Efficacy of chlorine dioxide, ozone, and thyme essential oil or a sequential washing in killing escherichia coli o157:h7 on lettuce and baby carrots

Lebensm.-Wiss. u.-Technol., 35, 720–729 (2002) Efficacy of Chlorine Dioxide, Ozone, and Thyme Essential Oil or a Sequential Washing in Killing Escherichia coli O157:H7 on Lettuce and Baby Carrots N. Singh, R. K. Singh*, A. K. Bhunia and R. L. Stroshine N. Singh, R. K. Singh: University, of Georgia, Department of Food Science & Technology, Food Science Building, A. K. Bhunia: Purdue University, Department of Food Science, West Lafayette, IN 47907-1160 (U.S.A.) R. L. Stroshine: Purdue University, Department of Agricultural and Biological Engineering, (Received July 23, 2002; accepted August 6, 2002) Chlorine dioxide (ClO2), ozone, and thyme essential oil has been found to be effective in reducing pathogens, including Escherichiacoli O157:H7, on selected produce. The efficacy of these sanitizers was evaluated, alone or through their sequential washing toachieve a 3 or more log reduction of mixed strains of E. coli O157:H7 on shredded lettuce and baby carrots. Samples sprinkleinoculated with mixed strains of E. coli O157:H7 were air-dried for 1 h at 2272 1C in a biosafety cabinet, stored at 4 1C for 24 h, andthen treated with different concentrations of disinfectants and exposure time. Sterile deionized water washing resulted inapproximately 1log reduction of E. coli O157:H7 after 10 min washing of lettuce and baby carrots. Gaseous treatments resulted inhigher log reductions in comparison to aqueous washing. However, decolorization of lettuce leaves was observed during long exposuretime. A logarithmic reduction of 1.48–1.97log10 cfu/g was obtained using aqueous ClO2 (10.0 mg/L for 10 min) ozonated water(9.7 mg/L for 10 min) or thyme oil suspension (1.0 mL/L for 5 min) on lettuce and baby carrots. Of the three sequential washingtreatments used in this study, thyme oil followed by aqueous ClO2/ozonated water, or ozonated water/aqueous ClO2 weresignificantly (Po0.05) more effective in reducing E. coli O157:H7 (3.75 and 3.99log, and 3.83 and 4.34 log reduction) on lettuceand baby carrots, respectively. The results obtained from this study indicate that sequential washing treatments could achieve 3–4logreduction of E. coli O157:H7 on shredded lettuce and baby carrots.
r 2002 Elsevier Science Ltd. All rights reserved.
Keywords: chlorine dioxide; ozone; thyme oil; E. coli O157:H7; lettuce; baby carrots vehicles of E. coli O157:H7. The E. coli O157:H7outbreaks occurring from the consumption of fresh-cut Pathogenic microorganisms of most concern in mini- vegetables (FCVs) have led investigators to search for mally processed fresh produce include Escherichia coli novel methods of controlling E. coli O157:H7 contam- O157:H7, Listeria monocytogenes, Shigella, Salmonella, and hepatitis A virus. These organisms have all been Water containing 50–200 mg/L of chlorine is widely implicated in outbreaks of food-borne illnesses linked to used in food processing plants to sanitize whole fruits the consumption of contaminated fresh vegetables and vegetables as well as fresh-cut produce, but this treatment only results in a reduction of bacterial populations of less than 2log cfu/g of fruits and O157:H7, which is known to cause hemorrhagic colitis and hemolytic uremic syndrome, has emerged as a food- borne pathogen of major public health concern L. monocytogenes on fresh-cut lettuce and cabbage meat, milk, fruit juices, and vegetables are possible treated with 200 mg/L of chlorine was 1.3–1.7 and0.9–1.2log cfu/g, respectively. In addition to suchlimited efficacy, the possible formation of carcinogenic *To whom correspondence should be addressed.
E-mail: rsingh@arches.uga.edu chlorinated compounds in water (chloramines and r 2002 Elsevier Science Ltd. All rights reserved.
All articles available online at http://www.idealibrary.com on chlorine in food-processing plants. Chlorine, even when are widely used in food industry and are considered used at low concentration, may cause taste and odor GRAS substances. Among them, thyme essential oil defect in treated products. Due to these problems, there has been reported to have antibacterial, antimycotic, is a great interest in developing alternative sanitizers for antioxidative and food preservative properties Research and commercial applications have revealed are inhibited by essential oils seems to involve different that chlorine dioxide, ozone, and natural antimicrobial modes of action. The most frequent inhibitions involve solutions can replace traditional sanitizing agents and phenolic components of oils that sensitize the phospho- lipid bilayer of the cell membrane, causing an increase Chlorine dioxide (ClO2) is a strong oxidizing agent and of permeability and leakage of vital intracellular has a broad biocidal effectiveness. It has about 2.5times or impairment of bacterial enzyme systems In addition, it does not react with nitrogen-containing compounds or ammonia to form dangerous chloramine reported promising results when naturally contaminated lettuce was washed with solutions con- react with phenolic compounds to produce foul smelling taining oil of basil, with comparable results to those and tasting chlorophenols as does chlorine Food preservation can be assisted not only by naturally has allowed the use of aqueous chlorine dioxide in occurring chemical agents with individual antimicrobial washing fruits and vegetables. Chlorine dioxide has been effects but also by interactions among multiple anti- used as a drinking water treatment agent since 1944 and microbial factors resulting in additive or synergistic efficacy of a combination of various GRAS chemicals chlorine dioxide in reducing the number of bacteria and moderate temperature to kill E. coli O157:H7 in present in poultry-processing water. It was found that 1.0 g/L peptone solutions. They reported that at 22 1C, 5mg/L chlorine dioxide was as effective as 34 mg/L complete inactivation of E. coli O157:H7 was observed after 20 min of exposure to 15mL/L lactic acid plus microorganisms inoculated onto the surface of a model 1.0 mL/L hydrogen peroxide, whereas a reduction of storage tank with a relative humidity (RH) above 90% 5log10 cfu/ml was observed with a treatment of 15mL/L were completely killed after a treatment by 10 mg/L chlorine dioxide gas for 30 min at 9–28 1C. evaluated a two-step disinfection process for control of Cryptosporidium parvum in 0.05mol/L treatment (5mg/L, 10 min) at 4 and 22 1C resulted in 1.1 phosphate buffer at pH 8 and temperature 22 1C. They and 0.8log reductions of L. monocytogenes, respectively, reported that the sequential treatment of oocytes by on cut lettuce. also studied the effects ozone followed by chlorine dioxide resulted in addi- of washing and chlorine dioxide gas treatment on the tional inactivation of C. parvum due to the synergism of survivability and attachment of E. coli O157:H7 on uninjured and injured green pepper surfaces. They The majority of this work has been conducted in culture obtained 3.03 and 6.45log reductions of E. coli media, consequently little is understood about their O157:H7 after treatments by 0.62 and 1.24 mg/L effectiveness in the food substrates. The present study chlorine dioxide, respectively, for 30 min at 22 1C and was done to evaluate the effectiveness of aqueous and 90–95% RH on surface-injured green peppers. Ozone gaseous forms of chlorine dioxide and ozone, and thyme has recently been declared as a generally recognized as essential oil as potential antibacterial surface treatments safe (GRAS) substance by an expert panel for use in to eliminate E. coli O157:H7 inoculated on shredded lettuce and baby carrots. The efficacy of these treat- encouraged broader use of this gas in food industry.
ments being applied individually and sequentially was The biocidal effect of ozone is caused by a combination of its high oxidation potential, reacting with organicmaterial up to 3,000 times faster than chlorine and its ability to diffuse through biological cell membranes. treated shredded lettucewith ozone and reported that bubbling ozone gas Romaine lettuce and baby carrots were purchased from decreased the natural microbial load by 1.5–1.9log in a local supermarket (West Lafayette, IN, U.S.A.) and 5min. Essential oils and extracts from some herbs and stored at 7 1C. The outer three or four leaves of lettuce spices have been shown to affect the growth and and core were removed and discarded. The remaining leaves and baby carrots were then rinsed with cold tap water for 1 min at 22 1C. Intact and unwilted portions of leaves were cut into pieces (3 Â 3 cm2, approximately 1 g each) using sterile knife and used in experiments. The was immediately dissolved in 1 L deionized sterile lettuce pieces and baby carrots were treated by UV-light water in a brown bottle. Two hundred milliliters of (30 W, 50 cm irradiation distance) in a class II biosafety chlorine dioxide solution was used for measurement cabinet (Labcono Corporation, Kansas city, MO, of ClO2 concentration following the procedures of the U.S.A.) for 30 min (15min for each side) to reduce the amperometric method. An aqueous solution of chlorine dioxide was prepared by dissolving, a certain volumeof chlorine dioxide gas into deionized sterile water usinga gas-sampling syringe. Before injecting the gas into the water, the gas was first dissolved in water in A three-strain cocktail of E. coli O157:H7 (C7927, the syringe by drawing some water in and out EDL933, and 204P) was used in this study. E. coli repeatedly. The chlorine dioxide concentration was O157:H7 C7927 was provided by Dr M. P. Doyle at the measured in triplicate and the data were recorded University of Georgia, Athens, GA, U.S.A. and strains EDL 933 and 204P were obtained from Dr A. K. Bhunia(Purdue University, IN, U.S.A). Bacterial cultures weremaintained at 5 1C on slants of tryptic soy agar (TSA) (Difco Laboratories, Detroit, MI, U.S.A.) and activated Ozone gas was generated using a laboratory corona by culturing in tryptic soy broth (TSB, pH 7.3) (Difco discharge ozone generator (Clear Water Tech, Inc., Laboratories) at 37 1C at least twice at 24 h intervals San Luis Obispo, CA, U.S.A.) from purified, extra- prior to being used in experiments. The 24 h cultures of dry oxygen feed gas. The oxygen carrier gas containing each bacterium were washed three times by centrifuging ozone was humidified by flowing through 75mL sterile (1,800 Â g, 10 min, 21 1C) with 0.1 mol/L sterile phos- deionized water in a 125mL gas-washing bottle (Chem- phate buffer, pH 7.0 (PBS) and the cell pellets were glass Inc., Vineland, NJ, U.S.A.). The humidified resuspended in the same buffer. A final inoculum and ozonated oxygen was passed through the cylinder (1 Â 109 cfu/mL) cocktail was prepared by mixing the at a 1 L/min flow rate using a diaphragm vacuum three bacterial suspensions in the same proportion, and pump (KNF Neuberger, Inc., Trenton, NJ, U.S.A.).
was used to inoculate the lettuce leaves and baby The concentration of ozone gas in the treatment cylinder carrots. Bacterial populations in the inoculum were was measured using a high-concentration ozone monitor determined by surface plating duplicate samples on TSA after serial dilution in 1.0 g/L peptone water. The plates tion, Inc., San Diego, CA, U.S.A.). For the production were incubated for 24 h at 37 1C before colony counts of ozonated water, the ozone-containing oxygen carrier gas was bubbled for a minimum of 15min at 22 1Cthrough 300 mL sterile deionized water in a 500 mL flaskat 1, 3, and 5ozone output levels. The flask was then connected to a reservoir containing 20 g/L potassium Shredded lettuce leaves, and baby carrots (100 g each) iodide (KI) solution capable of neutralizing excess were placed into sterile bags and then sprinkle inocu- ozone. The ozone solution was used within 2 min of lated with the mixed inocula of E. coli O157:H7 (1 mL) removing it from the gas stream. To determine the to obtain an initial level of 108 cfu/g. The samples were dissolved ozone concentration in water, a modified shaken vigorously for 3 min to evenly distribute the iodometric method was used. Ten millilitres of ozone- inoculum. To allow attachment of bacteria, inoculated treated water was added to 90 mL of a 20 g/L KI lettuce leaves and baby carrots were air-dried under a solution, acidified to pH of just below 2 with 1N H class II biosafety cabinet for 1 h at 2272 and 1 mL of starch indicator was added. The solution stored in a refrigerator at 4 1C for 24 h before exposing was titrated with 0.0005mol/L thiosulfate solution and the concentration of ozone was calculated accord-ing to All experimental workwith ozone was done in a chemical fume hood. The Chlorine dioxide (gas/aqueous) production concentration of ozone in the air and water was Chlorine dioxide gas was generated from a CDG laboratory generator (CDG Technology, Inc., NewYork, NY, U.S.A.) using 4% chlorine in nitrogengas (Matheson Gas Products Co., Joliet II). Thegenerated chlorine dioxide gas (115mg/L chlorine dioxide in nitrogen) was collected in 4.7 L Teflon PEP Thyme essential oil was obtained from Lebermuth Co., gas-sampling bag (Cole-Parmer Instrument Co., Vernon Inc. (Mishwaka, IN, U.S.A.) and stored at refrigeration Hills, IL, U.S.A.). The sampling bag was placed in temperature (571 1C). A suspension of thyme essential a light protected outer bag to prevent light decomposi- oil (0.1, 1.0, and 10 mL/L) was prepared by dispersing tion of chlorine dioxide. The concentration of chlorine appropriate amount of thyme essential oil in 1 L of dioxide gas was measured by a modified amperometric sterile deionized water. The suspension was thoroughly mixed by shaking vigorously for 5min at room of freshly generated chlorine dioxide gas (5mL) Gaseous treatment of lettuce and baby carrots water (9.7 mg/L for 10 min), and thyme oil suspension Chlorine dioxide gas treatments were carried out (1.0 mL/L for 5min) in the following order: in a 10 L Irvine Plexiglass cylinder with a stainless- (i) Two-step sequential washing: Aqueous ClO2/ozonated steel shelf, on which shredded lettuce leaves, and baby water; aqueous ClO2/thyme oil; ozonated water/aqu- carrots were placed. The following concentrations eous ClO2; ozonated water/thyme oil; thyme oil/ of chlorine dioxide gas were used: 0.5, 0.75, and aqueous ClO2; thyme oil/ozonated water.
1.00 mg/L for 5, 10, and 15 min, under 80% RH (ii) Three-step sequential washing: Aqueous ClO2/ozo- and 22 1C, respectively. A 60 mL plastic gas-sampling nated water/thyme oil; aqueous ClO2/thyme oil/ozo- syringe was used to deliver specific volumes of chlorine nated water; ozonated water/aqueous ClO2/thyme oil; dioxide gas into the cylinder containing the shredded ozonated water/thyme oil/aqueous ClO2; thyme oil/ lettuce leaves, or baby carrots. During treatment, the aqueous ClO2/ozonated water; thyme oil/ozonated chlorine dioxide gas inside the cylinder was circulated by a diaphragm vacuum pump (KNF Neuberger, Inc.,Trenton, NJ, U.S.A.), to ensure that chlorine dioxidegas was evenly distributed throughout the sample.
Procedure for enumeration of microorganisms During the treatment, the cylinder was covered with For enumeration of E. coli O157:H7 from shredded aluminum foil to prevent light decomposition of lettuce leaves/baby carrots, 10 g of the same was chlorine dioxide. Ozone gas treatment was also carried transferred into sterile stomaching bags (Fisher Scien- out in the same treatment cylinder as used for chlorine tific Inc., Pittsburgh, PA, U.S.A.), with the aid of a dioxide gas treatment. Inoculated lettuce, and baby sterile stainless-steel spatula, combined with 90 mL of carrots were treated with 2.1, 5.2, and 7.6 mg/L ozone sterile peptone water (1.0 g/L) and then pummeled in a gas for 5, 10, and 15 min, under 80% RH and 22 1C, Seward 400 Stomacher (Seward Medical Co., London, England) at medium speed for 2 min. One milliliter ofStomached sample was serially diluted in 9 mL of sterilepeptone water (1.0 g/L). Serially diluted samples were spread-plated (0.1 mL) in duplicates over a sterile Washing treatment of inoculated lettuce and baby polycarbonate filter membrane (Osmonics Co., West- carrots was performed by immersing shredded lettuce boro, MA, U.S.A.), which was previously placed on the leaves/baby carrots (10 g) in 200 mL of each treatment surface of a TSA plate. The coarse side of the membrane solution (aqueous chlorine dioxide 5.0, 10.0, and faced upwards. Plates were incubated at 37 1C for 4 h to 20.0 mg/L; ozonated water 5.2, 9.7, and 16.5 mg/L; repair injured cells. Then the membranes were gently and thyme oil suspension 0.1, 1.0, and 10.0 mL/L, and aseptically transferred onto Sorbitol Mac-Conkey for 1, 5, 10, and 15 min) in a sterile bag with gentle, agar plates (SMAC) (Oxide Ltd., Basingstoke, Hamp- continuous agitation using a shaker (New Brunswick shire, England) supplemented with cefixime-tullerite (CT) (Dynal, Inc., Lake Success, NY, U.S.A.) using (2272 1C). At the end of contact time, the respective sterile tweezers. The membrane-SMAC-CT plates were treatment solution was drained off, and the treated further incubated at 37 1C for 24 h. Presumptive E. coli samples were rinsed with 200 mL of neutralizing O157:H7 colonies were counted. For confirmation, two buffer solution (Neutralizing buffer, Difco, Detroit, colonies per plate were picked and confirmed by an E.
MI, U.S.A.) by shaking the bag for 30 s. For each coli O157:H7 Latex Test (Oxoid Inc., Ogdensburg, NY, aqueous treatment, two controls were prepared. The negative control consisted of shredded lettuce leaves/baby carrots without inoculation and aqueous treat-ment. The positive control consisted of the inoculated samples without aqueous treatment. Water washing was All the samples used for the enumeration of E. coli also done to compare removal effect of water washing O157:H7 by colony enumeration methods were pre- with different treatments. The inoculated leaves (10 g) pared in triplicate, and each test was replicated three were blended in a Stomacher bag containing 200 mL times, making a total of nine samples analysed for each sterile deionized water, and microbial counts were test parameter. Data were analysed using general linear model (GLM) and Duncan’s multiple range test (SASInstitute, Cary, NC, U.S.A.) to determine if significantdifferences (Po0.05) in populations of microorganisms Sequential washing of lettuce and baby carrots existed between mean values of treatments.
Based on the preliminary results of the current study, atwo-, and three-step sequential washing system wasadopted for the inactivation of inoculated E. coli O157:H7 from lettuce leaves and baby carrots. Themethod used for multi-step washing was similar to the one described for single-step washing. The multi-step Populations of E. coli O157:H7 on shredded lettuce and sequential process consisted of washing the samples with baby carrots surviving after treatments with aqueous aqueous chlorine dioxide (10 mg/L for 10 min), ozonated Effect of aqueous chlorine dioxide on the 5mg/L ClO2 in aqueous solution. Increasing the survival of Escherichia coli O157:H7 on shredded lettuce washing period from 1 to 15min with aqueous ClO2 (5.0 mg/L) showed no significant (Po0.05) reduction inthe population of E. coli O157:H7 on lettuce. However, a significant (Po0.05) reduction (1.69log10 cfu/g) in the population of E. coli O157:H7 was observed afterwashing for 15min compared to 1 or 5min washing of baby carrots. This may be caused by the penetration of microorganisms through cut edges into inaccessible sites of shredded lettuce leaves. Both the studies of disinfectants in the washing solutions could not pene- trate into the protective hydrophobic pockets, folds or minute cracks on the surface of leafy vegetables.
Washing of lettuce for 1 and 5min with aqueous ClO2 (10 mg/L) had no significant (Po0.05) effect on the population recovered, but increasing the washing time (10 min) resulted in a significant (Po0.05) decrease (1.67log10 cfu/g reduction) in the E. coli O157:H7 population. Similar results were also obtained with baby carrots The results obtained in this study showed that 15min exposure of shredded lettuce or Initial inoculation levels for lettuce and baby carrots are 8.12 baby carrots to 20 mg/L of aqueous chlorine dioxide caused a maximum reduction of 1.72 and 2.54log10 cfu/g Values are mean 7SD population recovered (log cfu/g) (n=3).
Values in the same row sharing a common letter are not of E. coli O157:H7, respectively. In general, greater reductions of 0.54, 1.06, and 1.39log of E. coli O157:H7were observed with baby carrots in comparison toshredded lettuce (0.26, 0.73, and 0.76log10 cfu/g reduc-tion) when using 5, 10, or 20 mg/L chlorine dioxide for E. coli O157:H7 was found on uninoculated lettuce 15min, respectively, as compared to the corresponding leaves or baby carrots. The initial population of mixed sterile deionized water, indicating that cells adhered less strains of E. coli O157:H7 was 8.12 and 7.85log10 cfu/g tenaciously to baby carrots surface as compared to the on lettuce and baby carrots, respectively. Washing shredded lettuce. This might be due to concentration of lettuce or baby carrots with sterile deionized water bacterial cells in the cut surfaces of the lettuce, which (control) for 10 min resulted in a significant (Po0.05) may have serious implications in relation to the efficacy reduction (0.93 and 1.15log10 cfu/g, respectively) of E.
of sanitization treatments. Once the pathogens are coli O157:H7 as compared to 1 or 5 min washing.
attached to the cut edges or entrapped in sides, it However, washing for 15min did not result in further becomes difficult to reduce the number of microorgan- significant (Po0.05) reductions in E. coli O157:H7. This isms by treatments. This was consistent with the findings suggests that washing had a limited effectiveness in reducing bacterial population regardless of the washing O157:H7 cells that attached to stomata and cut edges time. Standard washing of lettuce in tap water has been shown to result in the removal of an average of 92% ofthe naturally occurring microflora also found that water washing achieved only 1.5log reduction on green peppers. Our observations on the effectiveness of deionized sterile done to determine the efficacy of gaseous ClO2 in killing water (control) in removing pathogens from lettuce and E. coli O157:H7 on lettuce and baby carrots. Gaseous baby carrots concur with these reports. Increasing the ClO2 treatments of lettuce leaves with 0.5, 0.75, or concentration of ClO2 in deionized water (5.0 mg/L for 1 1.00 mg/L for 5min did not have any significant and 5min) resulted in a significant (Po0.05) decrease in (Po0.05) effect on reducing populations of E. coli E. coli O157:H7 population on lettuce and baby carrots O157:H7. Increasing the exposure time (from 5 to in comparison to washing with deionized water (control) 10 min) of lettuce leaves with gaseous ClO2 (0.5mg/L) did not result in any significant (Po0.05) decrease in that bacterial populations present on cucumbers were population of E. coli O157:H7. However, treatment of not greatly influenced by ClO2 treatment, even at lettuce leaves with gaseous ClO2 (0.75or 1.00 mg/L) for concentration of 5.1 mg/L. The effect of aqueous ClO2 10 min resulted in a significant (Po0.05) reduction (1.67 was in agreement with the results reported by and 1.91log10cfu/g reduction, respectively) of microbial who found that the initial microbial load population compared to 5min exposure time, but decreased by approximately 1log cycle for shredded increasing the exposure time (from 10 to 15min) did lettuce inoculated with L. monocytogenes at levels of not have any further significant (Po0.05) effect on the Effect of gaseous chlorine dioxide on the Effect of ozonated water on the survival of E.
survival of E. coli O157:H7 on shredded lettuce and coli O157:H7 on shredded lettuce and baby carrots Initial inoculation levels for lettuce and baby carrots are 8.10 and 7.72log10cfu/g.
Values are mean7SD population recovered (log cfu/g) (n=3).
Initial inoculation levels for lettuce and baby carrots are 8.10 Values in the same row sharing a common letter are not Values are mean7SD population recovered (log cfu/g) (n=3).
Values in the same row sharing a common letter are notsignificantly different (Po0.05).
efficacy of gaseous ClO2 to reduce the microbialpopulation. However, significant (Po0.05) difference during 1, 5, 10, or 15 min of washing. However, a in numbers of E. coli O157:H7 population on baby significant (Po0.05) reduction in microbial populations carrots was observed during 10 and 15min exposure to on baby carrots was observed after 10 min exposure to between 0.75and 1.00 mg/L gaseous ClO2 concentra- 5.2 mg/L ozonated water compared to 1 or 5 min. The tion, respectively. In general, somewhat greater reduc- reduced efficacy of ozonated water during lettuce tions in populations of E. coli O157:H7 on baby carrots washing might be due to more ozone demand of organic occurred as compared to reductions on lettuce treated with gaseous ClO2, indicating that microorganisms on also indicated that the type of organic material cut lettuce leaves are somehow protected from ClO2.
present during ozonation is more important than the The logarithmic reduction of E. coli O157:H7 on lettuce amount present. They reported that residual ozone and baby carrots using gaseous ClO2 contrasts with the levels in deionized water were significantly reduced in the presence of BSA. In the washing treatments reduction (3.03 and 6.45log) of E. coli O157:H7 on containing 9.7 or 16.5mg/L ozonated water, E. coli injured green pepper surface after treatments by 0.62 and 1.24 mg/L ClO2, respectively, for 30 min at 22 1C (Po0.05) (1.41, 1.42, and 1.68 and 1.8log10 reductions and at 90–95% RH. These differences could be due to cfu/g, respectively) on lettuce and baby carrots only differences in method of inoculation, initial microbial population, exposure time, and type of produce. The lettuce with ozone and reported that bubbling ozone gas color of lettuce leaves also changed (decolorize) during (49 mg/L, 0.5L/min) in a lettuce (1.0 g)–water (20.0 g) 15min exposure at 0.75mg/L gaseous ClO2. This may mixture decreased the natural microbial load by be due to the oxidation of chlorophyll content during longer exposure times at higher concentrations ofgaseous ClO2. Similar results were observed with1.0 mg/L gaseous ClO2 treatment on lettuce leaves for Results in w that the gaseous ozone exerts alethal effect toward E. coli O157:H7 inoculated ontolettuce and baby carrots. Ozone treatments (2.1 to 7.6 mg/L) inactivated E. coli O157:H7 by 0.79–1.79, and The antimicrobial effects of ozonated deionized water 1.11–2.64log cfu/g on lettuce and baby carrots, respec- on E. coli O157:H7 inoculated onto lettuce and baby tively. The bactericidal effect increases with concentra- carrots are presented in The results obtained in tion, and length of exposure to gaseous ozone on lettuce this study have shown that treatment with ozonated and baby carrots. Ozone treatment of lettuce leaves at water (5.2 mg/L) did not result in any significant 2.1, 5.2, or 7.6 mg/L did not decrease the population of (Po0.05) reduction in E. coli O157:H7 populations E. coli O157:H7 significantly (Po0.05) during 5 or Effect of gaseous ozone on the survival of Effect of thyme essential oil on the survival of E. coli O157:H7 on shredded lettuce and baby carrots E. coli O157:H7 on shredded lettuce and baby carrots Initial inoculation levels for lettuce and baby carrots are 8.00 Values are mean7SD population recovered (log cfu/g) (n=3).
Values in the same row sharing a common letter are not Initial inoculation levels for lettuce and baby carrots are 7.86 and 7.54log10 cfu/g.
Values are mean7SD population recovered (log cfu/g) (n=3).
Values in the same row sharing a common letter are notsignificantly different (Po0.05).
10 min of exposure; however, a significant reduction(1.42 to 1.79log cfu/g) in populations of E. coli O157:H7was observed after 15min of exposure. Decolorization 1.0 and 10.0 mL/L suspensions of basil essential oil of lettuce leaves was also observed during 10 or 15min resulted in 2.0 and 2.3log reductions of visible bacteria exposure at 5.2 and 7.6 mg/L of ozone concentration.
on fresh-cut lettuce, respectively. Increasing the con- Similarly, ozone treatment of baby carrots did not result centration of thyme oil (10.0 mL/L for 5, 10, or 15 min) in any significant (Po0.05) change in population of E.
resulted in a further decrease in microbial population on coli O157:H7 during 5 or 10 min exposure time at 2.1, lettuce and baby carrots, compared to deionized water 5.2, or 7.6 mg/L concentration level. However, the (control), 0.1 or 1.0 mL/L thyme oil concentration for increase in exposure time (15min) resulted in a the same period of time. In general, numbers of E. coli significant decrease (1.84 to 2.64log cfu/g) in population.
O157:H7 decreased only marginally with increasingexposure time (from 5to 15min), regardless of thymeoil concentration in the washing solution.
Effect of thyme essential oilThe effects of thyme essential oil washing of lettuce andbaby carrots, inoculated with E. coli O157:H7 are shown in Deionized water washing for 10 min resulted Results of the efficacy of washes containing aqueous in significant reductions (Po0.05) in population of ClO2, ozonated water, and thyme oil alone or their E. coli O157:H7 (7.00 and 6.61log cfu/g, respectively) on sequential treatments (two and three-step sequential lettuce and baby carrots in comparison to 1 or 5min washing), on the survival of E. coli O157:H7 on lettuce washing. However, increasing the washing time (15min) leaves and baby carrots can be seen in Washing did not result in any significant (Po0.05) change in of lettuce leaves with aqueous ClO2, ozonated water or microbial population. The results also revealed that thyme oil led to a significant (Po0.05) reduction in thyme oil washing (0.1 mL/L) did not lead to any numbers of E. coli O157:H7, as compared with the significant (Po0.05) difference in numbers of E. coli sterile deionized water (control). In general, reductions O157:H7 on lettuce and baby carrots as compared with ranged from 1.48–1.61log depending upon the type of deionized water washing. A significant (Po0.05) reduc- sanitizers used. Whereas, water washing reduced the tion (1.65and 1.90log cfu/g, respectively) in population population by 1.06log. Similar results were seen with of E. coli O157:H7 on lettuce and baby carrots was inoculated baby carrots (1.61–1.97log reduction). A observed when using thyme oil (1.0 mL/L for 5min) two-step sequential washing further reduced the popula- compared to deionized water or 0.1 mL/L, thyme oil for tions of E. coli O157:H7, as compared to single step- same period of time. Washing time from 5to 10 or washing. The efficacy of thyme oil/aqueous ClO2 or 15min did not result in any significant (Po0.05) thyme oil/ozonated water, however, proved to be reduction (thyme oil 1.0 mL/L) in microbial population.
significantly more effective (3.15and 3.00log reduction on lettuce and 3.47 and 3.25log reduction on baby Efficacy of washes containing different sanitizers (aqueous ClO2, ozonated water, or thyme oil suspension) alone or their sequential treatments on decontamination of shredded lettuce and baby carrotsinoculated with E. coli O157:H7 *Deionized water: 10 min; aqueous chlorine dioxide: 10.0 mg/L, 10 min; ozonated water: 9.3 mg/L, 10 min; thyme oil suspension:1.0 mL/L, 5min.
Values are mean7SD population recovered (log cfu/g) (n=3).
Values in the same row sharing a common letter are not significantly different (Po0.05).
carrots, respectively) in killing microbial population. It detailed study is anticipated. However, the use of plant is, however, interesting to note that during the three- extracts, aqueous ClO2, and ozonated water may step sequential washing treatments used in this study, adversely affect the organoleptic properties of food.
thyme oil followed by aqueous ClO2/ozonated water, Further studies are needed to determine the accept- or ozonated water/aqueous ClO2 were significantly ability of fruits or vegetables treated with lower (Po0.05) more effective in reducing E. coli O157:H7 concentrations of sanitizers to reduce contamination (3.75and 3.99log, and 3.83 and 4.34log reduction) on with lower microbial population. In addition, different lettuce and baby carrots, respectively. It could therefore food constituents may influence the sequential washing be inferred from the results of this study that thyme oil (synergistic action) of thyme oil/ozonated water/aqu- washing followed by other sanitizers is of great eous chlorine dioxide, and as they appeared to have an advantage. The populations of E. coli O157:H7 on important role in the interaction with sanitizers, such shredded lettuce were significantly decreased when effects on various fruits and vegetables need to be maximum reduction in E. coli O157:H7 populations onlettuce and baby carrots during two- or three-stepsequential washing by thyme oil followed by aqueous ClO2 or ozonated water might be due to the phenoliccomponents of thyme oil, which may increase the This research was supported by USDA-ARS grant in permeability and leakage of cell membrane, thereby support of the Food Safety and Engineering Project at enhancing the efficacy of other sanitizers.
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