Effect of low level ozone-enrichment on the quality and ... - chimia

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<strong>Effect</strong> <strong>of</strong> <strong>low</strong> <strong>level</strong> <strong>ozone</strong>-<strong>enrichment</strong> on the quality and condition
<strong>of</strong> citrus fruit under semi-commercial conditions
Metzger, C. 1 , Barnes, J.D. 1 , Singleton, I. 1 , Andrews, P. 2
1. Institute for Research on the Environment and Sustainability, Devonshire Building, Newcastle
University, Newcastle upon Tyne, NE1 7RU, England
2. MMG Global Citrus, Transfessa Road, Paddock Wood, Kent, TN12 6UT, England.
Abstract
This study examined the effect <strong>of</strong> simulated commercial storage in a <strong>low</strong> <strong>level</strong> (180-200 ppb)
<strong>ozone</strong>-enriched atmosphere on the <strong>level</strong> <strong>of</strong> waste resulting from Penicillium infection <strong>of</strong> citrus
products. Experiments conducted on waxed and unwaxed fruit <strong>of</strong> three varieties: “Hernandina”
clementines, “Navel” oranges and “Ellendale” mandarins revealed that after 35 days <strong>of</strong> storage in
an <strong>ozone</strong>-enriched atmosphere at 5°C the percentage <strong>of</strong> rotten fruit was significantly reduced (by
7%) . Subsequent experiments focusing on fungicide-treated versus wax and pesticide-free “Navel
oranges” again revealed <strong>ozone</strong>-treatment to yield a positive effect; treated fruit exhibiting
significantly less (27%) spoilage than equivalent controls (non-treated produce) after 35 days <strong>of</strong>
storage at 5°C;There were no commercially-relevant effects <strong>of</strong> <strong>low</strong>-<strong>level</strong> <strong>ozone</strong>-<strong>enrichment</strong> on fruit
quality parameters (juice percentage, soluble solids content and sugar/acid ratio). The results
clearly demonstrate that <strong>low</strong> <strong>level</strong> atmospheric <strong>ozone</strong>-<strong>enrichment</strong> can reduce the <strong>level</strong> <strong>of</strong> waste in
citrus stored in a semi-commercial environment.
Key-words: Ozone, postharvest, Penicillium, oranges, clementines, mandarins, citrus
Introduction
Ozone (the triatomic form <strong>of</strong> oxygen O 3 ) is one <strong>of</strong> the most powerful oxidants in nature (1) . It is
formed when a high-energy input splits molecular oxygen (O 2 ), into singlet oxygen (O), which then
combines with O 2 to form <strong>ozone</strong>. It is naturally produced by ultraviolet irradiation from the sun and
also during lightning discharges in storms (2) . It can be produced artificially, but cannot be stored
and needs to be produced continuously. The corona discharge method is most commonly used to
produce <strong>ozone</strong>, this operates by disassociating atoms <strong>of</strong> O 2 (using a high voltage electric current)
and subsequently producing <strong>ozone</strong> (3)
In its gaseous form, <strong>ozone</strong> has several characteristics that make it attractive for its use in the fresh
fruit industry: for example, <strong>ozone</strong> retards the production <strong>of</strong> fungal spores (4), (5), (6) controls odours (1) ,
reduces the <strong>level</strong>s <strong>of</strong> ethylene in storage conditions (7), (8) , improves firmness in some fruits (9), (10) ,
induces resistance to postharvest decay development, and rises the <strong>level</strong>s <strong>of</strong> antioxidants in the
fruit (11) . Most importantly <strong>ozone</strong> use does not leave residues on the fruit (12) which gives it a
significant advantage over conventionally applied pesticides.
The effectiveness <strong>of</strong> gaseous <strong>ozone</strong> to control infections by P. italicum and P. digitatum in citrus
fruit has been tested on several occasions. Ozone has the potential to replace or reduce traditional
fungicide treatments used on citrus fruit (such as imazalil), that leave residues on the fruit. One <strong>of</strong>
the main concerns is that the widespread use <strong>of</strong> fungicides will result the proliferation <strong>of</strong> resistant
fungal pathogens (13) . Indeed resistance to several fungicides including thiabendazole and imazalil
(14), (15) has been observed in fungal populations. The ability <strong>of</strong> <strong>ozone</strong> to inhibit sporulation could
avoid the generation <strong>of</strong> new resistant pathogens.
In this paper, we describe the effects <strong>of</strong> <strong>ozone</strong> storage on the quality and condition <strong>of</strong> citrus fruit in
a semi-commercial environment. We also looked at the potential <strong>of</strong> <strong>ozone</strong> to reduce pesticide
residues <strong>level</strong>s on fruit surfaces.
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Material and methods
Overview: The experiments were designed to determine the effect <strong>of</strong> <strong>ozone</strong> storage to reduce
spoilage in different citrus fruit types and varieties (Experiment 1 : waxed and unwaxed) and to
make an initial assessment <strong>of</strong> the potential for <strong>ozone</strong> to reduce fungal spoilage in fruit not
subjected to fungicide treatment (Experiment 2).
Experiment 1: 3 varieties <strong>of</strong> different types <strong>of</strong> citrus were used: Navel oranges, Hernandina
clementines and Ellendale mandarins. 2 boxes <strong>of</strong> each variety (1 waxed and 1 unwaxed) were
used for both the treatment (<strong>ozone</strong> exposed) and the control (no <strong>ozone</strong>). Each box contained
approximately 15 kg <strong>of</strong> loose fruit. All fruit was treated with imazalil and thiabendazole.
Experiment 2: oranges cv. Navel Late without any postharvest treatments were used, one box with
9 nets <strong>of</strong> 1.5kg each, and one box with 13 nets with 5 oranges each (for both treatment and
control).
In both experiments, the fruit was not artificially inoculated with Penicillium and we worked with the
natural <strong>level</strong>s <strong>of</strong> Penicillium infection found in most commercial packhouses.
Ozone generating and monitoring equipment
A corona discharge <strong>ozone</strong> generator (Bi<strong>of</strong>resh Ltd.) was connected to an UV monitor (Eco
Sensors, INC. Model UV-100) set to maintain a concentration <strong>of</strong> 180-200 ppb <strong>of</strong> <strong>ozone</strong>.
Storage conditions
Both treatment and control were stored at 5°C and 80-90% RH. Both experiments were conducted
for 35 days.
Variables measured
Rotten fruit count
At the end <strong>of</strong> the first experiment, the number <strong>of</strong> healthy and rotten fruit (showing evident
Penicillium infection) was counted in each box <strong>of</strong> fruit. For the second experiment, the number <strong>of</strong>
packs showing infected fruit was counted instead, fol<strong>low</strong>ing industry standards.
Juice analysis (experiment 1 only):
A sample <strong>of</strong> 2-4 fruits, chosen randomly, was taken at the end <strong>of</strong> the experiments. The fruits were
juiced and the percentage <strong>of</strong> juice was calculated, also a digital refractometer (Bellingham &
Stanley Ltd. model DR 103) was used to measure the <strong>level</strong> <strong>of</strong> soluble solids. An automatic titration
machine (Metrohm model 719 S Titrino) was used to measure the titratable acidity <strong>of</strong> the juice. The
values obtained were compared against commercial specifications.
Spore counts (experiment 1 only):
At the end <strong>of</strong> the first experiment the 9 most infected fruits (based on the amount <strong>of</strong> visible
mycelium per surface), from each batch (control and treatment) were selected. Three fruits at a
time were used to make a spore suspension (fruit was shaken for 15 mins in 200 ml <strong>of</strong> Tween
solution 0.01 % w/v). The number <strong>of</strong> spores released from the surface <strong>of</strong> fruit was counted using a
microscope and haemocytometer to give number <strong>of</strong> spores per mL <strong>of</strong> Tween solution.
Residue analysis
For experiment 1, one kilogram <strong>of</strong> fruit from both treatment and control (Waxed Navel Oranges)
was sent for pesticide residue analysis using a commercial laboratory.
Statistical Analysis
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A Chi squared test was used to compare the frequencies <strong>of</strong> healthy and rotten fruit. Control and
treatment were considered groups and rotten and healthy conditions. The <strong>level</strong> <strong>of</strong> significance
used to reject the Ho that the grouping did not affect the condition was 5%
Results and discussion
Experiment 1 (Fruit treated with postharvest fungicides and wax)
The net percentage <strong>of</strong> rotten fruit (all types and varieties) under control conditions (12.7%) was
more than double than under <strong>ozone</strong> <strong>enrichment</strong> (6.23%) (Fig.1a) Also, there were less rotten and
more disease free fruit resulting from <strong>ozone</strong> exposure (Fig.1b). This difference was statistically
significant when comparing frequencies <strong>of</strong> rotten and disease free fruits using a CHI squared test.
In general under <strong>ozone</strong>, there was less visible growth <strong>of</strong> the mycelium and less sporulation; this
fact has also been noted by Palou et al (5) .
A. Net percentage <strong>of</strong> rotten fruit
B. Frequency <strong>of</strong> rotten and healthy fruits
OZONE
CONTROL
14.00
700
12.00
600
10.00
500
Percentage
8.00
6.00
Number <strong>of</strong> Fruits
400
300
4.00
200
2.00
100
0.00
0
ROTTEN
DISEASE FREE
Figure 1. Results <strong>of</strong> experiment 1: a) net percentage <strong>of</strong> rotten fruit and b) frequency <strong>of</strong> rotten and disease
free fruit found during storage in <strong>ozone</strong> exposed and control (no <strong>ozone</strong>) fruit.
<strong>Effect</strong> <strong>of</strong> <strong>ozone</strong> storage on the <strong>level</strong> <strong>of</strong> rotten fruit in different citrus varieties
By varieties (Figure 2), the reduction in <strong>level</strong>s <strong>of</strong> rotten fruit caused by <strong>ozone</strong> exposure was
significant in Hernandina (both waxed and unwaxed) and Ellendale (only unwaxed). Ozone
exposure did not result in a significant reduction <strong>of</strong> rotten fruit <strong>level</strong>s in Navel (waxed and unwaxed)
or waxed Ellendale.
Only two varieties (unwaxed Navel oranges and waxed Ellendale mandarins) showed slightly
higher percentages <strong>of</strong> rotten fruit under <strong>ozone</strong>; however these differences were not significant.
The lack <strong>of</strong> significant effect on Navel oranges could have been due to two separate causes. First,
the number <strong>of</strong> oranges in a 15 kg box (40-50 units) is <strong>low</strong>er than a box <strong>of</strong> mandarin or clementines
<strong>of</strong> the same weight (100-120 units), making the size <strong>of</strong> the sample also smaller and more difficult to
draw statistically significant conclusions. In the other hand, the <strong>low</strong>er surface area (in relation to
their volume) <strong>of</strong> the oranges compared to the clementines or mandarins means that the total area
exposed to <strong>ozone</strong> is also <strong>low</strong>er.
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45
40
35
OZONE
CONTROL
% Rotten Fruit
30
25
20
15
10
5
0
Navel w axed Navel unw axed Hernandina
w axed *
Hernandina
unw axed *
Ellendale w axed
Ellendale
unw axed*
Figure 2. Ability <strong>of</strong> <strong>ozone</strong> exposure to reduce the percentage <strong>of</strong> rotten fruit by variety and wax coating, the
asterisk indicates significant difference.
<strong>Effect</strong> <strong>of</strong> wax coating on the frequency <strong>of</strong> rotten fruit showed by different varieties
The effect <strong>of</strong> <strong>ozone</strong> was evident only on Ellendale unwaxed fruit as good control <strong>of</strong> Penicillium was
observed for both waxed and unwaxed Hernandina. As the action <strong>of</strong> <strong>ozone</strong> is only superficial and it
does not kill pathogens developing under the surface <strong>of</strong> the fruit (12) , the lack <strong>of</strong> significant effect <strong>of</strong>
the <strong>ozone</strong> on the waxed mandarins could have been because <strong>of</strong> reduced contact <strong>of</strong> the mycelia
growing underneath the wax coating with the gaseous <strong>ozone</strong> in the air. However the effect is
variable as wax coating did not make a difference for the Hernandina clementines and more work
would be necessary to assess the effect <strong>of</strong> <strong>ozone</strong> on waxed and unwaxed fruit.
Spore counts
Even when the navel oranges did not show a significant difference in <strong>level</strong>s <strong>of</strong> rotten fruit, the
average amount <strong>of</strong> spores (ml) removed from the surface <strong>of</strong> fruit under control conditions was
more than double than that observed under <strong>ozone</strong> (Figure 3). This difference was statistically
significant and consistent with previous studies showing the ability <strong>of</strong> <strong>ozone</strong> to reduce sporulation
(4), (5) . However, this is the first time that this effect has been noticed in citrus at the <strong>low</strong>
concentrations we worked with (180-200 ppb). Higher concentrations can be damaging to humans
if exposed for more than 15 minutes (16) , but are more effective reducing sporulation completely:
Harding (4) used 1.0 ppm and Palou et al (5) used 0.3 ppm to achieve a reduction in sporulation. The
mechanism by which <strong>ozone</strong> can reduce fungal sporulation is not known at present but it would
appear that <strong>ozone</strong> is able to restrict the development <strong>of</strong> the reproductive life cycle <strong>of</strong> fungi. Other
work also indicates <strong>ozone</strong> can restrict sporulation <strong>of</strong> other fungi e.g.Botrytis (17)
The fruits that became rotten under <strong>ozone</strong> exposure could have become infected in the field, at
harvest or during postharvest operations. It can be assumed that the probability <strong>of</strong> both control and
treatment batches to have been infected in the field, harvesting or postharvest operations was the
same (the fruit was part <strong>of</strong> a single batch, harvested the same day, from the same plot, transported
and processed under the same conditions). So the <strong>ozone</strong> related reduction observed in terms <strong>of</strong>
rotten fruit is potentially due to the ability <strong>of</strong> <strong>ozone</strong> to either restrict fungal growth or <strong>low</strong>er the <strong>level</strong>s
<strong>of</strong> spores in the storage atmosphere, hence <strong>low</strong>ering the rate <strong>of</strong> re-infection. Harding (4) , when
using higher (1.0 ppm) concentrations <strong>of</strong> <strong>ozone</strong> reported a suppression <strong>of</strong> sporulation, but he found
that there was no effect <strong>of</strong> <strong>ozone</strong> on the amount <strong>of</strong> rotten fruit obtained.
Studies by other researchers (9) suggest that <strong>ozone</strong> improves the firmness <strong>of</strong> the citrus skin (skin
firmness was not measured in these experiments, but the fruit exposed to <strong>ozone</strong>, especially the
clementines were noticeably firmer to the touch). This effect on the firmness can also explain the
5.11 - 4

<strong>low</strong>er amount <strong>of</strong> rotten fruit under <strong>ozone</strong>. Fruits with stronger skin could be more difficult to infect
by Penicillium than weakened fruits. Other beneficial effects for the fruit include the ability <strong>of</strong> <strong>ozone</strong>
to destroy ethylene (18) . Reducing ethylene to very <strong>low</strong> concentrations during citrus fruit storage
results in less decay and prolongs shelf life (19) . Crisosto et al (7) and Skog and Chu (8) reported the
ability <strong>of</strong> <strong>ozone</strong> to reduce ethylene <strong>level</strong>s in fresh produce storage.
350000
300000
OZONE
CONTROL
250000
Spores/ml
200000
150000
100000
50000
Quality parameters
0
-=
Figure 3. Reduction in spore counts (ml)caused by <strong>ozone</strong> exposure. The error
bar shows the standard error <strong>of</strong> the mean.
The soluble solid content, tritatable acidity and juice percentage <strong>of</strong> the samples taken for each
variety were not different in commercial terms. All varieties in both environments showed <strong>level</strong>s
above the standards for the industry, and were not different between control and treatment. These
results coincide with those <strong>of</strong> Garcia et al (9) which studied the effect <strong>of</strong> <strong>ozone</strong> exposure on quality
parameters on oranges and mandarins.
Residue analysis
Lower <strong>level</strong>s <strong>of</strong> pesticide residues were found on fruit stored with <strong>ozone</strong>, the reduction was bigger
for imazalil and malathion than chlorpyrifos (Figure 4). The <strong>level</strong>s <strong>of</strong> residues in both environments
were well be<strong>low</strong> the MRLs (maximum residue limits). We have repeated this analysis for other
experiments (data not shown) and obtained similar results. These results show that <strong>ozone</strong> could be
useful in <strong>low</strong>ering the residues <strong>of</strong> fungicides on citrus fruit; further research is needed to check if
this affects the Penicillium control <strong>of</strong> imazalil, but the evidence shown in this study suggests that is
not the case. Ozone is known to transform pesticides in aqueous environments (3) , but very little
work has been carried out using gaseous <strong>ozone</strong>
0.8
0.7
OZONE
CONTROL
0.6
0.5
mg/kg
0.4
0.3
0.2
0.1
0
Chlorpyrifos Imazalil Malathion
Figure 4. Residue analysis results for three different pesticides. The fruit used for
the analysis was stored under <strong>ozone</strong> for 35 days. This results are from a
single analysis performed by a commercial laboratory.
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Experiment 2 (Fruit with no postharvest fungicide treatment)
In this experiment, the amount <strong>of</strong> nets containing rotten fruit was always <strong>low</strong>er under <strong>ozone</strong>. If the
1.5kg nets and the 5 fruits nets are taken separately the effect is not significant, but the overall
effect (27 % less rotten nets under <strong>ozone</strong>) is significant (Figure 5).
The results <strong>of</strong> this experiment show that <strong>ozone</strong> on its own can control Penicillium to a certain
extent but that it is probably not as effective as traditional treatments in controlling Penicillium rot
as a relatively high <strong>level</strong> <strong>of</strong> spoiled fruits were obtained in storage under <strong>ozone</strong>. For example, if the
fruit had been imazalil treated we could have expected a much <strong>low</strong>er loss to spoilage. However
this conclusion must be treated carefully as it is possible that the fruit used in this experiment had a
high Penicillium loading and even imazalil treated fruit may have shown high disease <strong>level</strong>s.
Overall though, <strong>ozone</strong> could be a powerful tool in combination with other residue-free postharvest
treatments in the search for alternatives to traditional fungicides.
12
OZONE
CONTROL
Number <strong>of</strong> nets with rotten fruits
10
8
6
4
2
0
1.5 kg nets 5 fruit nets Total *
Figure 5. 2 nd experiment results: Number <strong>of</strong> nets showing rotten fruits. The asterisk
shows a significant difference.
Conclusion
This work shows that <strong>ozone</strong> storage can <strong>low</strong>er natural <strong>level</strong>s <strong>of</strong> rot in a variety <strong>of</strong> citrus types
caused by Penicillium in commercial storage conditions potentially by a combination <strong>of</strong>
mechanisms including: a) <strong>low</strong>ering the amount <strong>of</strong> spores produced in the storage environment, b)
destroying ethylene and c) improving firmness/resistance to disease. There were no effects <strong>of</strong>
<strong>ozone</strong> in the quality <strong>of</strong> citrus fruit expressed in <strong>level</strong>s <strong>of</strong> soluble solids, titratable acidity and juice
percentage. Also <strong>ozone</strong> can reduce the <strong>level</strong> <strong>of</strong> pesticide residues found on fruit. Results indicate
that the best use <strong>of</strong> <strong>ozone</strong> is probably in combination with traditional treatments to improve overall
loss due to fungal spoilage: this could open the door to use <strong>of</strong> <strong>low</strong>er doses <strong>of</strong> pesticides in
conjunction with <strong>ozone</strong> storage or as part <strong>of</strong> other novel combination treatments.
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