Journal of Stored Products Research 87 (2020) 101605

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Journal of Stored Products Research

journal homepage: www.elsevier.com/locate/jspr

Application of ozone on rice storage: A mathematical modeling of the

ozone spread, effects in the decontamination of filamentous fungi and
quality attributes
Geovana D. Savi a, b, *, Thauan Gomes a, c, Sílvia B. Canever a, c, Ana C. Feltrin a, b,
Karim C. Piacentini d, Rahisa Scussel e, Daysiane Oliveira e, Ricardo A. Machado-de-Avila
e
,
c a, b, c
Maykon Cargnin , Elidio Angioletto
a
Laborato
rio de Desenvolvimento de Biomateriais e Materiais Antimicrobianos (LADEBIMA), Universidade do Extremo Sul Catarinense (UNESC), Criciúma,
SC, Brazil
b
Programa de Po ~o em Ci^

s-Graduaça encia e Engenharia de Materiais, Universidade do Extremo Sul Catarinense (UNESC), Criciúma, SC, Brazil
c
Departamento de Engenharia Química, Universidade do Extremo Sul Catarinense (UNESC), Criciúma, SC, Brazil
d
Departamento de Biotecnologia, Universidade de Sa ~o Paulo (USP), Sa
~o Paulo, SP, Brazil
e
Programa de Po
s-Graduaça~o em Ci^
encias da Saúde, Universidade do Extremo Sul Catarinense (UNESC), Criciúma, SC, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Rice is a food crop prone to contamination by toxigenic fungi. Ozone, as a decontamination strategy in
Received 4 September 2019 the industry, has attractive advantages over traditional food preservation techniques. A contribution to
Received in revised form the understanding of ozone as an antifungal agent in rice storage processes is performed in this study.
26 February 2020
For this, a mathematical model to predict the ozone spread in rice storage in silos was developed. In
Accepted 19 March 2020
Available online xxx
addition, the effect of ozone application on fungal decontamination and the influence on quality attri-
butes of stored rice were evaluated. This work links the ozone concentration gradient with fungal
elimination. A 3D finite element model and experimental measurements determined the ozone con-
Keywords:
Ozone
centration throughout the silo. In the inferior portion (where the ozone inlet occurs) showed strong fungi
Modeling reduction. In this case, an application of 0.393 kgO3 , m
3
rice caused 90% fungal elimination. The efficiency of
Storage ozone through the silo is different with relation to fungi reduction, which is compatible with the ozone
Fungi concentration gradient observed in physical model. The ozone treatment was not able to fully reduce the
Rice fungi growth in all the portions within the silo, which some fungi strains as Penicillium sp. remained
resistant to treatment. With regard to rice quality, starch modifications, lipid peroxidation, protein profile
and microstructure alterations did not reflect any significant alteration in the rice treated with ozone.
However, during the seed germination, the ozone showed significant inhibition of the coleoptile and
seminal root structure in higher exposure time. The use of ozone during the rice storage in silo scale
appear as a strong antifungal agent without causing damage to grain quality. However, the efficiency of
ozone throughout the silo is different in terms to fungi growth reduction and therefore, it is importance
of adopting strategies to homogenize the ozone spread.
© 2020 Elsevier Ltd. All rights reserved.

1. Introduction
Rice (Oryza sativa L.) is a staple food of the largest portion of the
population worldwide (FAO, 2018). Brazil is the largest producer of
* Corresponding author. Universidade do Extremo Sul Catarinense, Iparque -
Parque Científico e Tecnolo
gico, Rod. Gov. Jorge Lacerda, 3978, 88805-350, Cri-
ciúma, Santa Catarina, Brazil.
E-mail addresses: geovanasavi@gmail.com, ean@unesc.net (G.D. Savi).
rice outside the continent of Asia. It is among the ten main pro-
ducers in the world. In the Mercosul countries (Argentine, Brazil,
Paraguay, Uruguay and Venezuela), Brazil is the largest consumer of
rice with an estimated demand of 11.8 million tons and exports of
1.3 million tons for the 2018/2019 harvest (CONAB, 2018).
Rice crop is largely cultivated in subtropical environments,
which are considered favorable for fungal growth due to high
temperature and humidity. During storage in silos, it is prone to
contamination due to being an excellent food source for fungal

https://doi.org/10.1016/j.jspr.2020.101605
0022-474X/© 2020 Elsevier Ltd. All rights reserved.
2 G.D. Savi et al. / Journal of Stored Products Research 87 (2020) 101605
growth (Al-Zoreky and Saleh, 2019). The presence of toxigenic fungi
in rice grain from Brazil (Katsurayama et al., 2018; Savi et al., 2018)
and in other parts of the world (Al Husnain and AlKahtani, 2019;
Zhao et al., 2019) are reported in the current literature. The most
important fungal genera found in rice grain are Aspergillus, Peni-
cillium and Fusarium (Ferre, 2016). Some of these toxigenic species
produce mycotoxins in the food, during storage and also the pro-
cessing stage.
Mycotoxins are often found in rice grain, such as aflatoxin (Al-
Zoreky and Saleh, 2019; Nguyen et al., 2007), zearalenone (Savi
et al., 2018) and citrinin (Abd-Allah and Ezzat, 2005; Nguyen
et al., 2007), among others (Ferre, 2016). They are considered
serious health hazards. For example, aflatoxin B1 produced by some
Aspergillus species is possibly hepatotoxic, teratogenic and geno-
toxic (IARC, 1993). Zearalenone produced by Fusarium species can
cause significant changes in reproductive organs in animals and
humans due to high affinity for estrogen receptors (Zinedine et al.,
2007). Citrinin is produced by Penicillium species and besides to be
nephrotoxic, it is possibly carcinogenic in humans (Gupta et al.,
2018). Moreover, the co-occurrence of many kinds of mycotoxins
in food increase risks to human and animal health.
Due to the fact that fungi contamination in stored rice grains to
be a constant concern in the food industry, several preventive
strategies are used such as good agricultural practices in pre-
harvest, as well as appropriate storage condition in post-harvest.
Fungicides and other harmful chemicals are often used however,
there is an increasing public concern due to the potential risk of
residues that remain in food (Patriarca and Ferna
ndez Pinto, 2017).
The development of decontamination methods safe for use in
food is an attractive target for industry. Ozonization is the most
promising chemical method able to inactivate microorganisms and
degrade mycotoxins (Gomes et al., 2019; Trombete et al., 2017).
Ozone is an unstable molecule that rapidly decays to diatomic ox-
ygen, without leaving food residues, and has the GRAS (generally
recognized as safe) status (Gaou et al., 2005).
On the other hand, the structure and physical-chemical char-
acteristics of food are factors that influence the effectiveness of
applied ozone. The impacts of ozone treatment need to be studied
specifically for each food type. The food quality in terms of
composition, physicochemical properties of components (such as
starch and protein) and germination capacity of different food
grains are often studied (Zhu, 2018).
In order to microbiological elimination to be effective, a fumi-
gation system need to be properly evaluated in grain storage silos.
Therefore, an experimental study was developed to link the
reduction of filamentous fungi to mathematical model of the
propagation of ozone in different portion from the silo. The aim of
this study was to evaluate the ozone spread in an experimental silo
containing stored rice, as well as its effects regarding fungal
decontamination and grain quality.
2. Materials and methods
2.1. Sampling
The paddy rice grain samples were randomly collected from
local industries in southern Brazil during post-harvest after drying
and storage of three months during the 2018 crop year. Collection
was performed using a grain auger from different points of the bulk
batches. Rice samples were stored in polypropylene packaging bags
and then conditioned within the silo for the ozone treatment.
Fig. 1 illustrates the bench scale silo geometry that will be
shown in this paper. The silo consists of three regions: free flow at
the base (0.02 m) and at the top (0.136 m) and in the rice bed
(0.33 m). Ozonation of rice was performed at different exposure
times (30, 90 and 180 min) and the presence of filament fungi were
evaluated in different portions of the rice bed. The grain quality
attributes also were evaluated after the ozone treatment. SP, CP and
IP, representing the superior, central and inferior portion of the rice
bed, respectively. In each portion of the silo, rice grains (100 g) were
collected in duplicates to evaluate the fungi contamination. This
sampling was performed axially to link the results to a physical
model of ozone spread.
2.2. Modeling of ozone spread
A 3D model of ozone spread in the silo was developed based on
the principles of transport and ozone decomposition kinetics. This
is a strategy of this work to illustrate that the ozone propagation
influences axially the rice grain storage within silo. In this section
the governing equations, boundary conditions and hypotheses
adopted to solve and validate the model will be presented.
2.2.1. Governing equations and boundary conditions
Fig. 2 shows the silo (with the mesh used) and indications of the
governing equations and boundary conditions. The silo has the
same geometry as shown in Fig. 1.Where there is no porous me-
dium (free flow), the equations of motion for a single-phase fluid
were used:
vr
þ V , ðruÞ ¼ 0 (1)
vt
   2 
vu
r þ ru , Vu ¼
Vp þ V, m Vu þ ðVuÞT
mðV , uÞI (2)
vt 3
Equations (1) and (2) demonstrate the continuity and mo-
mentum equations, respectively.
These equations are applicable for a weakly compressible fluid
(density is evaluated at the pressure reference). This is a hypothesis
of this work, due to the low-pressure gradient.
In the rice bed, the flow was modeled by the Brinkman’s
equations:
vðεrÞ
þ V , ðruÞ ¼ 0 (3)
vt
     
r vu u 1  2
þ ðu , VÞ
¼
Vp þ V m Vu þ ðVuÞT
mðV , uÞI
ε vt ε ε 3
  

mK
1 þ Qbr u
(4)
These equations are used to compute fluid velocity and pressure
fields of single-phase flow in porous media in the laminar flow
regime (Auriault, 2009; Le Bars and Worster, 2006; Parvazinia et al.,
2006). The laminar flow regime was verified by monitoring the cell
Reynolds number throughout the silo.
In Equations (1)e(4), m and r are the dynamic viscosity of the
fluid (kg $ m
1 $ s
1) and density (kg $ m
3), on the basis of air at
25  C as previous reported (considering that the ozone is diluted).
The pressure is represented by p (Pa), K is the permeability of the
porous medium (m2), t is the time (s), ε is the porosity (dimen-
sionless) and u is the velocity vector (m $ s
1).
The effect of blocked pores on the flow was not considered; we
considered the rice bed as a porous medium, which consisted of a
pure gas phase for the flow of ozone.
For the modeling species transport, Equations (5) and (6) were
used, respectively, for the domains where there is rice bed (porous
medium) and where the flow is free. These equations are solved
 G.D. Savi et al. / Journal of Stored Products Research 87 (2020) 101605 3

Fig. 1. Geometry of bench scale silo and approach to linking ozone spread to fungal elimination.

The first term on the left in Equations (5) and (6) correspond to
the accumulation of species and the second term represents
convective transport due to a velocity field. On the right side of
Equations (5) and (6), the first term describes transport by diffusion
and dispersion, respectively. On the same side, the second term of
Equations (5) and (6) represents the chemical reaction. It was
considered a global chemical reaction for the interactions between
ozone and the rice bed (porous medium). In regions where there is
no porous medium, ozone self-decomposition has been considered.
In the outlet, it is assumed that convection dominates the mass
transport:


n ,
D , VCO3 ¼ 0 (8)

The symbol n refers to the unit normal vector oriented out-

wards. On the walls no-slip condition was assumed.
The use of a separation grille (separating the free flow from the
rice bed) ensured consistency with our assumption of uniform inlet
concentration.
Finite element simulations in COMSOL Multiphysics® were used
to solve the model, considering fully coupled equations. The silo
was considered isotropic and therefore we do not consider D and
Fig. 2. Indication of governing equations, boundary conditions and mesh illustration. Def as a function of the direction.
The Parallel Direct Sparse Solver (PARDISO) was chosen by
standard in COMSOL and used at its default settings. The mesh of
assuming dilute species.
the model was designed with 70,737 elements. The 3D geometry

  with mesh volume of 0.02107 m3 was used with tetrahedral
v εCO3
þ u , VCO3 ¼ V , Def VCO3 þ Rpm (5) (53,292), prism (10,208), triangular (6,436), quadrilateral (384),
vt
edge (397) and vertex (20) elements. The elaborated mesh was

adequate for the solution of the model considering minimum error
vCO3
þ u , VCO3 ¼ V , DVCO3 þ Rff (6) and computational effort. The discretization was linear for pres-
vt
sure/velocity and concentration. The convergence criterion adop-
In these equations, Rpm and Rff are expressions for the decom- ted was absolute residuals less than 10
5 for the mass and
position rate (mol $ m
3 $ s
1) in the porous medium and in free momentum conservation equations.
flow, respectively. CO3 is the ozone concentration (mol $ m
3). In
the free-flow domains, the diffusion coefficient (D) of the ozone in
2.2.2. Experimental procedure (model validation)
the air (1.44  10
5 m2 s
1) (Massman, 1998) was used. In the rice
Model validation was performed by experimentally determining
bed the Bruggeman model (Berson et al., 2011; Kong et al., 2015;
the parameters and comparing the numerical results with the
Tjaden et al., 2016) was used for effective diffusivity:
experimental data using a first order kinetic coefficient to fit the
model. For this, the ozone concentration in the silo outlet was
Def ¼ Dε1;5 (7)
sampled as a function of time. Thus, the ozone breakthrough curve
4 G.D. Savi et al. / Journal of Stored Products Research 87 (2020) 101605
was obtained.
Ozone demand can be divided into “finite demand” and “kinetic
demand” (Clayton et al., 2011). The finite demand (as the name
already says), extinguishes after the ozonation of the material.
However, even if the bed is ozonized for a long time, the kinetic
demand, which is persistent, will remain. Thus, to validate the
model, pre-ozonated grains were used, since in practice decon-
tamination of rice grain by ozone occurs at steady state.
The ozone was generated from dried oxygen (99.9%) through gas
generator (Brasil Ozo ^nio®-BRO3-PLUS2.1). The ozone concentra-
tion at the outlet was sampled using a continuous ozone monitor
(UV/vis spectrophotometer with the single-cell kinetics mode at
254 nm) (OZONE ANALYZER BMT 964 BT; BMT Messtechnik GmbH,
Germany). Inlet pressure was measured with a pressure gauge
(GDH 200-14; GHM Messtechnik GmbH, Germany) at a constant
value of 20 Pa.
In addition, the porosity and permeability were measured for
characterization of the transport factors of the silo. The volume
occupied by voids (water was inserted into the bed) determined
porosity. Permeability was measured by the decreasing pressure
method (Joseph et al., 2013; Kirkham, 1947).
Interactions of ozone with rice are complex and difficult to
understand completely. However, it is possible to experimentally
identify the effects of rice on ozone consumption. These effects are
expressed by the kinetic rate for Equations 9e12:
kpm
O3 þ riceƒƒƒƒƒƒƒƒ!ricereacted þ O3unreacted
Rpm ¼
kpm ,CO3
ksd
2 O3 ƒƒƒƒƒƒƒƒ! 3 O2
Rff ¼
ksd ,CO3
Equation (9) is an irreversible reaction between ozone and rice
bed. Equation (10) shows the kinetics proposed to describe the
reaction. Rpm is the rate law -that was shown in Equation (5)- (mol
$ m
3 $ s
1), and kpm (s
1) is the first order kinetic coefficient,
derived from experimental validation of the model. The kinetic
coefficient (kpm ) includes all factors that are associated with ozone
consumption in the porous medium.
The ozone self-decomposition (Equation (11)) is accounted for
Rff . The experiments were performed to determine the kinetics of
the ozone self-decomposition. It is a first order kinetics (Equation
(12)), and ksd is 1.07  10
3 s
1 at 25  C (Gomes et al., 2018).
2.3. Mycological analysis after ozone gas treatment
The rice sample (100 g) was removed from each portion in
duplicate after homogenization for the mycological assays. The
spread-plate technique was applied to evaluate the total fungal
count (Silva et al., 2010). Twenty five grams of each sample were
used to dilutions and aliquots of 0.1 mL of each dilution (10
1 to
10
4) were spread on the surface of PDA (potato dextrose agar)
medium plates containing 100 mg L
1 chloramphenicol (in dupli-
cate) and incubated for up 5 days at 28  C. The results were
expressed as colony forming units per gram (CFU $ g
1).
After fungal colony growth, the genera identification occurred
through sub-culture of the isolated strains from rice on culture
medium for macro-morphological observations. The malt extract
agar (MEA), glycerol nitrate agar (GN25) and czapek yeast extract
agar (CYA) media were used according to Pitt and Hocking (2009).
Thereafter, the microcultivation in czapek-dox was performed in
the selected colony for micro-morphological observations under a
light microscope (100  and 400  magnifications) (Samson et al.,
2006; Weber and Pitt, 2000). The genera identification was carried
out by means of available taxonomic keys and guides (Nelson et al.,
1983; Pitt and Hocking, 2009; Samson et al., 2006).
The moisture content (mc) was performed by the drying of
samples (2 g) in an oven (105  C) until they reached a constant
weight (AOAC, 2005). Water activity (aw) was measured using a
Model Aw43 apparatus (Etec, Sa ~o Paulo, Brazil). The assays were
performed in triplicate.
2.4. Rice grain quality after ozone gas treatment
Regarding grain quality assays, the samples were collected from
the inferior portion within the silo (where the ozone inlet occurs)
and after the higher exposure time was applied (180 min). These
are the extreme conditions of ozone-rice contact investigated in
this paper.
2.4.1. Rice starch extraction
A total of 250 g of rice grain taken prior and after treatment were
separately ground using a laboratory mill. The flour was diluted
with distilled water containing 0.16% sodium hydrogen sulphite
and remained in 4  C for 24 h. The methodology used for rice starch
extraction was according to Rupollo et al. (2011) and Vanier et al.
(2012). Thereafter, the water was drained off and then the slurry
was sifted using a 200-mesh sieve and then was washed thor-
(9)
oughly with distilled water. The filtrate slurry was allowed to stand
for 3 h after and only the settled starch layer was re-suspended in
(10)
distilled water and centrifuged at 1500 rpm for 30 min. Only the
white layer was re-suspended in distilled water and centrifuged
again at 1500 rpm for 30 min. This even procedure was performed
(11)
more twice. Finally, the starch was collected and dried in an oven at
40  C for 12 h (Rupollo et al., 2011; Vanier et al., 2012).
(12)
2.4.2. Carbonyl and carboxyl content
The titrimetric method was used to determine the carbonyl
content of the rice starch according to Smith (1967), Vanier et al.
(2012). A sample of rice starch (2 g) was added of distilled water
(100 mL) and the solution maintained in a boiling water bath over
20 min. Subsequently, the solution was cooled to 40  C and then the
pH level was set at 3.2 with 0.1 N HCl. Then, a hydroxylamine re-
agent (15 mL) was added to the mixture and placed in a 40  C water
bath for 4 h with slow stirring, remaining in the sealed flask. The
excess hydroxylamine was determined by titration by means of a
reaction with HCl, pH 3.2. A blank determination with only the
hydroxylamine reagent was performed in the same manner. The
carbonyl content was exhibited as the quantity of carbonyl groups
per 100 glucose units (COOH $ 100 GU
1).
For the determination of carboxyl content of the extracted
starch, a sample (2 g) was mixed with 25 mL of 0.1 N HCl, and the
slurry was stirred occasionally for 30 min. Then, it was vacuum-
filtered and washed with 400 mL of distilled water. The starch
was carefully moved and the volume was filled up to 300 mL with
distilled water. The starch was heated in a boiling water bath with
stirring for 15 min and then more distilled water was added up to
450 mL. It was then titrated to pH 8.3 with 0.01 N NaOH. Native rice
starch was used as blank sample of the determination. The carboxyl
content was exhibited as the quantity of carboxyl groups per 100
glucose units (COOH $ 100 GU
1). The carbonyl and carboxyl con-
tent of starch extracted of the rice were calculated as described by
Vanier et al. (2012), Smith (1967).
2.4.3. XRD, FT-IR and DSC/TG
X-ray diffractograms (XRD) of the isolated starches were
 G.D. Savi et al. / Journal of Stored Products Research 87 (2020) 101605
obtained using a Shimadzu LABX XRD 6000 (Japan) with the
accelerating voltage of 30 kV and 30 mA, copper (Cu) Ka radiation
was used with a nickel filter and data collection was carried out in
the 2q range 5-30 , according to Sandhu et al. (2012). The Fourier-
transform infrared spectroscopy (FT-IR) spectrum of the isolated
starches was obtained using a Shimadzu Spectrum IR Prestige 21
(Japan). The FT-IR spectra were taken in the range from 4000 to
400 cm
1 in the transmission mode using the KBr pellet technique.
Differential scanning calorimetry/thermogravimetry (DSC/TG)
analysis was carried out using a NETZSCH STA 449F3 thermal
analyzer under synthetic air atmosphere at heating rate of 5  C $
min
1 from room temperature to 400  C.
2.4.4. Rice protein analysis by SDS-PAGE
The water-soluble protein content of rice samples was deter-
mined by SDS-PAGE according to the method of Li et al. (2012) with
modifications. The water-soluble fraction was chosen since the
study of Jing et al. (2016) showed that only this protein fraction type
suffers from negative effects of high ozone exposure. The SDS-PAGE
was performed according to using 15% of separation gel (pH 8.8)
and 4% stacking gel (pH 6.8). Separately, 50 mg of control and
treated rice were ground in a mill and stirred in 1 mL of extraction
buffer (0.01 M Tris HCl, pH 6.8, including 10% (w/v) SDS, 5% (v/v) 2-
mercaptoethanol (2-ME), 10% (v/v) glycerol, 0.1% (w/v) bromo-
phenol blue). The samples were heated for 5 min at 100  C and then
centrifuged for 5 min at 8000 g. Aliquots of 20 mL were loaded into
each well and electrophoresis was performed at 50 V for 30 min
and then at 120 V until the end. The proteins were stained with
silver nitrate (Jing et al., 2016; Li et al., 2012).
2.4.5. Lipid peroxidation
The lipid peroxidation was determined according to
Micha1owicz et al. (2009) and Savi et al. (2014). Separately 0.5 g of
the control and treated rice samples were mixed with 0.1% tri-
chloroacetic acid (TCA) (5 mL). The mixture was centrifuged at
10,000 rpm for 10 min at 4  C. The supernatant was reduced to 1 mL
and added with 0.5% thiobarbituric acid (TBA) (4 mL) in 20% TCA
and then the solution was heated to 96  C for 30 min. The absor-
bance of the supernatant was measured at 532 nm. The lipid per-
oxidation was calculated by the content of malondialdehyde (MDA)
using thiobarbituric acid reactive substances (TBARS) (Michałowicz
et al., 2009).
2.4.6. SEM analysis
The rice grain and extracted starch before and after ozone
treatment were added into aluminum stubs with metal adhesive
glue and placed on the Au Coater holder. They were coated with a
1.40 nm layer of gold. Morphology surface of food was observed by
means of a Scanning Electron Microscope (SEM Zeiss EVO MA10,
UK) with the magnification rates and recorded as micrographies
(voltage from 0.5 V to 30 kV).
2.4.7. Rice seed germination
The rice seed before and after ozone treatment were aseptically
spread on the sterilized petri dish of 9 cm diameter containing
humid paper layers and placed in an incubator (30  C, 90% relative
humidity) for 7 days to germination. The tests were repeated four
times and the percentage germination was calculated according to
the modified method of the International Seed Testing Association
(1985). The coleoptile and seminal root of the rice seeds were also
measured.(Association, 1985).
2.5. Statistical analysis
The data was analyzed by analysis of variance (ANOVA)
5
considering the unpaired t-test or Bonferroni as post-tests as
needed. The main results were evaluated as mean ± standard de-
viation and values of P < 0.05 were statistically significant.
3. Results
3.1. Ozone spread in bench scale silo
Fig. 3 shows the ozone breakthrough curve (concentration of
ozone as a function of time at the silo outlet). The model was
compatible with the experimental data with R2 ¼ 0.97. For this, the
kinetic coefficient kpm was of 1.02  10
2 s
1. As the experiment
was performed with already ozonized grains, this coefficient rep-
resents the "kinetic demand" of ozone. Kinetic coefficients with
different orders were tested. The first order coefficient showed
model compatibility with the experimental data.
The values used in the equations for the model are shown in
Table 1. The use of the model is highlighted when the ozone con-
centration profile is shown (Fig. 4).
This concentration profile refers to the center of the silo, where z
is the axial coordinate. The steady state occurs after 500 s of
ozonization. There is a sharp drop in ozone concentration
throughout the silo. This is a common feature in systems where
there is a persistent consumption rate, and corroborates with the
proposition of this work, which establishes a first order kinetics for
ozone depletion. The ozone concentration is of the order of 10
1,
10
2 and 10
3 (mol $ m
3) for the portions IP, CP and SP,
respectively.
3.2. Effect of ozone treatment on mycological analysis
A total fungi load of 9.2  103 ± 0.8  103 CFU g-1 was found in
rice grain in the present study (Table 2). After ozone treatment,
there was strong reduction of fungi count in the rice samples. Fig. 5
shows reduction of fungi and their dependence on the longitudinal
axis of the silo and the time of exposure to ozone. In the inferior
portion (near ozone inlet) a strong fungi reduction occurs, reaching
reduction values of above 90% already in the early onset of ozone
application. In 180 min of ozone exposure, there was total reduc-
tion of fungal contamination. However, the behavior of ozone along
the silo is different in relation to fungi reduction. For example, after
30 min of exposure time to ozone, the superior portion reached up
to 42% reduction, but even increasing the exposure time, ozone
treatment was not able to completely reduce fungal growth. This
shows that increasing the ozone concentration (which in the SP is
of the order of 10
3 mol m3) is fundamental to maximize fungal
elimination.
Table 2 presents data of the fungi contamination reduction after
the ozone treatment at different time exposure (0, 30, 90 and
180 min) when compared to control (without ozone) in different
portions within of silo. Significant alterations of fungi contamina-
tion were found in terms of silo portions (F ¼ 205.56, df ¼ 3, 80,
P < 0.0001), time exposure (F ¼ 229.07, df ¼ 3, 80, P < 0.0001) and
the interaction of these two parameters (F ¼ 29.94, df ¼ 9, 80,
P < 0.0001). In terms of percentage, after 30 min of ozone exposure
time, the reduction was of 95%, 76% and 42% in the IP, CP and SP
inside of silo, respectively. Then, after 90 min of ozone exposure
there was reduction of 96%, 84% and 46% in same portions cited
above. Finally, in 180 min the reduction reached 100%, 94% and 82%
of fungal inhibition.
In our work, aw and mc also were evaluated before and after the
ozone treatment. No significant alteration in the values of mc were
observed among silo portions (F ¼ 2.83, df ¼ 3, 32, P ¼ 0.0541), time
exposure (F ¼ 0.30, df ¼ 3, 32, P ¼ 0.8280) and the interaction of
these two parameters (F ¼ 1.07, df ¼ 9, 32, P ¼ 0.4085). Similar
6 G.D. Savi et al. / Journal of Stored Products Research 87 (2020) 101605

Fig. 3. Comparison of the model and experimental data for the ozone concentration as a function of time in the silo outlet.

Table 1 remained mainly in SP and CP of the silo even after 180 min of
Parameters used for the model. ozone exposure. In contrast, the Aspergillus genera was fully
Parameter Value reduced after 180 min in all the portions within the silo (Table 2).
ε - Porosity 0.40 (dimensionless)
K- Permeability 2.21  10
10 (m2) 3.3. Effect of ozone treatment on grain quality
ksd Gomes et al. (2018) 1.07  10
3 (s
1)
kpm 1.02  10
2 (s
1) 3.3.1. Starch modification
DMassman (1998) 1.44  10
5 (m2 $ s
1)
There was no statistic change in the carbonyl and carboxyl
Def 3.78  10
6 (m2 $ s
1)
content after ozone treatment. Even after 180 min of ozone expo-
sure, no significant difference was evidenced between the carbonyl
and carboxyl content of starch extraction of treated and the control
rice (Table 3).
The starch crystallinity was studied by X-ray diffraction and
presented the A-type patterns (A) characteristic of cereal grain
starches, having peaks at 15 , 17, 18 and 23 , as shown in Fig. 6.
The diffraction patterns of ozone treated samples were similar to
control samples (without ozone).
The structural composition also was analyzed by FT-IR (Fig. 7).
The starch control (without ozone) display peaks at 3415, 2912,
1650, 1390 and 1022 cm
1 which were similar to starch ozone
treated at 3417, 2910, 1643, 1390, 1033 cm
1. The bands next at
3400 cm
1 correspond to OeH stretching (Fan et al., 2012;
Kacurakova and Mathlouthi, 1996; van Soest et al., 1994) and at
2920 cm
1 is associated to CH2 stretching (Fan et al., 2012;
Kacurakova and Mathlouthi, 1996; Kizil et al., 2002). The band at
1640 cm
1 is characteristic of COO-stretching (Fan et al., 2012;
Kacurakova and Mathlouthi, 1996; Kizil et al., 2002) vibration in a
carbohydrate group whereas the band next at 1390 cm
1 is CH2
bending, CeOeO stretch (Fan et al., 2012; Kacurakova and
Mathlouthi, 1996; Kizil et al., 2002). Finally, the band next at
1020 cm
1 characteristic to region amorphous, CeO of CeOeC of
rice starch in both of the samples (Fan et al., 2012; Kacurakova and
Fig. 4. Ozone profile concentration. Mathlouthi, 1996; van Soest et al., 1994).
The thermal analysis by DSC/TG is illustrated in Fig. 8 for both
samples, which shows that the thermal behavior was the same for
results were found to aw, among silo portions (F ¼ 0.30, df ¼ 3, 32, starch prior and after the ozone treatment, with a characteristic
P ¼ 0.8232), time exposure (F ¼ 2.55, df ¼ 3, 32, P ¼ 0.0731) and the peak of organic matter oxidation in 320  C and loss of mass of
interaction between this conditions (F ¼ 0.36, df ¼ 9, 32, P ¼ 0.9471) nearly 20%.
(Table 2).
The higher incidence of fungi genera found in rice grain 3.3.2. Grain physical-chemical modification
analyzed in the current study was the Penicillium, reaching values In our study, the rice grains were evaluated along with lipid
above of 90% in the control samples (without ozone). These strains peroxidation after 180 min of ozone treatment. However, the
 G.D. Savi et al. / Journal of Stored Products Research 87 (2020) 101605 7

Table 2
Effect of ozone treatment in stored rice in regard to fungi reduction and humidity factors.

Assay performed Ozone exposure time Total ozone dosage Control (without Ozone treatment in portions within silo P value vs.
(min) (kg O3 , m
3
rice ) ozone) Control*
Inferior Central Superior

Fungi colony county 0 0 91.8 ± 7.7 96.8 ± 5.6 92.7 ± 7.5 93.8 ± 16.7 NS
CFU $ g
1 (  102) 30 0.393 83.5 ± 7.2 4.6 ± 1.7 22.0 ± 7.5 54.0 ± 9.0 P < 0.001
90 1.180 89.2 ± 8.5 3.6 ± 1.5 14.7 ± 6.4 51.0 ± 8.9 P < 0.001
180 2.261 89.3 ± 12.2 0 5.3 ± 2.0 17.2 ± 2.5 P < 0.001
y
Moisture content (%) 0 0 11.8 ± 0.1 11.7 ± 0.2 11.9 ± 0.1 11.8 ± 0.3 NS
30 0.393 11.8 ± 0.2 11.8 ± 0.1 11.9 ± 0.04 11.8 ± 0.3 NS
90 1.180 12.1 ± 0.2 11.6 ± 0.1 11.7 ± 0.1 11.9 ± 0.3 NS
180 2.261 12.0 ± 0.1 11.0 ± 0.3 12.0 ± 0.3 11.7 ± 0.2 NS
Water activityy 0 0 0.68 ± 0.01 0.67 ± 0.01 0.67 ± 0.01 0.67 ± 0.01 NS
30 0.393 0.67 ± 0.01 0.67 ± 0.01 0.66 ± 0.01 0.66 ± 0.02 NS
90 1.180 0.67 ± 0.01 0.65 ± 0.003 0.66 ± 0.007 0.66 ± 0.01 NS
180 2.261 0.69 ± 0.01 0.68 ± 0.01 0.68 ± 0.01 0.68 ± 0.007 NS
Fungi Aspergillus 30 0.393 11 0 3 5 n.a.
generaz(%) Penicillium 89 6 29 59 n.a.
Aspergillus 90 1.180 10 1 3 5 n.a.
Penicillium 90 2 9 28 n.a.
Aspergillus 180 2.261 5 0 0 0 n.a.
Penicillium 95 0 5 19 n.a.

Note.*P < 0.001 ¼ statistically significant when compared to the control group by Bonferroni post-test or NS ¼ non-significant difference; yValues represented in mean ± SD; z
The values represent the percentage of growth compared to control (same line) specific for each fungal genera analyzed; n.a. ¼ not applicable.

Fig. 5. Fungal reduction (%) and steady state concentration gradient.

Table 3 The results of SDS-PAGE showed no difference in protein pat-

Carbonyl and Carboxyl content of starch extracted rice after ozone treatment for terns among control and ozone treated samples (Fig. 10). As ex-
180 min.
pected, a prominent band with a mass of around 15 kDa was
Treatment Carbonyl content (%)y Carboxyl content (%)y observed which according to Santos et al. (2013) corresponds to the
Control 0.056 ± 0.096 0.006 ± 0.013 Oryza sativa low molecular mass albumin protein (15e25 kDa).
O3 180 min 0.093 ± 0.129 0.024 ± 0.013 Regarding microstructure, in our study the ozone treatment did
Note.yResults are the means of three determinations ± standard deviations. not cause any physical alteration to the starch and whole grain, as
Data indicate no statistically significant when compared with control group P < 0.05 observed in Fig. 11.
by unpaired t-test (P ¼ 0.3052, df ¼ 8 and P ¼ 0.0974, df ¼ 8 for carbonyl and In the current study, even after 180 min of ozone exposure
carboxyl content, respectively).
reduction on germination capacity was not observed (F ¼ 0.4954,
df ¼ 15, P ¼ 0.6922), as showed in Fig. 12. In contrast, the ozone
exposure significantly change the growth of coleoptile and seminal
results showed no significant differences between the control and
root (F ¼ 13.18, df ¼ 3, 24, P < 0.0001), with a significant decrease
the ozone treated samples (Fig. 9).
8 G.D. Savi et al. / Journal of Stored Products Research 87 (2020) 101605
Fig. 6. X-ray diffraction of starch extracted rice prior and after the ozone treatment.
Fig. 7. FT-IR of rice starch prior to and after the ozone treatment.
Fig. 8. DSC/TG of rice starch prior and after the ozone treatment.
Fig. 9. Lipid oxidation of rice grain after ozone treatment for 180 min (data are shown
as mM TBARS $ g
1 of rice grain). The results were not statistically significant when
compared to the control (without ozone) by unpaired t-test (P ¼ 0.7007, df ¼ 13).
Fig. 10. SDS-PAGE profile of total protein patterns in rice grain, showing M: molecular
weight marker; B: blank sample; C: control (without ozone) and T: ozone treated.
(P < 0.05 and P < 0.001, respectively) after 180 min of ozone,
showing that the rice seed quality may be affected with a longer
ozone exposure.
4. Discussion
4.1. Ozone spread in bench scale silo
The compatibility of the model was with the experimental data
shows that approach with the first order kinetics allows repre-
senting the ozone depletion. In addition, in a recent work (Gomes
et al., 2019) demonstrated a first order coefficient for ozone
decomposition in a soybean silo. In this case, a coefficient of
1.33  10
2 s
1 was found. Also noteworthy, is that the coefficient
kpm is 10 times greater than ksd (ozone self-decomposition), indi-
cating that the interaction with the rice bed increases the ozone
decomposition rate significantly when compared to an inert bed.
 G.D. Savi et al. / Journal of Stored Products Research 87 (2020) 101605
Fig. 11. SEM images of microstructure of rice grain of the A) external and B) internal surface of the pericarp and C) morphology of rice starch in 1) control and 2) ozone treated
samples.
The consideration of the kinetic demand is valid because the
elimination of microorganisms occurs after the extinction of the
finite ozone demand. Thus, the coefficient kpm is suitable for rep-
resenting the ozone depletion at a steady state.
Beyond the afore mentioned aspects, it is important highlight
the profile of ozone as an indication that the microbiological
elimination is different along the silo, and the strategy of insertion
by the bottom of the silo should lead to a greater elimination of
fungi from the bottom up. It is evident that there is a need for
multiple injections of ozone at different positions throughout the
silo to homogenize the ozone spreading. Another strategy may
include continuous bed stirring up.
4.2. Mycological analysis after ozone treatment
A high fungal amount in unprocessed rice can lead to low quality
of sub-products and significant economic losses. The presence of
toxigenic fungi species can be indicative of potential mycotoxin
accumulation. The results of the present study are according to Savi
et al. (2018) that found in rice post-harvest after natural and arti-
ficial drying a total fungi load of 1.1  103 ± 1.2  103 CFU g
1 and
6  102 ± 1.2  103 CFU g
1, respectively. Previous studies describe
that in pre-harvest, in freshly harvested rice, the fungi load is
higher reading to index of 104 CFU g
1. It is likely that these values
were found due to higher mc and aw found in the pre-harvest
samples (reaching to approximately 20% of mc and 0.9 of aw)
when compared with the post-harvested samples (mc ¼ 13% and
9
aw ¼ 0.7) (Beber-Rodrigues and Scussel, 2013; Savi et al., 2018). Our
findings are according to values of mc 12% and aw 0.7, as expected
for stored rice. Despite this, the presence of fungi filamentous in
food may cause food deterioration, loss of seed germination,
alteration in nutritional value, apart from that some species can be
toxigenic and produce mycotoxin in rice. Therefore, the ozone has
been used as a strong treatment in various foods (Piacentini et al.,
2017; Savi et al., 2014; Savi and Scussel, 2014; Tiwari et al., 2010;
Zhu, 2018) and usually the treatment has shown favorable results in
the grain storage.
The stronger ozone capacities in inhibition of fungi growth were
achieved when longer exposure times were applied (180 min),
however the fungal reduction in the grain of the IP occurs faster,
because it is where the concentration of ozone is higher. The ozone
has a great potential for inactivating fungi genera commonly found
in rice, such as Aspergillus, Penicillium and Fusarium (Savi and
Scussel, 2014). The current study present results according to
Beber-Rodrigues et al. (2015) where Penicillium was the fungi
genera most resistant to ozone treatment in the rice (Table 2). In the
study of White et al. (2013), evaluating high-moisture maize
treated with ozone, the Aspergillus, Fusarium and Mucor were less
resistant to the effects of ozone when compared to Penicillium and
Rhizopus (Beber-Rodrigues et al., 2015).
Some fungal species are mycotoxigenic and already were
described in rice (Ferre, 2016). For example, the toxigenic Asper-
gillus sp. may produce aflatoxin B1, while Penicillium sp. has been
responsible for the production of citrinin. These mycotoxins have
10 G.D. Savi et al. / Journal of Stored Products Research 87 (2020) 101605
Fig. 12. A) Germination and B) Length (cm) of coleoptile and seminal root of rice seed
after ozone treatment in different times of ozone exposure. Symbols indicate statis-
tically significant when compared with the control group (*P < 0.05; ***P < 0.001) by
Bonferroni post-test.
carcinogenic, mutagenic and teratogenic effects on humans and
animal health (Gupta et al., 2018; IARC, 1993). Therefore, the
inactivation of Aspergillus and Penicillium genera with ozone rep-
resents a greatest impact on improving the storability of rice grain
in order to avoid the presence of these toxigenic fungi.
The fungi reduction is related to oxidation reactions of ozone
with fungi. Ozone and related chemical from its degradation may
react with spore coats, nucleic material and microbial intracellular
enzymes, leading to fungi reduction (Khadre et al., 2001). The
ozone also may induce oxidative stress due to reactive oxygen
species affecting the fungi cell development and lead to fungi
mortality (Savi and Scussel, 2014).
In the current study, the ozone treatment inhibited the fungi
growth in the rice samples, including potential toxigenic fungi
genera, reaching to total reduction in some portions within the silo,
showing to be a promising agent to maintain the quality of rice
storage.
4.3. Grain quality after ozone treatment
The ozone interaction with the grain composition and its
properties has been studied in order to evaluate if the effective
concentration used as antifungal may cause some adverse alter-
ation in the rice grain. The major component of rice is the starch
with approximately 90% of dry matter (Walter et al., 2008). Due to
this it is necessary to study the effects of ozone oxidation on
degradation of starch molecules. The ozone oxidizes the hydroxyl
groups of starch to carbonyl and carboxyl content, and can to cause
also alteration on the crystallinity and morphology. The increase in
levels of damaged starch contribute to the decrease in quality of
grains (Zhu, 2018).
In some studies, although the values of carboxyl content were
low, the grain treated with ozone exhibited higher values than the
control samples (without ozone). In the study of Goze
et al. (2016)
the higher value found was of 0.045% ± 0.034% in wheat starch after
ozone treatment. The same occurred with the findings of Savi et al.
(2014) where the carboxyl content reached 0.079% ± 0.045% after
high ozone exposure. According to our results, no significant dif-
ference was evidenced even after 180 min of ozone exposure.
The effects of ozone on the crystallinity changes during the
oxidation process may occur in the amylase and amylopectin chain
of starch and also cause alteration on their structural composition.
Our results are according to other results which no significant
difference was observed on the crystallinity of the starch granules
after the ozone treatment (Castanha et al., 2017; Sandhu et al.,
2012; Savi et al., 2014). Moreover, the thermal analysis by DSC/TG
also shown that the ozone treatment no change any property of
thermal characteristic of the starch. The results obtained in the
present study could be attributed to fact that the ozone is applied in
the silos when the storage grain still has husk, which provide a
strong protection for starch, not allowing the effects of ozone
oxidation to occur (Goze
et al., 2016).
The high potential of ozone oxidation can react with the double-
bonds associated with unsaturated lipids, forming free radicals
which may cause onset of rancidity in food. These effects were
already observed in wheat flour and corn treated with ozone (Qi
et al., 2016; Sandhu et al., 2011). Peroxide values of lipids extrac-
ted from wheat flour increased after 4.5 min of ozone exposure
(1500 mg kg
1, gas flow rate 2.5 L min
1) (Sandhu et al., 2011). In
corn, Qi et al. (2016) found that ozone treatment increased the fatty
acid values significantly (P < 0.05) after 180 min of exposure at
100 mg L
1. The results found in the present study could be due to
the matrix effect of rice that has lesser values of lipids in its
nutrition composition than other grains and therefore no suffer
significant oxidation.
Regarding to protein modifications, in contrast to Jing et al.
(2016) that showed a reduction of albumin content with a pro-
longed ozone exposure (100 ppb, for 8 h a day, during 66 days), the
findings showed that our ozone treatment (single-exposure) did
not affect the protein pattern content even after 180 min exposure
(Santos et al., 2013).
Moreover, the husk of rice grain did not show visible alterations
after ozone treatment and due to surface hardness, covering and
protection whole rice grain, the internal surface of grain also did
not suffer any change. The same may to be observed in the starch
grain, where there was no apparent damage after the ozone
treatment. Despite the ozone treatment in general not having an
effect on the morphology of cereals, the starch may often suffer
alterations. In study of Castanha et al. (2017), modification of potato
starch was observed using the ozone treatment (concentration of
47 mg L
1, gas flow 0.5 L min
1). After 30 min of ozone exposure,
the starch granules presented irregular shapes and some fissures in
_
its surface. Similar results also were related to Çatal and Ibano lu
g
(2012) when the corn and potato starch were evaluated after
ozone treatment (at a rate of 60 g h
1 for 1 h).
With respect to rice seed germination, the effects of ozone
depend on the applied ozone dose as well as exposure time and
also on the integrity of the grain. Savi et al. (2014) found that ozone
treatment at 3 h reduced the capacity by 12% of wheat grains
germination without affecting the coleoptile and seminal root of
grain. During the seed germination, two primary structures grow
and elongate: the coleoptile (protective covering enveloping the
shoot) and radicle (first root). Appearance of both loosely defines
the completion of germination. After germination, the coleoptile
and seminal root continue to grow and develop (Dunand and
Saichuk, 2015). The ozone treatment showed significant inhibi-
tion of the coleoptile (P < 0.05) and seminal root (P < 0.001)
structure in higher exposure time and it can lead to damage of plant
development. In this case, the treated grain with ozone could
 G.D. Savi et al. / Journal of Stored Products Research 87 (2020) 101605
present low quality in terms of germination.
5. Conclusion
The effect of ozone treatment on rice grain storage within an
experimental silo appear as a safe alternative to act as an antifungal
agent. The ozone was able to promote the inhibition of filamentous
fungi development including the possible toxigenic strain of Peni-
cillium sp. and Aspergillus sp. However, the efficiency of ozone
throughout the silo is different in relation to fungi growth reduc-
tion. This highlights the importance of adopting strategies to ho-
mogenize the ozone spread. The present study showed the
simulation of ozone propagation within the silo, which allows a
better understanding of fungal reduction in rice storage. The higher
ozone exposure caused total fungi reduction, and even with it, a
major alteration in grain quality attributes was not caused. The
ozone treatment deserves special attention to its application in rice
storage silos in the food industry in order to maintain quality grains
and to avoid the harmful effects cause by toxigenic fungi.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
CRediT authorship contribution statement
Geovana D. Savi: Conceptualization, Methodology, Formal
analysis, Investigation, Writing - original draft, Writing - review &
editing, Supervision, Project administration. Thauan Gomes:
Conceptualization, Methodology, Formal analysis, Investigation,
Writing - original draft, Writing - review & editing. Sílvia B. Can-
ever: Methodology, Investigation, Writing - original draft. Ana C.
Feltrin: Methodology, Investigation, Writing - original draft. Karim
C. Piacentini: Investigation, Writing - original draft, Writing - re-
view & editing. Rahisa Scussel: Methodology, Investigation,
Writing - original draft. Daysiane Oliveira: Methodology, Investi-
gation, Writing - original draft. Ricardo A. Machado-de-Avila:

Resources, Writing - original draft, Writing - review & editing.
Maykon Cargnin: Methodology, Investigation, Writing - original
draft. Elidio Angioletto: Conceptualization, Resources, Writing -
review & editing, Supervision, Project administration, Funding
acquisition.
Acknowledgments
The authors thank the MULTILAB-UNESC (Multiuser Laboratory)

gico
and the Laboratories of the Iparque-Parque Científico e Tecnolo
from the Universidade do Extremo Sul Catarinense. The Coordina-
tion for the Improvement of Higher Education Personnel (CAPES)
by financial research support with CAPES Thesis Award, 2015 edi-
tion, granted to Geovana D. Savi.
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