Osong Public Health and Research Perspectives 2017; 8(4): 260-263  https://doi.org/10.24171/j.phrp.2017.8.4.05
Aflatoxin M1 Contamination Levels in Cheeses Sold in Isfahan Province, Iran
Ali Sharifzadeha, Payam Ghasemi-Dehkordib, Mohsen Foroughic, Elham Mardanpour-Shahrekordid, and Shahin Ramazie
aDepartment of Microbiology, Faculty of Veterinary Medicine, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran, bCellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran, cFaculty of Veterinary Medicine, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran, dDeputy of Research and Technology, Shahrekord University of Medical Sciences, Shahrekord, Iran, eCellular and Molecular Research Center, Sabzevar University of Medical Sciences, Sabzevar, Iran
Correspondence to: Payam Ghasemi-Dehkordi, E-mail: payamghasemidehkordi@yahoo.com
Received: May 15, 2017; Revised: July 26, 2017; Accepted: July 31, 2017; Published online: August 31, 2017.
© Korea Centers for Disease Control & Prevention. All rights reserved.

This is an open access article under the CC BYNC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).


Aflatoxin M1 (AFM1)-contaminated dairy products pose serious human health risks, causing liver and renal failure if consumed. They are also related to decreased milk and egg production in infected animals. This study investigated the AFM1 contamination levels in cheeses sold in Isfahan province, Iran, by enzyme-linked immunosorbent assay (ELISA).


A total of 100 white cheese samples were randomly collected from supermarkets in Isfahan province and after extraction using dichloromethane were prepared for the ELISA.


Of the 100 samples, 52 (52%) were contaminated by AFM1, at levels ranging from 50.2 to 424.4 ng/kg. The remaining 48% of the samples had undetectable AFM1 levels (< 50 ng/kg). Based on the standard limit set by the European Commission and Iran, 8% (8/100) of the AFM1-positive samples (with concentrations between 250.2 and 424.4 ng/kg) had levels higher than the permissible value of 250 ng/kg.


Although the percentage of cheese samples in Isfahan province with AFM1 levels exceeding the national permissible limit was low, the examination of cheeses and the milk used for their production is nevertheless important for ensuring public health. Furthermore, optimum storage conditions of animal feed should be ensured, and livestock nutrition must be monitored for the presence of AFM1 and other aflatoxins.

Keywords: aflatoxin M1, cheese, enzyme-linked immunosorbent assay

Aflatoxins are highly toxic metabolites and the most carcinogenic substances produced by a certain group of Aspergillus species, namely, A. flavus, A. parasiticus, and A. nomius [1,2]. Foodstuffs and dairy products (e.g., infant milk formula, cheese, cream, butter, ice cream, and yogurt) that become contaminated by mycotoxins at various stages of the food-manufacturing process can be harmful to humans, once ingested [35]. In animals, aflatoxins exert acute and chronic effects, causing failure of various organs, especially the liver, kidney, and heart, as well as suppressed immunity and decreased milk and egg production [6]. There are six main aflatoxins: B1, B2, G1, G2, M1, and M2 [7]. The major aflatoxins M1 and M2 were first isolated from the milk of lactating animals that had been fed aflatoxin-contaminated feed. Aflatoxin M1 (AFM1) is the principal hydroxylated AFB1 metabolite in mammals [8,9]. AFM1 contamination in dairy products, including cheese and butter, displays variations according to geographical region, development level of the country, and season [10,11], and represents a worldwide concern [12]. The levels of AFM1 are increased in the grass, pasture, weed, and rough feeds consumed by livestock in the spring and summer seasons, which causes enhancement of AFM1 pollution in milk and milk products [13,14].

The most common techniques used for detecting AFM1 in milk and dairy products are enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC) [15]. The maximum level of AFM1 in cheeses that is permissible by the European Commission (EC) is 250 ng/L [16]. The presence of AFM1 in milk and dairy products is undesirable and poses a serious risk to human health, especially to children [12,17]. In Iran, dairy products containing milk, including cheeses, are widely consumed. Thus, this work aimed to determine by ELISA the AFM1 contamination levels in cheese samples collected from supermarkets in Isfahan province of Iran.


1. Sample preparation

Between January 2014 and March 2015, a total of 100 cheese samples were randomly collected from different supermarkets in the Isfahan province of Iran. All the samples (50–100 g slices) were immediately transported to the laboratory, where some were stored at 4°C before analysis and other fresh samples were immediately analyzed without freezing. The cheese samples (2 g each) were homogenized in 40 mL of dichloromethane for extraction by shaking for 15 minutes. Then, the suspension was filtered and 10 mL of the extract was evaporated at 60°C under nitrogen. The oily residue was redissolved and mixed thoroughly in 0.5 mL of methanol, 0.5 mL of phosphate-buffered saline (pH 7.2), and 1 mL of n-heptane. The mixture was then centrifuged for 15 minutes at 2,700 rpm. The upper heptane layer was completely removed, and 100 μL of the aliquat phase was brought to 10% methanol content and diluted in 400 μL of RIDASCREEN AFM1 kit buffer (R-Biopharm, Darmstadt, Germany). Finally, 100 μL of this solution was used for each well in the ELISA.


The concentration of AFM1 in the cheese samples was determined using the RIDASCREEN AFM1 iit according to the manufacturer’s recommendation. All reagents were first brought to room temperature (20°C–25°C) prior to use. The AFM1 kit reagents included AFM1 standard solutions (1.5 mL at levels of 0, 5, 10, 25, 50, 100, and 250 ng/L), peroxidase-conjugated AFM1, the substrate (urea peroxidase), the chromogen (tetramethylbenzidine), and the stop reagent containing 1 N sulfuric acid. In duplicate, test samples (100 μL for each well) were added alongside AFM1 standards into the wells of a microtiter plate that had been pre-coated with antibodies for AFM1. The plates were shaken gently on a rotator for a few seconds and then incubated at room temperature in the dark for 60 minutes. After washing with ELISA wash buffer (20×), 100 μL of peroxidase conjugate was added to the wells, and the plates were again incubated in the dark for 60 minutes at room temperature. At the end of the incubation period, the wells were washed with 250 μL of wash buffer to remove unbound conjugate. Subsequently, the substrate/chromogen (tetramethylbenzidine) was added to the wells and incubation was continued for 30 minutes in the dark. Finally, the stop solution (100 μL) was added to each well to terminate the reaction. The optical absorbance of each well was measured at 450 nm using a competitive Stat Fax-2100 ELISA plate reader (Awareness Technology, Palm City, FL, USA). The absorbance value of each AFM1 concentration was determined by interpolation from a linear calibration curve (Figure 1). The mean lower detection limit of the RIDASCREEN AFM1 test kit was 50 ng/kg.

3. Data analysis

The mean values and standard error of the mean were evaluated using IBM SPSS Statistics software, version 20.0 (IBM Co., Armonk, NY, USA).


The contamination levels and distribution of AFM1 in the 100 cheese samples collected from different supermarkets in Isfahan province (Iran) are shown in Tables 1 and 2.

Among these 100 samples, 52 (52%) were confirmed to be contaminated by AFM1 at levels ranging from 50.2 to 424.4 ng/kg (Table 2). Of these, 8 (8%) samples (AFM1 levels between 250.2–424.4 ng/kg) had more AFM1 levels (250 ng/kg) than that allowed by the EC and Iran. In addition, 48% of the 100 samples were considered AFM1-negative, with levels assumed to be below the 50 ng/kg detection limit of the assay kit.


In Iran, special industrial dairy products and traditionally made cheeses play a significant role in the human diet. In different studies by many researchers, various levels of AFM1 contamination in cheese and dairy products have been reported. In the present study, the levels of AFM1 in Iranian cheeses obtained from different supermarkets in Isfahan province (Iran) were determined by ELISA. In this study, 52% of the cheeses from Isfahan province were found to have AFM1 contamination, with 8% of these carrying levels above the limit set by the EC and Iran In a study by Rezaei et al. [18], 93.7% of the pasteurized milk, raw milk, and cheese samples in Arak, Iran were positive for the presence of AFM1, as measured by ELISA, with a total average concentration of 85.8 mg/L in milk and 30.39 mg/kg in cheese. In an HPLC study of dairy products from Punjab, Pakistan, AFM1 was detected in 61%, 78%, 59%, and 45% of the yogurt, white cheese, cheese cream, and butter samples, respectively, of which 47%, 15%, 11%, and 52%, respectively, were above the recommended limit set by the European Union [19]. In our present study of products in Isfahan, however, only 8% of AFM1-positive cheese samples exceeded the EC and nationally set permissible toxin level of 250 ng/kg. In Italian cheeses, 83% of samples showed detectable levels of AFM1 (> 25 ng/kg), most of them between 50 and 150 ng/kg [20], whereas only 52% of our Isfahan samples had detectable AFM1 (50.2–424.4 ng/kg). In a similar ELISA study on samples from Gilan province of Iran, AFM1 was detected in 23.33% (higher than the maximum permissible limit of 250 ng/L) and 63.33% (higher than 50 ng/L) of white cheeses and local yogurts, respectively [14,21]. Their findings on AFM1 contamination in white cheeses was less than that found in our present study (23.33% vs. 52%). ELISA showed 28.3% of white-brined Urfa cheese in Turkey contaminated by AFM1 (70.61–770.97 ng/kg), of which 10.2% exceeded the legal limit (250 ng/kg) established by the Turkish Food Codex [22]. Their results of cheeses exceeding the national permissible level closely resembled ours (10.2% vs. 8%). In an ELISA study on Turkish white Kashar and Tulum cheeses, 51.3% of all samples were contaminated with AFM1 in the range of 0.052–2.52 μg/kg [23], which was similar to the 52% contamination rate found in our present work.

The present study of Isfahan province white cheeses revealed a low-level presence of AFM1 (8%) that exceeded the national legal level for cheeses sold in Iran. Despite this low prevalence of cheese samples with exceeded AFM1 limit, the findings indicate the public health importance of examining for aflatoxin levels in milk, Iranian white cheese, and other dairy products. Moreover, checking for AFM1 levels in livestock feeds according to the national standards is essential, in addition to controlling the appropriate storage conditions of the animal feeds.


This study was approved and supported by the Research Deputy of Islamic Azad University of Shahrekord Branch (Grant No. 93/12/24). The authors would like to thank all the staff members of the Biotechnology Research Center of Islamic Azad University of Shahrekord Branch in Iran for their sincere support.

Fig. 1. Calibration curve of the absorbance values for standard aflatoxin M1 concentrations. Linearization of the calibration curve was performed using logit-log transformation.

Rate of aflatoxin M1 (AFM1) contamination in cheese samples sold in Isfahan province, Iran

AFM1 range (ng/kg)  Sample, n (%)  Mean Range
Not detecteda48 (48)--
50–10029 (29)62.450.2–99.6
100–25015 (15)156.8101.6–248.2
> 250b8 (8)288.2250.2–424.4
Total100 (100)133.250.2–424.4

AFM1 level < 50 ng/kg;

AFM1 ranges higher than the permissible level (250 ng/kg) in cheese.

Distribution level of aflatoxin M1 (AFM1) detected in cheese samples (Isfahan province, Iran)

 Positive52 (52)
 Negative (ND)48 (48)
 Exceed legal limit (> 250 ng/kg) 8 (8)
Concentration (ng/kg)a 169.13 ± 32.4 (50.2–424.4) 

Values are number (%) or mean ± standard deviation (range).

ND, no detectable AFM1.

For positive samples.

  1. Hedayati, MT, Pasqualotto, AC, and Warn, PA (2007). Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology. 153, 1677-92.
    Pubmed CrossRef
  2. Elkak, A, El Atat, O, and Habib, J (2012). Occurrence of aflatoxin M1 in cheese processed and marketed in Lebanon. Food Control. 25, 140-3.
  3. Caloni, F, Stammati, A, and Friggè, G (2006). Aflatoxin M1 absorption and cytotoxicity on human intestinal in vitro model. Toxicon. 47, 409-15.
    Pubmed CrossRef
  4. Khoshpey, B, Farhud, D, and Zaini, F (2011). Aflatoxins in Iran: nature, hazards and carcinogenicity. Iran J Public Health. 40, 1-30.
  5. Zain, ME (2011). Impact of mycotoxins on humans and animals. J Saudi Chem Soc. 15, 129-44.
  6. Sultana, N, and Hanif, NQ (2009). Mycotoxin contamination in cattle feed and feed ingredients. Pakistan Vet J. 29, 211-3.
  7. Mardani, M, Rezapour, S, and Rezapour, P (2011). Survey of aflatoxins in Kashkineh: a traditional Iranian food. Iran J Microbiol. 3, 147-51.
  8. Kang’ethe, EK, and Lang’a, KA (2009). Aflatoxin B1 and M1 contamination of animal feeds and milk from urban centers in Kenya. Afr Health Sci. 9, 218-26.
  9. Britzi, M, Friedman, S, and Miron, J (2013). Carry-over of aflatoxin B1 to aflatoxin M1 in high yielding Israeli cows in mid- and late-lactation. Toxins (Basel). 5, 173-83.
  10. Kazemi Darsanaki, R, Mohammad Doost Chakoosari, M, and Azizollahi Aliabadi, M (2013). Aflatoxin M1 contamination in milk and milk products in Iran: a review. J Chem Health Risks. 3, 13-20.
  11. Khodadadi, M, Khosravi, R, and Allahresani, A (2014). Occurrence of Aflatoxin M1 in pasteurized and traditional cheese marketed in Southern Khorasan, Iran. J Food Qual Hazards Control. 1, 77-80.
  12. Giovati, L, Magliani, W, and Ciociola, T (2015). AFM1 in milk: physical, biological, and prophylactic methods to mitigate contamination. Toxins (Basel). 7, 4330-49.
  13. Celik, TH, Sarımehmetoglu, B, and Kuplulu, O (2005). Aflatoxin M1 contamination in pasteurised milk. Veterinarski Arhiv. 75, 57-65.
  14. Aliabadi, MA, Issazadeh, K, and Darsanaki, RK (2012). Determination of Aflatoxin M1 levels in 1 white cheese samples by ELISA in Gilan province, Iran. Glob Vet. 9, 28-31.
  15. Darsanaki, RK, Aliabadi, MA, and Chakoosari, MMD (2013). Aflatoxin M1 contamination in ice-cream. J Chem Health Risks. 3, 43-6.
  16. Öztürk, B, Çelik, F, and Çelik, Y (2014). To determine the occurrence of Aflatoxin M1 (AFM1) in samples of Cyprus traditional cheese (Halloumi): a cross-sectional study. Kafkas Univ Vet Fak Derg. 20, 773-8.
  17. Atasever, M, Yildirim, Y, and Atasever, M (2014). Assessment of aflatoxin M1 in maternal breast milk in Eastern Turkey. Food Chem Toxicol. 66, 147-9.
    Pubmed CrossRef
  18. Rezaei, M, Fani, A, and Moini, AL (2015). Assessment of aflatoxin M1 levels in pasteurised milk, raw milk, and cheese in Arak, Iran. Toxin Reviews. 34, 61-5.
  19. Iqbal, SZ, and Asi, MR (2013). Assessment of aflatoxin M1 in milk and milk products from Punjab, Pakistan. Food Control. 30, 235-9.
  20. Anfossi, L, Baggiani, C, and Giovannoli, C (2012). Occurrence of aflatoxin M1 in Italian cheese: results of a survey conducted in 2010 and correlation with manufacturing, production season, milking animals, and maturation of cheese. Food Control. 25, 125-30.
  21. Issazadeh, K, Darsanaki, RK, and Khoshkholgh Pahlaviani, MRM (2012). Occurrence of aflatoxin M1 levels in local yogurt samples in Gilan Province, Iran. Ann Biol Res. 3, 3853-5.
  22. Kav, K, Col, R, and Tekinsen, KK (2011). Detection of aflatoxin M1 levels by ELISA in white-brined Urfa cheese consumed in Turkey. Food Control. 22, 1883-6.
  23. Hampikyan, H, Bingol, EB, and Cetin, O (2010). Determination of aflatoxin M1 levels in Turkish white, kashar and tulum cheeses. J Food Agric Environ. 8, 13-5.

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