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Hot water brushing: an alternative method to SO

fumigation for color retention of litchi fruits

Amnon Lichter

_{*, Orit Dvir}

_{, Ilana Rot}

_{, Miryam Akerman}

_{, Rafi Regev}

_,

Aharon Wiesblum

_{, Elazar Fallik}

_{, Giora Zauberman}

_{, Yoram Fuchs}

a_{Department of Posthar}₆_{est Science of Fresh Produce}_,_{Institute for Technology and Storage of Agricultural Products}_,_ARO_, The Volcani Center,PO Box6,Bet Dagan50250,Israel

b_{Department of Controlled Agriculture and En}₆_{ironmental Engineering}_,_{Institute of Agricultural Engineering}_,_ARO_, The Volcani Center,PO Box6,Bet Dagan50250,Israel

Received 26 July 1999; accepted 18 November 1999

Abstract

Distribution of high-quality litchi (Lychee chinensis Sonn.) fruits to global markets depends exclusively on postharvest treatments to suppress peel browning. The current standard treatment of litchi fruits in Israel includes fumigation with sulfur dioxide (SO2) followed by dipping the fruits in hydrochloric acid containing the fungicide prochloraz. As part of the effort to reduce the use of potentially hazardous chemicals in agriculture, we developed a new procedure that may enable SO2to be avoided. Instead of fumigation, litchi fruits are sprayed with hot water while being brushed in a revolving drum, after which the fruits are subjected to hydrochloric acid treatment. Fruits that are processed in this way maintain a uniform red color for at least 35 days, without apparent deterioration in external or internal quality, or taste. Physiological studies demonstrate that polyphenol oxidase (PPO) activity is reduced by the hot water brushing (HWB) procedure as compared with controls but not to the same extent as inhibition by SO2treatments. In addition to its effect on PPO activity, HWB may lead to reduced pH of the pericarp, or more uniform distribution of the acid in it. This result suggests that HWB may act by bruising the external layer of the pericarp allowing the peel to be uniformly exposed to the acid which may inhibit PPO activity and maintain the anthocyanins in their red-pigmented form. © 2000 Elsevier Science B.V. All rights reserved.

Keywords:Postharvest treatment; Polyphenol oxidase; Hot water

www.elsevier.com/locate/postharvbio

1. Introduction

Litchi (Lychee chinensisSonn.) fruits are in high demand as exotic commodities, because of their appealing natural color and rich taste and aroma. However, if the fruit is not treated after harvest, the red pericarp becomes brown and desiccated * Corresponding author. Tel.:+972-3-9683915; fax:+

972-3-9683622.

E-mail address:[email protected] (A. Lichter)

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before reaching the markets. One of the hypothe-sized mechanisms for the rapid browning is that it results from oxidation and polymerization of phe-nolic compounds, including the red anthocyanins, by polyphenol oxidase (PPO) (Akamine, 1960; Zauberman et al., 1989; Underhill et al., 1992). In undamaged tissue, PPO is presumed to be local-ized in the plastids, while anthocyanins accumu-late in the vacuole. The desiccation of the rind and formation of micro-cracks may be partly responsible for cell degradation, resulting in asso-ciation of PPO with its potential substrates (Un-derhill and Critchley, 1992, 1993a). It should be noted however, that there is no conclusive evi-dence for the role of PPO in anthocyanin degra-dation (Underhill and Critchley, 1994).

A number of methods have been devised over the years to retain the appealing color of litchi fruits (Holcroft and Mitcham, 1996; Ray, 1998). One of the most widely used methods involves fumigation of the fruits with SO2, several hours

after harvest (Swarts, 1983; Zauberman et al., 1989). This causes the red color to be bleached to yellow, which is slowly and partially restored to pink. SO2 interacts with the membranes, making

the rind pliable and leaky to solutes. Also SO2

directly reacts with anthocyanins rendering them colorless and stabilizing them against degradation (Timberlake and Bridle, 1975), and it is thought to inhibit litchi PPO. The dipping of litchi fruits in dilute hydrochloric acid was developed by Za-uberman et al. (1989) in order to restore the red color of the fruit after bleaching: the lower pH of the rind restores the red color to the colorless anthocyanins. This procedure was followed by dipping the fruits in prochloraz solution, but Fuchs et al. (1993) found that prochloraz was better dissolved directly in the HCl solution, and as such was more effective in preventing fungal development. After drying, the fruits are packed and can be cold-stored for several weeks. This procedure enables the marketing of attractive fruits, but SO2 represents health risks for allergic

people (Taylor, 1993) and therefore, SO2

treat-ments have been banned in the USA for every purpose other then control of gray mold in table grapes (Paull et al., 1995; Holcroft and Mitcham, 1996).

Alternative procedures to SO2 fumigation of

litchi fruits have been proposed, e.g. steam treat-ment (Kaiser et al., 1995) or hot benomyl dipping (Scott et al., 1982), but so far, no method has been widely accepted or established commercially. Our objective was to develop a procedure that will not compromise the quality of the fruits and that might be applied commercially upon demand from the market or public health authorities. The principle of brushing fruits and vegetables under hot water spray was reported recently for the postharvest treatment of bell peppers (Fallik et al., 1999) and mangos (Prusky et al., 1999). The apparatus adopted for litchi fruits is different from the systems used for other commodities and consists of a revolving drum internally covered with a bristle-brushing surface fitted with hot water nozzles. During 1998, approximately 500 kg of the cultivar ‘Mauritius’ from five different growers and in four different regions of the coun-try were treated by hot water brushing (HWB) and evaluated for quality. Combined with acid dipping, this procedure yielded high quality and safer litchi fruits.

2. Materials and methods 2.1. Litchi fruits

Litchi fruits (Lychee chinensisSonn. cv. Mauri-tius) were obtained from growers on the day of harvest, transported in an air-conditioned van and treated at the Volcani Center, 3 – 9 h after harvest, with the newly-developed hot water brushing (HWB) system or by fumigation with SO2 as previously described (Zauberman et al.,

1991). Immediately after HWB or after an overnight interval at 1.5°C following SO2

treat-ment, the fruits were dipped in an acid solution containing 4% food-grade HCl and 0.2% prochlo-raz (Sportak, AgrEvo UK Ltd., Hauxton, Cam-bridge, England). The fruits were dried under ambient conditions for 15 – 30 min and packed in 2 kg open cardboard boxes. Storage for 3 weeks (1.5°C, RH\95%) was followed by shelf-life sim-ulation for 3 or 5 days (18°C, RH 88 – 92%) after which the fruits were evaluated for quality.

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2.2. Fruit quality e6aluation

Brown discoloration of the fruit was deter-mined by examining the fruits (usually 2 kg per package) and assigning an index on a scale of 1 – 5 (1, 100% of the fruits with brown discol-oration; 5, no brown discoloration). As 5% of the fruits are equivalent to 0.2 index units (within the range of 1 – 5), the index was calcu-lated from the following formula: 5−(% of brown fruits)×0.2/5. Fruits with an index be-low 3.0 are considered commercially unaccept-able. Fruit firmness was evaluated manually according to three categories: 1, very soft; 5, soft; 10, firm. Brown patches on the rind were categorized as being either light or intense. Fruits with a brown patch only around the pedicel were assigned to a separate category. Ten fruits from each replicate were peeled, and their internal browning and taste were recorded.

2.3. Hot water brushing

Based on the technology described by Fallik et al. (1999), a modified machine has been de-veloped for hot water brushing (HWB) of small fruits. Instead of rolling brushes, a revolving drum, internally covered with a brushing sur-face, and hot water nozzles, was built. Litchi fruits were rinsed in hot water at 55°C and the angle of the drum was set to retain the fruits for 20 s inside it. The fruits were then dipped in HCl solution, dried, packed and stored as de-scribed above.

2.4. Prochloraz residues

To determine prochloraz levels, the fruits were treated by HWB or with SO2 followed by HCl/

prochloraz (4/0.2% v/v) dipping, or were un-treated. Twenty-five fruits in each of four replicates were submitted for chemical analysis (Plant Protection and Inspection Services, Pesti-cide Division, Bet Dagan, Israel). The samples were prepared for analysis according to FDA-PAM, 1994, for non-lipid products.

2.5. Leakage from peel discs

After the litchi fruits had been treated as spe-cified, the peel from four fruits per treatment was cut in half, peeled off from the flesh and lightly dried on a paper towel to remove free juice. A 10-mm cork borer was used to cut out 15 peel disks that were weighed and immediately placed in beakers containing 20 ml of 0.4 M mannitol solution. The beakers were incubated on a rotat-ing shaker (100 rpm) for 2.5 h at room tempera-ture (23°C). An El-Hamma TH27 conductometer (cell constant 0.99 cm) was used to measure solu-tion conductivity, and readings were taken in absolute mode on 300-mmho or 1-mmho scales. Total conductivity was assayed after freezing the peel disks in solution, boiling and cooling to room temperature.

2.6. Polyphenol oxidase acti6ity and determination of pericarp pH

Fruits were treated by HWB 6, 7.5 and 9 h after harvest and were dipped in acid 5 – 20 min after HWB. The fruits were stored at 20°C for 5 days to obtain an immediate assessment of their qual-ity, or were stored for 3 weeks at 1.5°C and then for 5 days at 20°C. Fruits were frozen before and after HWB, after acid treatment, and different time points during storage, as specified. Control treatments included fruits treated by HWB with-out the acid treatment, or by hot water dipping (55°C) for 20 s followed by acid dipping, or by SO2 fumigation followed by acid dipping. Three

fruits per treatment were thawed and peeled, and 2 g of pericarp tissue was homogenized in 0.1 M phosphate buffer, pH 6.6 and 0.5 g of insoluble polyvinyl pyrollidone (Merck) for 30 s with a polytron homogenizer (Kinematica GmbH, Kreins, Luzern, Switzerland; probe diameter, 20 mm) set at speed 6. The homogenate was cen-trifuged for 10 min at 8000×g in a Sorvall rotor SS-34 at 4°C. The supernatant was collected and centrifuged again in 1.5 ml tubes at 20 000×g for 10 min at 4°C. The supernatant was collected into a fresh tube and 0.75 ml was used for the PPO assay in duplicate. The PPO assay was conducted by adding 0.12 ml 4-methyl catechol (Sigma, St.

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Louis, MO, USA) freshly dissolved (0.25 g) in 2 ml of ethanol and 10 ml of water (final concentra-tion, 23 mM). A Contron spectrophotometer was used to follow changes in absorbance at 410 nm over 2 min, and the linear progress of the reac-tions was recorded between 30 and 90 s. Results were calculated as DOD410/g fw/min×1000. The

change in the pH of the buffer was determined in duplicate. To determine the pH of pericarp tissue, extraction was carried out as described, but with-out PVP and with distilled water instead of the phosphate buffer.

3. Results

3.1. E6aluation of fruit quality after hot water brushing

In general, the bright red color of fruits treated by HWB followed by acid dipping was more attractive than that of SO2-treated followed by

acid dipping (Fig. 1). Two parameters needed to be resolved — the timing of the treatment after harvest, and the length of the interval between HWB and acid dipping. These parameters were associated with the appearance of brown spots on the rind and fruit cracking. It was noticed that increasing the interval between harvest and HWB

led to increased browning. For example, in the first experiment conducted in the first week of the season fruits were treated by HWB 6, 7.5 and 9 h after harvest. As controls, fruits were treated with SO2 and acid dipping the following day, or were

dipped in 0.2% prochloraz solution, or dipped in hot water (55°C) for 20 s followed by acid treat-ment. The fruits were packed in 2-kg open card-board boxes and stored for 3 weeks at 1.5°C followed by 3 days at 20°C. Results from this experiment (Figs. 1 and 2) demonstrated that HWB was a viable alternative to SO2 for color

retention in litchi fruits. As expected, fruits that were dipped only in prochloraz solution became brown to black within a few days in storage. Fruits that were dipped in hot water prior to acid dipping varied in their appearance with the major-ity of the fruits showing brown spots. SO2

treat-ment produced fruits with an acceptable appearance, but their color ranged from yellow to pink – red and red (Fig. 1). In optimized condi-tions, the peel of the HWB-treated fruits had a brighter red hue, which was unspotted. However, a decline in appearance, in the form of limited-size brown spots, occurred when fruits were held at ambient temperature for 9 h compared to 6 h (Fig. 2). It should also be noted that the higher score assigned to SO2 fumigation (Fig. 2) was

based entirely on the amount of brown spots on

Fig. 1. The appearance of litchi fruits after hot water brushing (HWB, left). Also shown are fruits fumigated with SO2and dipped in HCl (middle) or fruits treated with HWB not followed by dipping in HCl (control, right). Storage time was 3 weeks at 1.5°C and 5 days at 20°C.

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Fig. 2. The appearance of litchi fruits after different posthar-vest treatments, rated according to the proportion of brown patches on the peel (index range, 1 – 5). HWB, hot water brushing; HWD, hot water dipping; HCl, hydrochloric acid (4%) dipping. The time of treatment after harvest is specified (h) and fruit appearance was evaluated after 3 weeks at 1.5°C and 3 days at 20°C. Bars represent standard deviation (S.D.) values.

the HWB treatment, following harvest, and in-creasing the interval between HWB and the acid dipping were both effective in reducing or elimi-nating fruit cracking. These practices had, how-ever, a cost in fruit quality (Table 2).

Table 1

Fruit cracking as a function of the interval following harvest or between hot water brushing (HWB) and acid dipping

Time after harvest Time from HWB to Cracked fruits HCl (min) (%)

(h)

Obser6ation I

6 3 18

6 8 9.5

6 11 2.5

9 20 0

Obser6ation II

3 4.5

3.2

3 20

0 5

0 15

Obser6ation III

4a ₀ ₁₂

4 2.3

5.2 0

7 5 3

Obser6ation IV–VIc

\1 Various

Various

a_{Sodium chloride (1%) was included in the acid solution for} the 4 h treatments.

b_{Sodium chloride (1%) was included in the hot water and in} the acid solution for the 7 h treatments.

c_{In the three later experiments of the season, less then 1%} fruit cracking was observed in the various postharvest treat-ment regimes.

the rind and did not account for the general impression, which was either in favor of HWB or at a similar score (not shown).

Another problem associated with HWB was fruit cracking. However, this appeared as a major problem only in the first three of the six harvests tested during the season (Table 1). Preliminary results obtained in observation I, 6 h after har-vest, revealed a high incidence of cracking (18%) for the 3 min interval between HWB and acid dipping, but when the interval was increased to 8 or 11 min, cracking declined to 9.5 and 2.5%, respectively. With 9 h from harvest to HWB and a subsequent 20-min interval to HCl dipping, fruit cracking was not encountered. In observation II (Table 1) cracking was evident only when HWB was applied 3 h but not 6 h after harvest and it was reduced by using a longer interval between HWB and acid dipping. Similar trends were ob-served in observation III, although interpretation of the results in this experiment is complicated by the addition of 1% sodium chloride either to the acid solution or to both the hot water and the acid. In three later experiments, cracking did not appear or was rare (not shown). Hence, delaying

Table 2

Prochloraz levels in litchi fruits

Treatment Prochloraz (mg/kg)a

Flesh

Pericarp Wholeb

Not detected

Control Not detected

0.99 HWB 6.6690.63 a 0.0590.0 c

0.0390.02 c

4.4990.24 b 0.67

SO2

a_{Four replicates were used, and means and standard errors} (SE) are given. Duncan’s multiple range test was preformed. Different letters denote significant differences (P=0.95).

b_{Calculated value for whole fruits assuming 15% of fresh} weight for pericarp (Holcroft and Mitcham, 1996).

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Table 3

Peel conductivity after postharvest treatments

Assay time after

Fruit treatment % Conductivity of totala

treatment

Experiment I

Control 2 h 16.090.5 b

89.291.1 a 2 h

SO2treatment

HWBb _{2 h} _16.19_{0.5 b}

16.190.4 b 2 h

HWDc

Experiment II

13.290.9 B 10 min

Control

HWB 10 min 16.390.5 A

17.590.7 A 40 min

HWB

a_{Four replicates were used and means and standard errors} (SE) are given. Duncan’s multiple range test was preformed and different letters denote significant (P=0.95) differences (small for experiment I and capital for experiment II).

b_{HWB, hot water brushing.} c_{HWD, hot water dipping.}

and the residual prochloraz in the peel or flesh was analyzed and compared with that in un-treated control fruits. The results of this experi-ment (Table 2) demonstrated that more than 99% of the residues in both treatments was concen-trated in the peel, but that almost 50% more prochloraz was found in the peel of fruits treated by HWB. The permitted level of prochloraz in Israel is 1 mg/kg for whole fruits, and the pericarp comprises 15% of the fresh weight of ‘Mauritius’ fruits (Holcroft and Mitcham, 1996). Thus, the calculated level of prochloraz may be below the statutory limit in SO2-treated fruits but marginal

for HWB fruits (Table 2).

3.3. Hot water brushing and ion leakage from peel disks

SO2 is known to increase ion leakage through

cell membranes and hence to facilitate accumula-tion of HCl in the cells, keeping the pigments red. To investigate whether HWB had a similar effect to SO2, peel disks were used to measure ion

leakage. The peel disks from SO2-treated fruits

were characterized by high conductivity compared to untreated fruits and HWB-treated fruits (Table 3). Ten minutes after HWB, the conductivity was higher by 3% as compared to untreated fruits but it is not obvious that this difference can account for the improvement in fruit color.

3.4. The effect of hot water brushing on PPO acti6ity

As expected, polyphenol oxidase (PPO) assays (Table 4) demonstrated that SO2 treatment

com-pletely eliminated PPO activity, irrespective of the acid treatment. Untreated fruits or fruits that had been treated by HWB but not subjected to dip-ping in HCl (dipped in 0.2% prochloraz solution), became brown to black within 1 – 2 days. Fruit samples were frozen before and after HWB and the peel PPO activity was compared. Six hours after harvest PPO activity was 31.590.3 before and 25.593.6 after HWB whereas 9 h after har-vest the activity was 38.791.3 before and 32.59 3.3 after HWB (see unit definition in Table 4). These results demonstrated that HWB by itself 3.2. Control of fungal de6elopment

Preliminary studies indicated that HWB fol-lowed by acid dipping failed to control fungal development on the peel. Exclusion of the fungi-cide prochloraz from the acid solution resulted in the appearance of fungal infection, mainly com-posed ofPenicilliumspp. which developed during the cold storage period and spread rapidly during shelf life (20°C). This suggests that the pathogens are deeply embedded in, or strongly adhere to, the rough surface of the pericarp. Nevertheless, it was of interest to find out whether the amount of prochloraz applied to the HCl solution could be further reduced. Non-quantitative observations, after HWB treatment followed by 10 days of storage at 20°C, suggested that effective control of fungal development may be achieved with 0.1% of prochloraz in the acid solution instead of 0.2%. Reducing the level of prochloraz to 0.05% re-sulted in a similar number of infected fruits, but with increased fungal colonization (results not shown). It was of interest to determine the resid-ual levels of prochloraz in litchi fruits treated by HWB, compared with the levels in those subjected to SO2 treatment. Fruits treated by HWB or

commercially treated with SO2 in the packing

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was not directly involved in retaining the pericarp color and in eliminating PPO activity. Hot water dipping (HWD) of the fruits at a temperature and for a duration comparable with HWB (55°C, 20 s) followed by acid treatment resulted in the reten-tion of the overall fruit color, although brown patches appeared frequently (Fig. 2). PPO activity for HWD was significantly higher than after HWB treatments at two of the four storage time points, suggesting that HWB improved the consis-tency of the treatment. Fruits that were treated by HWB followed by acid dipping had a distinct red color, which seemed to be more attractive than the color of SO2-treated fruits. However, brown

patches were occasionally observed on fruits and HWB treatment 9 h after harvest resulted in more brown patches than the treatment 6 h after. In all cases, PPO activity was higher in fruits treated 9 h after harvest as compared to 6 h after harvest (Table 4) but the differences were not statistically significant.

3.5. The pH of the pericarp

PPO assays were conducted in phosphate buffer (pH 6.6), but measurements proved that the pH of the extracts differed from 6.6, indicating that the buffer capacity was lower than that required to maintain a uniform pH (Fig. 3a). For that reason, the results shown in Table 4 can not be

compared independently of the pH and they reflect to some extent the degree of acidification of the peel. The pH of pericarp extracts from HWB with no acid dipping was the highest. HWB per-formed 6 h after harvest, followed by acid dipping resulted in lower pH of the buffered extract as compared to the 9 h treatment (Fig. 3a). The pH of extracts from fruits subjected to hot water dipping was higher than that of HWB/ acid-treated fruits. Pericarp tissue from fruits frozen after 5 days at 20°C was extracted in water and the pH was determined. Although the pH was much higher then expected, the trend shown in Fig. 3a for the water extracts was consistent with the pH obtained for the phosphate buffer and similar results were obtained for other storage times (not shown). These results indicated that HWB facilitated lowering of the pericarp pH as compared with hot water dipping. Because the acid treatment was reported previously to lower the pH of SO2-treated fruits to less then 3.0, fresh

pericarps were extracted in water in the following season and the pH was determined. Indeed, the results in Fig. 3b show that the pH of the fresh peel from acid treatments is much lower. In con-trast to what is shown in Fig. 3a, the pH of SO2-treated fruits was significantly lower than

that of HWB-treated fruits. The difference be-tween the pH of the pericarp after HWB and HWD was smaller and its significance is not obvious.

Table 4

Polyphenol oxidase activity after fruit storage under different regimes

PPO activity (DOD410/g fw/min×1000)a

5 7

Time after harvest (days) 21 215b

20 1.5

Storage temperature (°C) 1.5 1.520

−2.390.3 c

HWBc₊_{HCl, 6 h} _0.6_{90.7 b} _{1.591.0 c} _{1.090.0 b}

2.392.0 bc 1.891.6 b

HWB+HCl, 9 h 8.092.0 bc 6.594.5 b

HWB−HCl, 7 h 56.095.0 a 41.292.2 a 58.096.2 a 32.594.5 a

HWDd₊_{HCl, 7 h} _{8.591.0 b} _3.6_{91.8 b} _{15.090.5 b} _{2.792.7 b}

0.590.6 c −0.690.3 b

SO2+HCl 0.590.5 c −0.592.5 b

a_{Polyphenol oxidase (PPO) activity was measured spectroscopically with 4-methyl catechol as the oxidation substrate. Standard} errors (SE) were calculated for the mean of three replicates, each measured twice. Duncan’s multiple range test was used to assess the significance (P=0.95) of the differences for each storage time point separately.

b_{Fruits were stored for 3 weeks at 1.5°C and 5 days at 20°C.} c_{HWB, hot water brushing followed by dipping the fruits in HCl.} d_{HWD, hot water dipping followed by dipping the fruits in HCl.}

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Fig. 3. The pH of the fruit peel after different postharvest treatments. (A) pericarp obtained from fruits frozen after 5 days at 20°C. Clear columns, fruit peels extracted in water; dotted columns, pH of fruit peels extracted in phosphate buffer (100 mM, pH 6.6) for measurement of polyphenol oxidase activity (Table 4); (B) Fresh pericarp was removed from fruits kept 1 day at 20°C. Bars represent S.D. values.

remove dirt, dust and fungal spores from the fruit skin as well as to seal microscopic cracks, thus maintaining better keeping quality of the fruit (Fallik et al., 1999).

Our study suggests that the combination of hot water and mechanical brushing, followed by dip-ping the fruits in hydrochloric acid and prochlo-raz solution, may offer a viable alternative to SO2

fumigation. In addition to the obvious alleviation of health risks, the HWB procedure does not compromise fruit quality. However, limited browning of the peel, which was observed on fruits exposed to ambient temperature for pro-longed times prior to treatment, may necessitate strict management of harvested fruits. This prob-lem may be associated with increased activity of PPO during the pretreatment period, which may cause irreversible browning. Alternatively, rind desiccation may induce PPO or other enzymatic activities. The fact that cracking was diminished when fruits were kept longer at ambient tempera-ture, suggests that desiccation might have been involved and that water loss from the fruit in-creased its resistance to cracking. It is also impor-tant to note that fruit cracking is not inherent to HWB as it did not occur in fruits from some of the harvests. This may have been because of decreased turgor pressure of those fruits, but may also have been associated with the progress of the season and fruit ripening, or with horticultural practices.

The current state of the art does not permit exclusion of fungicidal treatment of litchi fruits if they are to be stored for several weeks. Cold storage can effectively control fungal development but after transfer to 18°C the rind becomes colo-nized by fungi. It is important to stress that HWB does not have a complete antifungal effect for litchi fruits, indicating that the fungi are inti-mately associated with the pericarp, possibly via fungal hyphae that penetrate the cuticle through micro-cracks and lenticles (Underhill and Simons, 1993). Obviously, finding alternative methods to control fungal development should be the focus of future research.

Without getting into the debate on the role of PPO in anthocyanin breakdown and peel brown-ing, it is possible to correlate reduced pericarp pH 4. Discussion

Hot water brushing has been established as a powerful method to improve the quality of vari-ous fruits and vegetables such as bell pepper (Fallik et al., 1999), and mango (Fallik et al., 1999; Prusky et al., 1999). HWB was shown to

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and PPO activity. In pH measurements of peri-carp obtained from frozen fruits, the HWB treat-ment resulted in more acidic pericarp than that from other treatments, although for frozen fruits, the absolute pH level of the peel was much higher then expected. For example, Underhill et al. (1992) measured a pH of 2.5 in the pericarp after SO2 treatment and acid dipping, whereas our

lowest value for frozen fruits was 4.1. In our second set of extractions (Fig. 3b) we obtained results that were consistent with those of Under-hill et al. (1992) and the average pH of HWB-treated fruits was closer to 3.0. Presumably, the diffusion between the pericarp and the flesh was responsible for the elevated pH observed for frozen fruits. According to Jiang et al. (1997) a pH of 4.2 or less in the pericarp may abolish PPO activity and stability and HWB may bring it below this threshold. The effect of pH is also reflected in the assays for PPO: the phosphate buffer was supposed to normalize the assay condi-tions but was affected by the acidification of the pericarp. Nevertheless, the pH range measured in the phosphate buffer (5.8 – 6.5) was within 55 – 90% of the activity range of purified PPO (Jiang et al., 1997). Whereas the effect of pH in the enzyme extracts may explain the smaller differ-ences in PPO activity (for 6 and 9 h after harvest or HWD), it can not account for the difference in activity observed between acid-treated and un-treated fruits. It is more likely that the exposure to acid destabilized the enzyme.

Two physical components are involved in HWB. The mechanical friction imposed on the rind warts by the brushes may cause uniform bruising of their peaks facilitating the entry of more acid in the subsequent dipping stage. The lower acidity and the increased uptake of prochlo-raz after hot brushing further support this hy-pothesis. Bruising did not cause greater leakage from the pericarp than occurred in the absence of treatment or under SO2 fumigation. Presumably,

once the peel is bruised, hydrochloric acid can penetrate the undamaged membranes whereas af-ter exposure to SO2the damage to the membranes

allows much greater solute leakage. In addition HWB may remove the dust and eliminate the formation of air bubbles that are trapped between

the fruit warts, allowing increased permeability of hydrochloric acid and prochloraz into the peel in the subsequent dipping stage.

Besides the mechanical component of brushing, application of the hot water spray clearly has a role as indicated by the increased dark patches on the rind caused by changing the temperature of HWB. This effect may be mediated by the re-moval of a hydrophobic layer from the fruit sur-face, or by a change in the structure of the cuticle. Hot water treatments of litchi fruits have been reported in combination with the fungicide beno-myl (Scott et al., 1982) or in the form of a steam treatment (Kaiser et al., 1995) followed by dip-ping the fruits in hydrochloric acid. However, steam treatment caused internal browning of the fruit flesh, although modifications to this method were reported to have commercial potential. The temperature used in HWB was not expected to impair PPO activity because the heat stability of PPO is well above 55°C (Underhill and Critchley, 1993b; Jiang et al., 1997).

Further research will be required to achieve better understanding of the HWB process. Com-mercial application of HWB will require study of the possibility of cold storage prior to HWB. Greater attention to health risks will require find-ing alternatives to fungicidal treatment and adop-tion of soluadop-tions such as hot water brushing to ensure the distribution of safer litchi fruits.

Acknowledgements

This paper is a contribution from the Agricul-tural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 413. This research was partly supported by grant No. 402-0171-98 from The Chief Scientist, The Ministry of Agriculture and Rural Development, and the Fruit Marketing Board of Israel.

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Holcroft, D.M., Mitcham, E.J., 1996. Postharvest physiology and handling of litchi (Litchi chinensisSonn.). Postharvest Biol. Technol. 9, 265 – 281.

Jiang, Y.-M., Zauberman, G., Fuchs, Y., 1997. Partial purifi-cation and some properties of polyphenol oxidase ex-tracted from litchi fruit pericarp. Postharvest Biol. Technol. 10, 221 – 228.

Kaiser, C., Levine, J., Wolstenholme, B.N., 1995. Vapour, heat and low pH dips improve litchi (Litchi chinensis Sonn.) pericarp colour retention. J. S. Afr. Soc. Hort. Sci. 5, 7 – 10.

Paull, R.E., Reyes, M.E.Q., Reyes, M.U., 1995. Litchi and rambutan insect disinfection: treatments to minimize in-duced pericarp browning. Postharvest Biol. Technol. 6, 139 – 148.

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fruits by hot benomyl and plastic film. Sci. Hort. 16, 253 – 262.

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decolorisa-tion and its role in lychee pericarp browning. Aust. J. Exp. Agric. 34, 115 – 122.

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Table 1

Fruit cracking as a function of the interval following harvest or between hot water brushing (HWB) and acid dipping Time after harvest Time from HWB to Cracked fruits

HCl (min) (%)

(h)

Obser6ation I

6 3 18

6 8 9.5

6 11 2.5

9 20 0

Obser6ation II

3 4.5

3.2

3 20

0 5

0 15

Obser6ation III

4a ₀ ₁₂

4 2.3

5.2 0

7 5 3

Obser6ation IV–VIc

\1 Various

Various

a_{Sodium chloride (1%) was included in the acid solution for} the 4 h treatments.

b_{Sodium chloride (1%) was included in the hot water and in} the acid solution for the 7 h treatments.

c_{In the three later experiments of the season, less then 1%} fruit cracking was observed in the various postharvest treat-ment regimes.

the rind and did not account for the general impression, which was either in favor of HWB or at a similar score (not shown).

Table 2

Prochloraz levels in litchi fruits Treatment Prochloraz (mg/kg)a

Flesh

Pericarp Wholeb

Not detected

Control Not detected

0.99 HWB 6.6690.63 a 0.0590.0 c

0.0390.02 c

4.4990.24 b 0.67

SO2

a_{Four replicates were used, and means and standard errors} (SE) are given. Duncan’s multiple range test was preformed. Different letters denote significant differences (P=0.95).

b_{Calculated value for whole fruits assuming 15% of fresh} weight for pericarp (Holcroft and Mitcham, 1996).

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Table 3

Peel conductivity after postharvest treatments Assay time after

Fruit treatment % Conductivity of

totala treatment

Experiment I

Control 2 h 16.090.5 b

89.291.1 a 2 h

SO2treatment

HWBb _{2 h} _16.1₉_{0.5 b}

16.190.4 b 2 h

HWDc

Experiment II

13.290.9 B 10 min

Control

HWB 10 min 16.390.5 A

17.590.7 A 40 min

HWB

b_{HWB, hot water brushing.} c_{HWD, hot water dipping.}

for HWB fruits (Table 2).

3.3. Hot water brushing and ion leakage from

peel disks

SO2 is known to increase ion leakage through

cell membranes and hence to facilitate accumula-tion of HCl in the cells, keeping the pigments red. To investigate whether HWB had a similar effect to SO2, peel disks were used to measure ion

leakage. The peel disks from SO2-treated fruits

3.4. The effect of hot water brushing on PPO

acti6ity

As expected, polyphenol oxidase (PPO) assays (Table 4) demonstrated that SO2 treatment

commercially treated with SO2 in the packing

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3.5. The pH of the pericarp

that of HWB-treated fruits. The difference be-tween the pH of the pericarp after HWB and HWD was smaller and its significance is not obvious.

Table 4

Polyphenol oxidase activity after fruit storage under different regimes

PPO activity (DOD410/g fw/min×1000)a

5 7

Time after harvest (days) 21 215b

20 1.5

Storage temperature (°C) 1.5 1.520

−2.390.3 c

HWBc_{+HCl, 6 h} _0.6₉_{0.7 b} _1.5₉_{1.0 c} _1.0₉_{0.0 b} 2.392.0 bc 1.891.6 b

HWB+HCl, 9 h 8.092.0 bc 6.594.5 b

HWB−HCl, 7 h 56.095.0 a 41.292.2 a 58.096.2 a 32.594.5 a

HWDd_{+HCl, 7 h} _8.5₉_{1.0 b} _3.6₉_{1.8 b} _15.0₉_{0.5 b} _2.7₉_{2.7 b} 0.590.6 c −0.690.3 b

SO2+HCl 0.590.5 c −0.592.5 b

b_{Fruits were stored for 3 weeks at 1.5°C and 5 days at 20°C.} c_{HWB, hot water brushing followed by dipping the fruits in HCl.} d_{HWD, hot water dipping followed by dipping the fruits in HCl.}

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remove dirt, dust and fungal spores from the fruit skin as well as to seal microscopic cracks, thus maintaining better keeping quality of the fruit (Fallik et al., 1999).

Our study suggests that the combination of hot water and mechanical brushing, followed by dip-ping the fruits in hydrochloric acid and prochlo-raz solution, may offer a viable alternative to SO2

Without getting into the debate on the role of PPO in anthocyanin breakdown and peel brown-ing, it is possible to correlate reduced pericarp pH

4. Discussion

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once the peel is bruised, hydrochloric acid can penetrate the undamaged membranes whereas af-ter exposure to SO2the damage to the membranes

allows much greater solute leakage. In addition HWB may remove the dust and eliminate the formation of air bubbles that are trapped between

the fruit warts, allowing increased permeability of hydrochloric acid and prochloraz into the peel in the subsequent dipping stage.

Acknowledgements

References

Akamine, E.K., 1960. Preventing the darkening of fresh ly-chees prepared for export. Tech. Rep. 127, Hawaii Agricul-tural Experimental Station, University of Hawaii, pp. 3 – 17.

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Akerman, M., 1993. The physiological basis of litchi fruit color retention. Acta Hort. 343, 29 – 33.

Holcroft, D.M., Mitcham, E.J., 1996. Postharvest physiology and handling of litchi (Litchi chinensisSonn.). Postharvest Biol. Technol. 9, 265 – 281.

Jiang, Y.-M., Zauberman, G., Fuchs, Y., 1997. Partial purifi-cation and some properties of polyphenol oxidase ex-tracted from litchi fruit pericarp. Postharvest Biol. Technol. 10, 221 – 228.

Kaiser, C., Levine, J., Wolstenholme, B.N., 1995. Vapour, heat and low pH dips improve litchi (Litchi chinensis

Sonn.) pericarp colour retention. J. S. Afr. Soc. Hort. Sci. 5, 7 – 10.