03a,xy 0.42 – 0.12a,xy 0.38 – 0.11a,xy 0.44 – 0.08a,xy 0.5 – 0.08a,x

100 0.23 – 0.06a,xy 0.45 – 0.07a,x 0.21 – 0.11a,xy 0.05 – 0.08b,y 0.17 – 0.18a,xy 0.17 – 0.16a,xy

150 0.24 – 0.09a,yz 0.42 – 0.01a,x 0.29 – 0.08a,xy 0.01 – 0.01b,z 0.11 – 0.06a,z 0.06 – 0.08a,x

200 0.16 – 0.13a,x 0.49 – 0.09a,x 0.18 – 0.1a,x 0.1 – 0.15ab,x 0.59 – 0.34a,x 0.62 – 0.4a,x

Iceberglettuce

50 0.16 – 0.01a,x 0.15 – 0.2a,x 0.22 – 0.18a,x 0.25 – 0.01a,x 0.46 – 0.05ab,x 0.43 – 0.15b,x

100 0.12 – 0.17a,x 0.15 – 0.09a,x 0.19 – 0.03a,x 0.25 – 0.11a,x 0.13 – 0.15b,x 0.34 – 0.34b,x

150 0.14 – 0.09a,x 0.25 – 0.1a,x 0.36 – 0.11a,x 0.39 – 0.39a,x 0.44 – 0.24ab,x 0.52 – 0.25b,x

200 0.12 – 0.14a,z 0.23 – 0.01a,z 0.09 – 0.09a,z 0.24 – 0.15a,z 0.77 – 0.25a,y 1.66 – 0.23a,x

Unit: log10 CFU/g – SE (average – standard error).aSynergistic effect values = (reduction achieved with the ultrasound treatment and the NaOCl treatment) – (reduction achieved by the

ultrasound + NaOCl treatment). Within the same column, means with different letters (a, b, or c) differ significantly ( p < 0.05). Within thesame row, means with different letters (x, y, or z) differ significantly ( p < 0.05).

Table 2. Maximum Load Values for Iceberg Lettuce Texture Measurements

After Combined Ultrasound and NaOCl Treatment

Ultrasound (min)Maximumload (N) 0 5 20 40 60 80 100

NaOCl (ppm)0 98.44 – 8.04a 99.51 – 6.33 100.67 – 7.93 96.97 – 5.42 98.46 – 5.41 104.19 – 2.25 101.17 – 2.1850 92.31 – 6.71 107.52 – 5.03 100.21 – 5.52 100.47 – 4.76 89.56 – 11.08 103.46 – 8.8 103.47 – 1.83100 96.52 – 5.52 99.13 – 11.42 102.74 – 5.94 92.24 – 12.1 91.3 – 2.09 96.82 – 7.91 98.39 – 9.15150 92.65 – 6.23 102.52 – 11.22 99.65 – 14.43 102.76 – 12.19 90.77 – 7.39 111.89 – 4.32 106.62 – 7.07200 98.21 – 9.05 103.48 – 11.24 96.58 – 8.92 94.07 – 10.37 101.24 – 0.94 103.91 – 3.22 98 – 18.53

aValues are mean – standard error.

4 LEE ET AL.

and NaOCl under different NaOCl concentrations and ul-trasound treatment times. There was no significant differencein maximum load values among all tested samples ( p > 0.05).The combined treatment did not significantly change iceberglettuce quality.

Discussion

Hurdle technology using a combination of physical andchemical techniques was conducted to compare the effects ofultrasound, NaOCl, or a combination of ultrasound andNaOCl in reducing the L. monocytogenes population oniceberg lettuce.

When ultrasound alone was applied to TSB, stainless steel,or iceberg lettuce inoculated with L. monocytogenes, therewere reduction effects for all treatment times. Increasedultrasound treatment time led to decreased numbers ofL. monocytogenes. The overall magnitude of reduction wasstainless steel > iceberg lettuce > TSB. This suggests that thetype of sample surface may influence antimicrobial activityand that ultrasound might help NaOCl remove L. mono-cytogenes by detaching the bacteria from flat surfaces such asstainless steel.

In this study, the treatment with ultrasound alone resultedin maximum reductions in L. monocytogenes of 0.20, 1.09,and 0.68 log10 CFU/g in TSB, on stainless steel, and iceberglettuce, respectively, with an ultrasound treatment time of100 min (Fig. 1). These results suggested that the treatmentwith ultrasound alone may be not effective for food industryapplications. Piyasena et al. (2003) reported that the bacte-ricidal effects of food treated with ultrasound alone is lo-calized and does not affect a large area. For this reason,ultrasound techniques should be refined to improve sterili-zation effectiveness by combining with other chemical sa-nitizers (Yuting et al., 2013).

NaOCl alone significantly reduced the number of L. mono-cytogenes in TSB, on stainless steel, and on iceberg lettuce.Also, NaOCl solutions were more effective against L. mono-cytogenes in TSB compared to stainless steel or iceberg lettuce.Mustapha and Liewen (1989) reported that 0–400 ppm chlorinetreatments for 1–5 minutes in TSB decreased L. monocytogenesby 3–4 log10 CFU/mL, whereas the same treatments on stain-less steel resulted in a decrease in L. monocytogenes in therange of 1–4 log10 CFU/g (Mustapha and Liewen, 1989). Thebactericidal effects of NaOCl on food were relatively lowcompared to TSB or stainless steel. This suggests that NaOClsolution is less effective on food items such as iceberg lettuce,which have folded leaves and hydrophobic pockets that make itdifficult to remove bacteria (Babic et al., 1996).

The goal of this study was to overcome the limitationsof ultrasound or NaOCl as an individual treatment againstL. monocytogenes on iceberg lettuce. The combination ofultrasound and NaOCl increased disinfection efficiency andshowed synergistic benefits, with the highest synergy valuesin TSB, on stainless steel, and on iceberg lettuce reaching0.99, 0.62, and 1.66 log10 CFU/mL for L. monocytogenes,respectively.

Synergy involves a multiple-damage mechanism in whichtwo different disinfection methods increase reduction effects bydamaging the microorganisms in different ways (Koivunen andHeinonen-Tanski, 2005). In this experiment, ultrasound attackscell membranes via localized heating and production of free

radicals (Piyasena et al., 2003), resulting bacteria detachingfrom the surface of food (Povey and Mason, 1998; Piyasenaet al., 2003; Demirdoven and Baysal, 2009). In the case ofultrasound alone, the damage may be minimal and repairable.However, when bacteria are treated with both ultrasound andNaOCl, the cumulative damage may be irreparable.

In the present study, all of the combined treatments hadsynergistic effects without causing significant texturechange. However, synergistic effects were not dependent onthe concentration of the sanitizer or ultrasound treatmenttime. Other researchers have studied the combined effects ofultrasound and sanitizer on killing and detaching foodbornepathogens in a variety of fresh produce. Zhou et al. (2009)reported that the reduction of the Escherichia coli O157:H7population on spinach leaves was 3.1 log10 CFU/g after acombined treatment of ultrasound and 200 ppm of chlori-nated water. This is an additional 1.1 log10 CFU/g reductioncompared to chlorinated water alone for 2 min. In addition,Sagong et al. (2011) reported that the combined treatment ofultrasound and organic acids for Escherichia coli O157:H7,Salmonella Typhimurium, and L. monocytogenes resulted inan additional 0.8–1.0 log10 CFU/g reduction compared tosingle treatments. Another study showed that SalmonellaTyphimurium on lettuce was reduced by 2.7 log10 CFU/gwhen ultrasound and 100 ppm chlorinated water were ap-plied. This reduction was 1.0 log greater than the reductionobtained by ultrasound or chlorinated water only for 10 min(Seymour et al., 2002).

Conclusions

The present study demonstrated that the combination ofultrasound and NaOCl resulted in synergistic benefits forreducing L. monocytogenes in TSB, on stainless steel, andon iceberg lettuce. The use of ultrasound alone againstL. monocytogenes is currently not feasible, but ultrasound canreduce the concentration of NaOCl required to eliminatebacteria. Therefore, combined ultrasound and NaOCl treat-ments could be used to effectively sanitize fresh-cut productssuch as iceberg lettuce against L. monocytogenes. However,reduction values and synergistic effects depend on the mi-crobial strain, which may be resistant to combined ultrasoundand NaOCl treatment (Piyasena et al., 2003). For this reason,further research on other foodborne pathogens should beconducted with the various conditions before applying thesetreatments in food-processing plants, which will enhance thesafety of minimally processed fresh produce.

Acknowledgments

This research was supported by Basic Science ResearchProgram through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & FuturePlanning (2013005051).

Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Sang-Do Ha, PhD

School of Food Science and TechnologyChung-Ang University

72-1 Nae-ri, Daeduk-myun, AnsungGyunggido 456-756, Korea

E-mail: sangdoha@cau.ac.kr

REDUCTION OF L. MONOCYTOGENES BY NAOCL AND ULTRASOUND 7