International Journal of Food Science and Technology 2006, 41, 513–519 513

Original article
Effectiveness of ozone for inactivation of Escherichia coli
and Bacillus cereus in pistachios

Meltem Yesilcimen Akbas1 & Murat Ozdemir2*

1 Department of Biology, Gebze Institute of Technology, PO Box 141, 41400 Gebze, Kocaeli, Turkey
2 Department of Chemical Engineering, Section of Food Technology, Gebze Institute of Technology, PO Box 141,
41400 Gebze, Kocaeli, Turkey
(Received 22 December 2004; Accepted in revised form 15 July 2005)

Summary The effectiveness of ozone for the decontamination of Escherichia coli and Bacillus cereus
in kernels, shelled and ground pistachios was investigated. Pistachios were inoculated with
known concentrations of E. coli and B. cereus. Pistachio samples were exposed to gaseous
ozone in a chamber at three different concentrations (0.1, 0.5 and 1.0 ppm) for various
times (0–360 min) at 20
C and 70% relative humidity. The effectiveness of ozone against
E. coli and B. cereus increased with increasing exposure time and ozone concentration. The
physico-chemical properties including: pH, free fatty acids and peroxide values, colour and
fatty acid composition of pistachios did not change significantly after the ozonation
treatments, except for the peroxide value of ground pistachios ozonized at 1.0 ppm for
360 min. Ozone concentration of 1.0 ppm was effective in reducing E. coli and B. cereus
counts in kernels and shelled pistachios, while ozone concentrations <1.0 ppm were found
to be appropriate in reducing the number of both bacteria in ground pistachios without
having any change in their physico-chemical properties.
Keywords Bacillus cereus, Escherichia coli, microbial decontamination, ozone, pistachio.

bacterium, found in intestines of animals and

Introduction
Pistachio nuts are Pistachia vera L. fruit seeds.
Kernels, shelled and ground pistachios are mar-
keted extensively all over the world. Imported
pistachios originate in Turkey, Iran and USA
(FAO, 1999). Raw pistachios are utilized for fresh
consumption or processed for use in desserts,
candies and ice creams.
Nuts are subjected to contamination with
pathogenic micro-organisms at various stages.
The traditional methods of tempering, cracking,
shelling and handling of nuts usually result in
heavy contamination. Escherichia coli and Bacil-
lus cereus, that cause food born illnesses, are the
major bacteria responsible for the contamin-
ation. Escherichia coli is a common Gram ())
*Correspondent: Fax: +90 262 653 8490;
e-mail: ozdemirm@gyte.edu.tr
doi:10.1111/j.1365-2621.2005.01099.x
 2006 Institute of Food Science and Technology Trust Fund
humans, and associated with sewage contamin-
ation of foods, water or poor personal hygiene.
Bacillus cereus is a Gram (+) spore-forming
organism, found widely in soil, and raw, dried
and processed foods. In the spore form, the
bacteria do not cause any illness, but the
bacteria come out of its spore state in proper
conditions and produce toxins which can cause
food poisoning. It was shown that some E. coli
on walnut hulls remains through the tempering
process and subsequently contaminates the nuts
(Meyer & Vaughn, 1969). Chipley & Heaton
(1971) found that shelled pecan samples could be
contaminated with E. coli during harvesting.
Studies with processed and non-processed Bra-
zilian cashews revealed contamination with
Bacillus species (Freire & Offord, 2002). In some
countries, the traditional methods used for the
pistachios, such as harvesting by hand, drying
514 Ozone treatment of pistachios M. Y. Akbas and M. Ozdemir
on the ground, soaking in water containing
sewage to soften the shells, cracking of shells by
hand and poor storage conditions (damp envir-
onments and high storage temperatures) cause
the possible bacterial contamination of the
pistachios with E. coli and B. cereus. This
contamination may pose a significant health
hazard for the consumers.
In 1997, an US expert panel affirmed ozone as
a GRAS (generally recognized as a safe)
substance (Graham, 1997). This affirmation
triggered broad usage of ozone gas in the food
industry. Ozone has been used mainly for
washing and storage of fruits and vegetables
(Hampson et al., 1996; Liangji, 1999). Recent
investigations involving the use of ozone for
dried foods have shown that ozone is a powerful
antimicrobial agent. It was shown that gaseous
ozone eliminated Bacillus spp. and Micrococcus
counts in cereal grains, peas, beans and whole
spices by up to 3 log units, depending on ozone
concentration, temperature and relative humidity
(Naitoh et al., 1987, 1988). Zhao & Cranston
(1995) used gaseous ozone as a disinfectant in
reducing microbial populations in ground black
pepper and they observed 3–6 log number
reductions in ground black pepper ozonized at
6.7 ppm for 6 h, depending on the moisture
content. However, there is no information
available for the use of gaseous ozone as a
disinfectant on pistachio nuts. Therefore, the
objectives of this study were to: (i) determine the
effect of different ozone concentrations and
exposure times for reducing pathogenic E. coli
and B. cereus in pistachios, and (ii) evaluate the
changes in physico-chemical characteristics of
pistachios exposed to different ozone treatments.
Materials and methods
Materials and ozonation system
Raw kernels, shelled and ground pistachios
(Pistachio vera L.) grown in Gaziantep, South
East region of Turkey, were obtained from a
local company (Callı Co., Istanbul, Turkey).
Ozonation was performed in a gas-tight plexi-
glass chamber (30 · 30 · 50 cm) placed in a
controlled temperature room kept at
20 ± 0.5
C. The relative humidity inside the
International Journal of Food Science and Technology 2006, 41, 513–519
chamber was maintained at 70 ± 3% RH with
saturated KCL solution as measured by a digital
humidity measurement device (Kimo, model HD
50, Bordeaux, France). The chamber was con-
nected to an ozone generator (MKD, model
Trio-10, Istanbul, Turkey) in which ozone was
produced from the air. The generator has a
capacity of producing 10 mg h)1 ozone, requi-
ring 5 W power at 220 V. Ozone concentration
was controlled by an Eco Sensor (model OS-3,
Santa Fe, NM, USA).
Microbial inoculation
Before ozonation, raw pistachios were microbi-
ologically analysed using culturing techniques on
selective media to establish whether they were
contaminated with E. coli and B. cereus. Pista-
chios were found to be free of E. coli and B.
cereus. Therefore, cultures of E. coli ATCC 8739
and B. cereus ATCC 6633 strains were suspen-
ded with sterile peptone water (0.1% w/v)
solution and pistachio samples (100 g) were
inoculated with the microbial cell suspensions
in sterile bags at a level of approximately 107
bacteria g)1. Pistachios were then mixed thor-
oughly and allowed to dry for 1 h at room
temperature to facilitate adherence of the bac-
teria to the pistachios. Contaminated samples
(100 g) were weighed out, divided into four Petri
dishes and placed into the ozonation chamber
after the desired ozone concentrations were
reached in the chamber.
Microbiological analysis
Raw, ozonized and non-ozonized samples (25 g)
were diluted with sterile peptone water (225 mL)
in stomacher bags (Seward, London, UK) and
homogenized with a stomacher (Tekmar Co.,
Cincinnati, OH, USA) for 2 min. Further deci-
mal dilutions were prepared in peptone water
solution and plated out on an appropriate
medium. Escherichia coli was enumerated by
the pour plate technique using TBX (Triptone
Bile X-glucuronide, Merck, Germany) agar after
the plates were incubated at 44
C for 24 h.
Bacillus cereus was enumerated by the surface
spread technique using MYP (Mannitol Egg
Yolk Polymixin, Merck, Germany) agar after
 2006 Institute of Food Science and Technology Trust Fund

the plates were incubated at 30
C for 24 h. The
plates having 30–300 cfu (colony forming
units) were counted. Results were expressed as
log cfu g)1 dry weight.
Physico-chemical analysis
Shells of pistachio samples were removed prior to
analysis. Kernels were ground in a blender and
crude lipids were extracted from the ground samples
with petroleum ether at 110
C in a Soxtec system
(Soxtec system HT 1043, Tecator, Hoganas, Swe-
den). Fatty acid esters were determined by the
esterification of purified lipids using BF3 – methanol
following hydrolysis by 0.5 N sodium hydroxide
(IUPAC, 1979) and analysed with a gas chroma-
tography (Thermoquest Trace GC, model 2000
Series, Milan, Italy) equipped with a flame ioniza-
tion detector and a fused silicagel capillary column
(30 m · 25 mm i.d., 0.2 lm thickness; Supelco,
Bellefonte, PA, USA) coated with SP-2330. The
oven temperature was programmed from 120
C
for 2 min initial hold and then 5
C min)1 increase
to a final temperature of 220
C. Injector and
detector temperatures were maintained at 240
C
and 250
C, respectively. Flow rate of helium,
carrier gas, was 0.5 mL min)1 and split ratio was
1:150. The rancidity of the samples was determined
by measuring free fatty acids (FFA, calculated as
per cent oleic acid) and peroxide values (meq kg)1)
according to the methods described in AOCS
(1986a,b).
pH values of the pistachio samples were
measured with a pH meter (Methrom, model
637, Bern, Switzerland) according to AOAC
(1985). The change in colour of the samples
was measured with a Minolta chromometer
(Minolta, model CR310, Osaka, Japan) where
L ¼ 0 to 100 indicates black to white, )a to +a
shows green to red, and )b to +b shows blue to
yellow.
Statistical analysis
All experiments were replicated three times. One-
way analysis of variance (anova) (Minitab Inc.,
2000) was performed to analyse physico-chemical
properties. Significant change (P < 0.05) between
treated and non-treated samples was determined
with Tukey’s HSD range test.
 2006 Institute of Food Science and Technology Trust Fund
Ozone treatment of pistachios M. Y. Akbas and M. Ozdemir 515
Results and discussion
Microbiological quality
Because pistachios were free of E. coli and
B. cereus, they were contaminated with E. coli
and B. cereus prior to ozonation experiments.
Contaminated pistachio samples were treated with
different ozone concentrations (0.1, 0.5 and
1.0 ppm) for 360 min (Fig. 1). At low ozone
concentrations (0.1 and 0.5 ppm), E. coli counts
were decreased by 2–3 log numbers in kernels and
shelled pistachios, respectively, whereas B. cereus
counts were reduced by 1.5–2 log numbers. How-
ever, populations of both bacteria were decreased
by 1.5 log in ground pistachios after being treated
with 0.1 and 0.5 ppm ozone for 360 min. At
1.0 ppm ozone concentration, E. coli counts in
kernels and shelled pistachios were reduced by
3.5 log numbers, while B. cereus counts were
reduced about 3 log numbers. Escherichia coli
and B. cereus counts were decreased about 2 log
numbers in ground pistachios after they were
treated with 1.0 ppm ozone for 360 min. This
showed that surface area of the pistachio samples
was a critical factor for the effectiveness of the
ozone treatment. The inoculated bacteria
remained protective in ground pistachios from
the oxidant effect of ozone, possibly because of
reduced penetration and interaction of ozone with
organic components such as lipids and carbohy-
drates in pistachios. Our results were consistent
with the findings reported by Naitoh et al. (1989)
and Zagon et al. (1992) who treated cereal flour
and ground pepper with gaseous ozone, respect-
ively. They found that surface area was an
important factor during the ozonation of cereal
flour and ground pepper because they required a
higher concentration of ozone and longer treat-
ment time than the whole cereal and pepper to
achieve the same degree of microbial deconta-
mination.
As far as the inactivation of E. coli and B. cereus
is concerned, both bacteria showed similar reduc-
tions at 0.1 and 0.5 ppm ozone concentrations. At
1.0 ppm ozone concentration, on the contrary,
inactivation of both bacteria was different. A
substantial reduction (more than 2.5 log) in pop-
ulation of B. cereus (except for ground pistachios)
occurred mostly in the initial 60 min of ozone
International Journal of Food Science and Technology 2006, 41, 513–519
516 Ozone treatment of pistachios M. Y. Akbas and M. Ozdemir

8
E. coli (0.1 ppm)
8 B. cereus (0.1 ppm)
7
7
6
6
Log (CFU g–1)

Log (CFU g–1)

5
5
4
4
3 3
2 2
1 1

0 0
0 60 120 180 240 300 360 0 60 120 180 240 300 360
Time (min) Time (min)

8 8
E. coli (0.5 ppm) B. cereus (0.5 ppm)
7 7

6 6
Log (CFU g–1)

Log (CFU g–1)

5 5

4 4

3 3

2 2

1 1

0 0
0 60 120 180 240 300 360 0 60 120 180 240 300 360
Time (min) Time (min)

8 8
E. coli (1.0 ppm) B. cereus (1.0 ppm)
7 7

6 6
Log (CFU g–1)

Log (CFU g–1)

5 5

4 4

3 3

2 2

1 1

0 0
0 60 120 180 240 300 360 0 60 120 180 240 300 360
Time (min) Time (min)

Figure 1 Comparative effects of ozone concentrations on microbial inactivation of Gaziantep Pistachios (–¤–, kernel; ÆÆÆ ÆÆÆ,
shelled; and - - - -, ground).

International Journal of Food Science and Technology 2006, 41, 513–519  2006 Institute of Food Science and Technology Trust Fund
 Ozone treatment of pistachios M. Y. Akbas and M. Ozdemir 517

treatment, while the same reduction in E. coli was
attained in 240 min when the pistachios were
treated with 1.0 ppm ozone concentration.
Physico-chemical quality
The effects of different ozone treatments (0.1 and
1.0 ppm for 360 min) on pH, FFA, peroxide and
colour values (L, a and b) of kernels, shelled and
ground pistachios are shown in Table 1. No
significant changes (P < 0.05) in pH values of
kernels, shelled and ground pistachios were ob-
served after they were treated with 0.1 and 1.0 ppm
ozone for 360 min. The FFA values, which give a
measure of hydrolytic rancidity involving the
enzymatic hydrolysis of fats into free fatty acids
and glycerol, did not change significantly
(P < 0.05) for kernels, shelled and ground pista-
chios. The peroxide value, a measure of the extent
of oxidative absorption and entrainment of oxygen
in fats or oils, did not change significantly
(P < 0.05) for kernels and shelled pistachios.
However, this value was significantly (P < 0.05)
higher for the ground pistachios treated with
1.0 ppm ozone for 360 min than that of the control
samples, possibly because of the destruction of
fatty acids in ground pistachios. No colour change
(P < 0.05) was observed in kernels, shelled and
ground pistachios after they were treated with 0.1
and 1.0 ppm ozone for 360 min.
Table 2 shows the comparative effects of differ-
ent ozone treatments on the fatty acid composi-
tions of pistachios. Five major fatty acids were
determined in pistachios. No significant change

Table 1 Comparative effects of different ozone treatments (0.1 and 1.0 ppm ozone for 360 min) on some of physico-chemical
qualities of Gaziantep pistachios* (kernel, shelled and ground)

Kernel Shelled Ground

Parameter† Control 0.1 ppm 1.0 ppm Control 0.1 ppm 1.0 ppm Control 0.1 ppm 1.0 ppm

pH 6.4a 6.4a 6.4a 6.4a 6.4a 6.4a 6.4a 6.4a 6.4a

FFA value‡ 0.9a 0.9a 1.2a 0.7a 0.7a 0.7a 3.5a 3.6a 3.7a
Peroxide value‡ 2.8a 2.9a 3.9a 2.7a 2.6a 2.7a 2.1a 2.6a 3.7b
L 47.8a 48.2a 49.1a 48.8a 48.7a 48.8a 41.8a 42.1a 42.2a
a 1.1a 1.3a 1.5a 1.6a 1.5a 1.5a )11.1a )10.8a )10.7a
b 15.3a 15.3a 15.5a 16.7a 16.8a 16.8a 35.1a 35.2a 35.4a

*Values with the same superscript letter within each row are not significantly different at P < 0.05.
†Each value is the average of triplicate samples.
‡FFA and peroxide values are expressed as titration volume % oleic acid and meq kg)1, respectively.

Table 2 Comparative effects of different ozone treatments (0.1 and 1.0 ppm ozone for 360 min) on fatty acid compositions of
Gaziantep pistachios* (kernel, shelled and ground)

Kernel Shelled Ground

Parameter† (%) Control 0.1 ppm 1.0 ppm Control 0.1 ppm 1.0 ppm Control 0.1 ppm 1.0 ppm

Crude lipid 46.60a 46.60a 46.60a 46.60a 46.60a 46.60a 48.20a 48.20a 48.20a
Palmitic, C16:0 10.04a 10.10a 10.19a 10.05a 10.05a 10.05a 9.73a 10.19a 10.03a
Palmiteloic, C16:1 0.72a 0.71a 0.72a 0.72a 0.72a 0.72a 0.66a 0.6 0.69a
Stearic, C18:0 2.72a 2.79a 2.70a 2.74a 2.74a 2.74a 2.89a 2.86a 2.86a
Oleic, C18:1 70.99a 71.01a 70.82a 71.24a 71.21a 71.27a 70.22a 69.95a 69.86a
Linoleic, C18:2 13.94a 13.48a 13.88a 13.95a 13.98a 13.98a 14.71a 14.82a 14.77a
SFA (16:0 + 18:0) 12.76a 12.80a 12.90a 12.79a 12.79a 12.79a 12.62a 12.79a 12.89a
USFA (16:1 + 18:1 + 18:2) 85.55a 85.08a 84.98a 85.91a 85.91a 85.97a 85.68a 85.44a 85.32a

*Values with the same superscript letter within each row are not significantly different at P < 0.05.
†Each value is the average of triplicate samples.
SFA, total saturated fatty acids; USFA, unsaturated fatty acids.

 2006 Institute of Food Science and Technology Trust Fund International Journal of Food Science and Technology 2006, 41, 513–519
518 Ozone treatment of pistachios M. Y. Akbas and M. Ozdemir
(P < 0.05) was observed between fatty acid
composition of ozonized and non-ozonized sam-
ples because low ozone concentrations (£1.0 ppm)
were used in ozone treatments. Our results agree
with those of Naitoh et al. (1987) who found that
the oxidation of lipids in dry foods rarely occurred
below 5 ppm ozone concentrations. However, aloe
powder, bee pollen and red ginseng powder
treated with a high ozone concentration
(18 ppm) for 8 h showed significant changes in
their fatty acid composition because of the
destruction of double-bonds in fatty acids (Byun
et al., 1997, 1998; Yook et al., 1997). Similarly,
Zhao & Cranston (1995) observed significant
changes in essential volatile oil constituents of
ground black pepper after being treated with
ozone at 6.7 ppm for up to 6 h.
Conclusions
This study has shown that ozonation was an
effective treatment for reducing E. coli and
B. cereus in kernels, shelled and ground pistachios.
Substantial reductions in E. coli and B. cereus
numbers in kernels, shelled and ground pistachios
were observed at the end of 360 min ozonation.
Ozone treatments did not change the pH, FFA
and peroxide values, colour and fatty acid com-
positions of pistachios significantly (P < 0.05),
except for the peroxide value of ground pistachios
treated with ozone at 1.0 ppm. Ozone concentra-
tions <1.0 ppm for ground pistachios and ozone
concentrations of 1.0 ppm for kernels and shelled
pistachios can be successfully used to reduce initial
counts of E. coli and B. cereus without affecting
any physico-chemical properties of pistachios. The
results from this study are believed to have
significant impact not only for the pistachio
processors, but also for the other nut processors.
It can be concluded that ozone treatment was a
promising alternative to the use of chemical
fumigants such as methyl bromide, ethylene oxide
etc. and gamma irradiation for the decontamina-
tion of kernels, shelled and ground pistachios.
Acknowledgments
We would like to present our thanks to Mr Ismail
Calli, the President of Calli Company, Istanbul,
Turkey, for providing kernels, shelled and ground
International Journal of Food Science and Technology 2006, 41, 513–519
pistachios. Financial support by the Gebze Insti-
tute of Technology (Project number: 2003-B-3)
and the Government Planning Organisation of
Turkey (DPT) is greatly acknowledged.
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