Journal of Food Engineering 119 (2013) 425–432

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Journal of Food Engineering

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Bactericidal effects of nonthermal low-pressure oxygen plasmaon S. typhimurium LT2 attached to fresh produce surfaces

0260-8774/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jfoodeng.2013.05.045

⇑ Corresponding authors. Address: Materials Science and Engineering Program,Texas A&M University, College Station, TX 77843-3122, United States. Tel.: +1 609964 6174; fax: +1 979 845 6446 (M. Akbulut).

E-mail addresses: lcisnero@tamu.edu (L. Cisneros-Zevallos), makbulut@mail.che.tamu.edu (M. Akbulut).

Ming Zhang a, Jun Kyun Oh b, Luis Cisneros-Zevallos c,⇑, Mustafa Akbulut a,b,⇑a Artie McFerrin Department of Chemical Engineering, Materials Science and Engineering Program, Texas A&M University, College Station, TX 77843-3122, United Statesb Materials Science and Engineering Program, Texas A&M University, College Station, TX 77843-3122, United Statesc Department of Horticultural Sciences, Texas A&M University, College Station, TX 77843-2133, United States

a r t i c l e i n f o

Article history:Received 25 March 2013Received in revised form 17 May 2013Accepted 31 May 2013Available online 19 June 2013

Keywords:Oxygen plasmaBactericidal effectSanitizationFresh produce

a b s t r a c t

This work investigates the feasibility of nonthermal low-pressure oxygen plasma on sanitization of spin-ach, lettuce, tomato and potato surfaces from Salmonella enterica subsp. enterica serovar Typhimuriumstr. LT2 (Salmonella typhimurium LT2). It was shown that the time of exposure and plasma power densitywere two critical parameters influencing the bactericidal efficiency. Surface roughness and hydrophobic-ity did not influence the sanitization of produce. Oxygen plasma was more effective than washing with3% H2O2 on eliminating S. typhimurium LT2 on spinach. Plasma treatment chemically changed a very thinsection of tomato wax cuticle layer by oxidation reaction and decomposition of carbon chains, whichcould readily and completely be removed by water. Overall, this study confirms that nonthermal oxygenplasma can be a new effective method of sanitization for fresh produce.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Fruits and vegetables play an important role in our diet andhealth by providing essential vitamins, minerals, and fibers. Hu-man pathogens can reach and attach fruit and vegetable surfacesduring growth, harvest, transportation and further handling fromanimal and human sources. Contaminated fruits and vegetablesin particular the ones that are consumed raw can lead to foodborneillnesses. In recent years, the number of documented outbreaks ofhuman infections associated with the consumption of contami-nated fruits and vegetables has increased (Berger et al., 2010;Van Boxstael et al., 2013).

Typical approaches for the sanitization of pathogenic microor-ganisms involve the use of heat, pressure, liquid or gas chemicaldisinfectants, and ionizing or non-ionizing radiation. However, be-cause of their fragile nature, high temperature methods are notsuitable for the decontamination of fresh produce. With outbreaksof foodborne illnesses occurring more frequently, the developmentof novel nonthermal methods to reduce and eliminate bacterialpathogens from fresh produce has received increasing attention(Parish et al., 2003a,b). To this end; X-rays, ultrasound, ultraviolet

light, oscillating magnetic fields, pulsed light, and high voltage arcdischarge based methods have recently been considered in thecontext of fresh produce safety (Zhang et al., 2011; Moosekianet al., 2012; Garcia Loredo et al., 2013; Odriozola-Serrano et al.,2013).

Among novel methods of bacterial sanitization, nonthermalplasma-based sanitization approaches has displayed promisingoutcomes in decontaminating living tissues and biomaterials fromvarious microorganisms (Ragni et al., 2010; Ermolaeva et al., 2011;Noriega et al., 2011). Effectiveness of nonthermal plasma in decon-taminating pathogenic bacteria is attributed to a combination ofeffects including the formation of electrons, ions, free radicalsand excited molecules, as well as UV radiation (Moisan et al.,2001; Laroussi and Leipold, 2004; Kong et al., 2009). The keyadvantages of nonthermal plasma technologies are their relativelysimple and inexpensive design, short processing times, absence oftoxicity, and lack of residue formation (Rossi et al., 2009; Rothet al., 2010; Rupf et al., 2010). Its effectiveness against pathogenicbacteria and the above mentioned advantages have prompted aninterest in the use of the nonthermal plasma-based approachesin food safety. For instance, Deng et al. (2007) has demonstratedthe applicability of nonthermal atmospheric plasma technologyfor the pasteurization of almonds. The technology was found toeffectively reduce Escherichia coli on almond by almost a 5 logfactor after 30-s treatment at 30 kV and 2000 Hz. Ragni et al.(2010) investigated the efficacy of resistive barrier discharge(RBD) plasma for decontamination of shell egg surfaces and

Vacuum Pump

Reaction Chamber

Process Controller

RF Power Generator

O2 gas

UV O

CO2

CO H2O

(A)

(B)

Fig. 1. Schematic illustration of (A) oxygen plasma system, and (B) plasmasanitization in reaction chamber.

426 M. Zhang et al. / Journal of Food Engineering 119 (2013) 425–432

observed reductions up to 2.5 log CFU/eggshell and 4.5 log CFU/eggshell for Salmonella enteritidis using air with low and high mois-ture contents, respectively, after 90 min of RBD plasma treatment.Noriega et al. (2011) investigated the efficiency of nonthermalatmospheric gas plasmas for decontaminating chicken skin andmuscle inoculated with Listeria innocua and observed a 1 log reduc-tion on skin, and a 3 log reductions on muscle under optimalconditions.

The studies on the efficiency of nonthermal plasma on bacteriaresponsible for foodborne illnesses when they are attached to freshproduce surfaces is very limited. Niemira and Sites (2008) reportedthe use of cold air plasma to inactivate human pathogens inocu-lated on golden delicious apples. The reductions of SalmonellaStanley and E. coli O157: H7 ranged from 2.9 to 3.7 and 3.4 to3.6 respectively, after treatment for 3 min at 30 kV and 60 Hz withflow rate of 40 l/min. Fresh produce surfaces may favor bacterialproliferation specially if rich in nutrients (Thunberg et al., 2002;Johnston et al., 2005), through biofilm formation and protectionin crevices (i.e. microscale roughnesses and valleys between theasperities of the produce surface) (Burnett and Beuchat 2000;Burnett et al., 2000). Thus, there is a need to correctly assess thefeasibility of nonthermal plasma in sanitizing fresh producesurfaces (Niemira, 2012).

In general, non-thermal plasma is generated from either atmo-spheric pressures or low pressures. Both atmospheric and lowpressure plasma generates same species and same electron densi-ties range (Schutze et al., 1998). Therefore, they have similar plas-ma sanitization mechanics (Moisan et al., 2001; Laroussi, 2005).The main advantage of the atmospheric-pressure plasmas is thatthey do not require vacuum systems to operate. However, underatmospheric conditions, higher voltages are required to generateplasma. To be specific, the voltage required to initiate the ioniza-tion decreases from �10,000 V to �100 V if the pressure reducesfrom atmospheric pressure to 10�3 atm for a 1-cm gap betweenelectrode plates (Lieberman and Lichtenberg, 2005). At highervoltages, often arcing occurs between the electrodes. The arcingmay damage and burn fragile surfaces such as fresh produce sur-faces. Considering that the low pressure vacuum packaging hasbeen used for packaging of many fresh produces (An et al., 2009)and the abovementioned points, the use of low-pressure plasmasanitization instead of atmospheric-pressure one has certainadvantages.

Accordingly, this paper investigated the bactericidal effect ofnonthermal low-pressure oxygen plasma on Salmonella entericasubsp. enterica serovar Typhimurium LT2 (S. typhimurium LT2) at-tached on fresh lettuce, spinach, tomato, and potato surfaces. Thismicroorganism was selected because data from the US-CDC food-borne outbreak surveillance system show that the most com-monly reported microorganisms associated with fresh producefoodborne illness outbreaks are Salmonella spp. (Sivapalasingamet al., 2004). Oxygen was selected as the gas source becauseoxygen was found to be one of the best sanitization agents(Bol’shakov et al., 2004). Furthermore, four possible combinationsof surface roughness and hydrophilicity including hydrophobicsmooth, hydrophobic rough, hydrophilic smooth, and hydrophilicrough surfaces were covered through the selected produce. This ispossible since spinach and potato are relatively rough, whiletomato and lettuce are relatively smooth, and spinach and tomatoare relatively hydrophobic, while lettuce and potato are relativelyhydrophilic. These properties can influence sanitization efficacy insome sanitization methods, especially liquid based methods(Ukuku and Fett, 2006; Fransisca and Feng, 2012). In the presentstudy, the efficiency of a nonthermal oxygen plasma methodwas compared with that of washing with aqueous solutions ofH2O2. Associated physicochemical changes upon the plasmatreatment were also reported.

2. Materials and methods

2.1. Preparation of produce surface

Spinach, lettuce, tomato and potato were purchased from a lo-cal grocery store (Wal-Mart, College Station, TX, USA). After mildlywashed for 30 s using 1 L of deionized water, the produce wasdried using tissue paper. Then, spinach, lettuce, tomato skin andpotato skin were cut into square pieces of 1 cm � 1 cm. The pro-duce pieces were immobilized on a silica wafer using a doublesided adhesive carbon tape.

2.2. Preparation of inoculum

Rifampicin-resistant S. enterica subsp. enterica serovarTyphimurium str. LT2 (S. typhimurium LT2; ATCC 700720) was ob-tained from the ATCC (Manassas, VA, USA) and maintained onslants of tryptic soy agar (TSA; Becton, Dickinson and Co., Sparks,MD, USA) at 5 �C. Working cultures were obtained by transferringa loop of culture from TSA slants to 9.0 mL of tryptic soy broth(TSB; Becton, Dickinson and Co.) and incubating aerobically with-out agitation at 37.5 �C for 24 h. After 24 h, a loop of culture wastransferred to a fresh 9 mL of TSB and incubated aerobically with-out agitation at 37.5 �C for 24 h. After incubation, the culture wastransferred to a 15 mL conical centrifuge tube (Thermo-Fisher Sci-entific, Inc.). Bacterial cells were collected by centrifugation at2191 � g in a Jouan B4i centrifuge (Thermo-Fisher Scientific, Inc.)for 15 min at 22 �C. The resulting pellet was suspended in 9.0 mLof Milli Q (MQ) water and washed by centrifugation for 15 minat 22 �C; the entire centrifugation and washing procedure was re-peated identically three times. After the final cycle, the pellet wassuspended in 9.0 mL MQ water and used immediately in the inoc-ulation experiments. This resulted in an inoculum concentration of8 ± 0.4 � 1010 CFU/ml, and determined via selective plating on TSA.Survivors were enumerated following 24 h aerobic incubation at37.5 �C.

2.3. Nonthermal oxygen plasma treatment

Plasma treatment experiments were performed by MarchCS-1701 Reactive Ion Etching system (March Plasma Systems,Inc., CA, USA). The system consists of four modules: a reactionchamber/process controller, a solid state radio frequency (RF)power generator, a vacuum pump and an oxygen source(Fig. 1A). By applying a strong RF electromagnetic field to the wafer

A A A

B

C

Fig. 2. Survival curve of S. typhimurium LT2 on spinach after plasma treatment for100 s as a function of plasma power density. Treatments with same letters are notsignificantly different based on Tukey’s test (p 6 0.05).

AA

BB

C

D

Fig. 3. Survival curve of S. typhimurium LT2 on spinach after plasma treatment at0.34 W/cm3 for different times of exposures. Treatments with same letters are notsignificantly different based on Tukey’s test (p 6 0.05).

M. Zhang et al. / Journal of Food Engineering 119 (2013) 425–432 427

platter, plasma is initiated in the system using oxygen gas. The sys-tem runs at an RF frequency of 13.56 MHz with a maximum poweroutput of 600 W. The chamber is 15 cm in interior diameter with2.5 cm spacing between electrodes. When the gas in the chamberis exposed to the oscillating electric field at high frequency and en-ergy, the electrons of the gas start to gain energy, eventually ioniz-ing to atoms. The initial ionization will provide more energy to thesystem to cause further ionizations in a chain reaction manner,ultimately filling the chamber with plasma, which could react withthe treated surface (Fig. 1B). The RF power applied and pressure ofthe chamber are two main parameters influencing the density andtemperature of the plasma (Chen and Chang, 2003). In the presentstudy, we investigated the effect of plasma power and exposuretime on the bactericidal properties of the oxygen plasma. To ensurethe reproducibility of the results, each experiment was repeated atleast three times.

Plasma chemistry of pure oxygen RF plasma has been well iden-tified. Mogul et al. (2003) studied emission spectrum for pure O2

plasma discharge powered by a 13.56 MHz radio frequency at100 W and 500 mTorr (67 Pa). They found the presence of excitedatomic oxygen, oxygen cation, dioxygen cation, and neutral exciteddioxygen in plasma. Bol’shakov et al. (2004) also found the similarresults when they studied oxygen plasma generated at 13.56 MHzin the range of 13–67 Pa and pressure and 100–300 W power.These emission spectrum results were also consistent with the pre-viously published oxygen spectra of oxygen plasmas (Carl et al.,1990; Tuszewski et al., 1995). Since the plasma in this paper wasgenerated under similar experiment conditions (13.56 MHz,34 Pa, 50–350 W), species in plasma in this paper is expected toconsist of excited atomic oxygen, oxygen cation, dioxygen cation,and neutral excited dioxygen as well.

2.4. Inoculation of produce surfaces

The experimental protocol for the inoculation of producesurfaces used in plasma treatment studies was as follows: Initially,100 ll of bacterial inoculum at 8 ± 0.4 � 1010 CFU/ml was addeddropwise and distributed evenly onto produce surfaces(1 cm � 1 cm pieces) and the produce surfaces were air-dried atroom temperature for �4 h. After the inoculated surface wasplaced in the chamber, the chamber was first evacuated at 13 Pa,and then the chamber was filled with O2 to 30 Pa with a gas flowrate of 10 sccm. After the plasma treatment at room temperature,the chamber was ventilated with air to reach atmospheric pres-sure. To compare the efficacy of plasma sanitization with that ofH2O2 washing, some of inoculated surfaces (spinach) were rinsedin 3% H2O2 solution for 600 s instead of the plasma treated. Bothof the treated produce surfaces were then used for bacterialcounting.

2.5. Enumeration of inoculum organisms

The numbers of S. typhimurium LT2 cells on the produce sur-faces were measured for each exposure time and power densityof plasma treatment. Once the treated surfaces were removed fromthe plasma chamber, these were put in 9 ml test tubes of 0.1% ofpeptone water. The tubes were shaken on a Mini Shaker (VWRInternational, LLC) at 900 rpm for 10 min. Serial dilutions of thesuspension were made and plated on TSA supplemented with80 lg/ml rifampicin. Survivors were enumerated following 24 haerobic incubation at 37.5 �C.

2.6. Water contact angle measurement

The relative hydrophobicity of produce surface was evaluatedusing water contact angle measurements. One drop of Milli-Q

water was placed on a produce surface and allowed to equilibratefor 30 s before making any measurements. A digital camera wasused to take the images for angle measurement. For each speci-men, at least three contact angle measurements were conducted.

2.7. Scanning electron microscopy (SEM)

SEM (JSM-7500F, JEOL, Peabody, MA, USA) was used for threepurposes in this study: to determine morphology (roughness) ofthe neat produce surfaces; to characterize the physical changeson the produce surface due to oxygen plasma treatment; and tocompare the bacterial attachment behavior of the produce sur-faces. To study the effect of surface property on bacteria adhesion,produce surfaces rinsed into S. typhimurium LT2 solution for 5 minand then dry in a hood at room temperature for overnight. SEMimages were obtained from there with or without the abovetreatment. Prior to the SEM studies, the samples were coated with

428 M. Zhang et al. / Journal of Food Engineering 119 (2013) 425–432

a 50-nm layer of Au by sputter coater (Cressington 105HR, Cres-sington, England) to ensure electrical conductivity.

2.8. Nuclear magnetic resonance (NMR)

Proton nuclear magnetic resonance (H NMR) was used to inves-tigate chemical changes induced by oxygen plasma treatment. To-mato skin with and without exposure to the oxygen plasma wasplaced in deuterated chloroform (CDCl3) and sonication for 5 min

Fig. 4. SEM micrographs of four produce surfaces with or without exposing to bactmagnification images were obtained. (For interpretation of the references to color in th

Num

ber o

f Sur

vivo

rs (L

og C

FU)

Food Surface

A A A

A

B

CC

C

((A)

Fig. 5. (A) Number of surviving S. typhimurium LT2 on different produce surfaces before ain number of surviving S. typhimurium LT2 on different food surfaces. Treatments with

to dissolve non-polar compounds and in deuterated water (D2O)to dissolve polar ones. The extracted compounds were transferredinto 5 mm NMR tubes and analyzed using a Bruker 400 MHz NMRspectrometer (Bruker, Billerica, MA, USA) at 298 K.

2.9. Statistical analysis

All experiments were replicated at least three times. Numbersof survival bacteria were converted to log CFU and means and

eria dispersion for 5 min. Red rectangles indicates the areas from which higheris figure legend, the reader is referred to the web version of this article.)

Red

uctio

n in

Num

ber

of S

urvi

vors

(Log

CFU

)

Food Surface

D

D

D D

B)

nd after plasma treatment of 600 s at 0.34 W/cm3. (B) Corresponding Log reductionssame letters are not significantly different based on Tukey’s test (p 6 0.05).

M. Zhang et al. / Journal of Food Engineering 119 (2013) 425–432 429

standard deviations were calculated. Analyses of variance wereconducted using JMP (SAS Cary, NC, USA) and mean separationtests performed using a post hoc Tukey’s test (p < 0.05).

3. Results and discussion

3.1. Effect of plasma energy density on bactericidal efficiency

Fig. 2 shows the number of surviving S. typhimurium LT2 onspinach surface as a function of actual power output of radiofrequency (RF) per unit volume of the reaction chamber (powerdensity) of plasma for a given time (t = 100 s). The number ofsurvivors decreased with increasing power density in a nonlinearmanner: At low power densities the bactericidal effect increasedweakly while at large power densities the bactericidal effect

A

B

C

D

H2O2Washing

Plasma(0.34 W/cm3)

WaterWashing

Num

ber o

f Sur

vivo

rs (L

og C

FU)

Control

Sanitization Method

Fig. 6. Number of surviving S. typhimurium LT2 for different sanitization methodson spinach surface. Duration of each treatment method was kept constant, i.e. anexposure time of 600 s. Treatments with different letters were significantlydifferent based on Tukey’s test (p 6 0.05).

Fig. 7. An illustration of tomato surface (A); SEM images of tomato before plasma treatmplasma treated for 600 s (E) and then rinsing in MQ water (F).

increased strongly (p < 0.05). Given higher plasma energy densitiesgive rise to higher intensities of UV irradiation, UV photons, andreactive species such as O, O�, and O2

� (Laroussi and Leipold,2004; Wu et al., 2012), it is reasonable that bactericidal effectincreased with increase power density. No visual damage wasobserved for power densities up to 0.57 W/cm3. However, the spin-ach surface showed some signs of etching at a power density of0.79 W/cm3 and an exposure time of 100 s. Thus, although higherenergy densities can allow better sanitization, care must be takento prevent damage on the produce surface.

3.2. Effect of plasma exposure time on bactericidal efficiency

Fig. 3 shows number of S. typhimurium LT2 on spinach surfaceas a function of time of plasma exposure at a power density of0.34 W/cm3 (output power of 150 W). The logarithmic number ofsurviving bacteria before the exposure (t = 0) was 6.3 ± 0.1. Thecomplete sanitization was achieved at 800 s (<1 log CFU). Novisible side effect was observed on the spinach after a plasmatreatment of 800 s at 0.34 W/cm3.The logarithmic numbers ofviable bacteria decreased linearly with increasing plasma exposuretime. This finding implies the number of surviving microorganismsdecreased, as an exponential function of time. This trend is consis-tent with other plasma sanitization studies involving other types ofsurfaces (Herrmann et al., 1999; Moisan et al., 2002). The half-lifeof the bacteria killing reaction is about 48 s at a power density of0.34 W/cm3. Accordingly, an exposure time of 317 s decreasesthe total number of surviving S. typhimurium LT2 to 1.0%, and anexposure time of 635 s decreases to 0.01%.

3.3. Effect of food surface on bactericidal efficiency

Tomato, lettuce, spinach, and potato were selected due to theirdifferent surface properties. The water contact angles are 107 ± 6for tomato, 81 ± 7 for spinach, 57 ± 4 for lettuce, and <30 for potato.SEM micrographs showed that tomato and lettuce were relativelysmooth while spinach and potato were relatively rough (Fig. 4).Rough and hydrophilic surface (potato) favored for bacteriaadhesion, while smooth and hydrophobic surface (tomato) hin-dered bacterial adhesion (Fig. 4). This behavior is consistent withthe previous studies also showing that these surface properties

ent (B); after plasma treated for 100 s (C) and then rinsing in MQ water (D); after

(A)

430 M. Zhang et al. / Journal of Food Engineering 119 (2013) 425–432

(roughness and hydrophobicity) can influence bacterial adhesion(Bruinsma et al., 2001; Wang et al., 2009).

To better understand the effect of produce surface properties onthe efficiency of plasma sanitization, we conducted plasma saniti-zation experiments using four different produce surfaces: tomato(smooth hydrophobic), lettuce (smooth hydrophilic), spinach(rough hydrophobic), and potato (rough hydrophilic). The numberof viable bacteria after drying and after plasma treatment for 600 sat 0.34 W/cm3 is shown in Fig. 5A. Initial viable bacteria were thesame for all surfaces (p < 0.05); and after treatments, all surfacesshowed lower bacteria counts, still the same for all surfaces(p < 0.05) with exception of a slight higher value for potatoes.However, when reporting reductions in numbers of the viable bac-teria, these were 3.0 ± 0.9, 2.7 ± 1.0, 2.2 ± 0.9 and 2.2 ± 0.9 for spin-ach, lettuce, tomato and potato, respectively with no significantdifference among them (p < 0.05). Accordingly, we confirm thatsurface properties of roughness and hydrophobicity have no im-pact on oxygen plasma sanitization. Upon 600 s plasma treatmentat 0.34 W/cm3, no visible damage was observed for all producesurfaces.

(B)

3.4. Comparison to hydrogen peroxide washing

Hydrogen peroxide treatment is one of the most common san-itization methods for vegetable and fruit surfaces (Parish et al.2003a,b; Ukuku and Fett, 2006). Therefore, we compared the effi-ciencies of the nonthermal oxygen plasma treatment method witha hydrogen peroxide treatment and water washing using spinachsurface (Fig. 6). Duration of each treatment method was kept con-stant, i.e. an exposure time of 600 s. The logarithmic number ofsurviving bacteria decreased from 6.3 ± 0.1 to 5.4 ± 0.1 after waterwashing. The survival number decreased to 4.2 ± 0.1 upon a 600 sexposure to 3% H2O2 while the number further decreased to3.4 ± 0.4 for the case of 600 s plasma treatment at 0.34 W/cm3.

When surface characteristics such as roughness and hydropho-bicity promote the formation of a physical barrier for aqueousmedia penetration, the effectiveness of H2O2 treatment decreases(Ukuku and Fett, 2006; Fransisca and Feng, 2012). We hypothesizethat the barrier is due to micro-air pockets which protect themicroorganisms from the sanitizer. On the other hand, the lengthscale (�angstrom) of gas reactive species is much smaller thanthe length scale of surface roughness of produce surfaces (severalmicrometers). Therefore, the crevices and micro-air pockets donot hinder the plasma sanitization. The difference in the efficiencyof plasma and peroxide treatment is attributed to thisphenomenon.

CH 3

CH 3

CH 3

CH 3

CH 2

CH

CH 2

CH 2

O O*

O2*

CO2

H2OCuticle layer

CO

OH

OHO OOH

OH

CO

OH

CO

OH

CO

OH

(C)

Fig. 8. (A) 1H-NMR spectrum of solution obtained through extraction of tomatoskin in CDCl3; (B) 1H-NMR spectrum of solution obtained through extraction oftomato skin in D2O. Black lines indicate ‘‘before plasma treatment’’, and red linesindicate ‘‘after the plasma treatment’’; and (C) An illustration of potential chemicalchanges taking place on produce surface upon oxygen plasma treatment. (Forinterpretation of the references to color in this figure legend, the reader is referredto the web version of this article.)

3.5. Physical changes occurring on produce surfaces after plasmatreatment

SEM was used to study the physical changes occurring onproduce due to the nonthermal oxygen plasma treatment (Fig. 7).Typical produce surfaces like tomato cuticle are covered by awax layer followed by a layer of cutin or cutin blends of wax andcell wall substances such as carbohydrates. Bellow the cuticle arethe epidermis and mesophyll cells (Fig. 7A). As observed inFig. 7B, tomato surfaces exhibited regular ridge-and-valley struc-tures. Because the outermost layer is covered with cuticle, it wasnot possible to directly visualize the upper epidermis. Upon anexposure of 100 s to oxygen plasma at 0.34 W/cm3 (output powerof 150 W), the surface roughness increased slightly and very smallamount of debris was observed on the surfaces (Fig. 7C). After rins-ing with water, the debris was almost completely removed andintercellular spaces became slightly more apparent (Fig. 7D). Therewas no sign of damage in epidermis at this point.

Upon an exposure of 600 s to oxygen plasma at 0.34 W/cm3,intercellular space could be observed through the translucentcuticle layer, indicating removal of the wax cuticle layer (Fig. 7E).In addition, the surface became roughness. When water rinsingfollow the plasma treatment, the epidermal cells seem to bedamaged, indicating a possible partial removal of the cuticle layerwith an exposure of 600 s to oxygen plasma at 0.34 W/cm3 (Fig. 7F)The damage was more evident after water washing, since the plas-ma treatment may convert hydrophobic groups from wax to smallmolecule weight hydrophilic forms and water can dissolve andetch away the hydrophilic groups easier.

M. Zhang et al. / Journal of Food Engineering 119 (2013) 425–432 431

In general, SEM studies suggest that short exposure times haveno major effects on the produce surface while long exposure timesmay partially damage the cuticle layer and epidermis cells.Depending on the produce type, the removal of the cuticle layermay reduce the storage and shelf life of produce (Kissinger et al.,2005; Saladie et al., 2007). However, given that coating of producesurfaces with edible wax or other coatings is becoming widespread(Park, 1999; Dávila-Aviña et al., 2012), if necessary, the removedcuticle layer can be substituted with an edible wax layer. Inessence, the time and power density of nonthermal plasmatreatment needs to be optimized for each produce surface toensure an acceptable level of cuticle/upper epidermis removaland bacterial sanitization.

3.6. Chemical changes occurring on produce surfaces after plasmatreatment

To characterize such chemical changes taking place onproduce surface upon nonthermal oxygen plasma treatment(e.g., tomato surface, 100 s, 0.34 W/cm3), we relied on NMR.NMR spectra obtained using CDCl3 extraction indicated that thesignal intensity of proton at �1–2 ppm, which is presumablydue to R-CH2CH3, R-CH(CH3)2, or R-C(CH3)3 groups, decreasedafter the plasma treatment (Fig. 8A). NMR spectra obtained usingD2O extraction showed that the signal intensity of proton at�2–3 ppm, which is presumably due to R-CO-CH3, R-CO-CH2CH3

or R-CH2COOH groups, increased after plasma treatment (Fig. 8B).These findings are consistent with previous studies focusing onthe chemical effects of oxygen plasma on mineral oils (Korzecet al., 1994; Jing et al., 2005), polymers (Hillborg et al., 2000;Calvimontes et al., 2011) and other organic materials (Li andHorita, 2000). Accordingly, NMR results suggest that when toma-to surfaces are treated by nonthermal oxygen plasma, the waxcuticle layer can be oxidized to form aldehyde and carboxylicacid groups and/or further oxidized (decomposition of carbonchains) to CO2 and H2O, which would be removed from thetomato surface (Fig. 8c).

4. Conclusion

In the present study, we have investigated the efficiency of non-thermal low-pressure oxygen plasma treatment on sanitization offresh produce surfaces and the associated physicochemicalchanges taking place on surface cuticle layers. It was shown thatthe time of exposure and plasma power density were two criticalparameters influencing the bactericidal efficiency of nonthermaloxygen plasma treatment. The half-life of the oxygen plasma kill-ing reaction of S. typhimurium LT2 was about 48 s at power densityof 0.34 W/cm3. The sanitization method worked equally well forrough hydrophobic (spinach), rough hydrophilic (lettuce), smoothhydrophobic (tomato) and smooth hydrophilic (potato) produce.For a given time, the bactericidal efficacy of nonthermal oxygenplasma was found to be 1 order of magnitude better than that of3% H2O2 treatment for S. typhimurium LT2 on spinach surface. Itwas also shown that oxygen plasma treatment (0.34 W/cm3) onlyaffects the wax cuticle layer under conditions of short- to interme-diate-exposure times. However for long exposure times, wax cuti-cle layer and upper epidermis cells may be damaged. Oxygenplasma changed the wax surface chemistry through oxidationreactions forming aldehyde and carboxylic acid, and by decompo-sition of carbon chains. Water rinsing could easily remove theseresidues from the produce surface. Overall, the nonthermal oxygenplasma treatment shows a potential for the efficient sanitization offresh produce surfaces.

Acknowledgement

This project was supported by Agriculture and Food ResearchInitiative Competitive Grant No. 2011-67017-30028 from theUSDA National Institute of Food and Agriculture.

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