Trends in Food Science & Technology 20 (2009) 17e26

Review

* Corresponding author.

0924-2244/$ - see front matter � 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.tifs.2008.09.005

Chlorine dioxide for

minimally processed

produce

preservation:

a review

Vicente M. Gomez-Lopeza,*,

Andreja Rajkovicb,

Peter Ragaertb, Nada Smigicb

and Frank Devlieghereb

aInstituto de Ciencia y Tecnologıa de Alimentos,

Facultad de Ciencias, Universidad Central de

Venezuela, Apartado 47097, Caracas 1041-A,

Venezuela (e-mails: vicente.gomez@ciens.ucv.ve;

vicente2709@yahoo.es)bLaboratory of Food Microbiology and Food

Preservation, Ghent University, Coupure Links 653,

9000 Gent, Belgium

Minimally processed fruits and vegetables (MPFV) have a short

shelf-life due to their metabolism and the action of spoilage

microorganisms. Chlorine dioxide is a powerful oxidizing

agent that can be used as decontaminant. It does not form sig-

nificant amounts of chlorinated by-products like as do chlo-

rine. This article revises the characteristics of chlorine

dioxide, the basis of its antimicrobial action, and its effect

on microorganisms and on the sensory quality and shelf-life

of fresh produce and mainly MPFV. Moreover, it discusses its

effects on the physiology and nutritional quality of MPFV,

and provides information on its toxicity and legal status.

IntroductionMinimally processed fruits and vegetables (MPFV) are

any fresh fruit or vegetables that have been physically

altered from its original form, but remain in a fresh state.Regardless of commodity, it has been trimmed, peeled,washed and cut into 100% usable product that is subse-quently bagged or pre-packaged (IFPA, 2006). Microorgan-isms present in MPFV can cause foodborne illnesses orspoilage, hence, the decontamination of MPFV can producesafer and more stable products. However the decontamina-tion process can promote sensorial and physiologicalchanges that will influence the shelf-life of MPFV.

The shelf-life extension of MPFV is nowadays a chal-lenge for the food industry, as well as for the research insti-tutions, where decontamination technologies are currentlyevaluated. The structure of the research for testing decon-tamination methods to prolong the shelf-life of MPFVshould encompass five different aspects, namely:

1) Reduction of the risk of foodborne infections andintoxications.

2) Decrease of the microbial spoilage.3) Preservation of fresh attributes.4) Preservation of nutritional quality.5) Absence of unacceptable levels of toxic residues or for-

mation of unacceptable levels of toxic by-products.

Depending on their goals, available research articleshave been focused in one or several, but not all these ap-proaches. Although the vast majority of published informa-tion elaborates effect of ClO2 on the reduction of the risk offoodborne infections and intoxications, the integration ofall five aspects that will determine potential applicationof ClO2 in food industry.

Reports describing aspects 2 and 3 of ClO2 applicationfollow in number. Decreasing spoilage microflora is impor-tant to prolong the shelf-life. Preserving fresh attributes isessential for a decontamination method to be applicable,since decontaminated MPFV should still have a fresh-look after the decontamination process. Most of the reportsdealing with foodborne pathogens did not take into accountthis essential aspect of MPFV preservation. Both levels ofresidual spoilage microflora and the level to which freshsensory attributes were maintained will determine the suc-cess of a decontamination treatment to prolong the shelf-life of MPFV.

Preservation of nutritional quality has been poorly stud-ied. It is likely that some of the novel decontaminationtechniques could decrease the nutritional quality of

18 V.M. Gomez-Lopez et al. / Trends in Food Science & Technology 20 (2009) 17e26

MPFV due to their oxidative capacities. It is important toquantify the degree of nutrient losses if they cannot beavoided, especially considering that MPFV are frequentlypurchased by health conscious consumers (IFPA, 2006)that expect produce of nutritional quality equivalent tothe fresh counterparts. Interestingly, there are evidencesthat minimal processing (Kang & Saltveit, 2002) andsome decontamination methods (Cantos, Espın, & Tomas-Barberan, 2001) can increase the content ofphytochemicals.

Finally, the toxicological aspects of the decontaminationtreatments for fresh and MPFV have been scarcely studiedtoo. Related studies however exist on the reaction betweendecontaminants and organic matter present in waterstreams. It is possible that the mild nature of this kind oftreatments, and consequently the low potential of by-prod-uct formation of most of them, have accounted for the lackof interest in this area.

This review is focused on the impact of ClO2 decontam-ination on the microbial, sensorial and nutritional qualitymainly of MPFV, and potential presence of toxic com-pounds. In this way, it attempts to provide summarized in-formation regarding the production of MPFV with extendedshelf-life and of high nutritional quality, while providingthe necessary level of product safety from the microbiolog-ical and chemical points of view.

Definition and generalitiesChlorine dioxide (ClO2) is one of the few compounds

that exists almost entirely as monomeric free radicals(WHO, 2000). Its use as disinfectant dates back to the be-ginning of the 20th century, when it was first used in Bel-gium for water disinfection (Tzanavaras, Themelis, &Kika, 2007). It is most commonly used as a bleaching agentat paper manufacturing plants, and in public water treat-ment facilities to make drinking water safer. In 2001,ClO2 and chlorite were used to decontaminate a numberof public buildings following the release of anthrax sporesin the United States (ATSDR, 2004). The interest in ClO2 indecontaminant of vegetables is largely based on its efficacy,which is less prone to impairment under low pH and in thepresence of organic matter, and its inertness towards ammo-nia to form chloramines (Beuchat, 1998). ClO2 is in a realsense gas, as it readily dissolves in water, without reactingwith it. The oxidation capacity of ClO2 is determined by theoxidation number of chlorine atom, being þ4. Therefore,ClO2 accepts 5 electrons when reduced to chloride ion, pro-viding 263 percent ‘available chlorine,’ which is more than2.5 times the oxidation capacity of chlorine in HOCl.However, due to its lower oxidation strength ClO2 ismore selective in its reactions, and oxidization and chlori-nation of organic compounds, will be performed at a slowerrate than with chlorine. In most cases, oxidation reactionpredominates (Fukayama, Tan, Wheelr, & Wei, 1986).ClO2 cannot be compressed and stored under the pressurebecause it is explosive, therefore the shipping of ClO2

gas is impossible, and it has to be generated on-site.Easy-to-use commercial systems exist on the market foron-site ClO2 generation. They generally consist of two sa-chets each one containing a precursor for ClO2 generation,which takes place upon mixing two components (Lee, Cost-ello, & Kang, 2004; Sy, McWatters, & Beuchat, 2005).

Basis of antimicrobial effectCell membrane has been identified as the primary target

of ClO2 on microbial cells. Although early work by Ber-narde, Snow, Olivieri, and Davidson (1967) performed onEscherichia coli indicated that the lethal lesion producedby ClO2 to microbial cells is directly related to protein syn-thesis, loss of permeability control was identified later byBerg, Roberts, and Matin (1986) as the primary lethal eventat the physiological level of ClO2 on bacterial cells. The ef-fect of ClO2 was related to non-specific oxidative damageof the outer membrane leading to the destruction of thetrans-membrane ionic gradient. Young and Setlow (2003)also proposed that some type of membrane damage couldbe the cause of Bacillus subtilis spores death, since ClO2

treated spores can undergo the initial steps in spore germi-nation, but cannot go further in this process. Peta, Lindsay,Brozel, and von Holy (2003) observed that cells of Bacilluscereus treated with ClO2 exhibited surface roughness andindentations, and were elongated, in contrast with controlcells, which were uniform rods with smooth surfaces. Elon-gation of cells might result from the inhibition of divisionand associated metabolic damage. Damage to genetic mate-rial is also a possibility since Alvarez and O’Brien (1982)demonstrated that ClO2 reacts with viral RNA, althoughin bacterial and fungal cells might not be the primaryfactor.

Chlorine dioxide gasThe use of ClO2 gas has been primarily limited to pulp

bleaching in the paper industry (Han, Applegate, Linton, &Nelson, 2003). Anyhow, numerous studies have reportedsuccessful application of gaseous ClO2 for food decontam-ination. The main advantage of ClO2 gas over aqueous san-itizers is that gas has more penetrability (Han, Linton,Nielsen, & Nelson, 2001), it could therefore reach microor-ganisms protected from aqueous disinfectants by surface ir-regularities or biofilms as discussed by Gomez-Lopez,Ragaert, Debevere, and Devlieghere (2008).

Factors affecting the efficacy of chlorine dioxide gasHan, Floros, Linton, Nielsen, and Nelson (2001) mod-

eled the effect of factors determining the inactivation ofE. coli O157:H7 on green peppers by ClO2 gas. The orderof significance from the most important to the least was:gas concentration (0.1e0.5 mg/l), time (7e135 min), rela-tive humidity (55e95%) and temperature (5e25 �C).Moreover, a synergistic effect of ClO2 gas concentrationand relative humidity was noticed. The reported effects ofgas concentration, time and relative humidity are in

19V.M. Gomez-Lopez et al. / Trends in Food Science & Technology 20 (2009) 17e26

agreement with results presented by Han, Guentert, Smith,Linton, and Nelson (1999) and Jeng and Woodworth(1990). Westphal, Price, Leighton, and Wheeler (2003)demonstrated that Bacillus thuringiensis spores swell withincreasing relative humidity. Swelling of a spore increasesthe diameter of channels for access of gases into sporesand may account for the greater kill effectiveness of sporesby gas-phase ClO2 at very high relative humidity. Kineticsof bacterial inactivation can be described by Weibull model(Mahmoud, Vaydia, Corvalan, & Linton, in press).

A very important factor for ClO2 influencing the effec-tiveness of gaseous is the amount of produce relative tothe amount of ClO2 applied (Yuk, Bartz, & Schneider,2006), the latter depends on the volume of the treatmentchamber and the gas concentration. The ClO2 degradationrate is dependant on the amount of organic matter. Ata given ClO2 concentration, a higher amount of sample ina small treatment chamber will degrade the disinfectantfaster than the case of a smaller amount of sample ina big chamber. Fast ClO2 degradation will decrease theamount of gas available for disinfection during treatmenttime.

Surface integrity and microorganism location are otherimportant factors. Han, Linton, Nielsen, and Nelson(2000) found that ClO2 gas treatment showed significantlymore log reductions (1.23e4.24 log) of E. coli O157:H7 onuninjured green bell pepper surfaces than on injured sur-faces. Du, Han, and Linton (2002) demonstrated that the in-activation of Listeria monocytogenes inoculated ontodifferent apple surfaces was more effective on bacteria at-tached to pulp skin than on bacteria attached to calyx orstem cavities.

Major types of ClO2 gassing experimentsWhen comparing studies on decontamination with ClO2,

it is important to consider the actual meaning of the re-ported ClO2 concentrations. These concentrations can in-crease, decrease, or keep constant depending on theexperimental setup. ClO2 concentration increases in exper-iments with gas releasing sachets (Lee et al., 2004; Sy,McWatters, et al., 2005; Sy, Murray, Harrison, & Beuchat,2005). In the experimental setup used by Gomez-Lopez,Devlieghere, Ragaert, and Debevere (2007) and Gomez-Lopez, Ragaert, Jeyachchandran, Debevere, and Devlie-ghere (2007), the sample is placed in a chamber to betreated for 10 min. Treatments start by injecting an airstream with ClO2 during 30 s. ClO2 concentration increasesduring the injection time, and afterwards decreases due toreaction with the present vegetable during the remainingtreatment time. A third experimental setup consists of aninstantaneous injection of gas to the treatment chamberwith a small amount of sample. In this setup used by Duet al. (2002), Du, Han, and Linton (2003), and Han, Sher-man, Linton, Nielsen, and Nelson (2000); the change ofClO2 concentration versus time was not reported, maybebecause the disinfectant concentration was constant due

to the low food amount to ClO2 ratio. Therefore, compari-sons between different studies should be taken with precau-tion due to the differences in gas evolution, and consideringthat single gas concentration values are generally providedonly as reference points.

Inactivation of pathogens in vitroStudies have proved the efficacy of ClO2 on a wide va-

riety of microorganisms and viruses. Chen and Vaughn(1990) demonstrated activity of ClO2 against rotavirusesand reviewed early works on its virucidal efficacy at alka-line conditions. ClO2 can also decrease the viability ofCryptosporidium parvum oocysts (Peeters, Ares, Massche-lein, Villacorta, & Debacker, 1989), but not until safe levels(Korich, Mead, Madore, Sinclair, & Sterling, 1990). Testswith conidial or sporangiosphore suspensions of four moldstreated with 3 or 5 mg/l ClO2 within 1 min demonstrated>2 log reductions (Roberts & Reymond, 1994). Also, Beu-chat, Pettigrew, Tremblay, Roselle, and Scouten (2005)demonstrated the efficacy of ClO2 to kill vegetative cellsas well as spores of B. cereus, whose counts were decreasedwithin 5 min by >5.4 and >6.4 log CFU/ml respectively,using 200 mg/ml ClO2. Recently, Kim, Khang, Beuchat,and Ryu (in press) proved that the type of acid used to gen-erate ClO2 and the pH affects the efficiency of ClO2 to in-activate B. cereus spores.

Inactivation of pathogens in vivoAqueous chlorine dioxide

ClO2 solutions have shown to be efficient to eliminatepathogens inoculated onto different fruit and vegetables(Table 1), although no complete elimination has been ob-tained. Although the early work by Zhang and Farber(1996) found discouraging results when liquid ClO2 wasused, reductions as high as 5 log CFU/ml have been latelyreported by other authors (Pao, Kelsey, Khalid, & Ettinger,2007). As in its gaseous counterpart, wounds limit aqueousClO2 efficacy; Han, Linton, et al. (2001) found that cells ofL. monocytogenes inoculated onto bell peppers were re-duced by 3.7 log CFU/5 g when surface was not injuredand by 0.44 when injured, after treatment with 3 mg/lClO2 solution. To increase the effectiveness of ClO2 solu-tion, the approach of sequential washings has been shownto be effective. Singh, Singh, Bhunia, and Stroshine(2002a) reported that washing shredded lettuce inoculatedwith E. coli O157:H7 for 5 min with 10 mg/l ClO2 reducedcounts by 1.20 log CFU/g, a second washing step of 5 minresulted in additional reduction, although a third one didnot show additional effect.

The results obtained in the study of Pao et al. (2007) re-vealed significant sanitizing ability of 20 mg/l ClO2 againstSalmonella enterica and Erwinia carotovora inoculatedonto the surface of tomato only when the fruit was freshlyinoculated with these microorganisms, reaching 5 log CFU/cm2 reduction. Once the inoculation suspension was dried

Table 1. Studies on the effect of liquid ClO2 on pathogenic microorganisms inoculated onto fruits and vegetables

Produce Microorganism [ClO2] (mg/l) Time (min) log Reduction Reference

Lettuce Listeria monocytogenes 5 10 0.8 Zhang and Farber (1996)Cabbage 0.8

Green bell pepper L. monocytogenes 3 10 3.7 Han, Linton, et al. (2001)

Lettuce Escherichia coli O157:H7 10 5 1.20 Singh et al. (2002a)

Lettuce E. coli O157:H7 20 15 1.7 Singh et al. (2002b)Baby carrot 2.5

Apple Enterobacter sakazakii 100 1 �4.49 Kim, Ryu, and Beuchat (2006)Tomato �3.59Lettuce 4.05

Apples Salmonella 5 10 z2 Huang et al. (2006)E. coli O157:H7 z1Salmonella z1

Tomato Salmonella enterica 20 1 5 Pao et al. (2007)Erwinia carotovora 10 1 5

Mungbean sprout Salmonella Typhimurium 100 5 3.0 Jin and Lee (2007)L. monocytogenes 100 5 1.5

Blueberry L. monocytogenes 15 120 4.88 Wu and Kim (2007)Pseudomonas aeruginosa 120 4.48S. Typhimurium 20 3.32Staphylococcus aureus 30 4.56Yersinia enterocolitica 60 3.69

20 V.M. Gomez-Lopez et al. / Trends in Food Science & Technology 20 (2009) 17e26

out on the fruit surface, no significant reduction was de-tected in comparison with regular tap water.

Gaseous chlorine dioxideA pioneering article on the use of ClO2 gas as disinfec-

tant was published by Jeng and Woodworth (1990) in an at-tempt to find a substitute of ethylene dioxide gas as sterilantof medical products. They proved that ClO2 gas can inacti-vate spores of different Bacillus species. Subsequent studieshave shown that gaseous ClO2 is effective in inactivatingdifferent microorganisms present on the surface of vegeta-bles and fruits (Table 2). In general, the level of decontam-ination achieved by gaseous ClO2 is higher than those bywashing methods, and can be as high as >8 logs (Han, Lin-ton, et al., 2000). Regarding aqueous and gaseous ClO2 ef-ficacy, Han, Linton, et al. (2001) found that gaseous ClO2 ismore effective than aqueous ClO2 to inactivate L. monocy-togenes inoculated onto both injured and uninjured greenpepper surfaces.

Spoilage and shelf-lifeAqueous chlorine dioxide

Reports of aqueous ClO2 decontamination (Table 3) andproduce shelf-life prolongation indicate variety of resultsranging from no significant inactivation (Costilow, Ueber-sax, & Ward, 1984) to 4.2 log reduction (Rodgers, Cash,Siddiq, & Ryser, 2004). Regarding shelf-life, Reina, Flem-ing, and Humphries (1995) reported significant reductionsin total aerobes and molds, but after storage at 10e12 �C,disinfected cucumbers did not have a longer storage life

than just water washed cucumbers. In contrast, Rodgerset al. (2004) reported significant reductions for mesophilicbacteria, yeasts and molds on shredded lettuce, and duringstorage at 4 �C for 9 days, samples washed for 5 min with 3or 5 mg/l ClO2 had lower counts of mesophilic bacteria,yeasts and molds than water washed controls.

Gaseous chlorine dioxideResearch on the effect of ClO2 gas on the spoilage mi-

crobiota of fruits and vegetables has yielded generally sat-isfactory results. Sy, Murray, et al. (2005) reported nosignificant differences in yeast and mold population of to-matoes and onions treated with up to 4.1 mg/l ClO2 for20e25 min. In the case of whole apples and peaches how-ever, the same treatment conditions produced 1.68 and2.65 log reductions respectively. When the concentrationof 8.0 mg/l ClO2 was applied for 120 min to different smallfruits the reductions in yeast and mold populations of2.06e2.32, 4.07e4.16 and 2.56 log CFU/g for blueberries,strawberries and raspberries were found, respectively (Sy,McWatters, et al., 2005). Also Gomez-Lopez, Ragaert,et al. (2007) reported >1 log reduction in aerobic platecounts of shredded Iceberg lettuce and white cabbagetreated at peak ClO2 concentrations of 1.74 and 1.29 mg/lfor 10 min.

ClO2 gas has been proved to be a tool to prolong theshelf-life of fresh and fresh-cut produce. A successful at-tempt to prolong the shelf-life of grated carrots usingClO2 gas was reported by Gomez-Lopez, Devlieghere,et al. (2007). A decrease of 1.7 logs in psychrotroph counts

Table 2. Studies on the effect of gaseous ClO2 on pathogenic microorganisms inoculated onto fruits and vegetables

Produce Microorganism [ClO2] (mg/l) Time (min) RH (%) log Reduction Reference

Green bell pepper Escherichia coli O157:H7 1.24 30 90e95 6.45 Han, Sherman, et al. (2000)Green bell pepper E. coli O157:H7 1.2 30 90e95 >8.04 Han, Linton, et al. (2000)Green bell pepper Listeria monocytogenes 3 10 90e95 >6 Han, Linton, et al. (2001)Apple L. monocytogenes 4.0 10 90 3.2e5.5 Du et al. (2002)

Lettuce E. coli O157:H7 1.00 15 80 2.31 Singh et al. (2002b)Baby carrot 3.08

Apple E. coli O157:H7 18.0 10 90e95 3.8e>7.0 Du et al. (2003)

Blueberry Salmonella 8.0 120 2.44e3.67 Sy, McWatters, et al. (2005)Strawberry Salmonella 8.0 120 3.76e4.41Raspberry Salmonella 8.0 120 1.54

Cabbage Salmonella 4.1 30.8 4.42 Sy, Murray, et al. (2005)E. coli O157:H7 20.5 3.13L. monocytogenes 29.3 3.60

Carrot Salmonella 4.1 30.8 5.15E. coli O157:H7 20.5 5.62L. monocytogenes 29.3 5.88

Lettuce Salmonella 4.1 30.8 1.58E. coli O157:H7 20.5 1.57L. monocytogenes 29.3 1.53

Apple Salmonella 4.1 25 4.21Tomato Salmonella 4.1 25 4.33Onion Salmonella 4.1 20 1.94Peach Salmonella 4.1 20 3.23

Apple Allicyclobacillus acidoterrestris 4.32 60 >5 Lee et al. (2006)

Blueberry L. monocytogenes 4 720 99.9 3.94 Popa et al. (2007)Salmonella 3.62E. coli O157:H7 4.25

Strawberry E. coli O157:H7 5 10 90e95 4.6 Mahmoud et al. (2007)L. monocytogenes 4.7Salmonella enterica 4.3

Lettuce E. coli O157:H7 5 10 90e95 3.9 Mahmoud and Linton (2008)S. enterica 2.8

Melon E. coli O157:H7 5 10 90e95 4.6 Mahmoud et al. (in press)L. monocytogenes 5 10 4.3Salmonella Poona 5 6 5

Experiments were performed at 20e23 �C.

21V.M. Gomez-Lopez et al. / Trends in Food Science & Technology 20 (2009) 17e26

in comparison to water washed samples, together with a lagphase of at least 2 days in the disinfected MPV resulted inone additional day of shelf-life. Mesophilic bacterialcounts, psychrotrophic bacterial counts, and yeast andmold counts in whole strawberries were reduced from3.9, 3.8 and 4.8 log CFU/g to less than 2 log CFU/g bytreating with 5.0 mg ClO2/l for 10 min. Furthermore, theshelf-life prolongation from 8 to 16 days was achievedwhen stored at 4 �C (Mahmoud, Bhagat, & Linton, 2007).ClO2 gas might be also helpful in controlling postharvestdecay of tomato. Fruits inoculated with E. carotovora in ar-tificial wounds, and treated with ClO2 at doses of 88 mg for

24 h or 99 mg for 2 h were free of decay after 3 days ofstorage at ambient temperature. In contrast, soft rot was ob-served at 91.3% of the wounds of the non-treated fruits af-ter the same storage time (Mahovic, Bartz, & Tenney,2007). Furthermore, Mahmoud and Linton (2008) foundlower levels of psychrotropic bacterial counts and yeastand mold counts on gassed shredded lettuce, which stayedlower compared to the untreated samples during storage at4 �C for 7 days. Finally, Mahmoud et al. (in press) reducedthe initial microflora of cantaloupe by >2 log CFU/cm2 andextended its shelf-life to 9 days compared to 3 days for theuntreated control when stored at 22 �C.

Table 3. Studies on the effect of liquid ClO2 on natural microflora of fruits and vegetables

Produce Microbial group [ClO2] (mg/l) Time (min) log Reduction Reference

Cucumber Yeast, molds, LAB 105 15 NS* Costilow et al. (1984)

Cucumber Total aerobes 5.1 3.46 Reina et al. (1995)Molds 1.32

Lettuce Mesophilic bacteria 5 5 4.2 Rodgers et al. (2004)Yeast 1.6Mold 1.2

Mungbean sprout Total mesophiles 100 5 NS Jin and Lee (2007)Blueberry Yeasts and molds 15 60 2.82 Wu and Kim (2007)Lettuce Psychrotrophs 5 2 >1 Mahmoud and Linton (2008)

Yeast and molds >0.9

Lettuce Aerobic plate count 20 20 NS Gomez-Lopez et al. (in press)Cabbage NSCarrot 1.89

*Non-significant reduction ( p¼ 0.05).

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In addition to lab-scale experiments, pilot studies arestarting to be available in the literature. Popa, Hanson,Todd, Schilder, and Ryser (2007) treated 30 lugs of blue-berries (approx. 9.1 kg/lug) with 18 mg/l, 0.13 mg/g ClO2

for 12 h. Reductions in counts of mesophilic aerobic bacte-ria, coliforms, E. coli, yeasts and molds were 2.33, 1.47,0.52, 1.63, and 0.48 logs respectively, compared withnon-gassed controls. Importantly, microbial inactivationwas homogeneous throughout the lot.

Sensory qualityAqueous chlorine dioxide

The little information available on the effect of liquidClO2 on the sensory quality of fruit and vegetables showsthat generally it does not affect sensory attributes. Shreddedlettuce treated with 5 mg/l ClO2 for 5 min could not be dif-ferentiated from controls by using triangle tests (Rodgerset al., 2004). Similarly, Wu and Kim (2007) reported nodamage of blueberries. However, Gomez-Lopez, Devlie-ghere, Ragaert, and Debevere (in press) reported that shred-ded lettuce treated with 20 mg/l ClO2 for 5 min wasdifferentiated from water washed controls by triangle tests,although this was not the case when shredded cabbage andgrated carrots were tested.

Gaseous chlorine dioxideDifferent reports have shown that ClO2 gas treatments

can decrease the sensory quality of MPFV, mainly inducingbrowning and bleaching. The careful selection of timeeconcentration combinations seems to be necessary to de-contaminate MPFV without causing damage to produces.

Browning in fruit and vegetables is generally producedby the oxidation of phenols into quinones that subsequentlypolymerize into brown pigments (melanins), a reaction cat-alyzed by polyphenol oxidase (Gomez-Lopez, 2005). It hasbeen reported that ClO2 oxidizes phenols (Napolitano,Green, Nicoson, & Margerum, 2005), so that the further

mechanism for the formation of melanins might be similaras in the enzymatic browning, although no O2 and polyphe-nol oxidase are necessary. Gaseous ClO2 can inducebrowning of cabbage immediately following treatment(Gomez-Lopez et al., in press; Sy, Murray, et al., 2005).A pre-treatment by immersion in a L-cysteine solution com-pletely inhibited browning (Gomez-Lopez, Ragaert, et al.,2007). Browning of shredded lettuce has been reported byGomez-Lopez et al. (in press), Mahmoud and Linton(2008), and Sy, Murray, et al. (2005), of peaches by Sy,Murray, et al. (2005), and of apples by Lee, Dancer, Chang,Rhee, and Kang (2006), Sapers, Walker, Sites, Annous, andEblen (2003) and Sy, Murray, et al. (2005).

Since ClO2 is widely used as a pulp bleaching agent inthe paper industry, Fu, Zhang, Wang, and Du (2007) hy-pothesized that it might useful to preserve color of vegeta-bles. These authors demonstrated that ClO2 can reducepolyphenol oxidase activity up to 37% in apple crude ex-tracts, which might be consequence of structural modifica-tion of the enzyme. However, in vivo results suggest that theoxidant capacity of ClO2 on phenols can overcome the anti-browning effect derived from polyphenol oxidaseinhibition.

Bleaching caused by gaseous ClO2 has been reported byseveral authors. The possible mechanism of bleaching pro-cess might be found in the oxidation of different oligosac-charides such as cellulose, hemicellulose and lignin presentin fruits and vegetables (Bunzel & Ralph, 2006; Rani &Kawatra, 1994). Cellulose has been used as a model com-pound for the explanation of bleaching by ClO2 and mainlythe oxidation of glycosidic bond and oxidative cleavage be-tween C-2 and C-3 carbons occurred according to the re-view by Fukayama et al. (1986). For hemicellulose it wasreported to react even more with ClO2 than cellulose (Fu-kayama et al., 1986). In lettuce, the color changed during15 min exposure to 0.75 mg/l gaseous ClO2, and 10 or15 min to 1.0 mg/l. This may have been due to the

23V.M. Gomez-Lopez et al. / Trends in Food Science & Technology 20 (2009) 17e26

oxidation of chlorophyll. The color change was not seenwhen milder treatment conditions were applied (Singh,Singh, Bhunia, & Stroshine, 2002b). Treating Iceberg lettuceleaves with 5 mg/l gaseous ClO2 for 10 min immediatelyaffected color, the yellow-green color completely changedto white-brown (Mahmoud & Linton, 2008). A slight whit-ening of julienne style carrots was reported by Sy, Murray,et al. (2005). Du, Fu, Li, and Xia (2007) reported more rapiddegradation of chlorophyll with increasing concentrations ofgas ClO2 in whole green bell peppers.

ClO2 induction of bleaching has been also reported inthe case of fruits. Bleached spots on treated strawberrieswere reported by Sy, McWatters, et al. (2005) within90 min after treatment, which were not as evident duringstorage. No effect of gassing blueberries with 0.13 mg/lClO2 for 12 h was observed after overnight storage at4 �C regarding appearance, aroma, texture, flavor, or over-all acceptability; although some bleaching of blueberryleaves was observed immediately after treatment (Popaet al., 2007). Wound sites of whole tomatoes treated with88 mg ClO2 were bleached and mildly sunken, whereas,stem scars, including the corky ring around the stem scarswere bleached as well (Mahovic et al., 2007).

Finally, some studies have not detected deleterious ef-fects of ClO2 gas on sensory quality of blueberries, rasp-berries (Sy, McWatters, et al., 2005), tomatoes, andonions (Sy, Murray, et al., 2005) immediately followingtreatment or after storage for 10 days at 8 �C (fruits) or21 �C (the rest). A ClO2 treatment with 5 mg/l for 10 minapplied to whole strawberries did not affect external colornor visual appearance neither immediately after treatmentnor during storage at 4 �C for 16 days (Mahmoud et al.,2007). Similarly, a treatment with 5 mg/l ClO2 did not af-fect cantaloupe skin color (Mahmoud et al., in press).

Effects on physiology and nutrientsVery different effects on the respiration rate of vegeta-

bles have been reported after ClO2 application. ClO2 gas in-creased the respiration rate of shredded white cabbage by22% (Gomez-Lopez, Ragaert, et al., 2007), but did not af-fect the respiration rate of grated carrots (Gomez-Lopez,Devlieghere, et al., 2007) and shredded Iceberg lettuce (Go-mez-Lopez, Ragaert, et al., 2007), and suppressed carbondioxide production by whole green bell peppers (Duet al., 2007).

With regard to nutritional quality, it is known that ClO2 canreact with phenolics (Napolitano et al., 2005). Since manyphytochemicals are in their essence phenolics, it is possiblethat ClO2 has an impact on the content of these compounds.As ClO2 is an oxidant, nutrients such as ascorbic acid couldbe easily oxidized. There is however a lack of research con-cerning this topic. Du et al. (2007) reported that, althoughthe loss of vitamin C in whole green peppers was initially ac-celerated after 10 days of storage in ambient with ClO2 gas at10 �C, it was however retarded after 20 days, resulting inhigher concentrations of vitamin C after 40 days.

Toxicity and toxic by-productsThe non-formation of chlorinated toxic by-products is

one important advantage of ClO2 over HOCl. Unlike theother disinfectants, the major ClO2 by-products, chloriteand chlorate ions, are derived from decomposition of thedisinfectant with no direct formation of organohalogenby-products. As opposed to chlorine, which reacts via oxi-dation and electrophilic substitution, ClO2 reacts only byoxidation; this explains why it does not produce organo-chlorine compounds (WHO, 2000). However, possiblypresent impurities (chlorine, chloramines, etc.) can lead tothe formation of chlorinated by-products (Richardsonet al., 2003).

Through the 8 years study of water treated with ClO2

Richardson et al. (2000) found only two chlorinated by-products: 1,1,3,3-tetrachloroprpanone and (1-chloroethyl)-dimethylbenzene; and no halomethans. Hua and Reckhow(2007) detected very low level of dihaloacetic acids and di-chloropropanone, while higher level of unknown total or-ganic halogen when the water samples were treated with1.5 mg/l ClO2. In all cases, chlorinated water had higherlevel of halogenated by-products. Similar, Sorlini and Col-livignarelli (2005) found that ClO2 produced 98% less tri-halomethanes than chlorine when added to surface waterscoming from ten different sources in Italy. Nevertheless,the toxicity of oxidation by-products has been much lessstudied than that of chlorination by-products, and addi-tional research in the field is needed.

Tsai, Huxsoll, and Robertson (2001) could not detectany residue of ClO2, chlorite or chlorate in potatoes storedin an atmosphere with ClO2. According to authors’ estima-tions, the ClO2 in potatoes could have theoretically reachedas much as 14 mg/l, but results showed that the combinedamount of chlorite and ClO2 was less than 0.07 mg/l andthat of chlorate also less than 0.07 mg/l; assuming ClO2

only penetrated skin deep (the detection limit of chloriteor chlorate was 0.1 mg/l).

Since ClO2 is most commonly used in drinking water dis-infection, majority of available studies assesses its toxicityand that of its by-products, mainly chlorite, in water. Studieshave found no adverse effects in humans living in areas withClO2-disinfected water. Such studies can however not be con-sidered conclusive due to methodological problems, accord-ing to the US Environmental Protection Agency (2000).Based on the inadequate availability of information, the Inter-national Agency for Research on Cancer determined that so-dium chlorite is not classifiable as to human carcinogenicity(IARC, 1991), and ClO2 as well (ATSDR, 2004).

A study showed that a solution of ClO2 and its by-productschlorite and chlorate administrated to rats during 90 days ata dosage estimated to be 120 times higher than the humandosage was not toxic (Qingdong, Guanming, & Li, 2006).The FAO/WHO expert committee on food additives, on thebasis of the available toxicological data, set acceptable dailyintake values for chlorite and chlorate respectively to 0.03and 0.01 mg/kg of body weight per day (JECFA, 2008).

24 V.M. Gomez-Lopez et al. / Trends in Food Science & Technology 20 (2009) 17e26

Legal frameworkIn European Union the Regulation EC 852/2004 (EU,

2004a) designates potable or clean water as the only decon-tamination agents allowed. Application of ClO2, or anyother chemical agent, for surface decontamination of freshproduce is in EU not legalized. In USA, fresh-produce pro-cessors are legally allowed to use ClO2 as disinfectant(FDA, 2008). A maximum of 200 mg/ml ClO2 is allowablefor sanitizing processing equipment and ClO2 concentrationof 3 mg/l in water is maximum allowable for contact withwhole produce. For peeled potatoes the maximum permit-ted is 1 mg/l (Parish et al., 2003). Treatment of producewith ClO2 must be followed by blanching, cooking, or can-ning (FDA, 2005; Parish et al., 2003). EC 853/2004 (EU,2004b) states that food business operators can use sub-stance, other than water, for surface decontamination onlyif the substance has been specifically approved. From theoccupational point of view, produce applications of ClO2

need specialized worker safety programs, and closed injec-tion systems for containment of concentrate leakage andfumes from volatilization (Suslow, 1997). It is of notethat the maximum permissible concentration of ClO2 inair is country dependent. It varies between 0.1 mg/m3 inRussia (Obvintseva & Gubanova, 2004) and 0.3 mg/m3

in USA (8-h time-weighted average concentration) (FDA,2005). These differences maybe in one part explained bythe symptoms and methods used in the determination.

ConclusionsChlorine dioxide is a strong oxidizing agent that has

great potential as a decontamination agent for minimallyprocessed fruits and vegetables. ClO2 has high effectivenessagainst a wide variety of microorganisms. Microbial cellmembrane has been identified as the primary target ofClO2 on microorganisms. It is less effective when microor-ganisms are hidden in produce wounds. It is useful to pro-long the shelf-life of MPFV, although in some cases the gascan induce browning and bleaching. It does not producesignificant amounts of toxic by-products. More research isneeded about its effect on the fruit and vegetable physiol-ogy, and on toxicology issues and nutrients’ stability.

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