COMPARISON OF ANOLYTE AND CHLORINATED WATER AS A DISINFECTING DIPPING TREATMENT FOR

STORED CARROTS

TILAHUN SEYOUM WORKNEH', G. OSTHOFF'.3, J.C. PRETORIUS2 and C.J. HUGO'

'Department of Microbiology. Biochemistry and Food Science 2Depattmenr of Soil, Crop and Clime Sciences

The University of the Free State Faculty of Nmral and Agricultural Science

P.O. Box 339 Bloemfontein 9300, South Afnca

Accepted for Publication October 3. 2002

ABSTRACT

Carrots (Daucus carota L.) were dipped in anolyte water for 5, 10 and 20 m'n or in 100 p g / d chlorine supplemented water for 20 min to study the flect of anolyte water as an environmentally friendly alternative disinfecting measure for carrots prior to packaging and storage. Packages of carrots were stored at 1 & 0.5C and ambient temperature (1 7.5-31.4C). The anolyte water dipping treatment was found to be as effective as chlorinated solutions in controlling growth of aerobic bacteria. molds, yeasts and colifonn bacteria during storage. There were no significant diflerences (P < 0.05) in microbiological changes on carrots dipped in anolyte water for 5, 10, and 20 min. Exposure of carrots to anolyte water for as shon as 5 min can be used flectively to reduce and limit growth of aerobic bacteria, molds, yeasts and colifonn bacteria. Losses in firmness and physiological weight were higher in carrots dipped in chlorinated water. Anolyte water treatments had no flect on total soluble solid content. pH value, firmness and the overall visual appearance of carrots.

INTRODUCTION

Postharvest decay is an important factor affecting quality of fresh commodities during storage. In moist storage conditions with a relatively higher temperature, postharvest decay is the most serious problem associated with

'Contact author: TEL: 027 51 4012216; FAX: 027 51 4480692; EMAIL: Osthofffi.sci@mail.uovs.ac.za

Journal of Food Quality 26 (2003) 463-474. All Righfs Reserved. 'Copyrighf 2003 by Food & Nurritwn Press, Inc.. Trumbull. Connecticut. 463

464 T.S. WORKNEH. G . OSTHOFF. J.C. PRETORIUS and C.J. HUGO

carrots, whereas loss of moisture is important in dry-hot air storage conditions. Much work has been done on the development of novel methods to control postharvest decay (Rodov er af. 1996; Fallik ef af. 1999). Postharvest hot water treatment has also been found to be effective in controlling postharvest contamination and chilling injury in various fruits, and decay in vegetables.

Synthetic fungicides are one of the effective ways to limit growth of microorganisms and control postharvest diseases associated with vegetables and fruits during storage (Eckert 1990). Several biological products including Aspire’, BioSave” and Yield Plus” are available for use as a biocontrol during storage of vegetables. However, the success of these products is limited due to the variability experienced in their effectiveness and lack of understanding of how to adopt “biological approaches” to a commercial setting (Wisniewski er al. 2001). High cost, availability and lack of understanding are the most important problems associated with the use of these materials and methods as postharvest decay control measures in developing countries. Chlorinated water is currently used in packaging-houses for the purpose of disinfecting and sanitizing fruits and vegetables in order to reduce postharvest decay (Nunes and Emond 1999). There is also a strong concern that the increased use of chemicals for postharvest disease control may aggravate health problems of rural populations, both adult and children ( M e w and Mekonnen 1998; Weibcerg 2000).

Prepackaging dipping of vegetables in chlorine supplemented water alone, or in combination with other treatments, are effective in limiting growth of microorganisms during storage (Delaquis et al. 1999; Prusky er al. 2001). However, the use of chlorine solution as a disinfectant has been facing challenge in developed countries. Recently a product (STEDS, Radical Waters, Midrand, South Africa) based on a Russian development, electrochemically activated water (ECA) was developed for use in dental unit water lines. The original development was applied in fields varying from agriculture, cooling towers, swimming pools, dermatology, dressing and cleaning of wounds and disinfecting of instruments (Leonov 1997; Bakhir 1997). ECA is considered totally harmless to human tissue, yet highly microbiocidal. The raw products are water and saline. These are fed into a special unit which then “activate” the water. The inventors described the “activation” process as a change of the molecular state of water from a stable to metastable state. Two kinds of ECA are produced, anolyte and catholyte. The anolyte is described as having an oxidation-reduction potential (ORP) in the region of + loo0 mV and catholyte an ORP of -800 mV. Current thinking around the concept is that the ORP of both solutions fluctuates between these values at a rate too rapid to measure. Some of the biocidal agents in the solutions are ClO,, HClO, Clz, C10-, HzOz, HO;, NaHO. Oz, 0,. 0,. H’ and ‘OH. The presence of the free radicals with their oxidizing effects in the solutions is considered of great importance. Higher organisms possess antioxidant defense systems whereas microorganisms generally do not. Anolyte

DISINFECTING CARROTS WITH ANOLYTE WATER 465

is thought to have the antimicrobial effect and catholyte a detergent or cleaning effect. It is further established that ECA solutions return to a stable, inactive state, pure water in fact, within a period of 48 h after production. In this study the potential to use as a postharvest disinfectant was explored. A chlorine supplemented water-dipping treatment was used as a control. The effect of an anolyte water-dipping treatment on the growth of aerobic bacteria, coliforms and fungi (molds and yeasts) on carrots, during storage, as well as the total soluble solid, firmness, physiological weight loss and pH was compared with standard control treatment.

MATERIALS AND METHODS

Sample Preparation and Postharvest Treatment

Carrots (cv Nantes) were topped in the field and manually harvested at a proper maturity stage and immediately transported to the laboratory. Carrots free from any defects were selected prior to washing with tap water at 4C. After washing, 36 kg of carrots were subdivided into 6 groups of 6 kg each for prepackaging treatments. Again each of the six subgroups were further divided into three samples containing approximately 2 kg carrots each. Carrots were then subjected to the following treatments: (1) dipping in anolyte water for an exposure duration of 5, 10 and 20 min, or (2) dipping in 100 pg/mL chlorine supplemented water for 20 min (the maximum time used without affecting quality during storage) (Nunes and Emond 1999) or (3) washing carrots for storage at 1C and ambient conditions in water as controls. After dipping treatments, carrots were dried and then packaged in commercial microperforated bags (Xtend" Fresh produce packaging technology, Israel) (Seyoum m al. 2001).

Anolyte water was prepared from potable water with 5% sodium chloride salt using the Ionyzer (Radical Waters, Midrand, South Africa) operating at a pressure of 50 kPa. The pH was adjusted to 6.8. containing 2.55% total dissolved solid. Immediately after preparation, anolyte water was used for dipping carrots without delay to avoid decay of the ionized properties.

Measurement of Physiological Weight Loss (PWL), Total Soluble Solids (TSS) and pH

Physiological weight loss and TSS ("brix) of carrots was determined according to the methods as described Waskar m al. (1999). The pH value of carrots was determined with a pH meter using the methods given by Mohammed et al. (1999).

466 T.S. WORKNEH. G. OSTHOFF. J.C. PIUXORIUS and C.J. HUGO

Measurement of Firmness

The firmness of carrots were measured with a constant load penetrometer (Model 1719, Stanhope Seta Limited, England) that was automatically controlled by a Seta-Matic penetrometer controller (Model 1720). A carrot was randomly selected from each of the 18 packages on each sampling time. After the middle portion of each carrot was cut from both sides, half a portion was carefully sliced longitudinally to have a uniform horizontal surface at the bottom when resting on the top of the penetrometer table surface. This was to secure stable functioning of carrot samples when a constant load of 50 g was dropped automatically on top for penetration with a needle into the carrot tissue. A constant load of 50 g was allowed for 10 s to puncture carrots and the depth of penetration in millimeter was recorded. Three measurements were made along the longitudinal direction for each treatment replication after which the average was calculated for each replication.

Microbiological Analysis

Microbial populations were estimated using the procedure as described by Brackett (1990). Three carrots were cut aseptically into pieces with sterile knives. Samples of 25 g were blended with 225 mL 0.1 % peptone water (pH 7.0) in a stomacher for 3 min. The slumes were serially diluted in 9 mL 0.1 % peptone water. To determine populations of total aerobic micro organisms (APC). duplicate samples were plated on plate count agar (PCA, Oxoid CM463, and pH 7.0 f 0.2) and incubated at 30C for 2 days. For the estimation of E. cofi and coliform populations, duplicate samples were plated on violet red bile agar (VBRA with MUG, Oxoid CM978) and incubated at 37C for 1 day. To determine populations of molds and yeasts, duplicate samples were plated on Rose-Bengal Chloramphenicol Agar Base (Oxoid CM549) and incubated at room temperature for 3 to 5 days. In all the cases pour plate methods were used. The presence of E. coli was detected with an UV light at 366 nm. The mean log10 of viable counts from two duplicate plates were noted. The estimation of the population of total aerobic bacteria, molds and yeasts and total coliforms at time 0 of storage was made prior to the washing treatment.

Data Analysis

The experimental design was arranged in a completely randomized design (CRD) to obtain a factorial ANOVA, with dipping treatment as main factor, and storage time as a split plot, with three samples from each treatment combination. Randomly, 18 packages of carrots were taken from each of the 6-treatment groups on each sampling time, and used for microbiological, PWL, TSS, firmness and pH analysis. Each replicate sample package was analyzed in

DISINFECTING CARROTS WITH ANOLYTE WATER 467

duplicate for microbiological growth. Statistical significant differences between the 6 treatments were determined by ANOVA using MSTAT-C software package (MSTAT, Michigan State Univ., East Lansing, MI) and the multiple comparison of the treatment means by Duncan's multiple range tests (Duncan 1955).

RESULTS AND DISCUSSION

The freshness quality of carrots was monitored through visual observation after 16 days of storage. The carrots dipped in anolyte and chlorinated water had no soft spots or signs of fungal or wet rot decay. Some bleaching by chlorinated water was observed on the surface of carrots while those dipped in anolyte water remained shiny and smooth. During storage at 1C. carrots dipped in anolyte water showed better visual appearance, when compared with the appearance of carrots dipped in chlorine supplemented water (Fig. 1). It therefore seems as if the absorbing tissue of carrots was not affected by anolyte water, but was etched by the chlorine. Although the effectiveness of chlorine and anolyte water is similar on reducing and suppressing growth of natural microbiological flora of carrots (Fig. 2, 3. and 4). marketability and consumer acceptance would be better for carrots having been dipped in the latter.

Physiological Weight Loss, Total Soluble Solid ('TSS) and pH and Firmness

The dipping time of carrots in anolyte water had no effect ( P < 0.05) on PWL, although the moisture loss seemed to be a little higher in carrots dipped in anolyte water for 20 min (2.81 % after 16 days) compared to those dipped for a shorter time (2.77% after 16 days). The PWL of carrots dipped in anolyte water was significantly lower (P < 0.05) than in carrots dipped in chlorinated water during storage at 1C (3.09% after 16 days). It might be that the tissue softening by chlorine (Table 1) could have resulted in a higher rate of moisture loss. The two-way interaction between dipping treatments and storage time on the PWL was significant (P < 0.001). The effect of exposure times (5-20 min) and associated type of dipping treatment (anolyte and chlorinated water) on carrots had no significant ( P 1 0.05) effect on the TSS content of carrots during 16 days of storage. TSS changed from 6.160 "Brix to between 6.73 and 7.80 "Brix after various dipping treatments and 16 days storage. There was no two- way significant interaction (P 2 0.05) between the dipping treatments and storage time on the response of TSS of carrots during the storage period of 16 days. Similarly, the exposure duration of carrots to anolyte water had no significant (P 2 0.05) effect on the pH value of carrots stored at 1C. The pH changed from 5.938 to between 6.300 and 6.507 after various dipping treatments

468 T.S. WORKNEH. G. OSTHOFF. J.C. PRETORIUS and C.J. HUGO

and 16 days storage. The overall dipping treatment effect on the pH value of carrots was significant (P C 0.001), although the difference shows no clear pattern among the treatments. The two-way interaction between disinfecting treatments and storage time on the changes of the pH value of carrots was found to be significant (P < 0.001). except for the control carrots stored at ambient temperature where the pH value remained higher and stable during the storage period, regardless of the postharvest treatment.

FIG. 1 . A PHOTOGRAPH SHOWING THE EFFECT OF CHLORINATED AND ANOLYTE WATER DIPPING TREATMENT ON THE VISUAL APPEARANCE QUALITY OF

CARROTS STORED AT 1C FOR 16 DAYS

Firmness is a postharvest quality parameter that shows the level of freshness of fruits and vegetables. Change in firmness of carrots is due to biochemical activities, associated with enzymatic activities, responsible for the degradation of cellulose. No substantial differences in firmness of carrots measured by depth of penetration of a constant load, as a result of disinfecting treatments, were observed (Table 1). Firmness changed from 4.257 mm penetration to around 3.7 mm penetration after 16 day storage. The dipping time of carrots in anolyte water had no significant effect (P 2 0.05) on their firmness during 16 days of storage at 1C.

DISINFECTING CARROTS WITH ANOLYTE WATER 469

TABLE 1 . CHANGES IN FIRMNESS OF CARROTS SUBJECTED TO DIFFERENT POSTHARVEST

TREATMENTS AND STORAGE AT 1C AND ROOM TEMPERATURE (17.5-31.4C)

Treatment Firmness (mm depthl50g force

day 0 day 8 Day 16 applied)

Anolyte, 5 min, MAP, 1C 4.251' 3.448& 3.761' Anolyte, 10 min, MAP, IC 4.257' 3.397" 3.750' Anolyte, 20 min, MAP, 1 C 4.2S7a 3.458" 3.713' Chlorinated, 20 min, MAP, IC 4.257' 3 .6Sb 3.858" Control, HzO, MAP, IC 4.257' 3.145* 3.028& Control, H20. MAP, RT 4.257" 3.043* 2.909' Control, H ~ O . RT ' 4.251' 5.752' 6.01 1'

Meall 4.257 3.700 3.862

'.b.C.d.C Mean data from three packages per treatment within a column followed by the same letter(s) are not significantly different according to Duncan's multiple range test ( P < 0.05). LSD Value = 0.417, SE = 0.144 and CV = 0.006.

Microbiological Changes

Analyses of microbial populations in the packaged carrots are shown in Fig. 2, 3 and 4. The anolyte and chlorinated water dipping treatments significantly decreased (P < 0.001) the numbers of natural microbiological flora of carrots immediately after dipping. There was no significant difference (P < 0.05) between anolyte and chlorinated water in killing the microbial population immediately after dipping treatment. However, washing carrots with potable tap water significantly reduced the initial microbial populations in carrots. Low numbers of microorganisms, responsible for postharvest decay, were noted as a result of washing and dipping treatments. The high number of microorganisms before washing was due to the amount of soil adhering to the surface of carrots. The number of the microorganism started to increase during the second week of storage and this determines the level of contamination during short or long storage if the washing treatment does not immediately follow the harvest. This is in agreement with results of Madden (1992) who recommended washing with water and disinfecting of fresh produce to prevent contamination. and the population counts were also in the same order as those reported by Brackett (1992) and Garg et al. (1990).

The use of anolyte and chlorinated water dipping treatments reduced (P < 0.001) the population of microorganisms and suppressed their growth during storage. Dipping carrots in anolyte and chlorinated water resulted in the total number of aerobic bacteria to stay below 2.4 log cfu/g during 16 days of storage at 1C. The interactive effect of disinfecting treatment with storage duration had

470 T.S. WORKNEH. G. OSTHOFF. J.C. PRETOWS and C.J. HUGO

a highly significant (P < 0.001) effect on the populations of total aerobic bacteria. Anolyte water dipping is therefore an effective step in reducing and limiting the growth of aerobic bacteria even at 5 min exposure time.

The dipping time (5, 10, 20 min) of carrots in anolyte water had no significant ( P < 0.05) effect on the estimated population of total aerobic bacteria (Fig. 2). and there was no significant (P < 0.05) difference in the number of aerobic bacteria associated with carrots dipped in anolyte water and carrots dipped in chlorinated water during storage at 1C. In comparison, carrots washed in tap water and stored at 1C showed the presence of high amounts of aerobic bacteria (Fig. 2). Prepackaging treatments therefore had a significant (P < 0.001) effect on the reduction and limitation of growth of aerobic bacteria during the storage period. The multiple comparison of the means showed that there was a significant ( P < 0.001) difference in the population of aerobic bacteria in control carrots and carrots dipped in either anolyte or chlorinated water prior to packaging.

The two-way interaction between prepackaging treatment and storage time on the population of molds and yeasts were highly significant (P < 0.001). However, there was no significant (P < 0.05) difference in the estimated population of molds and yeasts in carrots dipped in anolyte water for all three exposure times, as well as chlorine supplemented water. Dipping carrots in anolyte water for 5 . 10 and 20 min had statistically the same level of effect in reducing the population and limiting the growth of molds and yeasts during the storage at refrigeration temperature (Fig. 3).

The population of coliform bacteria was highly reduced (P < 0.001) after prepackaging treatment in carrots dipped in either anolyte water or chlorine supplemented water during the 16 days of storage at 1C (Fig. 4). Mostly, the total number of coliform bacteria in packages of carrots dipped in anolyte water and chlorinated water remained below 1.0 x 10' cfu. g-' during 16 days of storage, and the two-way interaction between prepackaging treatments and storage time was highly significant (P < 0.001) on the population of total coliforms. The 20 min exposure time of carrots in anolyte water showed no significant (P 2 0.05) increase in its effectiveness in reducing the population and controlling the growth of coliform bacteria compared to the short 5 min exposure (Fig. 4). There was also no significant (P 1 0.05) difference in the population of total coliforms associated with carrots dipped in anolyte and chlorinated water during 16 days of storage. None of the plates showed the presence of E. coli, the most prominent fecal coliform. during the investigation.

The results presented in Fig. 2, 3 and 4 implied that a short dipping time of 5 min in anolyte water would be sufficient to disinfect carrots prior to packaging and storage. This would be useful in the food industry, as time efficiency is one of the important factors in a processing line during postharvest handling of commodities.

DISINFECTING CARROTS WITH ANOLYTE WATER 47 1

0 2 4 6 8 10 12 14 16 18 Days affer packaging

FIG. 2. GROWTH OF TOTAL AEROBIC MICROORGANISMS IN PACKAGES OF CARROTS SUBJECTED TO DIFFERENT POSTHARVEST TREATMENTS AND

Standard deviation is indicated (lower than symbol sizes were omitted). STORED AT 1C AND AMBIENT TEMPERATURE (17.5-31.4C)

--+--Anolyte,5min,MAP, IC - - . D - - . Anolyte. lOrnin.MAP, IC

-Control, MAP.RT

LSDn nq = 0.217

0 2 4 6 8 10 12 14 16 18

Days after packaging

FIG. 3. GROWTH OF MOLDS AND YEASTS IN PACKAGES OF CARROTS SUBJECTED TO DIFFERENT POSTHARVEST TREATMENTS AND STORED AT 1C AND AMBIENT

Standard deviation is indicated (lower than symbol sues were omitted). TEMPERATURE (17.5-31.4C)

472 T.S. WORKNEH, G. OSTHOFF, J.C. PRETORIUS and C.J. HUGO

7 . a 5. 6 -

- -*--AnoIyle,Smin,MAP. 1C

-+-Anolyte,20min,MAP, IC +Chbrinatcd.Mmin,MAP. I ( +Control, MAP, 1C -Contml,MAP,RT

---.-.- Anotyte. IOmin.MAP, IC

FIG. 4. GROWTH OF TOTAL COLIFORh4 MICROORGANISMS IN PACKAGES OF CARROTS SUBJECTED TO DIFFERENT POSTHARVEST TREATMENTS AND

Standard deviation is indicated (lower than symbol sizes were omitred). STORED AT 1C AND AMBIENT TEMPERATURE (17.5-31.4C)

CONCLUSIONS

Anolyte water was tested for its potential to control the postharvest decay of carrots. Results from this study showed that the anolyte water reduced the population and limited growth of microorganisms, i.e., bacteria, molds, yeasts and coliforms on carrots during storage at lC, as effective as did the chlorine solution. Dipping in chlorinated water is normally carried out for up to 20 min (Nunes and Emond 1999) while dipping in anolyte water, for as short as 5 min, resulted in equal disinfecting. After 16 days of storage, carrots dipped in either anolyte or chlorinated water were shiny and free from any signs of deterioration, whereas spots of bleaching on carrot surfaces were visually observed due to dipping in chlorinated solutions. Exposure of carrots to different dipping time in anolyte water had no significant effect on the total soluble solid, pH and firmness of carrots during 16 days of storage at 1C.

DISINFECTING CARROTS WITH ANOLYTE WATER 473

ACKNOWLEDGMENT

The authors are grateful to Mr. W. Kruger for his assistance during the production of carrots on his farm. We are also grateful to the Agricultural Research Training Project (ARTP), Alemaya University, Ethiopia for funding.

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