LWT - Food Science and Technology 61 (2015) 516e523

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LWT - Food Science and Technology

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

Dissipation of chlorpyrifos-methyl by Saccharomyces cerevisiae during

wheat fermentation
c*, Rada D. Ðurovi

Tijana M. ÐorCevi
c-Pej
cev
Institute of Pesticides and Environmental Protection, Belgrade, Serbia

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

Article history: The dissipation of chlorpyrifos-methyl during wheat fermentation by Saccharomyces cerevisiae was
Received 6 June 2014 investigated. The effect of pesticide on the yeast growth, and on the efficiency of wheat fermentation,
Received in revised form was studied. Obtained results indicate that growth of yeast in medium was significantly inhibited by
21 October 2014
chlorpyrifos-methyl applied in higher than 5MRL concentration (growth inhibition 53%), reaching in-
Accepted 19 December 2014
hibition of 88.3 and 90.1% at the two highest doses (15MRL, 20MRL). The presence of chlorpyrifos-methyl
Available online 31 December 2014
in wheat, overall negatively affected the yeast fermentation expressed through pH alteration, number of
yeast cells mg
1 and CO2 production. Further in order to evaluate chlorpyrifos-methyl dissipation during
Keywords:
Pesticides
preparation of wheat substrate, the effect of sterilization on reduction of pesticide in wheat was
Dissipation determinate. Dissipation of pesticide was extremely high at all fortification levels (MRL, 5MRL and
Fermentation 15MRL), reaching 79.4, 79.2 ad 78.8% respectively. During the incubation period in samples without yeast
Yeast there was no further reduction of the chlorpyrifos-methyl with time on different temperatures. Finally,
Wheat activity of yeast itself caused pesticide reduction for approx. 14.8, 18.8 and 19% respectively, without
significant differences regarding fortification levels, or variations of incubation parameters. Overall,
maximum reduction of approximately 94.2, 98.0 and 97.8% at three fortification levels respectively was
reached during fermentation.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction undesirable residues can remain on grains and can be transferred to

Consumption of wheat flour and its products is increasing
everyday throughout the world, leading to tremendous increment
of wheat crop cultivation and production. In order to obtain good
yield and satisfactory harvest with minimal lost during storage,
intensive usage of pesticides is required. Nowadays, in many major
wheat-producing countries a large proportion of stored cereal grain
is protected against insect attack by numerous residual contact
insecticides, and active substances with a long persistence of
insecticidal activity, such as organophosphates, are most
commonly used for this purpose (Arthur, 1996).
The control of pesticide residues in wheat grain is generally
based on MRLs (Maximum Residual Limits). Although the correct
use of pesticides does not cause problems of public concern in
health and environmental areas, if inappropriate abusive treat-
ments are applied without respecting safety recommendations,
* Corresponding author. Banatska 31b, 11080 Belgrade, Serbia. Tel./fax: þ381
113076133.
E-mail address: tijana.djordjevic@hotmail.com (T.M. ÐorCevi

c).
cereal food products. In presence there are increasing concerns over
the use of organophosphorus insecticides linked to health, as those
are the main residues detected in wheat (Balinova, Mladenova, &
Obretenchev, 2006). One of the most commonly used organo-
phosphate is chlorpyrifos-methyl(O,O-dimethylO-(3,5,6-trichloro-
2-pyridyl)phosphorothioate), a nonsystemic, broad-spectrum
insecticide with contact, stomach, and respiratory action (Tomlin,
2003), listed as a product unlikely to present acute toxicity in the
WHO's recommended classification of pesticides by hazard (WHO,
1997). The main degradation pathway of chlorpyrifos-methyl is
hydrolysis to 3,5,6-trichloro-2-pyridinol, while degradation in-
volves both chemical hydrolysis and microbial activity.
As microbial degradation is considered to be one of the most
significant process determining the fate of chlorpyrifos-methyl and
other organophosphates in nature (Lu, Wu, Newman, Faber, & Gan,
2006; Yang, Liu, Guo, & Qiao, 2006; ), and in the context of recent
increased interest in obtaining better information about the
pesticide residue levels in “processed food”, it is toxicologically
interesting to study the potential loss of pesticide during fermen-
tation in order to obtain information about the real intake of

http://dx.doi.org/10.1016/j.lwt.2014.12.044
0023-6438/© 2014 Elsevier Ltd. All rights reserved.
 T.M. ÐorCevi
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pesticides. Numerous publications already indicated that various
food processing techniques can significantly reduce the pesticide
residues in foods, and this was extensively reviewed by Kaushik,
Satya, and Naik (2009). These authors established that there was
reduction of pesticide residue levels due to processing techniques
in most of the food materials, including whole wheat grain, wheat
and wheat flour. Although fermentation is one of the oldest simple
biotechnological process and reduction of pesticide residues during
fermentation has been continuously studied in different food
commodities (Bo & Zhao 2010; Jung et al., 2009; Navarro et al.,
2007), there is very few data regarding pesticide residues dissipa-
tion during cereal fermentation process.
Therefore, the objective of the present study was to investigate
the dissipation of chlorpyrifos-methyl during wheat fermentation
by baker's yeast as one of the key baking ingredient especially in
homemade baking, but also in industrialized processes. In the
experiment the fermentation conditions were optimized according
to RSM (Response Surface Methodology) in order to obtain the
highest pesticide dissipation level. The effect of chlorpyrifos-
methyl on the yeast growth, and further on the efficiency of
wheat fermentation, was firstly studied in order to determine the
fermentation rate in samples contaminated with pesticides.
Spelt wheat (Triticum spelta L.) used in this study is an alterna-
tive culture of major cereal grain cultivated throughout the world. It
shows a very good adaptability (Laghetti, Piergiovanni, Volpe, &
Perrino, 1999), and, even with low fertilizing, spelt wheat gives a
good harvest with significant mineral uptake (Moudrý & Dvora

cek,
1999). As the nutritive value of spelt wheat is high and it contains
all basic components which are necessary for human beings, it is
not surprising that this culture with undemanding growing re-
quirements is nowadays more grown in the European countries.
Currently it is widely used for fermented food products such as
pasta products, muesli and flakes.
As cereal grains are naturally contaminated by moulds, yeasts
and bacteria (Russo, Beleggia, Ferrer, Pardo, & Spano, 2010), mi-
croorganisms of grains might be found in products made with flour
such as yeast-raised dough. Since during bakery products prepa-
ration flour is not subjected to sterilization, the occurrence and
number of certain types of microorganisms may vary significantly
in dependence on a combination of available substrates and specific
technological parameters. In this study milled wheat samples were
sterilized in an autoclave prior inoculation to avoid contamination
with wild strains in order to determine the individual effect of used
fermentative microorganism strain (Saccharomyces cerevisiae) on
chlorpyrifos-methyl residues. Concerning possible degradation of
different pesticides during sterilization, pasteurization, autoclaving
and boiling (Abou-Arab, 1999; Kontou, Tsipi, & Tzia, 2004; Lalah &
Wandiga, 2002), the effect of applied thermal treatment by steril-
ization on chlorpyrifos-methyl residues in wheat was as well
investigated.
2. Materials and methods
2.1. Starter culture and growth conditions
A yeast strain e S. cerevisiae (collection of the Laboratory of
Microbiology, Faculty of Technology and Metallurgy, Belgrade,
Serbia) used in the study was maintained at 4e6  C on YPD broth
(Torlak Institute, Belgrade, Serbia). When used for wheat fermen-
tation, cells were incubated 5 h in YPD broth at 30  C (cell density of
9 log cfu mL
1, cells diluted 1:10 gave an O.D.620nm of ca. 0.069)
harvested by centrifugation on 9580 g for 10 min at 4  C (Velocity
14R, Dynamica, Salzburg-Mayrwies, Austria), washed with
50 mmol L
1 phosphate buffer pH 7.0 (SigmaeAldrich Chemie
517
GmbH, Taufkirchen, Germany), and re-suspended in sterile distilled
water to its original volume.
2.2. Analytical standard and working solutions
Stock solution (2.0 mg mL
1) of analytical standard of
chlorpyrifos-methyl (Dow Agro Sciences, Indianapolis, Indiana,
USA, purity 99.9%) in acetone was stored at
18  C and working
solutions were prepared daily by diluting with sterile distilled
water.
Acetone, methanol, ethyl-acetate and anhydrous sodium sulfate
(99.0%), were purchased from J.T. Baker (Deventer, Holland), while
florisil (60e100 mesh) was purchased from Serva Electrophoresis
GmbH (Heidelberg, Germany). Before use, sodium sulfate was dried
24 h at 130  C and florisil activated 4 h at 600  C and then 5 h at
130  C.
2.3. Effect of chlorpyrifos-methyl on S. cerevisiae growth
Cells of yeast were incubated 5 h on 30  C in YPD broth fortified
with pesticide (at 1.5, 3, 6, 15, 30, 45 and 60 mg mL
1 rates i.e.
1
/2MRL, MRL, 2MRL, 5MRL, 10MRL, 15MRL and 20MRL for
chlorpyrifos-methyl in wheat), and growth was determined
directly by plating on YPD agar (Torlak Institute, Belgrade, Serbia)
and indirectly by measuring the O.D.620nm (UV/visible spectro-
photometer LKB Biochrom Novacpec II, Biochrom Ltd., Cambridge,
UK).
2.4. Effect of chlorpyrifos-methyl on efficiency of wheat
fermentation
Wheat mash used as a substrate in this study was prepared
from uncontaminated grain Triticum spelta manufactured in
organic production by Jevti
c farm, Backo Gradiste, Serbia. Wheat
grains were milled, fortified at 0, 3, 15 and 45 mg kg
1 rates of
chlorpyrifos-methyl (i.e. MRL, 5MRL and 15MRL for wheat) and
homogenized for 24 h. Spiked samples were introduced to in-
dividual Pyrex bottles (100 mL) (Corning B.V. Life Sciences,
Amsterdam, The Netherlands) loosely sealed with screw caps and
subjected to sterilization in an autoclave for 15 min at 121  C
(103 kPa) (Sanyo Lab Autoclav MLS 3780, Sanio Electric Co, Ltd.,
Osaka, Japan). After cooling, slurry was prepared by adding
distilled water (1:1). Previously prepared cultures of S. cerevisiae
were inoculated individually at 8% (v/w) levels (final cell number
in the substrates of ca. 8 log cfu g
1). Fermentations were carried
out at 23, 30 and 37  C for 24, 48 and 72 h (FOC 225l Incubator,
Velp Scientifica, Usmate, Italy), and samples were analyzed for
fermentation efficiency. The following analyses were made: col-
ony count (yeast cells mg
1), determination of CO2 production
and pH measurements. S. cerevisiae cells present in fermented
wheat mash were enumerated using YPD agar. 10 g of fermented
slurry was mixed with 90 mL sterile distilled water and ho-
mogenized for 30 min by horizontal shaker (IKA Werke HS 501
Digital, Staufen, Germany). Further dilutions were made and pour
plated in sterilized YPD agar plates, incubated at 30  C for 48 h
and the colonies were counted using a colony counter (Scan 100
Manual Colony Counter, Interscience, Saint Nom, France). Carbon
dioxide production i.e. mass of CO2 lost was measured directly by
weighing the fermentation vessels during incubations. For the
measurement of pH InoLab 730 pH meter (WTW, Weilheim,
Germany) was used.
518 T.M. ÐorCevi
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2.5. Effect of yeast fermentation on chlorpyrifos-methyl dissipation
in wheat
Wheat mash was prepared as described previously (2.4.), with
fortifications at 3, 15 and 45 mg kg
1 of chlorpyrifos-methyl. After
sterilization in an autoclave samples were distributed in three
groups.
First group of samples was analyzed for chlorpyrifos-methyl
residue immediately after autoclaving to check the effect of steril-
ization on pesticide dissipation.
From second group of sterilized samples, slurry was prepared
with distilled water (1:1), and cultures of S. cerevisiae were inocu-
lated at 6, 8 and 10% (v/w) levels. Fermentation was carried out at
23, 30 and 37  C for 24, 48 and 72 h and samples were analyzed for
chlorpyrifos-methyl residue.
The third group of sterilized samples, a control substrate, was
held under the same conditions as previously prepared for
fermentation (on 23, 30 and 37  C for 24, 48 and 72 h), but without
starter, to check pesticide spontaneous chemical degradation.
2.6. Sample preparations for pesticide analysis
For chlorpyrifos-methyl residue GCeMS analyses, samples were
prepared according analysis procedure described in our previous
 ability (growth inhibition of 53%) with a dramatic reduction of
work (ÐorCevi
c, Siler-Marinkovi

c, Ðurovi
c-Pej
cev, Dimitrijevi

c-
Brankovi
c, & Gaji
c-Umiljendi
c, 2013a, 2013b). For recovery assay
uncontaminated wheat grain and fermented wheat samples were
fortified at 0.6, 3, 15 and 45 mg kg
1 rates of chlorpyrifos-methyl
prior analysis procedure.
2.7. Instrumetation
A gas chromatograph-mass spectrometer (GC/MS) (CP-3800/
Saturn 2200, Varian, Melbourne, Australia) with
30 m  0.25 mm  0.25 mm, VF-5ms column (Varian, Melbourne,
Australia) was used as a detection device The GC was programmed as
follows: initial temperature was 170  C, then increased to 260  C at
9  C min
1 and held for 3.5 min. The carrier gas (helium, 99.999%)
flow rate was in constant flow mode at 1.1 mL min
1. The ion trap
mass spectrometer operated in the electron impact/selected ion
monitoring (EI/SIM) mode. Ion (m/z) 286 was used for quantifica-
tion, while ion (m/z) 125 was used for confirmation. The ion trap and
transferline temperatures were set to 210  C and 250  C.
Fig. 1. Effect of chlorpyrifos-methyl on S. cerevisiae growth in YPD broth presented as (a) reduction of CFU mL
1 and O.D.620 nm, and (b) percentage of growth parameters in-
hibition. Data are expressed as the mean ± SEM (n ¼ 3). (Legend: C log10 CFU ml
1, , O.D.620nm).
2.8. Statistical analysis
All experiments were performed in triplicates. Statistical anal-
ysis of data from determination of effect of chlorpyrifos-methyl on
S. cerevisiae growth and effect of pesticide on fermentation effi-
ciency were processed by Statistica 8.0 (StatSoft Inc., Tulsa, Okla-
homa, USA). The whole RSM procedure i.e. data regarding
optimization of fermentation conditions in order to obtain the
highest pesticide dissipation level, were analyzed using ReliaSoft's
DOEþþ software (ReliaSoft Corporation, USA).
3. Results and discussion
3.1. Effect of chlorpyrifos-methyl on S. cerevisiae growth
The effect of pesticide on the growth rate of S. cerevisiae is
presented on Fig. 1. The results indicate that yeast growth was not
significantly inhibited by chlorpyrifos-methyl applied in less than
5MRL concentration. Although low inhibition of growth did start on
½MRL concentration and in the presence of MRL and 2MRL
remained on slightly elevated level (12.4e21.2% of inhibition), no
relevant differences in comparison to control were found for those
three lowest pesticide concentrations. At 5MRL concentration of
chlorpyrifos-methyl cell stress was reflected on colony-forming
CFU/mL following pesticide application at the two highest doses
(growth inhibition of 88.3 and 90.1%). As contamination with
chlorpyrifos-methyl at concentration five time higher than
permitted cause reduction of yeast cell number (CFU) to approx.
50%, while concentration twenty-fold higher than MRL has an
extremely negative effect on S. cerevisiae, it could be expected for
the efficiency of wheat fermentation to significantly decrease in the
presence of higher contamination with chlorpyrifos-methyl.
It is known that the occurrence and growth of yeasts during
fermentation is influenced by many factors, including pesticides
residues (Fleet, 2003). As expected, many fungicides were shown to
have caused a inhibition of S. cerevisiae growth (Bi Fai & Grant,
2009; Cu s & Raspor, 2008; Santos, Simo ~ es, & Sa

-Correia, 2009;
Shin, Kim, Park, Kim, & Rhee, 2003), although there are in-
vestigations which showed that residues of some did not influence
the course and rate of yeast growth (Cabras et al., 1999; Chaves

pez et al., 2004; Jawich, Lteif, Pfohl-Leszkowicz, & Strehaiano,
Lo
2006), as in all cases growth resumptions, with an increasing dose-
dependent period of latency, were observed (Santos et al., 2009).
 T.M. ÐorCevi
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c-Pejcev / LWT - Food Science and Technology 61 (2015) 516e523 519

Current literature also provides a discrete number of studies
reporting the effects of herbicides on yeast biological parameters. It
was stated that herbicide treatments were able to affect yeast
growth by affecting the enzymatic activity of catalase and super-
oxide dismutase (SOD), as well as inducing oxidative modifications
of proteins (Braconi et al., 2006; 2008). Finally, it is also evident
that, although presence of insecticides malathion, chlorpyriphos
and deltamethrin in media did not show considerable affect on
yeast growth, inhibition of yeast growth was however observed in
samples containing insecticide endosulfan (Sharma, Satya, Kumar,
& Tewary, 2005). Results obtained in this study are confirmation
of how different pesticides could impact specific micro biodiversity
including food interest microorganisms like S. cerevisiae, resulting
in lowering of viable counts in growth mediums contaminated with
those agrochemicals.
Regarding obtained results, three chlorpyrifos-methyl concen-
trations (3 mg kg
1 (MRL), 15 mg kg
1 (5MRL) and 45 mg kg
1
(15MRL)) were further used for wheat fortification in order to
evaluate influence of pesticide on fermentation and effect of
fermentation on chlorpyrifos-methyl dissipation.

Fig. 2. Effect of chlorpyrifos-methyl on fermentation activity of S. cerevisiae in wheat substrate. Data are expressed as the mean ± SEM (n ¼ 3). (Legend: C 0 mg kg
1 of
chlorpyrifos-methyl, B 3 mg kg
1 of chlorpyrifos-methyl, A 15 mg kg
1 of chlorpyrifos-methyl, D 45 mg kg
1 of chlorpyrifos-methyl).
520 T.M. ÐorCevi
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3.2. Effect of chlorpyrifos-methyl on efficiency of wheat
fermentation
Results presented on Fig. 2 show that the presence of
chlorpyrifos-methyl in all three applied concentrations, overall
negatively affected the yeast fermentation in wheat by S. cerevisiae.
Obtained P-values (ANOVA, a ¼ 0.01) of 1.68*10
7 for pH, 1.10*10
10
for CFU and 1.60*10
7 for CO2 production, for fortification at
different concentration levels (from 0 to 45 mg kg
1) indicated that
this insecticid had significant effect on all fermentation responses.
However, although contamination on all three concentration rates
caused significant decrease in the number of cells per g of
fermentation substrate, this was least expressed in presence of the
smallest used amount of pesticide i.e. at MRL level (3 mg kg
1), thus
pH lowering in those samples, especially after 48 and 72 h of
fermentation, and CO2 production, especially after 24 h of
fermentation, were not significantly negatively affected. Further
fortification with higher concentrations leaded to more expressive
reduction in CFU number, as well as less pH alterations and less
carbon dioxide production.
There is a lack of publications for comparison of obtained re-
sults, as most of researches concerning impacts of pesticides on
yeast fermentation are focused on influence of fungicides on
fermentation of grape must. Due to extensive use of fungicides in
the treatment of several fungi-caused diseases of grapes for vini-
fication, numerous studies are focused on the connection between
fungicide residues and stuck and sluggish alcoholic and malolactic
 s &
fermentations (Angioni et al., 2005; Cabras et al., 1999, 2000; Cu
Raspor, 2008; Gonza
lez-Rodríguez, Cancho-Grande, Torrado-Agra-
sar, Simal-G
andara, & Mazaira-Pe
rez, 2009; Ruediger, Pardon, Sas,
Godden, & Pollnitz, 2005) and therefore the negative effect on the
aromatic composition of wines (García et al., 2004; Oliva, Navarro,
Barba, Navarro, & Salinas, 1999). Unexpectedly, in most, if not all,
cases, fungicide residues were found to have little or no effect on
yeast fermentation through the winemaking process. And while
most of authors were focused on influence of fungicides at MRL
concentration and/or recommended application rate on fermen-
tation of grape must (Cabras et al., 2000; Gonza
lez-Rodríguez et al.,
2009; Ruediger et al., 2005), others tested impact of higher con-
centrations, up to 1.5 and 5 MRL (Cabras et al., 1999; Cu  s &
Raspor, 2008), and still obtained similar results with lack of nega-
tive effect on fermentation. Concerning that, and the results ob-
tained in this study, it could be concluded that various yeast species
response differently in presence of different amounts of various
pesticides, especially in diverse fermentation substrates, thus those
interactions should be always tested prior further experiments
dealing with dissipation of pesticides during yeast fermentation.
3.3. Effect of yeast fermentation on chlorpyrifos-methyl dissipation
in wheat
With described analysis procedure, developed in our previous
work (Ðurovi
c & ÐorCevi
c, 2010), and confirmed for efficiency for
organophosphate pesticide extraction from wheat substrate altered
by fermentation (ÐorCevi
c et al., 2013a, 2013b), chlorpyrifos-
methyl was efficiently extracted from fermented and unfer-
mented samples and measured accurately. Recoveries at four
fortification levels (0.6, 3, 15 and 45 mg kg
1) were for unfermented
wheat samples 84.3, 89.8, 85.3 and 84.4% with good reproducibility
i.e. RSD% of 7.5, 1.9, 5.2 and 0.2%, and for fermented wheat samples
86.9, 88.2, 82.8 and 85.3% with RSD% of 6.1, 4.6, 2.7 and 2.5%. The
limit of detection (LOD) and quantification (LOQ) were determined
according to IUPAC recommendations (Currie, 1999), and were
0.007 mg kg
1 and 0.04 mg kg
1, respectively, thus it is obvious that
the presented method is sensitive enough for determination of this
pesticide at concentration levels much below its MRL value.
In order to evaluate chlorpyrifos-methyl reduction during
preparation of wheat substrate, the effect of thermal processing by
sterilization on reduction of pesticide in fortified wheat samples
analyzed immediately after autoclaving was first determinate. As
can be seen from the obtained results (Fig. 3), after thermal pro-
cessing at 121  C (103 kPa) for 15 min, reduction of chlorpyrifos-
methyl was extremely high at all fortification levels, reaching
79.4, 79.2 ad 78.8% respectively for samples fortified with MRL,
5MRL and 15MRL of pesticide. Mentioned results correspond with
one obtained by Uygun, Senoz, and Koksel (2008), Uygun, Senoz,
Ozturk, and Koksel (2009) who pointed out that the rates of
degradation and volatilization of chlorpyrifos-methyl residues
were increased by the heat, and not just by extremely high ones
(205  C), but by significantly lower ones (40  C). It is obviously that,
as Holland, Hamilton, Ohlin, and Skidmore (1994) mentioned,
processes involving heat can increase volatilization, hydrolysis or
other chemical degradation and thus reduce residue levels. As
commonly the loss of pesticide residues from heated substrate
fortified with pesticides obtain through physico-chemical pro-
cesses, e.g., evaporation, co-distillation and thermal degradation
which may vary with the chemical nature of the individual pesti-
cides (Sharma et al., 2005), it is not surprising that obtained high
reduction of chlorpyrifos-methyl occurred, considering that this
pesticide have significantly high vapor pressure. Regarding results,
it could be concluded that possible chlorpyrifos-methyl contami-
nation in wheat, in amount over MRL, could be reduced nearly up to
80% by autoclaving.
Further, during the incubation periods on different tempera-
tures without yeast inoculums, there was no significant additional
reduction of the chlorpyrifos-methyl. Although the percentage of
remaining pesticide was consistently lower than the value of the
respective control (for approx. 1.5%, without significant differences
among variations of incubation parameters), the differences were
not statistically significant (t-test, P < 0.05) (Fig. 3). Obtained results
are in agreement with proved stability of chlorpyrifos-methyl in
wheat stored at ambient conditions for longer period of time after
Fig. 3. Dissipation of chlorpyrifos-methyl residues during sterilization of wheat sub-
strate and under fermentation conditions during incubation time without yeast. First
column in series e samples fortified with 3 mg kg
1 concentration of chlorpyrifos-
methyl; second column in series e samples fortified with 15 mg kg
1 concentration
of chlorpyrifos-methyl; third column in series e samples fortified with 45 mg kg
1
concentration of chlorpyrifos-methyl. According to Student's t test (P < 0.05) the dif-
ferences between dissipation during sterilization and dissipation during
sterilization þ dissipation during incubation, differences between samples fortified at
different levels, as well as differences between dissipation among variations of incu-
bation parameters, were not statistically significant. (Legend: , dissipation during
sterilization, - dissipation during incubation).
 T.M. ÐorCevi
c, R.D. Ðurovi
c-Pejcev / LWT - Food Science and Technology 61 (2015) 516e523 521

Fig. 4. Dissipation of chlorpyrifos-methyl residues during yeast fermentation of S. cerevisiae in wheat substrate. (a) samples fortified with 3 mg kg
1 concentration of chlorpyrifos-
methyl; (b) samples fortified with 15 mg kg
1 concentration of chlorpyrifos-methyl; (c) samples fortified with 45 mg kg
1 concentration of chlorpyrifos-methyl.
522 T.M. ÐorCevi
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post-harvest treatments (Fleurat-Lessard, Vidal, & Budzinski, 1998).
The half life for this insecticide was already in several early re-
searches stated to be 4e5 months on 30  C and 50% relative hu-
midity (Desmarchelier et al., 1980), which is in agreement with
later findings on the behavior of residual chlorpyrifos-methyl with
ambient temperature (Balinova et al., 2006), and somewhat higher
than half-life of 2 months measured, under conditions of elevated
humidity, by Fleurat-Lessard et al. (1998). This finding can be useful
in establishing the appropriate conditions for the laboratory stor-
age of analytical samples.
Overall, considering sterilization in autoclave and spontaneous
chemical degradation of chlorpyrifos-methyl, there was reduction
of pesticide residues of about 82% for all fortification levels, without
involving yeast fermentation.
In order to determine effect of yeast fermentation on
chlorpyrifos-methyl dissipation in wheat samples, the fermenta-
tion condition was optimized using a Box-Behnken design con-
taining three levels for each parameter toward the highest pesticide
reduction level as response. Results presented on Fig. 4 indicated
that, after sterilization, further degradation by S. cerevisiae obtained
at all three fortification level, reaching maximum reduction of
approximately 94.2, 98.0 and 97.8% respectively in samples fortified
with MRL, 5MRL and 15MRL. Considering already mentioned
reduction of pesticide residues prior fermentation (79.4, 79.2 and
78.8% respectively), it can be concluded that activity of S. cerevisiae
itself caused chlorpyrifos-methyl residue reduction in wheat sam-
ples for approx. 14.8, 18.8 and 19% respectively, without significant
differences regarding fortification levels, or differences among
variations of incubation parameters. It could be presumed that,
even though the presence of chlorpyrifos-methyl, especially at
higher concentrations, caused significant decrease in the number of
cells per g of fermentation substrate during fermentation (Fig. 2),
there were still a sufficient number of cells in all of the samples to
allow enzymatic activity which could lead to pesticide degradation.
Somewhat higher reduction rate in samples fortified with 5MRL
and 15MRL could be connected with pH dependent hydrolytic
degradation of chlorpyrifos-methyl (being more rapid at higher
pH), as in those samples presence of used amount of pesticide
reduced fermentation efficiency, hence pH of samples maintained
at higher level (Fig. 2). There are some works showing the ability of
S. cerevisiae cells to adsorb inorganic and organic contaminants as
pesticides (Aksu & Do € nmez, 2003; Gomes, Fragoso, Riger, Panek, &
Eleutherio, 2002; Goyal, Jain, & Banerjee, 2003; Razmkhab et al.,
2002), which is attributed to the cell wall polysaccharides (Ballou,
1988). Ruediger et al. (2005) also mentioned that reductions of
pesticide concentrations by microorganisms could possibly be due
to the absorption onto the bacterial cell walls, rather than chemical
or biological degradation. However, concerning that during the
extraction procedure in presented study cells of yeast remained in
wheat samples (enabling the pesticide extraction from the cell
wall), it is more likely that, in studied yeast fermentation, reduction
of chlorpyrifos-methyl occurred as a result of enzymatic activity, i.e.
biological degradation which may, as Boethling (1993) noted, either
occur immediately or only after a period of adaptation to the
chemicals. Considering that the biochemistry of degradation of all
organophosphorus compound by most of the microorganisms
seems to be identical (a structurally similar enzyme called organ-
ophosphate hydrolase or phosphotriesterase catalyzes the first step
of the degradation (Singh & Walker, 2006)), it could be presumed
that same or similar was degradation pathway of chlorpyrifos-
methyl by S. cerevisiae.
Overall, results obtained in this study are in correlation with
numerous publications concerning role of microbes in pesticide
degradation in different food commodities (Rajashekar et al., 2007;
Sharma et al., 2005). This is, surely, one more confirmation that
food processing techniques, at domestic and industrial level, can
significantly reduce the pesticide residues in food.
4. Conclusion
Although nowadays MRLs are a credible and useful means for
regulating the acceptable pesticide use, food processing studies are
critical in understanding the real dietary consumption of pesticides.
The present study demonstrated high reduction of chlorpyrifos-
methyl in wheat during sterilization applied prior fermentation
processes, and during fermentation by S. cerevisiae. Reduction of
chlorpyrifos-methyl in wheat was highly impacted by elevated
temperature during sterilization, thus it could be concluded that
this pesticide is considerably unstable during thermal processing
for 15 min up to 120  C, and this could be effective tool for mini-
mizing the residual contamination from one of the most commonly
used storage insecticide in final product through reduction to
nearly 80%. Further, yeast fermentation was responsible for addi-
tional reduction of chlorpyrifos-methyl enhancing dissipation for
approximately 14e19%, thus yeast fermentation itself without
heating could be tool for minor lowering of the residual pesticide
contamination in final product.
Although food processing techniques seems to offer a suitable
means to tackle the current scenario of unsafe food, based on
presented results and due to real health risk in case of exposure to
chlorpyrifos-methyl at higher dosage levels, the limits established
for wheat are adequate and changing regulatory guidelines and
recommendations for MRL is not recommended.
Acknowledgments
This study was carried out as a part of the project No TR31043,
supported by the Ministry of Education and Science of the Republic
of Serbia.
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