Aflatoxin M1 Contamination in Pasteurized Milk in Mashhad, Iran

Document Type : Research Paper


School of Pharmacy, and Medical Toxicology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran


     The aim of this study was to evaluate aflatoxin M1 (AFM1) contamination in pasteurized milk samples in Mashhad, Iran. One hundred and ten milk samples from different supermarkets were collected during three months in spring and investigated by Enzyme Linked Immuno Sorbent Assay (ELISA). AFM1 was found in 100% of the milk samples. About 5.4% of the samples contained AFM1 greater than the maximum tolerance limit (0.05 μg/l) accepted by European Union. There was not a significant difference among the mean value of AFM1 in three months.


1. Introduction

     Milk is a good source of many nutrients, and it is used extensively even as a main food in many countries. However, milk could also be a source of toxic substances such as aflatoxin M1 (AFM1). Aflatoxins are a group of naturally occurring toxins produced by moulds such as Aspergillus flavus. When some animals ingest aflatoxin B1 (AFB1)-contaminated feed, it is metabolized to AFM1 and transferred to food materials such as milk and eggs [1, 2]. AFM1 remain stable when milk is heat-treated; there is no evidence that cold storage, concentrating or drying changes the level of AFM1 [3, 4].

     Although the potency of AFM1 is less than that of its parent compound, it is also known to be hepatotoxic and carcinogenic [5, 6]. More recently, aflatoxin exposure early in life has been associated with impaired growth, particularly stunting [7]. Therefore, the presence of AFM1 in milk and dairy products may pose a threat, mainly towards children who are considered to be the major consumer of milk and dairy products in many countries [8]. This study was carried out to evaluate the prevalence of pasteurized milk contamination with AFM1 in Mashhad, Iran and to compare the results with maximum tolerance limit (0.05 μg/l) accepted by some European countries [9].

     For measurement of AFM1, we used ELISA because immunochemical assays are reliable, rapid, simple, specific and sensitive methods for the routine analysis of mycotoxins in food and feed materials [10, 11].


 Table 1. Concentration of aflatoxin M1 in milk samples collected in different months.











0.016 ± 0.009





0.017 ± 0.011





0.022 ± 0.01

  *Samples were collected in 2006.


2. Materials and methods


2.1. Material

     One hundred and ten pasteurized milk samples from different supermarkets in Mashhad were collected during spring (March, April, May, 2006).

2.2. Methods

     The method is based on the modification of Alborzi et al. [12]. Milk samples were centrifuged in 20 °C for 10 min. with 3500 g. After discarding the upper cream layer, the lower phases were used for quantitative test. The AFM1 quantity was determined by Ridascreen AFM1 test (R-biopharm, Germany) which is a competitive enzyme immunoassay based on antigen-antibody reaction. Sample solutions of 100 μl were added to wells that coated antibodies to AFM1 and after mixing, incubated for 60 min. at the room temperature in dark. Then the liquid was poured out of the wells and wells were washed with buffer solution. In the next state, 100 μl of enzyme conjugate washed with buffer and 50 μl of the enzyme substrate and 50 μl of chromogen were added to wells and incubated for 30 min. at the room temperature in the dark. Bond enzyme conjugate converted the chromogen to a blue product and then 100 μ1 of the stop solution was added to wells which lead to a yellow discoloration of the chromogen. The AFM1 measurement was performed photometrically at 450 nm.


2.3. Statistical analysis

     The results are expressed as meanESD. Data were analyzed by ANOVA. Sequential differences among means were calculated at the level of p < 0.05, using Tukey contrast analysis or Dunn's test as needed.


3. Results and discussion

     The results of our study are shown in Table 1. AFM1 in March, April and May samples ranged from 0.008 to 0.034, 0.009 to 0.039 and 0.013 to 0.089 μg/l, respectively. There are differences in maximum permissible limit of AFM1 in various countries, and many including Iran have no legal limit for AFM1 in milk.

     AFM1 was found in 100% of milk samples. About 5.4% of the samples had AFM1 greater than maximum tolerance limit (0.05 μg/l) accepted by European Union. There was not a significant difference among the mean value of AFM1 in the three months.

     There is little documented data about the occurrence of milk AFM1 in Iran (Table 2). In Tehran, from 73 samples were analyzed. AFM1 detected in 60 samples [13]. All contaminated samples had a level of AFM1 above the European limit. In Sarab, a city in north west of Iran, from 111 samples, AFM1 was observed in 85 samples. In 40% of positive samples, AFM1 levels were higher than the European limit [14]. In Shiraz, 624 samples analyzed. 100% of the samples were contaminated and 17.8% of samples had AFM1 greater than the European limit [12].

     Another study in Tehran showed that from 128 samples, 78% was higher than the European limit [15]. The changes in milk contamination in Iran may be due to differences in the time of sampling, method of determination and level of cowsheds sanitation in cities.


Table 2. The prevalence of milk contamination in cities of Iran.




Number of Samples

Samples above 0.05 mg/l (%)






















    Our results showed that contamination with AFM1 in our region is lower than other regions reported in Iran, most probably because of low contaminated food collections used by cows, industrialization of cowsheds, training the personnel and the season of our sampling (spring). Some studies indicated seasonal effect influences AFM1 occurrence and a higher incidence of AFM1 contamination during cold seasons than hot ones [16, 17]. Therefore, the results obtained by us were in agreement to prior studies.

     According to results obtained in Iran, the incidence and contamination levels of AFM1 in some cities of Iran seem to be a serious problem for public health, especially infants and children. So, to produce high quality milk, it is essential to keep feeds free from contamination to AFB1. The concentration of AFB1 in animal feed can be reduced by good manufacturing and good storage practices. If preventive measures fail, however, AFB1 can be reduced in food by blending with feed that has lower concentrations or by chemical, physical or biological treatment [18]. Also, increasing the intake of antioxidants and vitamins with the diet in order to prevent carcinogenesis should be involved in the preventive strategies [19].

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