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 DOD
410
g fwmin × 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 SO
2
-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
SO
2
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 SO
2
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. SO
2
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 SO
2
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 SO
2
and 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.
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 18
3 6
8 9.5
6 2.5
11 9
20 Obser6ation II
4 3
4.5 3.2
3 20
5 6
15 6
Obser6ation III 4
a
12 5
4 2.3
5.2 7
b
7 5
3 Obser6ation IV–VI
c
\ 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 mgkg
a
Flesh Pericarp
Whole
b
Not detected Control
Not detected 0.99
HWB 6.66 9 0.63 a
0.05 9 0.0 c 0.03 9 0.02 c
4.49 9 0.24 b 0.67
SO
2 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.
Table 3 Peel conductivity after postharvest treatments
Assay time after Fruit treatment
Conductivity of total
a
treatment Experiment I
Control 16.0 9 0.5 b
2 h 89.2 9 1.1 a
2 h SO
2
treatment HWB
b
2 h 16.1 9 0.5 b
16.1 9 0.4 b 2 h
HWD
c
Experiment II 13.2 9 0.9 B
10 min Control
HWB 16.3 9 0.5 A
10 min 17.5 9 0.7 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 mgkg 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 SO
2
-treated fruits but marginal for HWB fruits Table 2.
3
.
3
. Hot water brushing and ion leakage from peel disks
SO
2
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 SO
2
, peel disks were used to measure ion leakage. The peel disks from SO
2
-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 SO
2
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 31.5 9 0.3 before and 25.5 9 3.6 after HWB whereas 9 h after har-
vest the activity was 38.7 9 1.3 before and 32.5 9 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 of Penicillium spp. 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 SO
2
treatment. Fruits treated by HWB or commercially treated with SO
2
in the packing house, were dipped in acid prochloraz solution
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 SO
2
-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 HWBacid-
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 SO
2
-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
SO
2
-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 DOD
410
g fwmin×1000
a
5 7
Time after harvest days 21
21 5
b
20 1.5
Storage temperature °C 1.5
1.5 20 −
2.3 9 0.3 c HWB
c
+ HCl, 6 h
1.0 9 0.0 b 1.5 9 1.0 c
0.6 9 0.7 b 2.3 9 2.0 bc
1.8 9 1.6 b HWB+HCl, 9 h
8.0 9 2.0 bc 6.5 9 4.5 b
HWB−HCl, 7 h 56.0 9 5.0 a
41.2 9 2.2 a 58.0 9 6.2 a
32.5 9 4.5 a HWD
d
+ HCl, 7 h
8.5 9 1.0 b 3.6 9 1.8 b
15.0 9 0.5 b 2.7 9 2.7 b
0.5 9 0.6 c −
0.6 9 0.3 b SO
2
+ HCl
0.5 9 0.5 c −
0.5 9 2.5 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.
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 SO
2
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
