ORIGINALRESEARCH Survey of aflatoxin M1 in milk and dairy products

consumed in Burdur, Turkey

FATMA SAHINDOKUYUCU KOCASARI,1* FULYA TASCI2 andFIRDEVS MOR1

1Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Mehmet Akif Ersoy, 15030Burdur, Turkey, and 2Department of Food Hygiene and Technology, Faculty of Veterinary Medicine, University of MehmetAkif Ersoy, 15030 Burdur, Turkey

*Author forcorrespondence. E-mails:fatmasa@mehmetakif.edu.tr; fsahindokuyucu@hotmail.com

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The aim of this study was to investigate the occurrence of aflatoxin M1 (AFM1) in dairy product samplesin Burdur city. A total of 315 samples of dairy products were collected during 2008. Of the 315 samplesanalysed, AFM1 in 246 samples (78.1%) was found to range from 5.5 to 800 ng ⁄kg. In addition, AFM1

levels of 16 raw milk, two pasteurised milk, only one milk powder and three white cheese samples wereabove the Turkish Food Codex. It is concluded that the occurrence of AFM1 in dairy products may beconsidered as a possible hazard for public health.

Keywords Aflatoxin M1, Milk, Dairy products.

INTRODUCT ION

Aflatoxins are a group of mycotoxins produced bycertain fungi, especially Aspergillus flavus (A. fla-vus) and Aspergillus parasiticus (A. parasiticus)(Wang et al. 2010). These fungi are ubiquitous andcan occur in a wide range of agricultural commodi-ties (Prado et al. 2008). Aspergillus flavus onlyproduces B aflatoxins, while A. parasiticus pro-duces both B and G aflatoxins (Van Egmond1989). The formation of aflatoxins depends on thefoods on which the moulds grow and the condi-tions of heat and humidity during the growth, har-vesting and storage of the crops (Yaroglu et al.2005).Aflatoxins are toxic, carcinogenic, teratogenic

and ⁄or mutagenic effects in human and animals(IARC 1993). Aflatoxin B1 (AFB1) presents thehighest degree of toxicity for animals, followed bythe aflatoxins M1 (AFM1), G1 (AFG1), B2 (AFB2)and G2 (AFG2) (Sassahara et al. 2005). AFM1 isthe main monohydroxylated derivate of AFB1,forming in the liver by means of cytochromeP450-associated enzymes (Zinedine et al. 2007). Itcan be found in milk and dairy products from dairycattle that have consumed feed contaminated withAFB1 (Dashti et al. 2009).AFM1 could be detected in milk 12–24 h after

the first AFB1 ingestion, reaching a high level aftera few days. When the intake of AFB1 is finished,

the AFM1 concentration in the milk decreases toan undetectable level after 72 h (Van Egmond1989). Many researchers reported that there was alinear relationship between the amount of AFM1 inmilk and AFB1 in feed consumed by the animals(Bakirci 2001). About 0.3–6.2% of AFB1 in ani-mal feed is transformed to AFM1 in milk. The ratiovaries from animal to animal, from day to day, aswell as from one milking to the next (Martins andMartins 2004).Milk and milk products are a good source of

many nutrients such as proteins and calcium andare mainly consumed by children (Prado et al.2008). However, these products may be contami-nated with residues and pose a risk to humanhealth. AFM1 is relatively stable during pasteurisa-tion, sterilisation, preparation and storage of vari-ous dairy products (Wiseman and Marth 1983).The International Agency for Research on Cancer(IARC) of the World Health Organisation (WHO)included AFB1 as primary and AFM1 as secondarygroups of carcinogenic compounds (IARC 1993).For this reason, many countries have carried outstudies concerning the incidence of AFM1 in milkand dairy products and proposed maximum levelsfor this mycotoxin (Galvano et al. 2001; Zinedineet al. 2007). The Turkish Food Codex (TFC) legallimits for AFM1 in milk and dairy products are50 ng ⁄L and 500 ng ⁄kg, respectively (TurkishFood Codex 2008). The European Commission

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(EC) has set a limit of 50 ng ⁄L for AFM1 in milk and dairyproducts (EC 2006), while the United States Food and DrugAdministration (US FDA) has set a limit of 500 ng ⁄L forAFM1 in milk and dairy products (FDA 1996).The purpose of this study was to investigate the presence and

levels of AFM1 in raw milk, pasteurised milk, milk powder,white cheese, butter, yoghurt and ice cream samples consumedin Burdur and to compare the results with Turkish legal regula-tion for AFM1.

MATER IALS AND METHODS

MaterialsIn 2008, a total of 315 samples consisting 45 samples each ofraw milk, pasteurised milk, milk powder, white cheese,yoghurt, butter and ice cream were obtained randomly fromfarms and markets in various districts of Burdur. The sampleswere transported to the laboratory in refrigerated containers(4 �C) and stored at )20 �C until analysis.

MethodsThe quantitative analysis of AFM1 in the samples was deter-mined by competitive enzyme-linked immunosorbent assay(ELISA) method according to the procedure described byR-Biopharm GmbH, Germany (RIDASCREEN, Aflatoxin M1

30 ⁄15, Art No.: R1111).

Samples preparationPreparation of samples was conducted according to the instruc-tions of the RIDASCREEN kit (R-Biopharm GmbH 1999).

Milk samplesThe milk samples were centrifuged at 3500 g for 10 min at10 �C. After centrifugation, the upper cream layer was com-pletely removed and aliquot of the lower phase was carefullypoured off with a Pasteur pipette. The skimmed milk (defattedsupernatant) was used directly in the test (100 lL).

Milk powder samplesThe milk powder samples (10 g each) were diluted in 100 mLof warm deionised water and were stirred for 5 min. After-wards, the samples were prepared as described previously formilk samples.

Cheese samplesThe white cheese samples (2 g each) were homogenised andextracted with 40 mL dichloromethane. The suspension was fil-tered and 10 mL of the extract was evaporated at 60 �C under anitrogen stream. The residue was dissolved in 0.5 mL metha-nol, 0.5 mL of buffer phosphate and 1 mL n-heptane. Aftercentrifugation for 15 min at 2700 g, the upper heptane layerwas completely removed. The lower methanol–aqueous phasewas carefully poured off using a Pasteur pipette. One hundredmicrolitre of this aliquot was diluted with 400 lL of Ridascreen

buffer 1. An aliquot of this solution was used in the test(100 lL).

Butter samplesThe butter samples (5 g each) were added to 25 mL of metha-nol ⁄water (70:30) and shaken for 10 min. Then, the sampleswere centrifuged at 3500 g for 10 min at 10 �C. The upperlayer was filtered through a paper filter and 5 mL of the filtratewas added with 15 mL distilled water and 2 mL buffer phos-phate. Then, the entire amount of the solution passed throughthe column (Rida Aflatoxin Column Art. No: R 5001 ⁄5002).Clean-up procedure was conducted according to the instruc-tions of the kit. One hundred microlitre of this aliquot wasdiluted with 900 lL of Ridascreen buffer 1. An aliquot of thissolution was used in the test (100 lL).

Yoghurt samplesThe yoghurt samples were heated to 80 �C for 3 min to inacti-vate the living yoghurt bacteria and were then pasteurised (Ak-kaya et al. 2006). Then, the samples were cooled down toroom temperature and diluted in 1:10 in PBS-buffer (pH 7.2).They were homogenised by stirring. The diluted yoghurtsamples were used directly in the test (100 lL).

Ice cream samplesThe ice cream samples (2 g each) were diluted in 2 mL warmdeionised water and were centrifuged at 3500 g for 10 min at10 �C. The upper oily layer was completely removed. Thelower defatted supernatant was used directly in the test(100 lL).

ELISA test procedureOne hundred microlitre standard solutions and prepared samplesin separate wells were added and incubated for 30 min at roomtemperature in the dark. At the end of incubation, the liquid in thewells was poured out, the microwell holder was tapped upsidedown, and an absorbent paper was used to remove the remainderof the liquid. Thewells were washed twice with 250 lL ofwash-ing buffer. After thewashing steps, 100 lLof the diluted enzymeconjugate was added to the wells and incubated for 15 min atroom temperature in the dark. At the end of incubation, the wellswere washed three times with 250 lL of washing buffer. Then,100 lL of substrate and chromogen were added to each well andmixed thoroughly and incubated for 15 min at room temperaturein the dark. Following that, 100 lL of the stop solution wasadded to each well and mixed. The absorbance was measuredat 450 nm by an ELISA (ELX-800; Bio-Tek Instruments,Winooski, VT,USA) against air within 15 min.

EvaluationThe samples were evaluated according to the RIDAWIN com-puter program, prepared by R-Biopharm. The levels of afla-toxin standards used were 0, 5, 10, 20, 40 and 80 ng ⁄kg.According to the test preparation record, the lower detection

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limit was 5 ng ⁄L for milk, 50 ng ⁄kg for milk powder, cheeseand yoghurt, 25 ng ⁄kg for butter and 10 ng ⁄kg for ice cream.The statistical analysis was performed by Minitab StatisticalProgram (Minitab� Statistical Software to Analyze, Conventry,UK).

RESULTS

The occurrence and distribution of AFM1 concentration milkand dairy products are summarised in Tables 1 and 2. Of the315 samples analysed, 78.1% (246) were found to be contami-nated with AFM1. AFM1 levels in 16 (35.5%) raw milk sam-ples, 2 (4.4%) pasteurised milk samples, 1 (2.2%) milk powdersample and 3 (6.7%) white cheese samples were found to behigher than the maximum acceptable levels (milk: 50 ng ⁄L,dairy products: 500 ng ⁄kg) of the TFC (Turkish Food Codex2008). However, none of the butter, yoghurt and ice creamcontaminated samples exceeded the legal limit of 500 ng ⁄kg.

D I SCUSS ION

Milk and dairy products can be the source of aflatoxins (Tekin-sen and Ucar 2008). The occurence of AFM1 in milk,

especially cow’s milk, makes it a particular risk for humansbecause of its importance as a foodstuff for adults and espe-cially children. Therefore, the presence of AFM1 in milk andmilk products is considered to be undesirable (Prandini et al.2009). There are many studies about the contamination of milkand milk products with AFM1 (Tables 3 and 4).In this study, the occurrence of AFM1 in milk and dairy

products was high, as 78.1% (246) of the samples were posi-tive. The overall incidence of AFM1 contamination found inthis study might be attributed to the fact that the analysed sam-ples were collected at the cold period when dairy animals werefed with relatively higher amounts of concentrated feeds.Previous studies have reported different levels of AFM1 in

milk and dairy products (Tables 3 and 4). However, someresearchers (Lin et al. 2004; Atanda et al. 2007) reported noAFM1 in dairy product samples. These differences may beexplained by different analytical methods, geographical region,climatic factors and the seasonal variability (Tekinsen and Ucar2008; Var and Kabak 2008; Fallah 2010).A number of analytical methods have been reported for the

determination of AFM1 in milk and dairy products (Wanget al. 2010). Of significant importance are thin layer chromato-graphy (TLC), ELISA, high-performance liquid chromatography

Table 1 Occurrence of AFM1 in milk and dairy product samples

SamplesTestedn

Positiven (%)

Contamination (ng ⁄L or ng ⁄ kg)Exceed regulationa

n (%)Range Mean ± SD

Raw milk 45 41 (91.1) 15.3–80 45.3 ± 3.5 16 (35.5)Pasteurised milk 45 30 (66.7) 5.5–57 19.1 ± 2.9 2 (4.4)Milk powder 45 42 (93.3) 60–800 204.3 ± 23.1 1 (2.2)White cheese 45 40 (88.9) 55–600 199.4 ± 21.9 3 (6.7)Butter 45 39 (86.7) 25–320 95.6 ± 10.9 0Yoghurt 45 20 (44.4) 50–360 176.6 ± 21.4 0Ice cream 45 34 (75.6) 10–136 32.6 ± 4.1 0

SD, standard deviation.aThe Turkish limit for AFM1 is 50 ng ⁄L for milk, 500 ng ⁄ kg for dairy products.

Table 2 Distribution of AFM1 in milk and dairy product samples

Samples

Distribution of samples (ng ⁄L or ng ⁄ kg) n (%)

5–10 11–25 26–50 51–100 101–250 251–500 >500

Raw milk 0 10 (22.2) 15 (33.3) 16 (35.5) 0 0 0Pasteurised milk 16 (35.5) 5 (11.1) 7 (15.6) 2 (4.4) 0 0 0Milk powder 0 0 0 16 (35.5) 12 (26.7) 13 (28.9) 1 (2.2)White cheese 0 0 0 11 (24.4) 19 (42.2) 7 (15.6) 3 (6.7)Butter 0 1 (2.2) 15 (33.3) 9 (20) 13 (28.9) 1 (2.2) 0Yoghurt 0 0 0 6 (13.3) 10 (22.2) 4 (8.9) 0Ice cream 2 (4.4) 13 (28.9) 14 (31.1) 4 (8.9) 1 (2.2) 0 0

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Table 3 The occurrence of AFM1 in raw milk and pasteurised milk samples reported in previous studies

Samples CountryTestedn

Positiven (%) Range (ng ⁄L) References

Raw milk Portugal 31 25 (80.6) 5–50 Martins and Martins (2000)Turkey 90 79 (87.8) 12.5–123.6 Bakirci (2001)Spain 92 5 (5.4) 14–24.9 Rodriguez Velasco et al. (2003)Argentina 56 6 (10.7) 12–30 Lopez et al. (2003)Libya 49 35 (71.4) 30–3130 Elgerbi et al. (2004)Brazil 42 10 (24) 295–1975 Sassahara et al. (2005)Iran 72 72 (100) 4.3–91.8 Tajik et al. (2007)Pakistan 168 168 (100) 10–700 Hussain and Anwar (2008)Indonesia 113 65 (57.5) 5–25 Nuryono et al. (2009)Syria 74 70 (95) 20–690 Ghanem and Orfi (2009)

Pasteurised milk Argentina 16 8 (50) 10–17 Lopez et al. (2003)Taiwan 44 40 (90.9) 2–83 Lin et al. (2004)Turkey 85 75 (88.2) 5.2–127.6 Celik et al. (2005)Iran 624 624 (100) <45–>80 Alborzi et al. (2006)Colombia 120 83 (69.2) 10.7–213 Diaz and Espitia (2006)

121 96 (79.3) 10.6–288.9Morocco 54 48 (88.8) 1–117 Zinedine et al. (2007)Iran 128 128 (100) 31–113 Oveisi et al. (2007)Syria 10 10 (100) 8–765 Ghanem and Orfi (2009)Iran 50 50 (100) <50–>80 Ghazani (2009)Iran 91 66 (72.5) 13–250 Fallah (2010)

Table 4 The occurrence of AFM1 in dairy product samples reported in previous studies

Samples CountryTestedn

Positiven (%) Range (ng ⁄ kg) References

Milk powder Italy 92 50 (54.3) <1–79.6 Galvano et al. (2001)Argentina 5 4 (80) 10–14 Lopez et al. (2003)Taiwan 24 – – Lin et al. (2004)Turkey 21 19 (90.5) 0–705 Deveci and Sezgin (2005)Turkey 80 50 (62.5) 1–>601 Elmali et al. (2008)Syria 8 1 (13) 12 Ghanem and Orfi (2009)

Cheese Libya 20 15 (75) 110–520 Elgerbi et al. (2004)Turkey 100 82 (100) 51–800 Sarimehmetoglu et al. (2004)Turkey 23 9 (39.1) 11–106 Gurses et al. (2004)Turkey 200 10 (5) 100–600 Yaroglu et al. (2005)Turkey 25 17 (68) 51–400 Yapar et al. (2008)Kuwait 40 32 (80) 23.8–452 Dashti et al. (2009)Iran 72 59 (81.9) 30–120 Fallah (2010)

Butter Turkey 92 92 (100) 10–7000 Tekinsen and Ucar (2008)Turkey 10 3 (30) 40–70 Var and Kabak (2008)Iran 31 8 (25.8) 13–26 Fallah (2010)

Yoghurt Italy 120 73 (60.8) <1–32.1 Galvano et al. (2001)Portugal 48 2 (4.2) 43–45 Martins and Martins (2004)Turkey 104 68 (65.4) 1–100 Akkaya et al. (2006)Nigeria 11 – – Atanda et al. (2007)Turkey 20 16 (80) 54–263 Var and Kabak (2008)Iran 68 45 (66.1) 15–119 Fallah (2010)

Ice cream Nigeria 6 – – Atanda et al. (2007)Iran 36 25 (69.4) 15–132 Fallah (2010)

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(HPLC) and gas chromatography (GC) (Rosi et al. 2007; Wanget al. 2010). ELISA methods are rapid, simple, specific, sensi-tive and are even in portable format. These have become themost common, as well as quickest methods for routine analysisof mycotoxins in food and feed materials (Rosi et al. 2007).Therefore, the ELISA method was used for detecting AFM1 inmilk and dairy products in the present study.

MilkThe incidence and levels of AFM1 in raw milk samples werehigher than in pasteurised milk. In addition, the AFM1 inci-dence of exceeding the legal limit in raw milk was higher thanin pasteurised milk.In this study, the incidence of AFM1 in raw milk samples

was rather high; 91.1% (41) of the samples were positive at lev-els ranging from 15.3 to 80 ng ⁄L. AFM1 levels in 16 (35.5%)of 41 raw milk samples were found to be higher than the maxi-mum tolerable limit (50 ng ⁄L) accepted by the TFC (TurkishFood Codex 2008). In comparison with previous studies, theincidence and contamination levels of AFM1 in raw milk werehigher than those detected in Portugal (Martins and Martins2000), Spain (Rodriguez Velasco et al. 2003), Argentina(Lopez et al. 2003) and Indonesia (Nuryono et al. 2009); theywere lower than those found in Iran (Tajik et al. 2007), Paki-stan (Hussain and Anwar 2008) and Syria (Ghanem and Orfi2009). In surveys conducted in Turkey (Bakirci 2001), Libya(Elgerbi et al. 2004) and Brazil (Sassahara et al. 2005), theincidence of AFM1 in raw milk was lower, but the contamina-tion levels were higher.In this study, AFM1 was detected in 66.7% (30) of pas-

teurised milk samples at levels ranging from 5.5 to 57 ng ⁄L.Also, AFM1 levels in 2 (4.4%) of 30 pasteurised milk werefound to be higher than the maximum tolerable limit(50 ng ⁄L) accepted by the TFC (Turkish Food Codex 2008).In comparison with previous studies, the incidence and con-tamination levels of AFM1 in pasteurised milk were higherthan that found in Argentina (Lopez et al. 2003); they werelower than these detected in Taiwan (Lin et al. 2004), Turkey(Celik et al. 2005), Colombia (Diaz and Espitia 2006), Mor-occo (Zinedine et al. 2007), Syria (Ghanem and Orfi 2009)and Iran (Alborzi et al. 2006; Oveisi et al. 2007; Ghazani2009; Fallah 2010).

Milk powderThe highest incidence and levels of AFM1 among analysedmilk and dairy products were found in milk powder. AFM1

was detected in 93.3% (42) of milk powder samples at levelsranging from 60 to 800 ng ⁄kg. Also, AFM1 levels in 1 (2.2%)of 42 milk powder samples were found to be higher than themaximum tolerable limit (500 ng ⁄kg) accepted by the TFC(Turkish Food Codex 2008). The incidence and contaminationlevels of AFM1 in milk powder samples analysed in this studywere similar to the results reported by Deveci and Sezgin(2005); they were also higher than the results reported by other

researchers (Galvano et al. 2001; Lopez et al. 2003; Elmaliet al. 2008; Ghanem and Orfi 2009).

White cheeseIn this study, the incidence of AFM1 in white cheese sampleswas rather high; 88.9% (40) of the samples were positive at lev-els ranging from 55 to 600 ng ⁄kg. AFM1 levels in 3 (6.7%) of40 white cheese samples were found to be higher than the max-imum tolerable limit (500 ng ⁄kg) accepted by the TFC (Turk-ish Food Codex 2008). The incidence and contamination levelsof AFM1 of white cheese samples analysed in this study werelower than the results reported by Sarimehmetoglu et al.(2004); they were also higher than the results reported by otherresearchers (Elgerbi et al. 2004; Gurses et al. 2004; Yaparet al. 2008; Dashti et al. 2009; Fallah 2010). In this study, thecontamination levels of AFM1 in white cheese samples weresimilar to the results reported by Yaroglu et al. (2005), but theincidence of AFM1 was lower.

ButterIn this study, the incidence of AFM1 in butter samples washigh; 86.7% (39) of the samples were positive at levels rangingfrom 25 to 320 ng ⁄kg. However, none of the contaminatedsamples exceeded the legal limit of 500 ng ⁄kg (Turkish FoodCodex 2008). In this study, the incidence and contaminationlevels of AFM1 in butter samples were lower than the resultsreported by Tekinsen and Ucar (2008), but were higher thanthe results reported by other researchers (Var and Kabak 2008;Fallah 2010).

YoghurtThe incidence of AFM1 in yoghurt samples was the lowestamong the analysed milk and dairy products. The possible rea-son for this is that some strains of lactic acid bacteria existingin yoghurt are effective in AFM1 decontamination from milk(Fallah 2010). In this study, AFM1 was detected in 44.4% (20)of yoghurt samples at levels ranging from 50 to 360 ng ⁄kg.However, none of the contaminated samples exceeded the legallimit of 500 ng ⁄kg (Turkish Food Codex 2008). As seen inTable 4, the contamination levels of AFM1 in Italy (Galvanoet al. 2001) and Portugal (Martins and Martins 2004) werelow. In this study, the contamination levels of AFM1 in yoghurtsamples were higher than the results reported by other research-ers (Akkaya et al. 2006; Var and Kabak 2008; Fallah 2010),but the incidence of AFM1 in yoghurt samples was lower.

Ice creamA very few studies have been concerned with the occurrence ofAFM1 in ice cream samples. In Turkey, there are no data avail-able or studies carried out on AFM1 in ice cream. In this study,the incidence of AFM1 in ice cream samples was high; 75.6%(34) of the samples were positive at levels ranging from 10 to136 ng ⁄kg. However, none of the positive samples exceededthe legal limit of 500 ng ⁄kg (Turkish Food Codex 2008). In

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this study, the incidence and levels of AFM1 in ice cream weresimilar to the results reported by Fallah (2010).

CONCLUS IONS

The results of this study revealed that the incidence and levelsof AFM1 in milk and dairy products in Burdur area were highand could be considered to be hazardous to the public, espe-cially children. For this reason, milk and dairy products shouldbe controlled and monitored continuously for AFM1. Also, ani-mal feeds should be analysed regularly for aflatoxins and espe-cially, storage conditions of the animal feeds and feedstuffsmust be controlled. Alternatively, the contaminated feedsshould be mixed with either aflatoxin absorbents or noncontam-inated feeds to dilute the concentration of aflatoxin levels.Therefore, these methods could be used to prevent and reducethe risk of aflatoxins in animal feeds.

A C KNOWL EDG EMEN T S

This study was supported by the University of Mehmet Akif Ersoy(Project No: 0003-NAP-07).

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