Food Science and Technology

ISSN 0101-2061

Inactivation of E. coli and B. subtilis spores in ozonized cassava

starch
Emanuele Oliveira Cerqueira AMORIM1, Alline Artigiani Lima TRIBST1,
Pedro Esteves Duarte AUGUSTO1,2, Marcelo CRISTIANINI1*
Abstract
In the present study, the efficacy of ozone inactivation of B. subtilis spores and E. coli in cassava starch was evaluated.
Cassava starch with 18 and 30% moisture content was processed with ozone at concentrations of 40-118 ppm and
exposure times of
15-120 minutes. The processing at 113 ppm/120 minutes (maximum exposure level to ozone evaluated) at 18% of
moisture content did not cause significant reduction of B. subtilis spores and caused the reduction of only 2 decimal of E.
coli. On the other hand, when the ozonation process was carried out for 120 minutes at 30% of moisture content, 3.6
decimal reduction of B. subtilis was achieved at 40 ppm of ozone and total B. subtilis load reduction (>5 log cycles) was
observed at 118 ppm of ozone. Similarly, total E. coli load reduction (>7 log cycles) was achieved at 40 ppm of ozone
exposure for 60 minutes. Therefore, the results indicate that the ozone efficacy against microorganisms in cassava starch was
mainly dependent on the sample moisture content and to ozone concentration and exposure time. Moreover, it was
observed that ozone is a promising technology to reduce microbial counts in dried food.
Keywords:
ozonation;
technologies.

food

processing;

non-thermal

1 Introduction
To d a ys c o n s u m er s h a v e d em a n de d p r o
c e s s e d products very similar to fresh food (TRIBST;
SANTANA; DE MASSAGUER, 2009). For such reason, the
development of non-thermal
methods
for food
processing has been extensively studied e.g. highhydrostatic pressure, irradiation, UV radiation, pulsed
electric field, pulsed light, ultrasound, and high-pressure
homogenization, and ozone (DIELS; WUY TACK;
MICHIELS, 2003; TRIBST;
SANT ANA; DE
MASSAGUER, 2009; KLOCKOW; KEENER, 2009).
It is expected that products processed by such
technologies maintain the nutrients and sensory
characteristics better than thermal process. Among these
technologies, ozone stands out for its antimicrobial
properties and for being totally degradable in oxygen with no
waste/toxic products (TIWARI et al., 2008,
2010); thus it is generally recognized as safe (GRAS) for food
applications (GUZEL-SEYDIM; GREENE; SEYDIM,
2004; DHILLON et al., 2009; TIWARI et al., 2010). An
additional advantage is the lower cost of ozone equipment
(GUZEL- SEYDIM; GREENE; SEYDIM, 2004).
The ozone is a high oxidative compound with a broad
antimicrobial spectrum; it is able to inactivate bacterial
vegetative cells and spores, yeast, molds, and viruses and to
kill insects in stored grains and degrade mycotoxins (TIWARI
et al.,
2010). Ozone inactivates microorganisms by the progressive
oxidation of vital cellular components. The microbial
surface is the primary target of ozonation with the oxidation
of the polyunsaturated fatty acids and consequent loss of
selective permeability and cell disruption. Additionally,
ozone causes
Food Sci. Technol, Campinas, 33(2): 289-294, Apr.-June
2013

the oxidation of sulfhydryl groups and amino acids of

enzymes, peptides, and proteins including nucleic acids and
vital enzymes (KHADRE; YOUSEF; KIM, 2001; GUZELSEYDIM; GREENE; SEYDIM, 2004). In general, Gram
negative bacteria are more sensitive to ozone treatment than
Gram positive ones (KHADRE; YOUSEF; KIM, 2001).
However, several other factors affect the ozone efficacy
including the strain of the microorganism, age of the culture,
density of the treated population, and presence of ozonedemanding medium components and method of applying
ozone (KHADRE; YOUSEF; KIM, 2001)
In spores, ozone degrades the coat layer components
thus exposing the cortex and the core to oxidation by ozone
(KHADRE; YOUSEF, 2001; TIWARI et al., 2010). The
molecular ozone and the reactive by-products of ozone
decomposition
2
3
(free radicals: OH, O and HO ) have antimicrobial activity.
Due to its high efficacy, low concentration and short contact
time are normally enough to achieve the desired microbial
inactivation.
Ozone has been previously used in food industry to
inactivate microorganisms in packages and stainless steel
surfaces (KHADRE; YOUSEF, 2001), for microbial
decontamination of poultry carcasses, on chilled water
treatment and recycling (GUZEL-SEYDIM; GREENE;
SEYDIM, 2004; KHADRE; YOUSEF, 2001), for fish shelflife extension (PASTORIZA et al.,
2008), for fruit juice processing (TIWARI et al., 2009a, b),
for sanitation of fruit and vegetables (ACHEN; YOUSEF,
2001;
BIALKA;
DEMIRCI,
2007;
NAJAFI;
KHODAPARAST, 2009; KIM; YOUSEF; CHISM, 1999;
KOSEKI; ISOBE, 2006), and
33

Received 10/7/2012
Accepted 14/2/2013 (005780)
1
Department of Food Technology DTA , School of Food Engineering FEA, University of Campinas UNICAMP, Rua Monteiro Lobato, 80,
CP 6121, Cidade Universitria Zeferino Vaz, CEP 13083-862, Campinas, SP, Brazil, e-mail: [email protected]
2
Technical School of Campinas COTUCA, University of Campinas UNICAMP, Campinas, SP, Brazil
*Corresponding author
DOI: http://dx.doi.org/10.1590/S0101-20612013005000043

34

Food Sci. Technol, Campinas, 33(2): 289-294, Apr.-June

2013

Amorim et al.

for decontamination of dry products as seeds, grains, and

pepper (TIWARI et al., 2010, AKBAS; OZDEMIR, 2006;
EMER; AKBAS; OZDEMIR, 2008; IBANOGLU, 2001,
2002; KETTERINGHAM et al., 2006; ZHAO; CRANSTON,
1995).
In addition to ozone antimicrobial potential, this gas can
also be used to change physicochemical characteristics of
corn, sago, and tapioca starch (CHAN; BHAT; KARIM,
2009) and wheat flour (LI et al., 2012) by oxidation
improving their functional performance and presenting better
results than those obtained by the traditional oxidation
process (using chlorine).
Cassava starch is an important raw material in the food
industry due its absence of cereal flavor, typically
observed in corn starch, its viscoamylographic standard
during cooking (DEMIATE et al., 2005), its pasta low
temperature, and its syneresis low susceptibility (SILVA et
al., 2003).
Considering that cassava starch is extracted from plant
root, it is potentially contaminated with many
microorganisms, mainly bacterial spores. Therefore, some
processes are required to guarantee the microbial stability
of cassava starch before its use as raw material in the food
industry. Due to low temperature gelation, the thermal
process application for microbial inactivation of cassava
starch is not feasible (CEREDA; VILPOUX, 2005); therefore,
the development of a non-thermal process is necessary.
The Bacillus subtilis is considered an ideal target for
the chemical process of microbial inactivation due to its
safety and high resistance to chemical compounds
(MOREAU; ORANGE; FEUILLOLEY, 2008). In addition
to the analysis of the spore forming microorganisms, which
are potentially starch pathogens or spoilers, the evaluation of
Escherichia coli was carried out considering the the Brazilian
food legislation (BRASIL, 2001), which establishes a
maximum limit of coliforms able to growth at 45 C of 102
CFU/g of starch.
Therefore, based on the ozone properties as a sanitizer
and the requirements of a non-thermal process to stabilize
cassava starch, this study aimed to evaluate the efficacy of
ozone inactivation of B. subtilis spores and E. coli in cassava
starch.

2 Materials and methods

2.1
Starch
microorganisms

and

Cassava starch was supplied by the company Corn

Products Brasil (Conchal, Brazil). It was previously
sterilized using irradiation (10 kGy) performed by the
Brazilian Radiation company (EMBRARAD- Cotia, Brazil)
to inactivate its native microorganisms.
The lyophilized strains of Bacillus subtilis ATCC 6633
and Escherichia coli ATCC 8739 were donated by Andr
Tosello Research and Technology Foundation FAT
(Campinas, Brazil www.fat.org.br).
2.2

Cell

culture

suspensions
The E. coli and B.subtilis cultures were activated using
nutrient broth and kept under refrigeration on nutrient agar
slants.

Ozonization of cassava starch

Petri dishes containing nutrient agar were inoculated

with E. coli in and incubated at 37 C for 24 hours. To prepare
E. coli suspension, the cell mass was harvested in sterile
peptone water and used on the same day. The B. subtilis
spores suspension was prepared as described by Wuytach,
Boven and Michiels (1998), with few modifications: B. subtilis
sporulation was carried out on nutrient agar supplement with
20 mg.L1 of manganese sulphate, incubation was carried
out at 30 C for 14 days and, after harvesting, spores were
three times centrifuged at 10,000 rpm for 10 minutes at 10
C. A heat shock at 70 C for 20 min was carried out before
B. subtilis count. Both microorganisms were plate counted
on nutrient agar and incubated at 30 C for
48 hours (B. subtilis) or 37 C for 24 hours (E.
coli).
2.3 Ozonation
Ozone was produced from concentrated O2
air
(Model NewLife Elite, AirSep Corporation, New York,
United States) using an Interozone generator (Line
DEVOC, Model SGOZ123, Interozone Group of Brazil, Jundia, Brazil) with 5 gh1
of production capacity. The ozonized air flow was 14 m3h1,
and the ozone concentration in the gas was continuously
monitored by an ozone analyzer (Model UV-100, Eco
Sensors, Inc., Santa Fe, USA). Ozonized air was injected in a
stainless steel powder mixer of 25 L through two air inlet
devices located in the mixer lid. The spinning blades
promoted an efficient contact between starch and ozonized
air.
2.4 Ozone treatment of cassava starch inoculated with E.
coli
1 kg of cassava starch (initial moisture of 11%) was
inoculated with 100 mL of E. coli suspension reaching a
final count of 106-108 CFUg1 and moisture of 18%
(maximum limit of starch moisture allowed by the Brazilian
law). For assays using cassava starch moisture at 30%
(moisture of starch immediately after cassava centrifugation
carried out in the starch industry just before the drying
processing), the samples were previously humidified with
sterile water, followed by inoculation with
100 mL of E. coli suspension. After inoculation, cassava
starch rested for one hour before ozonation to enable the
adherence of microorganism to starch.
The starch of 18% moisture content was processed at
ozone concentration of 40 and 113 mg.L 1 for 30, 60,
90, and 120 minutes. The starch of 30% moisture content
was processed at ozone concentration of 40 mg.L1 for 30,
60, 90, and 120 minutes or at ozone concentration of 118
mg.L1 for
15, 30, 45, and 60 minutes. The 113 and 118 mgL1 were
the higher ozone levels reached in the equipment for the
samples at
18 and 30% of moisture. This small variation in the
maximum ozone concentration can be attributed to few
oscillations in the oxygen concentration and ozone generated
in the equipment.

Survivor counts of E. coli were performed on nutrient

agar just after the ozonation process. The control samples
were obtained by exposing previously inoculated cassava
starch to air (without ozone) under the same conditions used
for the ozonized samples. Initial count was determined by
starch plating immediately after inoculation. The samples
were incubated at

37 C for 24 h. The Number of Decimal Reductions (NDR)

of each process was determined according to Equation 1.
NDR = log(initial count) log (survivors count)

(1)

2.5 Ozone treatment of cassava starch

inoculated with B. subtilis
1 kg of cassava starch was inoculated with B. subtilis
spores suspension at the same conditions described for E. coli.
B. subtilis final count was 107 CFUg1, and cassava starch
moisture was 18 and 30%. The inoculated sample was
allowed to rest for 1 hour for the adherence of microorganism
to starch, and the starch of
18% moisture content was then ozonized at ozone
concentration of 118 mg.L1 for 30, 60, 90, and 120 minutes,
and starch of 30% moisture content was processed at 40 and
118 mg.L1 for 30, 60,
90,
and
120
minutes.
The B. subtilis enumeration was carried out after a heat
shock (70 C/20 minutes) for spore activation. The samples
were plated on nutrient agar and incubated at 30 C for 48
hours. The control samples were obtained by exposing
previously inoculated cassava starch to air (without ozone)
under the same conditions used for the ozonized samples.
Initial count was determined by starch plating immediately
after inoculation. The Number of Decimal Reductions (NDR)
of each process was determined by Equation 1.
2.6
analysis

Statistical

The analysis of variance (ANOVA) was performed to

compare the effects of the different treatments, and the
Tukey test was used to determine their differences at a 5%
confidence level. Statistical analyses were carried out using
STATISTICA 5.0 software (StatSoft, Inc., Tulsa,
Okla.,U.S.A). All of the tested conditions were evaluated in
triplicate. The results were presented as mean standard
deviation.

3 Results and discussion

3.1
E.
inactivation

coli

The E. coli inactivation on ozonized 18% moisture

cassava starch is shown in Figure 1. Statistical analysis
indicated that 30,
1 ozone caused
60, and 90 minutes of contact time with 40
mg.L
about one decimal reduction, with no differences between

them (p > 0.05). On the other hand, 120 minutes of contact

at this same concentration caused about 2 decimal reduction,
and it was different from other conditions evaluated (p <
0.05). The results of cassava starch ozonized at the
concentration of
113 mg.L1
showed 1-2 decimal reduction, with no
significant differences between different ozone contact times.
Considering the control sample results (<0.5 NDR), it was
observed that ozone was able to reduce E. coli counts in 18%
moisture cassava starch, but these reductions (<2 decimal
reductions) were not enough to guarantee the cassava starch
safety when used as food raw material. Moreover, no
significant differences were observed between E. coli
inactivation in the samples ozonized at 40 and 113 mg.L1,
which probably indicates ozone saturation at concentration of
40 mg.L1.
The effect of starch humidification on the ozone efficacy
was evaluated through ozonation process carried out on
previously humidified starch at 30% moisture (moisture of the
starch after centrifugation). The E. coli inactivation at this
condition is shown in Table 1.
The control treatment (carried out with no ozone
injection) showed 1.3-1.6 decimal reductions, indicating
that at 30% moisture, the microorganism was more sensitive
in non-ideal growth media or its recovery after the process
was reduced. Comparing the results of control and ozonized
samples, on the other hand, it was observed that ozonation was
able to inactivate the microorganism.
The results showed that increases in ozone concentration
and exposure time improve E. coli inactivation during the
process. Processes applying ozone concentration at 40 mg.L
1
for 60 minutes or 118 mg.L1 for 30 minutes are enough
to guarantee an adequate level of E. coli inactivation assuring
the safety of foods with this starch as raw material.
E. coli inactivation by ozone has been previously
studied by several authors in different products including
water, fruits, vegetables, meat, and dry products
(MUTHUKUMARAPPAN; HALAWEISH; NAIDU, 2000;
ZHAO; CRANSTON, 1995; KHADRE; YOUSEF; KIM,
2001; AKBAS; OZDEMIR ,
2006, 2008b; EMER; AKBAS; OZDEMIR, 2008). These
studies were carried out using different equipment and
strains of E. coli, but, in general, it was observed that higher
inactivation occurred in water or products with high moisture
(MUTHUKUMARAPPAN; HALAWEISH; NAIDU, 2000).

The use of 0.35 ppm of ozone resulted in 5 decimal

reductions of E coli in water, and the use of 18 ppm for
50 minutes resulted in total load inactivation of E coli O157:H7

Table 1. Inactivation of E. coli in ozonized 30% moisture cassava

starch.
Time
(minutes)

NRD at different ozone concentration

*0 mgL1
40 mgL1
118 mgL1

Figure 1. Inactivation of E. coli in ozonized 18% moisture

cassava starch. Vertical bars represent the standard deviation of
each value.

15
30
45
60
90
120

1.26
1.45
1.51
1.64

3.72
>7
>7
>7

3.57
>6
>7
>7
-

*Results of control sample. These values are the NDR average of two
processes.

in several foods (MUTHUKUMARAPPAN; HALAWEISH;

NAIDU, 2000). The concentration of 1.0 ppm of ozone applied
for 360 minutes resulted in 3.5 decimal reduction in dried
figs (AKBAS; OZDEMIR, 2008b). For pistachio (AKBAS;
OZDEMIR, 2006), red pepper (AKBAS; OZDEMIR, 2008b),
and black pepper (EMER; AKBAS; OZDEMIR, 2008), similar
inactivation level (about 2 decimal reductions) was obtained
after ozonation for 120-360 minutes.
Therefore, the presence of water in food is very important
to reach high microbial inactivation level by ozone, as
observed in the present study. Similarly, previous results
showed that microbial inactivation by ozone on black pepper
was improved at high moisture content (ZHAO; CRANSTON,
1995). It is known that ozone is a powerful oxidative
compound that reacts with microorganism and organic
materials
(KHADRE;
YOUSEF;
KIM,
2001;
MUTHUKUMARAPPAN; HALAWEISH; NAIDU,
2000). Thus, the increase in the organic material content
may have affected the ozone efficacy.
Additionally, the ozone gas is rapidly decomposed in
oxygen (MUTHUKUMARAPPAN; HALAWEISH; NAIDU,
2000). It is possible that ozone partial dissolution in the food
free water results in the increase in the antimicrobial half-life,
which can become more effective. Another possible effect is
that moisture can improve the gas-product contact area or
promote water free- radical formation that was effective for
microbial inactivation in addition to the ozone effects.
Comparing the results obtained for E. coli inactivation in
the cassava starch and other dry products (AKBAS;
OZDEMIR,
2006, 2008b; EMER; AKBAS; OZDEMIR, 2008), it was
observed that, for the other products, lower ozone
concentration of
0.5-1 mgL1 was required to reach the same level of
inactivation (i.e., 40 times smaller than that required for
cassava starch). Additionally to the differences in the
moisture content and water activity of these foods and
cassava starch, the differences between the results obtained
can be explained by different ozonized air flow and the
amount of product ozonized, which interfere in the ozone
available amount and also on ozone mass/ product mass ratio.
Moreover, the E. coli adherence in cassava starch for one
hour before ozonation and the powder small
dimension with rough surface can also affect ozone
activity.
3.2
B.
inactivation

subtilis

Considering the results obtained for E. coli inactivation

in ozonized 18% moisture cassava starch and the expected
higher ozone resistance of B. subtilis, its inactivation in 18%
moisture cassava starch was evaluated only at the ozone
concentration
of 118 mg.L1 (which was the maximum ozone concentration
reached in the ozonation equipment). No B. subtilis spore
reduction was observed in cassava starch of 18% moisture
content, even after ozonation at 118 mg.L1 concentration
for 120 minutes (data not shown). This indicates that, at this
cassava starch moisture, ozone was not effective for B.

Figure 2. Inactivation of B. subtilis in ozonized 30% moisture

cassava starch. Vertical bars represent the standard deviation of
each value.

explained by the high resistance of the spore coat and outer

membrane to the oxidative mechanisms of the ozone
(AKBAS; OZDEMIR, 2008a, b; MUTHUKUMARAPPAN;
HALAWEISH; NAIDU, 2000), compared with oxidation of
vegetative cell envelope and membrane.
The effect of 30% moisture starch on the ozone efficacy
against B. subtilis was also evaluated, and the results are
shown in Figure 2. The results showed that the control
processes caused no significant reduction on B. subtilis
spores. On the other hand ozone promoted B. subtilis spores
inactivation at the concentration of 40 and 118 mg.L1, and it
was higher at higher ozone concentration (p < 0.05). In
addition, increased contact time up to 60 and 120 minutes
caused a significant increase in spore inactivation at ozone
concentration of 40 and 118 mg.L1, respectively. Again, with
the increase in the starch moisture, a significant increase in
the microbial inactivation was observed. Data on B. subtilis
spores in water also demonstrated higher level of spore
inactivation, with 3 log reductions after processing with
0.7 ppm of ozone, indicating that the water content is
important for spore inactivation.
It is interesting to highlight that by increasing ozone
concentration by three times (40 mg.L1 to 118 mg.L1) it
was able to reduce 50% of process time aiming to achieve the
same level of B. subtilis spores inactivation, which can be a
possible solution when low time processing is required.
Previous data on ozone treatment of wheat and wheat
flour showed low level of inactivation of total aerobic
microorganism and yeast and moulds (<1 decimal reduction)
using ozonated water at concentrations up to 16.5 ppm
(IBANOGLU, 2001,
2002; LI et al., 2012). These results corroborate the findings
that food with low water content possible slows ozone
reaction speed and therefore reduces the disinfection ability
of the ozone gas.

4 Conclusion

The ozonation process can be effective for E.coli and

B. subtilis inactivation in cassava starch, and it is highly
dependent on starch moisture. At low starch moisture (18%),
the process was not enough to guarantee microbial
inactivation;
subtilis
inactivation.

Comparing the results obtained for B. subtilis and E.

coli inactivation (113 mg.L1 ozone concentration), it was
confirmed that Bacillus spores are more resistant to ozone
than E. coli, as previously described by Akbas and Ozdemir
(2008a, b) and Muthukumarappan, Halaweish and Naidu
(2000). This can be

however, at 30% moisture starch, it was possible to reach

>5 decimal reductions, ensuring safe products. E coli were
less resistant to ozone treatment, and it was inactivated
after
30 minutes treatment with 118 ppm of ozone. On the other
hand,
90 minutes are required to reach an appropriated inactivation
of B. subtilis. Therefore, the application of ozone at these
conditions makes cassava starch a safe product to be used as a
raw material in the food industry.

Acknowledgments
The authors would like to thank the So Paulo Research
Foundation (FAPESP) for financial support to the project
#
2008/10942-5, the Brazilian National Research Council
(CNPq) for the E.O.C Amorim fellowship, and the Corn
Products Brasil for the cassava starch donation.

Reference
s
ACHEN, M.; YOUSEF, A. E. Efficacy of ozone against Escherichia
coli
O157:H7 on apples. Journal of Food Science, v. 66, n. 9, p.
13801384,
2001.
http://dx.doi.org/10.1111/j.13652621.2001.tb15218.x
AKBAS, M. Y.; OZDEMIR, M. Effectiveness of ozone for
inactivation of Escherichia coli and Bacillus cereus in pistachios.
International Journal of Food Science and Technology, v. 41,
n. 5, p. 513-519, 2006. http://dx.doi.org/10.1111/j.13652621.2005.01099.x
AKBAS, M. Y.; OZDEMIR, M. Application of gaseous ozone
to control populations of Escherichia coli, Bacillus cereus and
Bacillus cereus spores in dried figs. Food Microbiology, v.
25,
n.
2,
p.
386-391,
2008a.
http://dx.doi.org/10.1016/j.fm.2007.09.007
AKBAS, M. Y.; OZDEMIR, M. Effect of gaseous ozone on
microbial inactivation and sensory of flaked red peppers.
International Journal of Food Science and Technology, v. 43,
n. 9, p. 1657-1662, 2008b. http://dx.doi.org/10.1111/j.13652621.2008.01722.x
BIALKA, K. L.; DEMIRCI, A. Utilization of gaseous ozone for the
decontamination of Escherichia coli O157:H7 and Salmonella
on raspberries and strawberries. Journal of Food Protection, v.
70, n. 5, p. 1093-1098, 2007.
BRASIL. Ministrio da Sade. Resoluo RDC n 12, de 02 de janeiro
de
2001. Aprova o regulamento tcnico sobre padres
microbiolgicos para alimentos. Dirio Oficial da Repblica
Federativa do Brasil, Braslia, DF, 10 jan. 2001.
CEREDA, M. P.; VILPOUX, O. Uso de lmpada ultravioleta
Germetec para reduo de contedo microbiano de amido
comercial de mandioca. Centro de Tecnologias para o
Agro-negcio, Universidade Catlica Dom Bosco, 2005. Nota
prvia.
CHAN, H. T.; BHAT, R.; KARIM, A. A. Physicochemical and
functional properties of ozone-oxidized starch. Journal of
Agricultural and Food Chemistry, v. 57, p. 5965-5970, 2009.
http://dx.doi. org/10.1021/jf9008789
DEMIATE, I. M. et al. Viscographic characteristics of
chemically m o d i f i e d c a s s a v a s t a r c h e s a s s e s s e d
b y RVA . P u b l iti o UEPG - Cincias Exatas e da Terra, v.
11, n. 1, p. 7-17, 2005.
DHILLON, B. et al. Development and evaluation of an ozonated
water system for antimicrobial treatment of durum wheat.
Journal of Food Science, v. 74, n. 7, p. E396-403, 2009.
http://dx.doi. org/10.1111/j.1750-3841.2009.01275.x
DIELS, A. M. J.; WUYTACK, E. Y.; MICHIELS, C. W.
Modeling inactivation of Staphylococcus aureus and Yersinia

enterocolitica by high-pressure homogenization at different

temperatures. International Journal of Food Microbiology, v.
87, n. 1-2, p. 55-62,
2003. http://dx.doi.org/10.1016/S0168-1605(03)000503
EMER, Z.; AKBAS, M. Y.; OZDEMIR, M. Bactericidal activity of
ozone against Escherichia coli in whole and ground black
peppers. Journal of Food Protection, v. 71, n. 5, p. 914-917,
2008.
GUZEL-SEYDIM, Z. B.; GREENE, A. K.; SEYDIM, A. C. Use of
ozone in the food industry. LWT - Food Science and
Technology, v. 37, n. 4, p. 453-460, 2004.

IBANOGLU, S. Influence of tempering with ozonated water on

the selected properties of wheat flour. Journal of Food
Engineering, v.
48,
n.
4,
p.
345-350,
2001.
http://dx.doi.org/10.1016/S02608774(00)00177-1
IBANOGLU, S. Wheat washing with ozonated water: effects
on selected flour properties. International Journal of Food
Science and Technology, v. 37, n. 5, p. 579-584, 2002.
http://dx.d oi. org/10.1046/j.1365-2621.2002.00593.x
KHADRE, M. A.; YOUSEF, A. E. Decontamination of a
multilaminated aseptic food packaging material and stainless
steel by ozone. Journal of Food Safety, v. 21, p. 1-13,
2001. http://dx.d o i . org/10.1111/j.1745-4565.2001.tb00304.x
KHADRE, M. A.; YOUSEF, A. E.; KIM, J-G. Microbiological aspects
of ozone applications in food: a review. Journal of Food Science,
v. 66, n. 9, p. 1242-1252, 2001. http://dx.doi.org/10.1111/j.13652621.2001. tb15196.x
KETTERINGHAM, L. et al. Application of aqueous ozone for
treating pre-cut green peppers (Capsicum annuum L.). Journal
of Food Engineering, v. 76, p. 104-111, 2006.
http://dx.doi.org/10.1016/j. jfoodeng.2005.05.019
KIM, J.-G.; YOUSEF, A. E.; CHISM, G. W.Use of ozone to
inactivate microorganisms on lettuce. Journal of Food
Safety,
v.
19,
p.
17-34,
1999.
htt p://dx.d o i. or g/10.1111/ j.1745-4565.1999. tb00231.x
KLOCKOW, P. A.; KEENER, K. M. Safety and quality assessment
of packaged spinach treated with a novel ozone-generation
system. LWT - Food Science and Technology, v. 42, n. 6, p.
1047-1053, 2009.
KOSEKI, S.; ISOBE, S. Effect of ozonated water treatment on
microbial control and on browning of Iceberg Lettuce (Lactuca
sativa L.). Journal of Food Protection, v. 69, n. 1, p. 154-160,
2006.
LI, M. et al. Evaluation the quality characteristics of wheat flour
and shelf-life of fresh noodles as affected by ozone treatment.
Food
Chemistry,
v.
135,
p.
2163-2169,
2012.
http://dx.doi.org/10.1016/j. foodchem.2012.06.103
MOREAU, M.; ORANGE, N.; FEUILLOLEY, M. G. J. Nonthermal plasma technologies: New tools for
biodecontamination. Biotechnology Advances, v. 26, n. 6, p. 610617, 2008. http://dx.doi. org/10.1016/j.biotechadv.2008.08.001
MUTHUKUMARAPPAN, K.; HALAWEISH, F.; NAIDU, A. S.
Ozone.
In: NAIDU, A. S. Natural Food Antimicrobial Systems. CRC
Press, 2000. http://dx.doi.org/10.1201/9781420039368.ch29
NAJAFI, M. B. H.; KHODAPARAST, M. H. H. Efficacy of ozone
to reduce microbial populations in date fruits. Food Control, v.
20,
p.
27-30,
2009.
http://dx.doi.org/10.1016/j.foodcont.2008.01.010
PASTORIZA, L. et al. Use of sterile and ozonized water as a
strategy to stabilize the quality of stored refrigerated fresh
fish. Food Control, v. 19, p. 772-780, 2008.
http://dx.d oi.org/10.1016/ j. foodcont.2007.08.003
SILVA, G. O. et al. Amidos nativos e modificados: propriedades e
aplicaes em alimentos. Boletim da Sociedade Brasileira de
Cincia e Tecnologia de Alimentos, v. 37, p. 101-106, 2003.
Suplemento.
TIWARI, B. K. et al. Effect of ozonation on the rheological
and colour characteristics of hydrocolloid dispersions. Food
Research International, v. 41, n. 10, p. 1035-1043, 2008.
http://dx.d oi. org/10.1016/j.foodres.2008.07.011
TIWARI, B. K. et al. Anthocyanin and colour degradation in
ozone treated blackberry juice. Innovative Food Science and

Emerging Technologies, v. 10, n. 1, p. 70-75, 2009a.

http://dx.doi. org/10.1016/j.ifset.2008.08.002

TIWARI, B. K. et al. Effect of ozone processing on anthocyanins

and ascorbic acid degradation of strawberry juice. Food
Chemistry, v. 113, n. 4, p. 1119-1126,
2009b.
http://dx.d oi.org/10.1016/ j. foodchem.2008.08.085
TIWARI, B. K. et al. Review: Application of ozone in grain
processing.
Journal of Cereal Science, v. 51, p. 248-255, 2010.
http://dx.doi. org/10.1016/j.jcs.2010.01.007
TRIBST, A A. L.; SANTANA, A. S.; DE MASSAGUER, P. R.
Review: Microbiological quality and safety of fruit juices
past, present and future perspectives. Critical Reviews in
Microbiology,

v. 35, n. 4, p. 310-339, 2009. PMid:19863382.

http://dx.d oi . org/10.3109/10408410903241428
WUYTACH, E. Y.; BOVEN, S.; MICHIELS, C. W. Comparative
Study of Pressure-Induced Germination of Bacillus subtilis
Spores at Low and High Pressures. Applied and Environmental
Microbiology, v. 64, n. 9, p. 3220-3224, 1998.
ZHAO, J.; CRANSTON, P. M. Microbial decontamination of
black pepper by ozone and the effect of the treatment on
volatile oil constituents of the spice. Journal of the Science of
Food and Agriculture, v. 68, n. 1, p. 11-18, 1995.
http://dx.doi.org/10.1002/ jsfa.2740680103