Determination of aflatoxin M1 levels in Turkish white ...

WFL Publisher Science and Technology Meri-Rastilantie 3 B, FI-00980 Helsinki, Finland e-mail: [email protected]

Journal of Food, Agriculture & Environment Vol.8 (1) : 13-15. 2010

www.world-food.net

Determination of aflatoxin M1 levels in Turkish white, kashar and tulum cheeses Hamparsun Hampikyan 1*, E. Baris Bingol 2, Omer Cetin 2 and Hilal Colak 2 1

Beykent University, The School of Vocational Studies, 34500, Buyukcekmece, Istanbul, Turkey. 2 Istanbul University, Faculty of Veterinary Medicine, Department of Food Hygine and Technology, 34320, Avcilar, Istanbul, Turkey. *e-mail:[email protected], [email protected]

Received 1 September 2009, accepted 7 January 2010.

Abstract Aflatoxins are a group of highly toxic secondary metabolic products of some Aspergillus spp. Aflatoxin M1 (AFM1) is the hydroxylated metabolite of aflatoxin B1 and may be found in milk and dairy products such as cheese. This study was conducted to determine the AFM1 levels in Turkish white, kashar and tulum cheeses. For this purpose, 80 cheese samples were randomly obtained from markets and bazaars in Istanbul and analyzed for AFM1 by ELISA. While 39 of 80 cheese samples (48.7%) were negative for AFM1, 18 white cheese, 12 kashar cheese and 11 tulum cheese samples, in total 41 samples (51.3%) were found to be contaminated with AFM1 in the range of 0.052-2.52 µg/kg. The mean AFM1 values of examined white, kashar and tulum cheese samples were detected as 0.42, 0.25 and 0.38 µg/kg, respectively. Key words: Aflatoxin M1, white cheese, kashar cheese, tulum cheese.

Introduction Mycotoxins are the secondary toxic metabolites of fungi, synthesized during the end period of the logarithmic growth phase and have no obvious role in fungi metabolism or growth 1. Of all mycotoxins, aflatoxins are a group of highly toxic metabolic products named as aflatoxin B1(AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) 2, 3. Aflatoxins are produced by certain species of Aspergillus, especially Aspergillus flavus, A. parasiticus and A. nomius, that contaminate plants and plant products and are contaminants of animal feeds particularly in critical temperature and humidity conditions before or during harvest or because of inappropriate storage 4, 5. A. flavus produces only B aflatoxins, while the others produce both B and G aflatoxins 6. Aflatoxins have sub-acute and chronic effects such as liver cancer, chronic hepatitis, jaundice, hepatomegaly and cirrhosis in humans 7, 8. International Agency for Research on Cancer (IARC) included AFB1 as primary and aflatoxin M1 (AFM1) as secondary groups of carcinogenic compounds 9 . AFM1 is the monohydroxylated derivate of AFB1, metabolized by cytochrome P450 enzyme system in liver and excreted into the milk of lactating livestock which have ingested feedstuffs contaminated with AFB110, 11. AFM1 appears in milk 12-24 h after ingestion of the first AFB1-contaminated ration 12. Milk and dairy products are fundamental components in the human diet, and may be the principle way for entrance of aflatoxins into the human body. The consumption of contaminated milk and dairy products especially cheese is an important risk factor for consumers in particular for infants and young children 12, 13. Aflatoxins may be found in cheese originating from three possible sources: a) presence of AFM1 in milk with which cheese are Journal of Food, Agriculture & Environment, Vol.8 (1), January 2010

manufactured, as a consequence of food contaminated with AFB1 eaten by dairy cattle; b) synthesis of AFB1, B2, G1, and G2 by fungi which grow on cheese such as Aspergillus spp. and c) the use of dried milk with AFM1, to enrich the milk employed in cheese production. When cheese is manufactured from AFM1contaminated milk, the toxin can be carried over into both whey and cheese 13, 14. Due to their frequent occurrence and toxicity, guidelines and tolerance levels of aflatoxins have been set in several countries including Turkey. According to Turkish Food Codex (TFC) maximum residue limits have been set for AFM1 in cheese as 0.5 µg/kg 15. In Turkey, 40–50 cheese varieties are known but only three of them have national and economic value: Turkish white (named as Beyaz Peynir originally), kashar (like Kashkaval or Kasseri cheeses) and tulum (ripened in goat skin bags or plastic material) cheeses 16. Therefore, this study was conducted to determine the presence and levels of AFM1 in Turkish white, kashar and tulum cheeses consumed in Istanbul and to compare the obtained results with maximum AFM1 tolerance limits accepted by the TFC. Material and Methods Samples:A total of 80 samples of cheeses (30 white cheeses, 30 kashar cheeses and 20 tulum cheeses) commercialized in Istanbul were randomly obtained from markets and bazaars. The samples (approximately 250 g) were transported to the laboratory within an insulated container at about 4°C.

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Analysis of AFM1: Extraction of AFM1 and ELISA test procedure were performed according to the instructions of the test kit (Ridascreen Art No: R1101, R-Biopharm) manual 17. In our previous study, we compared the performance of the test kit for the determination of AFM1 in Turkish white, kashar and tulum cheeses against an HPLC method and we found that the results obtained by ELISA were related to those by HPLC 18. Therefore, this ELISA method was used in this study for the determination of AFM1in samples. Sample preparation: Two grams of cheese samples were homogenized (Ultraturrax, Janke & Kunkel) and extracted with 40 ml dichloromethane. The suspension was filtered, and then 10 ml of the extract was evaporated at 60°C under nitrogen. The oily residue was redissolved in 0.5 ml methanol, 0.5 ml phosphate buffer (Na2HPO4 1.427% w/v, KH2PO4 0.907 w/v, ratio 8:2, pH 7.2) and 1 ml n-heptane and was mixed thoroughly. After centrifugation for 15 min at 2700 rpm, 100 µl of the methanolic phase was brought to 10% methanol content by addition of 400 µl Ridascreen buffer 1. An aliquot (100 µl per well) of this solution was used in the test. ELISA test procedure: The AFM1 standards and test samples in duplicate were added to the wells of micro-titer plate pre-coated with antibodies for AFM1 and incubated at room temperature in dark for 60 min. After the washing step, AFM1 peroxidase conjugate (supplied with the kit) was added to the wells and plate was incubated again for 60 min at room temperature in dark. The unbound conjugate was removed during washing. Subsequently, 50 µl each of substrate (urea peroxide) and chromogen (tetramethyl-benzidine) were added to the wells and incubated for 30 min in dark. The reaction was stopped by 100 µl of 1 M H2SO4 and the absorbance was measured at 450 nm in ELISA plate reader (ELX 800, Bio-tek Inst.). The mean values of the absorbance for the standards and the samples were evaluated according to the R1-DAVIN.EXE computer program prepared by R-Biopharm. The mean lower detection limit was 50 ng/kg for cheese. Results and Discussion The distribution and evaluation of AFM1 levels in Turkish cheese samples are given in Tables 1 and 2. AFM1 was detected above

measurable level (50 ng/kg) in 18 white cheese (60.0%), 12 kashar cheese (40.0%) and 11 tulum cheese (55.0%) samples. All together AFM1 was found in 41 cheese samples, corresponding to 51.3% of the total examined samples, ranging between 0.052 and 2.52 µg/kg. AFM1 levels in 8 (10.0%) samples, consisting of 4 (13.3%) white cheese, 2 (6.7%) kashar cheese and 2 (10.0%) tulum cheese samples, were found to exceed the legal limit of 0.5 µg/kg established by TFC according to the new regulations 15. (This limit was 0.25 µg/kg for cheese in old version of TFC.) The mean AFM1 values of examined white, kashar and tulum cheese samples were 0.42, 0.25 and 0.38 µg/kg, respectively (Table 2). The presence of AFM1 in milk and dairy products is a hazard to human health because aflatoxins are carcinogenic, teratogenic and mutagenic. Therefore, studies have been conducted in different countries 1, 6, 19-23 and also in our country to examine the presence and levels of AFM1 in milk and dairy products. Several surveys were performed in order to determine the AFM1 levels in cheeses which were produced in our country. Demirer et al. 24, Kivanc 25 and Ozkalp 26 reported that AFM1 was not found in examined cheese samples. Oruc and Sonal 27 examined AFM1 levels in milk and cheese from Bursa, Turkey and found AFM1 in 89.5% of 57 cheese samples with ranges of 0-180 ng/kg. Seven of the samples were found contaminated over maximum permissible level of AFM1 in TFC. Gunsen and Buyukyoruk 28 analyzed 130 cheese samples in Bursa and determined an average of 0.142 µg/ kg AFM1. The amounts of AFM1 residue in 15% of the samples were found over maximum permissible level of 0.25 µg/kg given in TFC (old edition). Sarimehmetoglu et al. 29 detected AFM1 contamination in 327 (81.75%) of 400 cheese samples. The numbers of cheese samples that contained AFM1 over the legal limits of 0.25 µg/kg were 110 (27.5%). Gurses et al. 30 analyzed 63 cheese samples in Erzurum for the occurrence of AFM1. In 28 (44.44%) of 63 samples, AFM1 was detected in concentrations between 7 ng/kg and 202 ng/kg. In a study of Tekinsen and Eken 31, 132 kashar cheese samples were analyzed for AFM1 and 82.6% of the samples contained AFM1 (50-690 ng/kg) and 36 cheese samples (27.3%) exceeded the maximum tolerable limit of the TFC. In another study 193 white cheese samples were analyzed by Ardic et al. 32 and AFM1 was found in 82.4% of the samples (52 – 860 ng/kg). According to these results 26.4% of the samples exceeded the TFC.

Table 1. Distribution of AFM1 levels in Turkish type cheese samples. Detected level (µg/kg) ND*(0.50

White cheese (n =30) 12 3 4 3 2 2 4

Kashar cheese (n =30) 18 4 1 2 2 1 2

Tulum cheese (n = 20) 9 2 1 3 2 1 2

Total

%

39 9 6 8 6 4 8

48.7 11.3 7.5 10.0 7.5 5.0 10.0

*ND Not detected.

Table 2. Evaluation of AFM1 in Turkish type cheese samples. Cheese type White Kashar Tulum

n 30 30 20

Number (%) 18 (60) 12 (40) 11 (55)

Positive samples Meanb±SE 0.42±8.2 0.25±7.5 0.38±9.4

Rangea 0.052 – 2.52 0.060 – 1.15 0.057 – 1.36

>0.5 µg/kgc (%) 4 (13.3) 2 (6.7) 2 (10.0)

SE Standard error, n Number of samples, a Minimum – maximum values (µg/kg), b Mean values (µg/kg), c Turkish legal limit for cheese.

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In conclusion, cheeses may contain AFM1 in different levels, because of the presence of AFM1 in milk. Regardless of different research results on the ratio of AFM1 distribution in cheese, consensus of opinion is to accept that AFM1 residues in cheese are hazardous for public health. Because of this, it is important to minimize the mould contamination of the feeds, to prevent animals eating the contaminated feeds and to analyze the feeds periodically for AFB1 level. Also, in order to prevent consumer health, routine controls and survey researches have to be performed periodically and seriously for the detection of AFM1 contamination in milk and dairy products. References 1

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