Food Control 20 (2009) 603–605

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Food Control
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Aflatoxin M1 in raw, pasteurized and powdered milk available in the Syrian market
I. Ghanem *, M. Orfi
Atomic Energy Commission of Syria, Biotechnology Department, Damascus P.O. Box 6091, Syrian Arab Republic

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

Article history: The incidence of contamination of aflatoxin M1 (AFM1) in milk samples collected from the Syrian market
Received 18 January 2008 was investigated by using the competitive enzyme linked immunosorbent assay (ELISA) technique. A total
Received in revised form 24 August 2008 of 126 samples composed of raw cow milk (74 samples), raw sheep milk (23), raw goat milk (11), pasteur-
Accepted 29 August 2008
ized cow milk (10) and powdered milk (8) showed that 80% of tested samples were contaminated with
various levels of AFM1 ranging from >20 to 765 ng/l. Percentages of AFM1-contaminated samples exceed-
ing the American, Syrian and European tolerance limits were 22%, 38% and 52%, respectively.
Keywords:
The range of contamination was relatively higher in pasteurized milk than in raw cow and sheep milk.
Aflatoxin M1 (AFM1)
Milk
80% of AFM1-contaminated pasteurized cow milk samples exceeded the European tolerance limit with a
ELISA range of contamination between 89 and 765 ng/l. Percentages of contaminated raw cow, sheep and goat
milk exceeding the European tolerance limit were 59%, 24% and 14%, respectively.
Milk powder was almost free of AFM1 contamination with only one sample containing a concentration
lower than the European tolerance limit (12 ng/l).
Extrapolation of aflatoxin B1 (AFB1) from AFM1 levels of contamination in milk samples indicates that
contamination in dairy cattle feeds may range from 0.5 to 47.8 lg/kg.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction (Stoloff et al., 1991). In Syria, the Ministry of Health established

the maximum acceptable limit of AFM1 at 200 ng/kg for fluid milk
Milk and dairy products are fundamental components in the and 50 ng/kg for powder milk (FAO, 2004).
human diet, and may be the principle way for entrance of aflatox- The purpose of this survey was to detect the levels of AFM1 in
ins into the human body (Galvano et al. 1998). Of all the mycotox- raw cow, sheep, and goat milk, and in pasteurized milk, and milk
ins, aflatoxin B1 (AFB1) is considered to be the most toxic and powder sampled at different locations in Syria, and to compare
carcinogenic. Aflatoxin M1 (AFM1) may be found in the milk of ani- these levels with maximum AFM1 limits adopted by European,
mals that are fed on AFB1-containing feed (Van Egmond, 1991; USA, and Syrian health organisations.
Wood, 1991, chap. 6). Ingested AFB1 is metabolized at the hepatic
level and the hydroxylated metabolites of aflatoxin M1 (AFM1) and
2. Materials and methods
aflatoxin M2 (AFM2) are excreted through the milk, faeces and ur-
ine (Goto & Hsieh, 1985; Guerre, Bailly, Bernard, & Burgat, 2000).
2.1. Sampling
Recently, the demonstrated toxic and carcinogenic effects of
AFM1 have lead WHO-IARC to re-consider its carcinogenicity clas-
A total of 126 samples were collected from the north, south and
sification and to change it from group 2 to group 1 (IARC, 1993,
east of Syria, during April 2005–April 2006. These samples were
2002).
composed of raw cow milk 74 samples, raw sheep milk 23 samples,
Regulatory limits throughout the world are influenced by eco-
raw goat milk 11 samples, pasteurized milk 10 samples and full-
nomic considerations and may vary from one country to another
cream milk powder 8 samples. Collected samples were transported
(Stoloff, van Egmond, & Park, 1991; Van Egmond, 1989). The Euro-
to the laboratory in refrigerated containers (+4 °C) and stored at
pean Community and Codex Alimentarius has prescribed that the
20 °C until analysis.
maximum limit of AFM1 in liquid milk and dried or processed milk
products is 50 ng/kg (Codex Alimentarius Commission, 2001; Euro-
2.2. Sample preparation
pean Commission Regulation, 2001). According to USA regulations
the level of AFM1 in milk should not be higher than 500 ng/kg
For raw milk, 5 ml were incubated for 30 min at 4 °C, and cen-
trifuged at 3000g for 10 min. The milk serum below the fat layer
* Corresponding author. Tel.: +963 11 2132580; fax: +963 11 6112289. was sampled and directly assay for AFM1 using a specific ELISA
E-mail address: eghanem@aec.org.sy (I. Ghanem). KIT (Romer Labs, 2005).

0956-7135/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2008.08.018
604 I. Ghanem, M. Orfi / Food Control 20 (2009) 603–605

For full-cream milk powder, 9.1 g of the powder was dissolved

in 100 ml double-distilled water, the solution was warmed up to
about 50 °C and homogenized using a magnetic stirrer. Then, the
sample was prepared as described above for raw milk sample.

2.3. Analysis of AFM1 in samples by the competitive ELISA

The quantitative ELISA kit AgraQuant Aflatoxin M1 (Romer

Labs, Singapore) was stored at 2–8 °C. Before its use the kit was
left for 2 h at room temperature to bring it to room temperature.
The KIT was used according to the manufacturer’s instruction
(Romer Labs, 2005) as follows: Into an AFM1-antibody-coated mi-
cro titer plate an AFM1 standards at the concentrations 0, 5, 10,
30, 50 and 100 ng/l1 (supplied with the KIT) were pipetted into
each well (100 ll/well/standard). Test samples were also pipetted Fig. 1. Calibration curve of aflatoxin M1. B/B0: represents absorbance at 450 nm for
the sample, or the standard divided by absorbance at the same wavelength for the
in duplicate (100 ll/well/sample). The plate containing the sam- control.
ples was incubated at room temperature for 60 min using a titer
plate shaker at a speed of approximately 100 rpm. Following a
washing step with washing solution (supplied with the KIT),
AFM1 conjugate was added to the wells, and the plate was incu- 3.2. AFM1. incidence in milk samples
bated again at room temperature for 30 min on a microtiter plate
shaker at a speed of approximately 100 rpm. The plate was The occurrence of AFM1 in raw cow, sheep and goat milk, and in
washed with the washing solution in order to remove the un- milk powder is presented in Table 1. Of the 126 samples analyzed,
bound conjugate. A 100 ll of substrate solution was added into 80% were found to be contaminated with AFM1.
the wells and the reaction was allowed to proceed in the dark The range of contamination levels varied among different cate-
for 40 min at room temperature, at the end of which a blue colour gories of milk samples. AFM1 in raw cow, raw sheep milk, raw goat
is developed. The reaction was stopped by adding 100 ll of stop milk and pasteurized milk ranged from 20 to 690, 6 to 634, 8 to 54
solution to the wells, and the colour changed from blue to yellow. and 8 to 765 ng/kg respectively. While mean values of contamina-
The absorbance was measured at 450 nm in Multiskan Ascent tion were 143, 67, 19 and 492 ng/kg, respectively.
ELISA Plate Reader (Thermo Labsystems, USA), and the absorption Pasteurized cow milk showed the highest range of contamina-
intensity was found to be inversely proportional to AFM1 concen- tion of 8–765 ng/kg with a mean value 492. Pasteurization plants
tration in the samples. in Syria usually collect milk from different farmers at different
The log–logit AFM1 sheet supplied with the KIT was used to locations in the country and collected milk is pooled and pasteur-
generate a standard curve and to calculate the concentration of ized. No testing of milk for contamination with AFM1 is done prior
AFM1 in the samples. to pasteurization. Only one milk powder sample out of the tested
samples (8) showed a low level (12 ng/kg) of contamination with
2.4. Calculation of extrapolated values of AFB1 concentration in feeds AFM1 milk powder. Milk powder in Syria is imported mostly from
European countries which implement a strict maximum tolerance
The values of AFB1 in cattle feeds were extrapolated from back level of AFM1 in milk (50 ng/kg). This may explain the finding that
calculation of the values of AFM1 obtained from analysis of milk milk powder samples tested in the present study were almost free
samples. The calculation was based on the assumption that only of contamination with AFM1.
1.6% of ingested AFB1 is converted to AFM1 by dairy cattle. The The US regulation has prescribed a limit of 500 ng/kg for AFM1
formula used for calculation was AFB1 ðlg=kgÞ ¼ AFM1ðng=kgÞ100
1:61000 in milk and dairy products. However, European Communities and
(Forbisch, Bradley, Wagner, Long-Bradley, & Hariston, 1986; Codex Alimentarius have fixed the limit to a maximum of 50 ng/
Rastogi, Dwivedi, Khanna, & Das, 2004). kg of AFM1 (Codex Alimentarius Commission, 2001; European
Communities, 1992; European Commission Regulation, 2001; Rast-
ogi et al., 2004). In Syria, the maximum tolerance limit of AFM1 is
3. Results and discussion
200 ng/kg for liquid milk (FAO, 2004). Table 2 shows percentages of
samples in which levels of contamination of AFM1 exceeds the
3.1. Performance of analytical method
European Communities/Codex Alimentarius, US and Syrian regula-
tions. In total 52%, 38% and 22% of AFM1-contaminated samples
The standard curve obtained in the present study for AFM1
detection by using the competitive ELISA test is depicted in
Fig. 1. The resolution of the curve within the range 5–10 ng/l was
Table 1
insufficient to enable reliable interpolation of AFM1 concentrations
Concentration of AFM1 in different milk samples, and extrapolated AFB1 concentra-
based on their corresponding absorbance values, i.e. B/B0% values tion in cattle feeds
(Fig. 1). Therefore, concentration values of AFM1 obtained from
Sample Samples Positive AFM1 Extrapolated
the curve within this range were not considered reliable. However,
category analyzed samples contamination(ng/l) AFB1 (lg/kg)
the standard curve for the range 10–100 ng/l was reliable, and
within this range the inverse correlation between AFM1 concentra- Range Mean ± SD Range
a
tions in the standard samples and their correspondent absorbance Raw cow milk 74 70 (95%) 20–690 143 ± 53.22 1.3–43.1
values at 450 nm was good (Fig. 1). Raw sheep milk 23 13 (57%) 6–634 67 ± 18.43 0.37–39.6
Raw goat milk 11 7 (64 %) 8–54 19 ± 13.8 0.5–3.37
In our study, the recovery of AFM1 in spiked milk samples was Pasteurized milk 10 10 (100%) 8–765 492 ± 212.56 0.5–43.8
found to be 84% (CV = 3.12), 92% (CV = 1.23) and 98% (CV = 1.03) Milk powder 8 1 (13 %) 12 12 0.7
for spiking concentration of 50, 240, 300 ng/l, respectively. All Total 126 101 (80 %) 0.37–43.8
experiments were made in triplicate. a
Values in parenthesis indicate % of contaminated samples.
 I. Ghanem, M. Orfi / Food Control 20 (2009) 603–605
Table 2
AFM1 contamination, in different kinds of milk samples, exceeding limits established
by the EC/Codex, Syrian and US regulations
Sample Positive Exceeding EC Exceeding Syrian Exceeding US
category samples regulations regulations regulations
No Range No Range No
(ng/l) (ng/l)
Raw cow 70 41(59%)a 57–690 30(43%) 422–690 15(21%) 543–690
milk
Raw sheep 13 3(23%) 60–634 1(8%) 634 1(8%)
milk
Raw goat 7 1(14%) 54 – – – –
milk
Pasteurized 10 8(80%) 89–765 7(70%) 345–765 6(60%)
milk
Total 101 53(52%) 54–765 38(38%) 345–765 22(22%) 543–765
a
Values in parentheses indicate % samples exceeding the prescribed limits.
exceeded the EU/Codex, US and Syria regulations, respectively. All
tested pasteurized milk samples were contaminated with AFM1, of
which 80%, 70% and 60% exceeded the EU/Codex, US and Syrian
recommended limits, respectively. The only AFM1-contaminated
milk powder sample reported in the present study was well below
the lowest recommended limit, i.e. EU/Codex limit. AFM1 contam-
ination in sheep and goat milk was relatively lower than that in
raw and pasteurized cow milk. Percentages of sheep and goat milk
samples exceeding the EU/Codex prescribed limits were 14%,
whereas, 8% of sheep milk exceeded the Syrian and US limits, with
no goat milk samples exceeding those limits. The low incidence of
AFM1 contamination of sheep and goat milk is probably related to
the fact that sheep and goat in Syria are raised on range lands and
freely graze the range plants for major part of the year, they are fed
on stored grains for two to three months only. Whereas, cows are
kept in dairy farms and fed on manufactured feedstuffs made of
various stored grain products and byproducts of agricultural indus-
try. The latter type of feedstuffs is prone to fungal infection and to
subsequent contamination with aflatoxins during storage. In this
respect an earlier study showed that the goat and sheep cheese
from Syria was free of AFM1 contamination (Haydar, Benelli, & Bre-
ra, 1990).
Several studies around the world have addressed the issue of
AFM1 contamination of milk and milk products. To put our results
in perspective, it would be helpful to compare them to those of the
sort reported elsewhere. For raw milk samples, the levels of AFM1
contamination reported in the present study where somewhat
higher than those reported in Turkey where 47% of the analyzed
samples (129 sample) contained AFM1 at levels exceeding the EU
accepted limit (Unusan, 2006). As for pasteurized milk, the magni-
tude of AFM1 contamination in the present study is comparable to
that reported in Morocco where 88% of the tested pasteurized milk
samples exceeded the EU accepted limit (Zinedine et al., 2007). A
study on randomly selected samples of raw cow milk from North
African countries showed a high level of contamination with
AFM1 ranging between 30 and 3130 ng/l (Elgerbi, Aidoo, Candlish,
& Tester, 2004). In a study conducted in India the incidence of
contamination of AFM1 in infant milk, milk based cereal weaning
food and liquid milk samples was almost in the magnitude of
87% (Rastogi et al., 2004), with 99% of contaminated samples
exceeding the EU/Codex recommended limits. On the other hand,
a Greek study showed that levels of AFM1 in milk were far below
the tolerance level (Kaniou-Grigoriadou, Eleftheriadou, Mourati-
dou, & Katikou, 2005).
It is possible to extrapolate AFB1 contamination levels in feed
stuff based on AFM1 Contamination levels in milk samples
605
(Forbisch et al., 1986). Results of such extrapolation based on
AFM1 concentrations found in milk samples tested in the present
study (Table 1) indicates the likelihood that feeds provided to dairy
cattle in Syria contains higher concentrations of AFB1 than those
prescribed by the European Communities, i.e. 5 lg/kg (Rastogi
Range et al., 2004). It is therefore important to monitor the levels of
(ng/l) AFB1 in feedstuffs of dairy animals in Syria and to devise mecha-
nisms to improve their quality in such a way that reduces AFM1
contamination of milk and milk products.
634
4. Conclusion
543–765 The present study showed that there is a risk of milk being con-
taminated with AFM1 which consequently affects the public health
in Syria, since almost all the population, including infants and chil-
dren, consume milk and milk products daily. Accordingly, the lev-
els of AFM1 in milk and milk products should be controlled and
monitored continuously. It is also important to maintain low levels
of AFB1 in feeds of dairy animals.
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