Biotechnological

Communication

Biosci. Biotech. Res. Comm. 9(4): 804-808 (2016)

Evaluation of fumonisin and zearalenone levels in wheat

of silages in Golestan Province, Northeastern Iran

Hamidreza Yazdi

, Hamid Reza Joshaghani

, Mojgan Nejabat

, Mohammad Mostakhdem

Hashemi

, Nasser Behnampour

, Arazgaldi Chogan

, Ziba Abbasinejat

and Farhad Niknejad

Cereal Health Research Center, Golestan University of Medical Science, Gorgan, Iran

Laboratory Sciences Research Center, Golestan University of Medical Sciences, Gorgan, Iran

Department of Biochemistry and Biophysics, Metabolic Disorders Research Center, Gorgan Faculty of

Medicine, Golestan University of Medical Sciences, Gorgan, Golestan province,Iran

Health Management and Social Development Research Center, Golestan University of Medical

Sciences,Gorgan, Iran

ABSTRACT

Mycotoxins are secondary fungal metabolites that can contaminate stored foods and lead to various complications.

Golestan province has a humid climate so on it is considered as a high risk area for fungal contamination of wheat

products, therefore this study was aimed to evaluate the levels of fumonisin (FB) and zearalenone (ZEA) contami-

nation in wheat, stored in the province’s silages which has a humid climate. 35 samples of stored wheat were col-

lected from silages of 14 cities in the province. FB and ZEA were extracted from samples and later their levels were

measured using enzyme-linked immunosorbant assay method by the commercially available kits. Fungal myco ora

of the samples was identi ed too. 31 out of 35 samples were contaminated by ZEA, while only 3 samples had FB

contamination. Temperature or relative humidity had no effect on the prevalence or concentration of these toxins.

The mean ± SD for ZEA and FB were 3.77±2.46 ng/g and 0.034±0.11 ng/g, respectively which were lower than the

maximum tolerated level for mycotoxins in food and feed. 29 out of 35 samples (82.84%) had one of four fungi

types which went under detection. The detected levels of FB and ZEA in wheat samples of this province are lower

compared with other similar studies in Iran and particularly northeastern of Iran. Despite the low concentrations in

our study, it is recommended to monitor the occurrence of ZEA and FB in wheat and wheat products to ensure safety

and consequently improved public health.

KEY WORDS: ZEARALENONE, FUMONISINS, WHEAT, ELISA

804

ARTICLE INFORMATION:

*Corresponding Author: niknejad@goums.ac.ir

Received 21

Nov, 2016

Accepted after revision 25

Dec, 2016

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2015: 3.48 Cosmos IF : 4.006

reserved.

Online Contents Available at: http//www.bbrc.in/

Hamidreza Yazdi et al.

HIGHLIGHTS

• Fumonisin and Zearaleone are two of abundant

mycotoxins in cereals

• Positive correlations were seen between mycotox-

ins and health problems such as cancers

• Mycotoxins monitoring in stored cereals may have

potent  nancial and health bene ts

INTRODUCTION

Mycotoxins are toxic secondary metabolites produced

by various mould fungi such as Aspergillus, Fusarium,

and Penicillium which are naturally developed in food

products. They generally grow on agricultural prod-

ucts due to pre/post-harvest states and transportation

or storage conditions. The Fusarium fungus species are

the most frequent pathogens in crops in humid climate.

Fumonisins (FB) and zearalenone (ZEA) are two myco-

toxins, produced by these species which can have seri-

ous effects on the safety of foods and feeds. ZEA binds to

the mammalian estrogen receptors induces oestrogenic

effects in mammals and interferes with conception, ovu-

lation, implantation, fetal development, and viability of

newborn animals. While B1, B2 and B3 are the most

common types of fumonisins, fumonisin B1 (FB1) is the

most toxic form which contaminates wheat and causes

many diseases in both animals and humans (Shier, 2001,

Šegvić Klarić, 2009, Pérez-Torrado, 2010, Shirima, et al.,

2013, Arroyo-Manzanares, 2014, Feizy, 2014).

Fumonisins are absorbed through the digestive sys-

tem and affect cell surface by interfering with the bio-

synthesis of sphingolipids, resulting in accumulation of

sphingosine and membrane dysfunction (Stockmann-

Juva, 2008). These compounds act as an accelerator for

cancer and cause mutations through frequent accumula-

tion of sphingoid free base (Bhandari, 2002). Epidemio-

logic studies have reported a direct correlation between

FB intake and esophageal cancer (EC) in South Africa

(Shirima, et al., 2013) and China (Yoshizawa, 1994).

FB1 is also thought to be a risk factor for EC, especially

in high risk areas (Yli-Mattila, 2010) such as Golestan

province, located in northeastern Iran (Roshandel, et al.,

2012).

It also has been categorized by the International

Agency for Research on Cancer as a possible human

carcinogen (Group 2B) (IARC, 2004). Wheat is one of

the most important cereal crops for human consumption

(Chehri, 2010) and mycotoxin contamination of crops,

particularly wheat, maize, peanuts, and rice can have

serious economic and environmental consequences.

Moreover, the food and agriculture organization (FAO)

estimated that 25% of the world

’

s crops are affected

by mycotoxins annually, with an average loss of 1 bil-

lion metric tons of food and food products each year

(Schmaile, 2009). There are several methods available

for mycotoxin analysis including ELISA (Sebaei, 2012

and Arroyo-Manzanares, 2014). Therefore, this study

was aimed to use the ELISA technique to determine FB

and ZEA levels in wheat grains collected from silages of

Golestan province, Iran.

MATERIAL AND METHODS

Samples were collected from 35 wheat silages in 14

cities of Golestan province, according to the guideline

provided by the standard and industrial research insti-

tute of Iran (ISIR) number 2087, in a way that for every

1500 tons of wheat, 1 Kg should be taken as sample.

The samples were  lled in well-packed sterile plastic

bags and then stored in 2-8 °C until the time of analysis.

Questionnaires were also designed for each silo which

contained the following items: silo location, structural

type of silo (metal, concrete, etc.), storage temperature,

relative humidity (%), total capacity of the silo and the

planting and harvesting area of the stored products.

The fungal myco ora of current wheat samples were

determined as following: Each sample (20 g) was surface

disinfected for 2 minutes with 0.2% sodium hypochlo-

rite solution and rinsed three times with sterile distilled

water. From each sample, 40 grains were randomly

selected and then put in Petri plates (90 mm diameter,

10 grains/dish) containing Sabouraud’s dextrose agar

(Merck, Darmstadt, Germany) with 5% chloramphenicol

in duplicate. Petri plates were incubated at 25°C for 6 to

10 days. Each pure culture was characterized and identi-

 ed based on their morphological and microscopic char-

acteristics using the keys of Pitt and Hockings (1997)

and Raper and Fennel (1965).

First, 50 – 100 jars of wheat were ground into a  ne

powder and then 3 jars of this powder were added to 3

ml of 80% methanol and mixed for 15 minutes at room

temperature. It was later centrifuged for 10 minutes at

2000*g and the supernatant liquid was used as the sam-

ple for further FB detection test.1 gram of well-pulver-

ized wheat powder was added to 4 ml of 60% methanol

and then mixed for 15 minutes at room temperature.

After centrifugation for 10 minutes at 2000*g, the super-

natant liquid was used as the sample for ZEA detection

test.

In order to measure FB, 50μl of the obtained superna-

tant from the FB extraction step were added to 150μl of

dilution buffer provided by the FB measurement ELISA

kit (Euro Proxima, Cat number: 5121FUM), according to

the manufacturer’s instruction. For ZEA measurement,

25μl of the obtained supernatant from the ZEA extrac-

tion step was added to 475μl of dilution buffer provided

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EVALUATION OF FUMONISIN AND ZEARALENONE LEVELS IN WHEAT OF SILAGES 805

Hamidreza Yazdi et al.

Table1: The descriptive characteristics of the investigated wheat silages in the Golestan province, Iran

Ave. Temperature

and (Range) (C°)

Ave. relative humidity

and (Range) (%)

Ave. capacity (tons)

and (Range)

Ave. period of wheat

storage (year)

Type of silo

31.37±2.85

26-37

35.51±7.77

21-58

18700

2500-100000

1 Metal: 34.5%

Concrete: 8.5%

Traditional: 37%

Mechanized: 20%

Table 2: The comparison of FB and ZEA contamination in

wheat silages located in the east and west of the province

East (n=17) West (n=18)

p-value

ZEA (mean ± SD) 3.1059 ± 1.8084 4.4111 ± 2.8569 0.118

FB (mean ± SD) 0 ± 0 0.67 ± 0.1534 0.083

Table 3: The occurrence of fungi in the samples

Fungi type No %

Aspergillus  avus 16 45.71%

Aspergillus niger 2 5.71%

Penicillium spp 7 20%

Yeast 4 11.42%

Total 29 82.84%

by the ZEA measurement ELISA kit (EuroProxima, Cat

number: 5121ZON), according to the manufacturer’s

instruction. The tests were performed in duplicate and

the results were reported as ng/g.The data were analyzed

by SPSS (version 21) statistical software. The normality

of data distribution was evaluated using Kolomogrov-

Smirnov Shapiro-Wilk and Speraman correlation coef-

 cient test was used to investigate the relationships

between mentioned toxin levels and other parameters

due to the non-parametricity of the data.

Overall, 35 wheat silages were investigated in this

study and Table 1 shows the basic descriptive character-

istics of all the tested silages from the Golestan province,

Iran. It also has to be mentioned that the average period

of wheat reservation for all the silages was one year

(table 1).

RESULTS

FB contamination was found in only three samples with

concentrations equal to 0.4 ng/g, while 31 samples (88%)

were contaminated by the ZEA mycotoxin. The ZEA-free

samples were related to four silages which were also free

of FB contamination. The found FB levels in the samples

were ranging from 0 to 0.4 ng/g with a mean ± SD of

0.034±0.11 ng/g. The levels of ZEA in the tested samples

were ranging from 0 to 10.40 ng/g with a mean ± SD

of 3.77±2.46 ng/g.Overall, there were 4 silages without

any toxic samples and it was also found that all the FB

contaminated samples were accompanied by ZEA con-

tamination.Further analysis was performed to explore

the probable difference of contamination between the

silages located in the East or West of the province and

no signi cant variation was detected (table 2).

The Pearson correlation coef cient test showed no

relationship between the presence and level of myco-

toxin contamination with temperature or relative

humidity percentage in the tested silages. Also, the type

or capacity of the silages had no signi cant effect on the

level of these toxins. While, all three FB contaminations

were found in traditional silages (the silages without any

developed structure or building), contamination-free

silages were of metal and mechanized types. However,

the analysis showed no signi cant statistical difference

between these types of silages.

Table 3 shows the frequency and percentage of the

samples owning various types of fungi (table 3).

According to the guidelines of Iranian institute of

standard and industrial research (ISIR) number 2087 for

food and feed’s maximum tolerated level of mycotoxins,

the maximum permitted levels of FB1 and ZEA should

not exceed 5 ng/g and 200 ng/g, respectively. Although

in this study ZEA contamination was present in 88% of

the collected samples, all of them were at levels lower

than the cutoff point reported by the ISIR guidelines.

Moreover, the amount of the three FB contaminated

samples was notably lower than the maximum permit-

ted level.

806 EVALUATION OF FUMONISIN AND ZEARALENONE LEVELS IN WHEAT OF SILAGES BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Hamidreza Yazdi et al.

DISCUSSION

In our study, the presence or rate of mycotoxin contami-

nation was not associated with temperature or relative

humidity percentage of silages. Also, the type or capac-

ity of the silages had no signi cant effect on these tox-

ins. However, all three FB contaminations were related

to traditional silages and also the silages without any

toxins were metal and mechanized types, but the sta-

tistical analysis showed no signi cant difference which

can suggest the effect of other non-mentioned factors in

the establishment of mycotoxin contamination. Josha-

ghani (2013) conducted a study on the myco ora of

fungal contamination among the same wheat silages in

this province and found that 10 out of 34 samples were

contaminated with a atoxin B

with hazardous levels

only in one case. They reported Aspergillus niger hav-

ing the highest frequency in their samples while in our

study, Aspergillus  avus was the most prevalent fungi in

the samples and also 82.84% of wheat samples showed

the presence of fungi. According to the toxicogenity

potency of some of these moulds, the risk of mycotoxin

increase exist when long-lasting reservation of wheat in

un t circumstances.

There was no signi cant difference between the mean

levels of ZEA and FB from silages located in the West

and East of the province which is against their potential

role in cancer pathogenesis, since there is a big differ-

ence in the frequency of cancer between the East and

West of Golestan province (Pourshams, et al., 2005,

Islami, et al., 2009). In a study by Bettencourt (2005),

the contamination level of 60 maize meal and  our sam-

ples was assessed in Sao Paolo, Brazil and the average

concentration of these toxins in maize meal and  our

was found to be 6170 ng/g and 2740 ng/g, respectively

which are remarkably higher than our  ndings. Šegvić-

Klarić (2009) studied on the prevalence of ZEA contami-

nation in household cereals and foods reported that 92%

of foods had ZEA toxins and a high prevalence for ZEA

compared to FB, which were similar to our  ndings.

Curtui (1998) analyzed 55 wheat and corn samples des-

tined for animal consumption and demonstrated that all

wheat samples and 13% of corn samples were contami-

nated by ZEA which are in agreement with our  ndings.

Feizy (2014) reported 12.6% FB

contamination and

no ZEA contamination in wheat samples of Mashhad,

Iran, which are different from our  ndings. In the study

of Chehri (2010), FB

was found as the dominant type of

FB toxin with 68.2% prevalence rate, ranging 22-455

ng/g which are extremely higher compared to the total

FB level in this study. Hedayati (2006) analyzed the

samples obtained from 12  our producing factories and

showed that all the granaries had ZEA contamination

with minimum concentration of 29 ng/g, which is in

agreement with our results. In another study in Iran by

Roohi (2012), 42  our samples from bakeries and con-

fectioneries were assessed for FB contamination and 18

samples from bakeries and 17 samples from the stud-

ied confectioneries had FB contamination. Alizadeh and

others (2012) analyzed 132 grain silo samples (66 rice

and 66 corn) and reported 50% and 40.9% FB

contami-

nation in the corn and rice samples, respectively. Despite

the heterogeneity of our samples with the former study,

there was still a very high concentration of FB

(223.64

ng/g for corn and 21.59 ng/g for rice). In two parallel

studies by Yazdanpanah and others (2006) and Shephard

and others (2000) on corn samples of Mazandaran and

Isfahan province in Iran, Mazandaran province had a

higher level of FB in comparison to Isfahan which had

results similar to our  ndings.

Wheat and other starchy grains are the staple food

of many countries all over the world, especially Asian

and African countries. Also, the use of grains in ani-

mal feeding further highlights the issue of their safety.

Furthermore, development of advanced technology

and possibility to store foods and particularly grains,

propounds the importance of considering food storage

safety. Therefore, more extensive studies are required to

evaluate the levels of mycotoxin contamination in these

foods for improved public and environmental health.

CONCLUSION

Low levels of mycotoxins are detected in the silages of

Golestan province. ZEA contamination is more preva-

lent compared to FB, while none of the silages exceeds

maximum tolerated levels of mycotoxins according to

the safety of food and feed guidelines. Furthermore,

there is an important necessity to monitor food products

before consumption.

ACKNOWLEDGEMENTS

The authors would like to thank the Deputy of Research

and Technology of Golestan University of Medical Sci-

ences, Iran for  nancial support.

CONFLICT OF INTEREST

The authors declare that they have no con ict of

interest.

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