Food and Chemical Toxicology 47 (2009) 1624–1625

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Food and Chemical Toxicology

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Aflatoxin M1 contamination in pasteurized milk in Tabriz (northwest of Iran)

Mohammad Hosein Movassagh Ghazani *
Department of Food Hygiene, Faculty of Veterinary Medicine, Islamic Azad University, Shabestar Branch, Shabestar, Iran

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

Article history: Aflatoxin M1 (AFM1) appears in milk as a direct result of the ingestion of food contaminated with afla-
Received 11 February 2009 toxin B1 by cattle. The role of milk in human nutrition is well-known. The aim of this study was to eval-
Accepted 9 April 2009 uate Aflatoxin M1 contamination in pasteurized milk samples in Tabriz city (Iran) by ELISA (Enzyme
Linked Immunosorbent Assay). Fifty pasteurized milk samples from different supermarkets in Tabriz city
were collected during 6 months (July to December 2008). AFM1 was found in 100% of the examined milk
Keywords: samples. Sixty-two percent of the samples had AFM1 greater than the maximum tolerance limit (50 ng/l)
Aflatoxin M1
accepted by European Union. It can be concluded that AFM1 levels in the samples purchased in Tabriz
Pasteurized milk
ELISA
city, appear to be a serious public health problem at the moment. To achieve a low level of AFM1 in milk,
Tabriz dairy cows’ feed samples from various cows’ herds must be controlled periodically for aflatoxin and kept
away from fungal contamination as much as possible.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction 2. Materials and methods

A total of 50 pasteurized milk samples from different supermarkets in Tabriz

Aflatoxin may be produced by three species of Aspergilus – Asper-
gilus flavus, Aspergilus parasiticus, and rare Aspergilus nomius – that
contaminate plants and its products. A. flavus produces only B afla-
toxins, while the others produce both B and G aflatoxins (Unusan,
2006). Aflatoxins M1 and M2 are the hydroxilated metabolites of
aflatoxin B1 and B2 and may be found in milk products obtained
from live stock that have ingested contaminated feed (Unusan,
2006).
Aflatoxins are both acutely and chronically toxic for animals
and humans, and can produce dangerous illnesses including acute
liver damage, liver cirrhosis, tumor induction and are also terato-
gen (Deshpandeh, 2002; Alborzi et al., 2006).
It has been stated, in fact, that the contamination of milk and
milk products with AFM1 displayed variations according to geogra-
phy, country and season. The pollution level of AFM1 was differen-
tiated further by hot and cold seasons, due to the fact that grass,
pasture, weed, and rough feeds were found more commonly in
spring and summer than in winter (Galvano and Galofaro, 1996;
Pittet, 1998; Celik et al., 2005). AFM1 is resistant to thermal inac-
tivation; pasteurization, autoclaving and other varieties of food
processing procedures are not effective in the reduction of this tox-
in (Deshpandeh, 2002; Park, 2002). This study was carried out to
evaluate the prevalence of pasteurized milk contamination with
AFM1 in Tabriz city (Iran).
* Corresponding author. Tel.: +98 4712225311; fax: +98 4712226557.
E-mail address: Movassagh2@yahoo.com
city were collected randomly during 6 months (July to December 2008).
The milk samples were centrifuged in 10 °C for 10 min with 3500g. After centrifu-
gation, upper cream layers were completely discarded and the lower phases were fre-
ezed for the quantitative test. The quantity of AFM1 was determined by I’ screen
aflatoxin M1 test (Tecna, Italy) which is a competitive enzyme immunoassay based
on antigen–antibody reaction. Sample solutions of 100 ll were added to the wells
to occupy the binding sites proportionately then mixed gently and incubated for
45 min at room temperature (20–25 °C). The liquid was poured out of the wells and
the wells filled with 250 ll washing buffer and poured out the liquid again. This wash-
ing step repeated four times. In the next stage 100 ll of enzyme conjugate were added
to occupy the remaining free binding sites and incubated for 15 min at room temper-
ature and repeated washing step. Then 100 ll of developing solution was added to
each well and incubated for 15 min at room temperature. By using a multichannel pi-
pet, 50 ll of stop solution was added to each well. The measurement of AFM1 was
done photometrically at 450 nm against air blank within 60 min in ELISA reader (Sun-
rise, USA). Then data were analyzed by chi square test.
3. Results
The standard curve for AFM1 detection by competitive ELISA is
given in Fig. 1. As can be seen from the figure, the calibration curve
was found virtually linear in the 5–250 ng/l range. The detection
limit was found to be 5 ng/l. The occurrence of AFM1 in pasteur-
ized milk samples is shown in Table 1. The mean value was
50.55 ± 3.73 ng/l. There was a high incidence rate of AFM1, with
50 (100%) milk samples being contaminated. Although 19 (38%)
were below the limit, the remaining 31 (62%) were well above
the limit permitted by EU.

0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2009.04.011
 M.H. Movassagh Ghazani / Food and Chemical Toxicology 47 (2009) 1624–1625 1625

tion (p < 0.01) (Alborzi et al., 2006; Gholampour Azizi et al., 2007;
Nakajima et al., 2004; Elgerbi et al., 2004). The milk samples in
Libya had the same contamination as our findings (Gholampour
Azizi et al., 2007; Elgerbi et al., 2004).
According to results obtained in Iran and other countries, inci-
dence and contamination levels of AFM1 in Iran and some of the
countries seem to be a serious problem.
It is known that contamination of AFM1 in milk is a result of
exposure of AFB1 to dairy cattle through feed-stuffs (Unusan,
2006). The wide variations in AFM1 levels among studies could
be related to geographic and climatic differences but also to differ-
ences in feeding systems, and farm management practices.
This study showed that contamination with AFM1 in Tabriz is
more than standard levels.
Fig. 1. Calibration curve of AFM1. In Iran, especially in Tabriz, pasteurized milk is used more than
the other milk products by all the age groups including infants and
children, and it should be considered that, total daily aflatoxin in-
Table 1 take from other foods could be an important risk factor for public
Occurrence of AFM1 in pasteurized milk samples.
health as well.
Range of AFM1 concentration (ng/l) Pasteurized milk, n (%) It is concluded that dairy farmers must be educated by the
<50 19 (38) government authorities on potential health consequences of
50–80 Exceeding EC regulations 22 (44) aflatoxins.
>80 9 (18)
Range 0–259
Conflict of interest statement

The authors declare that there are no conflicts of interest.

Table 2
The prevalence of milk contamination in other studies.
Acknowledgements
Location Milk samples Number of contaminated milk Percent
(number) samples >50 ng/l The author gratefully acknowledges the contribution of Dr. A.R.
Babol 78 78 100 Ahmadzadeh, Mr. E. Torabei, Mr. K. Ashtianei and Tabriz Blood
(Iran) Transfusion Organization to this work.
Japan 208 207 99.5
Libya 49 35 71.4
Tabriz 50 31 62 References
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