Food and Chemical Toxicology 46 (2008) 3287–3289

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Food and Chemical Toxicology

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Aflatoxin M1 levels in UHT milk and kashar cheese consumed in Turkey

K. Kaan Tekinsßen a,*, H. Semih Eken b
a
Department of Food Hygiene and Technology, Faculty of Veterinary Science, Selçuk University, 42 075 Konya, Turkey
b
Army Commandership, B Type Food Control Detachment Command, 22 800 Kesßan Edirne, Turkey

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

Article history: In the present study, 100 UHT milk and 132 kashar cheese samples were analyzed for aflatoxin M1
Received 3 June 2008 (AFM1). They were obtained from retail outlets in five big cities (Istanbul, Izmir, Konya, Tekirdag, Edirne).
Accepted 17 July 2008 The occurrence and concentration range of AFM1 in the samples were investigated by competitive
enzyme-linked immunosorbent assay (ELISA) method. Sixty-seven percent of the UHT milk samples
and 82.6% of the kashar cheese samples contained AFM1. The positive incidence of AFM1 in the UHT milk
Keywords: samples and the kashar cheese samples ranged from 10 to 630 ng/kg and from 50 to 690 ng/kg, respec-
Aflatoxin
tively. AFM1 levels in 31 (31%) UHT milk and 36 (27.3%) kashar cheese samples exceeded the maximum
UHT milk
Kashar cheese
tolerable limit of the EC and the TFC. AFM1 levels in the samples show that there is a presence of high
ELISA aflatoxin level that constitutes a human health risk in Turkey. Therefore milk and dairy products have
to be controlled continuously for presence of AFM1 contamination by the Turkish public health
authorities.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction
Aflatoxin M1 (AFM1) is a hepatocarcinogen found in milk of ani-
mals that have consumed feeds contaminated with aflatoxin B1
(AFB1), the main metabolite produced by stroge fungi of the genus
Aspergillus, particularly A. flavus, A. parasiticus and A. nomius
(Galvano et al., 2001; Moss, 1998; Oliveira and Ferraz, 2007). Sev-
eral research workers reported that there is a linear relationship
between the amount of AFM1 in milk and AFB1 in the feed which
is consumed by these animals (Bakirci, 2001). Studies have clearly
demonstrated that AFM1 causes toxic and carcinogenic effects,
therefore this toxin, classified by the International Agency for Re-
search on Cancer (1993) of WHO as a Group 2 human carcinogen.
Milk and dairy products are a good source of many nutrients
such as proteins and calcium. However, milk and milk products
are the most potent source of aflatoxin among foods. Therefore, hu-
mans are potentially exposed to these metabolites and it is gener-
ally assumed that neither stroge nor processing provides a
reduction of AFM1 content (Unusan, 2006). AFM1 is relatively sta-
ble in raw and processed milk products, and unaffected by pasteur-
ization and ultra-high-temperature (UHT) treatment or processing
into cheese (Unusan, 2006; Yaroglu et al., 2005). Recently, it was
reported that the consumption of milk and dairy products has a
tendency to increase in the young generation in Turkey. In relation
countries have carried out studies about the incidence of AFM1
in milk and dairy products and proposed maximum levels for this
mycotoxin (Galvano et al., 2001; Kamkar, 2005). The European
Community (EC) and the Turkish Food Codex (TFC) legal limit for
AFM1 in milk and cheese are 50 ng/kg and 250 ng/kg, respectively
(Commission Regulation, 2006; Turkish Food Codex, 2002).
Although there is some information about the occurence of
AFM1 in kashar cheese there is not enough information about
the occurence of AFM1 in UHT milk in Turkey (Table 3). The pres-
ent investigation was undertaken to gather on the aflatoxin M1
levels of UHT milk and kashar cheese consumed in Turkey and to
compare the results with the maximum AFM1 tolerance limits
which are accepted by the TFC.
2. Materials and methods
2.1. Materials
A total of 100 samples of UHT milk and 132 kashar cheese samples were ob-
tained randomly from supermarkets between September 2007 and January 2008
in five big Turkish cities (Istanbul, Izmir, Konya, Tekirdag, Edirne). The samples
were transported to the laboratory in an insulated container at about 4 °C and ana-
lyzed upon arrival.
2.2. Methods

to that milk and dairy products a possible source of AFM1 could

also be very hazardous to them (Tekinsßen and Uçar, 2008). Many
* Corresponding author. Tel.: +90 332 2233568; fax: +90 332 2410063.
E-mail address: kktekinsen@selcuk.edu.tr (K.K. Tekinsßen).
The samples analysed for AFM1 using the competitive ELISA (RIDASCREEN Afla-
toxin M1, R-Biopharm) procedure as described by R-Biopharm GmbH (1999). Most
of the reagent used were contained in the RIDASCREEN AFM1 test kit; which in-
cluded microtiter plate coated with capture antibodies, AFM1 standard solutions
(1.3 ml each 0 ng/l, 5 ng/l, 10 ng/l, 20 ng/l, 40 ng/l, 80 ng/l), peroxidase conjugated
AFM1, substrate (urea peroxidase), chromogen (tetramethylbenzidine) and stop

0278-6915/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2008.07.014
3288 K.K. Tekinsßen, H.S. Eken / Food and Chemical Toxicology 46 (2008) 3287–3289
reagent contains 1N sulphuric acid. Methanol, n-heptane and dichloromethane
used were provided by Merck. Phosphate Buffer Solution (PBS) was prepared by
mixing 0.55 g sodium dihydrogen phosphate hydrate with 2.85 g disodium hydro-
gen phosphate-2-hydrate and 9 g sodium chloride and filling up to 1000 ml distilled
water.
2.2.1. Preparation of the milk samples
Ten milliliter of the milk samples were chilled to 10 °C and the centrifuged for
10 min at 3500 rpm. The upper oily phase was completely collected. An aliquot
(100 ll per well) of the lower oil-free phase was used in the test.
2.2.2. Preparation of the cheese samples
40 ml dichloromethane was added to the triturated cheese sample (2 g) in a
screw-top glass vial and shaken for 15 min. The suspension was filtered and
10 ml of the filtrate was evaporated at 60 °C under nitrogen. The oily residue was
dissolved with the addition of 0.5 ml methanol, 0.5 ml PBS buffer and 1 ml heptane,
and then mixed thoroughly and the centrifuged for 15 min at 2700 rpm. The upper
heptane phase was completely collected. An aliquot of 100 ll of the lower phase
(methanolic-aqueous) was taken up and was diluted with 400 ml Ridascreen buffer
1. An aliquot (100 ll per well) of this mixture was used in the test.
2.2.3. Test procedure
A sufficient number of microtiter wells were inserted into the microwell holder
for all standards and samples. One hundred microliter standard solution and pre-
pared samples in separate wells were added and incubated for 30 min at room tem-
perature in the dark. At the end of incubation, the liquid in the wells was poured
out, and the microwell holder was tapped upside down on an absorbent paper to
remove the remainder of the liquid. The wells were washed twice with 250 ll
washing buffer. One hundred microliter of the enzyme conjugate (peroxidase con-
jugated AFM1) was added to each well and incubated 15 min at room temperature
in the dark. At the end of incubation, the wells washed three times with 250 ll
washing buffer. One hundred microliter substrate/chromogen were added to each
well and incubated for 15 min at room temperature in the dark. Following the addi-
tion 100 ll of the stop reagent to each well, the absorbance was measured at
450 nm in ELISA reader (ELX-800, Bio-Tek Instruments, USA).
2.2.4. Evaluation
According to the test preparation record, the lower detection limit was 10 ng/l
(10 ppt) for milk and 50 ng/l (50 ppt) for cheese. The recovery rate in milk is 95% with
a mean coefficient of variation of 15%. In case of cheese, the recovery rate is approx-
imately 102% and the mean coefficient of variation is approximately 11%. The samples
were evaluated according to the RIDAVIN computer program, prepared by R-Biop-
harm, and the statistical analysis was performed by Mstat Statistical Program.
3. Results
The occurrence and the distribution of AFM1 concentration in
UHT milk and kashar cheese samples obtained are presented in Ta-
bles 1 and 2, respectively.
Table 1
Occurrence and distribution of aflatoxin M1 in UHT milk samples
Sample Number of sample
Range (ng/kg)
<10 10–25 26–50 51–100 >100
UHT milk 33 (33) 12 (12) 24 (24) 18 (18) 13 (13)
<10: range of negative samples (): indicates percent.
*
EC and the TFC limits in milk is 50 ng/kg.
Table 2
Occurrence and distribution of aflatoxin M1 in kashar cheese samples
Sample Number of sample
Range (ng/kg)
<50 50–100 101–250 251–500
Kashar cheese 23 (17.4) 26 (19.7) 47 (35.6) 26 (19.7)
<50: range of negative samples (): indicates percent.
*
EC and the TFC limits in cheese is 250 ng/kg.
As shown in Tables 1 and 2, AFM1 was found above the detect-
able level in 67% (67/100) of the UHT milk samples and in 82.6%
(109/132) of the kashar cheese samples, respectively. AFM1 levels
in 31 (31%) UHT milk and 36 (27.3%) kashar cheese samples were
found to be higher than the maximum acceptable limits (milk:
50 ng/kg, cheese: 250 ng/kg) of the TFC. The concentrations of
AFM1 in UHT milk and kashar cheese samples were in the positive
range of 10–630 ng/kg and 50–690 ng/kg accordingly. However, as
shown in Tables 1 and 2 36% and 55.3% of the samples contained
AFM1 at the level 10–50 ng/kg and 50–250 ng/kg in UHT milk
and kashar cheese samples, respectively.
4. Discussion
At the present study, AFM1 levels were determined in UHT milk
and kashar cheese which are mostly consumed in the big cities of
Turkey. As depicted in Tables 1 and 2, AFM1 was detected in 67% of
the UHT milk samples and in 82.6% of the kashar cheese samples,
respectively. These results are in parallel with the findings of some
previous reports (Aycicek et al., 2005; Günsßen and Büyükyörük,
2003; Gürbay et al., 2006; Sarımehmetoglu et al., 2004; Unusan,
2006) which pointed out the presence of AFM1 in more than
60% of the UHT milk and kashar cheese samples in Turkey (Table
3). These reports and the present findings suggest that the milk
which is processed into dairy products may contain a high concen-
tration of AFM1 and/or be contaminated with Aspergillus spp. The
results confirm the findings of some researchers (Blanco et al.,
1988; López et al., 2003) who reported a higher incidence of
AFM1 contamination during cold seasons than hot ones and of
some scientists who were pointing out that 47% of the UHT
milk samples (Unusan, 2006) and 13–33% of kashar cheese samples
(Aycicek et al., 2005; Günsßen and Büyükyörük, 2003;
Sarımehmetoglu et al., 2004; Yapar et al., 2008) exceed the legal
limits for AFM1 levels. However, as seen on Table 3 a number of
researchers (Blanco et al., 1988; Gürbay et al., 2006; Gürbüz et
al., 1999; Kardesß, 2000; Razza, 2006; Yaroglu, 2002; Yaroglu et
al., 2005) reported a lower incidence or not detected of AFM1 in
UHT milk and kashar cheese. The variations on the findings may
be attributable to the fact that milk to be processed may contain
different levels of AFM1 according to the seasonal changes and
the different processing techniques and analysis methods (Blanco
et al., 1988; Tekinsßen and Uçar, 2008). Moreover, differences in
the hygiene and storage conditions at the dairies and retail points
are other key factors on the variations of the results (Tekinsßen and
Tekinsßen, 2005; Tekinsßen and Uçar, 2008).
Positive samples Exceed legal limit* (%)
Mean (ng/kg) % Min. – Max. (ng/kg)
67 ± 11 67 10–630 31
Positive samples Exceed legal limit* (%)
Mean (ng/kg) % Min. – Max. (ng/kg)
>500
10 (7.6) 194 ± 15 82.6 50–690 27.3
 K.K. Tekinsßen, H.S. Eken / Food and Chemical Toxicology 46 (2008) 3287–3289 3289

Table 3
Aflatoxin M1 contents of UHT milk and kashar cheese reported in previous studies

Sample Country Samples tested Mean (ng/kg) Positive samples Exceed legal limit* (%) References
% Min. – Max. (ng/kg)
UHT milk Spain 47 – 29.8 20–100 4.26 Blanco et al. (1988)
Portugal 70 – 85.7 5–61 2.86 Martins and Martins (2000)
Greece 17 – 82.3 >5 – >50 0 Roussi et al. (2002)
Turkey 24 – 58.3 10–50.5 4.17 Gürbay et al. (2006)
Pakistan 79 – 11.3 29.3–102.8 7.59 Razza (2006)
Turkey 129 108.2 58.1 <10–543.6 47.1 Unusan (2006)
Brazil** 12 58 ± 44 83.3 10 – >200 50 Oliveira and Ferraz (2007)
Kashar cheese Turkey 60 ND 0 – 0 Gürbüz et al. (1999)
Turkey 50 ND 0 – 0 Kardesß (2000)
Turkey 100 – 9 >100 – >250 2 Yaroglu (2002)
Turkey 125 206 ± 15.9 68.8 10–740 32.55 Günsßen and Büyükyörük (2003)
Turkey 100 – 85 51 – >800 34 Sarımehmetoglu et al. (2004)
Turkey 53 – 88.7 <1 – >250 13.2 Aycicek et al. 2005
Turkey 200 272 ± 59 6 120–800 1 Yaroglu et al. (2005)
Turkey 25 – 52 51 – >400 24 Yapar et al. (2008)
*
EC and the TFC limits in milk and cheese are 50 ng/kg and 250 ng/kg, respectively.
**
From goat origin ND: AFM1 was not detected.

Especially in this study, it is important that AFM1 incidence of
exceeding legal limit (31%) in UHT milk samples was relatively
much higher than some other countries (Spain, Portugal, Greece,
Pakistan) (Table 3). It has been indicated that many countries in
Europe showed relatively low levels of contamination of AFM1 in
milk samples because of a result of stringent regulation of AFB1
in dairy cattle feed (Trucksess, 1999, 2006).
In conclusion, the presence of high levels of AFM1 in the UHT
milk and kashar cheese in Turkey seems to be still a main concern
for public health. For this reason, milk and dairy products have to
be controlled continuously for presence of AFM1 contamination by
the Turkish public health authorities.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
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