Received: 7 May 2018 Revised: 5 October 2018 Accepted: 26 October 2018

DOI: 10.1111/jfs.12580

ORIGINAL ARTICLE

Deoxynivalenol screening in wheat-derived products in Gran

Asunción, Paraguay
Andrea Alejandra Arrúa Alvarenga1 | Juliana Moura Mendes Arrua2 |
3 1
Cinthia Carolina Cazal Martínez | Pablo David Arrúa Alvarenga |
1
Danilo Fernández Ríos | Pastor Enmanuel Pérez Estigarribia4 | Man Mohan Kohli3

1
Universidad Nacional de Asuncion, San
Lorenzo, Paraguay Abstract
2
Centro Multidisciplinario de Investigaciones Fusarium graminearum is one of the most important pathogens affecting wheat production in
Tecnológicas, Dirección General de Paraguay. This fungus decreases yield and grain quality; the most important consequence is the
Investigación Científica y Tecnológica,
production of mycotoxins from the trichothecene group, secondary metabolites toxic to humans
Universidad Nacional de Asunción, San
Lorenzo, Paraguay and other animals. The toxin reported in Paraguay is deoxynivalenol, which has effects on the
3
Cámara Paraguaya de Exportadores y gastrointestinal tract and immune system. To determine the presence of deoxynivalenol in flour
Comercializadores de Cereales y Oleaginosas, and baked goods, samples of wholemeal flour and white flour, white and wholemeal bread, and
Asunción, Paraguay bran crackers were carried out in the Gran Asunción Area. The presence of deoxynivalenol was
4
Facultad Politécnica, Universidad Nacional de determined by monoclonal antibodies through the use of quantitative test strips. The results
Asunción, San Lorenzo, Paraguay
were variable and below those established by international standards.
Correspondence
Andrea Alejandra Arrúa Alvarenga Universidad Practical applications
Nacional de Asunción, Centro
In the developing countries, the control of the quality and safety of food is practically nonexis-
Multidisciplinario de Investigaciones
Tecnológicas, Dirección General de tent. In Paraguay, there is no legislation on the maximum levels of deoxynivalenol in wheat and
Investigación Científica y Tecnológica. San derivatives. As this cereal and its derivative products are highly susceptible to contamination by
Lorenzo, Paraguay. Dirección postal: 1039.
Fusarium and its mycotoxins, it is a priority to initiate controls and establish bases for legislation
Email: aaarrua@gmail.com
on maximum acceptable limits in these products. Also highlight the need at country level of the
Funding information
Instituto de Biotecnología Agrícola (INBIO) application of high efficiency and sensitive techniques for quantification mycotoxins in food.

1 | I N T RO D UC T I O N A substantial problem associated with Fusarium is mycotoxins,

Wheat is the main winter agricultural commodity in Paraguay, having
reached a key position in the country's productive system, because it
contributes millions of dollars to the national economy and stubbles of
excellent quality to cover the soil as part of the zero tillage system; its
sowing in the winter is considered fundamental (Kohli, 2015). During its
cultivation, wheat is exposed to the attack of pathogenic microorgan-
isms that affect the yield, quality of the grains, and the food products
derived from them (Arrúa Alvarenga et al., 2014; Martinez et al., 2014).
One of the most significant phytopathogenic fungi currently associated
to crop in Paraguay is Fusarium sp. (Quintana de Viedma, 2004), respon-
sible for the disease called Fusarium head blight or white spike. It has
been suggested that the increase in frequency and severity of Fusarium
infections in the field is mainly due to the adoption of the zero tillage
system, as this hemibiotrophic pathogen is able to survive in crop
residues, weeds, and soil (Pereyra & Dill-Macky, 2008).
toxic secondary metabolites, responsible for syndromes that occur
when ingesting products contaminated by them called mycotoxicosis,
both in humans and in other animals (Antonissen et al., 2014; Edite
Bezerra da Rocha, Freire, Erlan Feitosa Maia, Izabel Florindo Guedes, &
Rondina, 2014; Walter, Nicholson, & Doohan, 2010). The most impor-
tant mycotoxins produced by this genus belong to the trichothecene
group, being deoxynivalenol (DON) (Leslie & Summerell, 2006;
Martinez et al., 2014). Deoxynivalenol or vomitoxin is one of the 100
trichothecenes discovered to date; it belongs to the sesquiterpene
family and it is the most common contaminant of the group associated
with grains and foods (Mugrabi de Kuppler, Steiner, Sulyok, Krska, &
Oerke, 2011). The Fusarium sp. species isolated from wheat in
Paraguay are: Fusarium graminearum, Fusarium semitectum, Fusarium
avenaceum, Fusarium equiseti and Fusarium culmorum (Arrúa Alvarenga
et al., 2014; Quintana de Viedma & Morel, 2004), Fusarium oxysporum
and Fusarium verticilloides (Quintana de Viedma & Morel, 2004). The

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https://doi.org/10.1111/jfs.12580
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predominant species is F. graminearum with a frequency of 85–90%.
Previous studies indicate that DON is the most relevant wheat myco-
toxin in Paraguay, found in 100% of the grain samples analyzed in
concentrations ranging from 0.247 to 10.13 ppm (Quintana de
Viedma, 2004). Recently, levels of between 0.31 and 1.14 ppm of
DON were reported in wheat grain from experimental lines (Arrúa
Alvarenga et al., 2014). Gran Asunción concentrates more than 10%
of the population of the country; and per-capita consumption of
cereals and derivatives is 78 kg per person in the metropolitan area,
81 kg in urban areas, and 72 kg in rural areas (DGEEC, 1998).
Due to the high consumption of these foods, the population is
exposed to the negative effects of mycotoxins, especially within a sys-
tem where the controls are nonexistent. In Paraguay, during the grow-
ing season in the winter, the optimal conditions for the development
of the fungus and the production of mycotoxins are achieved; there-
fore, it is necessary to study the levels of DON present in grains and
derived products that are sold in distribution centers in the country. In
Paraguay, there is currently no legislation regarding the maximum per-
missible DON limits in cereals and derivatives, nor any institution to
monitor the presence of this mycotoxin in foods. The aim of the pre-
sent study was to evaluate the presence of DON in wheat products
destined for human consumption in Gran Asunción, Paraguay.
2 | MATERIALS AND METHODS
2.1 | Food samples
Forty-two samples of food products destined for human consumption
were evaluated: four of integral bread, nine of white bread, three of
crackers of national production; six of white flour four-zero-type, and
11 of wholemeal flour type from distribution centers of the main cities
of the Gran Asunción. Nine samples of imported crackers were also
included. Products for export were not considered in this study.
1,000 g of white bread and whole bread were collected; a package
was taken from the crackers, and a pack of 1 kg was taken from the
flours. All samples were taken in triplicate.
2.2 | Deoxynivalenol quantification
Each sample was reduced to 15 g for the detection of DON. For the
quantification of DON, 5 g of each product per sample were milled in
duplicate. The quantification was done by means of the DON-V Test
Strips and the Vertu Lateral Flow Reader (Vicam, Milford,
TABLE 1 Levels of DON in ppm in wheat derived products for human consumption analyzed in Gran Asunción
Brown flour White flour White bread
N 33 18 27
Min 0.08 0.01 0.02
Max 1 0.235 0.55
Median 0.180 0.103 0.100
Mean 0.237 0.114 0.136
SD 0.203 0.059 0.099
Detection limit 0.2 ppm.
ARRÚA ALVARENGA ET AL.
Massachusetts, USA), per the manufacturer's instructions. Detection
limit 0.2 ppm; range of lectures 0–10 ppm (Vicam, 2011).
The experimental design was completely randomized. Mean con-
centration of DON was calculated for the different samples pro-
cessed. The normal Shapiro–Wilk test (Shapiro & Wilk, 1965) was
conducted for each type of sample studied. Nonparametric Kruskal–
Wallis variance analysis (Kruskal & Wallis, 1952) was used for the
comparison of groups with a 95% confidence interval. Mann-Whitney
pairwise analysis (Mann & Whitney, 1947) was used to generate p
values, which were corrected by sequential Bonferroni test for signifi-
cance (Brown & Rothery, 1993; Di Rienzo et al., 2011).
3 | RE SU LT S
The mean results obtained in the measurements are shown on
Table 1. Levels of DON were variable. The maximum content of DON
was present in imported bran crackers, 11.86 ppm.
Regarding the analysis of variance, significant differences were
observed in the analyzed samples in relation to the different products
and their DON contents. The white bread and domestically manufac-
tured bran crackers (U: 35 and p < .01); white bread and imported
bran crackers (U: 225 and p < .05); white bread and wholemeal flour
(U: 225 and p < .01); white flour and domestically manufactured bran
crackers (U: 27.5 and p < .01); wholemeal flour and domestically man-
ufactured bran crackers (U: 14.5 and p < .05); imported bran crackers
and wholemeal flour (U: 232.5 and p < .01); and finally, white flour
and wholemeal flour (U: 127.5 and p < .01); other comparisons not
presented significative differences. In all local products, maximum
DON levels remained below those recommended by international reg-
ulations (FAO, 2014; FDA, 2010).
4 | DI SCU SSION
In the results obtained, it can be verified that the DON content was
variable and related to the type of product studied. The imported
whole-grain crackers had the highest contamination levels with a max-
imum of 11.86 ppm on average, above international standards that
establish a maximum of 1 ppm of DON for products derived from
cereals intended for human consumption.
In the preliminary draft of maximum DON levels, the FAO Codex
Alimentarius Commission recommends the following limits: 2 mg/kg
for raw cereal grains, 1 mg/kg in semiprocessed products based on
Imported bran Domestically manufactured
crackers bran crackers
27 9
0 0
11.86 0.13
0.040 0.020
1.215 0.038
2.751 0.049
ARRÚA ALVARENGA ET AL.
wheat, maize, and barley, and 0.5 mg/kg in cereal-based products for
infants and young children (Alim, Iqbal, Selamat, & Ariño, 2016).
In a study carried out in Argentina, 156 samples of different
baked products obtained from supermarkets in Luján were studied,
and the varying levels of DON were determined by gas chromatogra-
phy. French bread was the product with the highest levels of DON,
ranging between 7 and 271 ng/g. As for bran crackers, DON values
ranged between 8 and 85 ng/kg, which were lower than the ones
obtained in this study (Pacin, Ciancio Bovier, Cano, Taglieri, &
Hernandez Pezzani, 2010).
DON is found in wheat grains due to contaminations that occur
during its cultivation, being wheat flour one of the main vehicles for
the introduction of this mycotoxin to the industry, for the most part
as basic raw material to produce various baked goods (Denardi de
Souza, Caldas, Primel, & Furlong, 2015).
In Brazil, the presence of DON, HT-2 toxin, and T-2 was studied
by HPLC-DAD in various types of bread. The presence of DON was
detected in 99% of the studied samples, while the percentages of HT-
2 and T-2 were lower—84 and 81%, respectively. In all, 22% of the
products studied presented DON levels above those established by
Brazilian regulations (Tralamazza, Bemvenuti, Zorzete, de Souza
Garcia, & Corrêa, 2016).
Also in Brazil, the industrialization process of the wheat grain and
its derived baked goods was the object of another study, which con-
cluded that the fraction with highest DON content is bran. In wheat
bran and flour, 35% of the samples analyzed showed DON levels
above the limits set by regulations instated in Brazil after the study
had been performed (Savi et al., 2016).
DON is not removed during milling, but is distributed and concen-
trated in certain fractions; the concentration is lower in fractions des-
tined for human consumption. It has been mentioned that the
variation of DON content in the different fractions is due to the diffi-
culty of the fungus to penetrate the grain, therefore the content of
the mycotoxin in the external parts is greater as the bran layer
behaves like a barrier that prevents the development of the pathogen
(Ríos et al., 2009; Wu & Wang, 2015). Similarly to what was observed
in this work, DON levels in white flour were significantly lower than
those observed in grain and bran.
Although it is the first time that this type of study is carried out in
Paraguay in products destined for human consumption, in an earlier
work carried out on wheat grain, HPLC levels of DON were detected
at levels ranging from 0.247 to 10.13 ppm (Quintana de Viedma,
2004). At present, Paraguay lacks any regulations regarding accept-
able levels of DON in flour and baked goods, but the United States
Food and Drug Administration establishes DON levels of 1 ppm for
products derived from wheat intended for human consumption and
5 ppm for products destined for animal consumption. The levels
detected are below said limit in all local products analyzed. In this
study, the presence of DON in samples of wholemeal bread, white
bread, national and imported whole meal cookies, wholemeal flour
and white flour was confirmed. It is important to consider the need
for a national legislation that establishes acceptable limits of DON in
products intended for human consumption, and the implementation
of management systems at the field level to prevent contamination
with F. graminearum and DON.
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In this study, the presence of DON in samples of wholemeal
bread, white bread, local and wholemeal flour, and white flour was
confirmed; the results were variable, and below those established by
international standards.
ACKNOWLEDGMENTS
The authors would like to thank Instituto de Biotecnología Agrícola
(INBIO) for financial support; Cámara Paraguaya de Exportadores y
Comercializadores de Cereales y Oleaginosas (CAPECO) for critically
reviewing the manuscript.
ORCID
Andrea Alejandra Arrúa Alvarenga https://orcid.org/0000-0002-
9489-2120
Juliana Moura Mendes Arrua https://orcid.org/0000-0001-5483-
3612
Cinthia Carolina Cazal Martínez https://orcid.org/0000-0001-5964-
1003
Danilo Fernández Ríos https://orcid.org/0000-0002-8394-532X
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How to cite this article: Arrúa Alvarenga AA, Moura Mendes
Arrua J, Cazal Martínez CC, et al. Deoxynivalenol screening in
wheat-derived products in Gran Asunción, Paraguay. J Food
Saf. 2018;e12580. https://doi.org/10.1111/jfs.12580