Abstract: In this study, total number of 220 milk and cheese samples were analysed, consisting 120 pasteurised milk samples from 8 different firms, 30 raw milk samples and 70 Turkish white brined cheese samples from 7 different firms which were collected from Ankara region, Turkey. Aflatoxin M₁ (AFM₁) was determined with an Enzyme Linked Immunosorbent Assay (Elisa) using the I’screen Afla M₁ ELISA kit. Sample preparations were done according to the instructions of the Tecna kit. Mean levels of AFM₁ was 0.025±0.003 ppb in the milk samples. Regarding the Turkish white brined cheese samples, mean level of AFM₁ was found to be 0.023±0.004 ppb. The data revealed that while mean AFM₁ levels found in pasteurised milk of two firms were higher than Turkish Food Codex (TFC) values for the other milk was within TFC values. In raw milk and cheese samples all the AFM₁ levels were within the TFC.

INTRODUCTION

Milk is a source of many nutrients like proteins and calcium and consumed as a main food in many countries. However, milk could also be a source of aflatoxin M₁ (AFM₁) and humans, especially infants and young children who consume contaminated milk and milk products are at high risk for AFM₁ toxin (Karimi et al., 2007; Yapar et al., 2008). Aflatoxins are toxic and carcinogenic metabolic products of Aspergillus (A. flavus, A. parasiticus and A. nomius) (Kim et al., 2000; Gurses et al., 2004; Ozdemir, 2007; Tajik et al., 2007; Yapar et al., 2008; Nuryono et al., 2009). Aflatoxin M₁ (AFM₁) occurs on food and feed during growing, harvesting and storage. Aflatoxin contamination may occur after ingestion or contamination of milk and milk products with fungi (Celik et al., 2005; Ozdemir, 2007). As a result of ingestion of feed stuffs by the animal, aflatoxin B₁ (AFB₁) may occur in milk and dairy products. After AFB₁ ingestion, it can be converted to hydroxylated form, AFM₁ and can be detected in milk after 12-24 h (Lopez et al., 2003; Gurses et al., 2004; Tajik et al., 2007; Ozdemir, 2007).

Aflatoxins are highly toxic, mutagenic, teratogenic and carcinogenic compounds (Yentur et al., 2006). Although AFM₁ is less mutagenic than AFB₁, it is also known to be carcinogenic and classified by the International Agency for Research on Cancer (IARC, 1993) as 2nd class carcinogen Lopez et al., 2003; Karimi et al., 2007; Tajik et al., 2007; Yapar et al., 2008; Nuryono et al., 2009). AFM₁ is resistant to thermal inactivation; therefore, it is stable after pasteurization. The process of milk to cheese also results in disregarded destruction of AFM₁. Thus, if raw milk contains AFM₁, cheese made from such milk will also contain AFM₁ (Lopez et al., 2003; Gurses et al., 2004; Sarimehmetoglu et al., 2004; Yaroglu et al., 2005; Ozdemir, 2007). Since, liquid pasteurised milk and cheese are being consumed by various age groups and especially infants, determining the AFM₁ in these products is important to protect consumers and it is an important public health concern (Franco et al., 1998; Mohamadi et al., 2009).

In European countries the maximum level of AFM₁, should not exceed 50 ng kg^-1 for liquid milk and milk products. In USA, the AFM1 level should not be greater than 500 ng kg^-1 in milk. In Turkey, according to the Turkish Food Codex, limits for AFM₁ are 50 ng L^-1 (0.05 ppb) and 500 ng L^-1 (0.5 ppb) for milk and cheese, respectively (Anonymous, 2008). AFM₁ level can be determined by chromatographic methods in milk and milk products (Franco et al., 1998; Aycicek et al., 2005). Although, these methods are reliable methods, they are expensive and lengthy. In recent days, Enzyme Linked Immunosorbent Assay (ELISA), a simple, cheap, sensitive and quick method is preferred in routine work (Gurses et al., 2004; Karimi et al., 2007; Tajik et al., 2007; Ghiasian et al., 2007). In the study it was aimed to determine AFM₁ levels in raw milk, pasteurised milk and Turkish white brined cheese samples consumed in Ankara region, Turkey with ELISA method.

MATERIALS AND METHODS

Total 220 milk and cheese samples; consisting 120 pasteurised milk samples from A, B, C, D, E, F, G, H firms, 30 raw milk samples (I) and 70 Turkish white brined cheese samples from J, K, L, M, N, O, P firms were investigated in case of AFM₁ levels. Raw milk samples were collected from open air markets, pasteurised milk and cheese samples were collected from supermarkets in Ankara region. Samples were carried to laboratory in a cold chain for the detection of AFM₁. AFM₁ was determined with an ELISA using the I’screen AFLA M₁ ELISA kit (TECNA S.r.1., Trieste, Italy) (Tajik et al., 2007; Anonymous, 2008) Sample preparations were done according to the instructions of the TECNA kit.

The kit was stored at 4°C and all the reagents were brought to room temperature, 2 h before use and all reagents were returned to 4°C after use. Milk samples were refrigerated and centrifuged at 2-8°C for 10 min at 3000xg. The fat from the skimmed milk was separated and the skimmed milk was used.

The cheese samples were weighted and 2 g of the samples were used. About 15 mL of dichloromethane was added to the sample and extracted by shaking the vial for 15 min. The suspension was filtered and 3.75 mL of the suspension was transferred to a glass tube and evaporated at 60°C under a nitrogen stream.

The residue was dissolved in 750 μL of the extraction solution and mixed by vortex for 1 min. After then, 750 μL hexane was added and vortexed for 1 min. The suspension was centrifuged for 15 min at 2000xg. The upper hexane layer was removed and 50 μL of the methanolic/acqueous phase was taken and 200 μL dilution buffer was added. This final suspension was used in the test.

The AFM₁ standards and milk samples were added to 96 wells microplate coated with AFM₁ antibodies and incubated for 45 min at room temperature. The kit reagents consisting of the enzyme conjugate solution, developing solution and stop solutions were added to the wells, respectively and washed several times in the appropriate order and incubation times according to the instructions. The absorbance was measured at 450 nm.

Statistical analyses: Student’s t-test and One Way ANOVA test were conducted for statistical evaluation (Daniel, 1991).

RESULTS AND DISCUSSION

The levels of AFM₁ in pasteurised milk, raw milk and cheese samples were determined. The concerning values are shown in Table 1-3. The results of the analyses were evaluated within the Turkish Food Codex. Table 1 shows that the mean level of AFM₁ was 0.025±0.003 ppb in the milk samples. The maximum and minimum levels were determined as 0.26 and 0.0, respectively.

The aflatoxin mean level of D and H firm were found higher than the Turkish Food Codex Standard value (0.05 ppb). The mean levels of the aflatoxin M₁ in pasteurised milk of other firms and raw milk were found within the Turkish Food Codex Standard value. The differences between mean levels of aflatoxin M₁ in these samples and the TFC values were statistically significant (p<0.001).

Table 2 shows that the level of average AFM₁ was 0.023±0.004 ppb in the Turkish white brined cheese samples. The maximum and minimum levels were determined as 0.24 and 0.0, respectively. The mean levels of the AFM₁ in all of the samples were found within the Turkish Food Codex (TFC) Standard. The difference between the mean levels of AFM₁ in all of the cheese samples and the TFC values (0.5 ppb) were found statistically significant (p<0.001). This TFC value (0.5 ppb) was for AFM₁ in terms of risky food stuffs.

Table 1:	Statistical analysis for level of AFM1 (ppb) in milk samples
*p<0.001 (the difference between the levels of aflatoxin M1 was found statistically significant amongst the groups)

Table 2:	Statistical analysis for level of AFM1 (ppb) in cheese samples
*p<0.001 (the difference between the levels of aflatoxin M1 was found statistically significant amongst the firms)

Table 3:	Levels of AFM1 in pasteurised milk, raw milk and cheese samples
*Exceeding TFC standard value

The data revealed that the mean levels of AFM₁ found in milk samples of A, B, C, E, F, G firms and raw milk (I) were within the TFC standard values. However, the AFM₁ mean level of D and H firm were found higher than the TFC standard value. Furthermore, the AFM₁ mean levels determined in all of the cheese samples were within the TFC value. Levels of AFM₁ in pasteurised milk samples, raw milk samples and cheese samples are shown in Table 3.

The aflatoxin levels in the milk and cheese samples were differing from each other surveys performed in Turkey. Celik et al. (2005) have analysed a total of 85 pasteurised milk samples with ELISA and they have determined that in 27 samples AFM₁ level was determined to exceed the Turkish Food Codex levels (Celik et al., 2005). Kirecci et al. (2007) have reported that in 18 fresh milk samples of total 20 samples investigated, AFM₁ level was higher than the acceptable levels according to the Turkish Food Codex. These findings have higher rates than our study. Gurbay et al. (2006) have investigated a total of 27 commercial milk samples collected from supermarkets in Ankara and analysed for AFM₁ and they have reported that in only 1 sample AFM₁ level was over the permissible level of European Countries as well as in Turkey. Gurbay et al. (2006) reported only one sample contaminated with AFM₁. The reason for this low rate is thought to be because of the milk samples were mostly UHT milk samples.

The AFM₁ level in milk samples has been investigated in several countries. Dashti et al. (2009) have studied with 321 milk samples in Kuwait. Of these samples 177 were fresh milk samples and it had reported that all fresh milk samples except one were contaminated with AFM₁ ranging from 4.9-68.7 ng kg^-1 and eight samples were reported to exceed the European Unions regulatory limit (Dashti et al., 2009). Nuryono et al. (2009) have investigated AFM₁ levels in 113 fresh milk in Indonesia and although 65 samples were found to be contaminated with AFM₁, none of the samples exceeded the European Limit levels (Nuryono et al., 2009). Karimi et al. (2007) have investigated 110 pasteurised milk samples, obtained from Iran, in case of AFM₁ levels and AFM₁ was found in 100% of milk samples. They have determined that about 5.4% of the samples had AFM₁ greater than maximum tolerance limit (0.05 μg L^-1) accepted by European Union (Karimi et al., 2007). Tajik et al. (2007) investigated 72 raw milk and 72 pasteurised milk samples and detected 9 raw milk samples exceeded the level of European Union and none of the pasteurised samples exceeded this limit (Tajik et al., 2007). In the study all the raw milk samples none of the samples exceeded the European Limit levels as well as TFC values. This finding is in accordance with Nuryono et al. (2009) however, Dashti et al. (2009) and Tajik et al. (2007) have found that 4.5 and 12.5% of the raw milk samples exceeded the levels, respectively. In pasteurised milk samples we have determined that 15 samples (12.5%) are contaminated with AFM₁ in an unacceptable level according to TFC. Karimi et al. (2007) have also studied pasteurised milk and 4.5% of the samples were exceeding the limit values.

The AFM₁ level in the milk was significantly affected by the geographical region, the country and the season (Sarimehmetoglu et al., 2004). The differences in our results could be because of these reasons. In the process of milk to cheese, AFM₁ insufficient destruction was reported in literature. Thus, cheese was thought to be a potential risk hazard as well as milk and we have studied Turkish white brined cheese samples that are consumed widely in Turkey. Ardic et al. (2009) have studied with 193 Turkish white brined cheese samples and 51 samples were detected to have higher AFM₁ than the acceptable limit (Ardic et al., 2009). Sarimehmetoglu et al. (2004) have investigated AFM₁ in cheese samples by ELISA and of the 400 cheese samples in 110 samples, the AFM₁ level was reported to be higher than the limits of the Turkish Food Codex. About 100 of the 400 samples were Turkish white cheese samples and they have determined that 27% of the white cheese samples had higher AFM₁ levels than the acceptable levels of the Turkish Food Codex (Sarimehmetoglu et al., 2004). Aycicek et al. (2005) have determined the aflatoxin levels in some dairy and food products consumed in Ankara region and in a total of 223 samples, they have investigated 94 samples of white cheese. In 12 white cheese samples, the AFM₁ were higher than the acceptable limits of Turkish Food Codex (Aycicek et al., 2005). Kirecci et al. (2007) have investigated 20 white cheese samples determined that 4 samples had higher AFM₁ than the acceptable limit (Kirecci et al., 2007). Gurses et al. (2004) have studied with 23 white cheese samples of total 63 cheese samples and determined that 9 of the white cheese samples were AFM₁ positive (Gurses et al., 2004). Yapar et al. (2008) investigated the AFM₁ levels in different type of cheese produced in Turkey. Of the cheese samples, 25 samples were Turkish white cheese samples and in 7 of the Turkish white cheese samples, AFM₁ level was determined to exceed the Turkish Food Codex levels (Yapar et al., 2008). Yaroglu et al. (2005) have analysed 200 white cheese samples for AFM₁ level and 2 samples were reported to have higher AFM₁ levels than the acceptable levels of the Turkish Food Codex (Yaroglu et al., 2005).

The literature available on the occurrence of aflatoxins in cheese indicates higher levels of contamination however; the findings are in accordance with Yaroglu et al. (2005). Researchers from different countries have also investigated AFM₁ in cheese samples. Kamkar (2005) has studied the presence of AFM₁, using the thin layer chromatography in Iranian feta cheese samples and of the 80 samples analyzed, 82.5% were found to be contaminated with AFM₁. (Torkar and Vengus, 2008) have studied in Slovenia with 40 cheese samples and they have determined that 4 samples were contaminated with AFM₁ and concluded that AFM₁ presence in Slovenian cheese does not represent a serious risk for human health (Torkar and Vengus, 2008).

The differences in the results are thought to be because of following reasons. The AFM₁ levels in the cheese significantly affected by cheese manufacturing procedures, different milk contaminants, type of cheese, conditions of cheese ripening and the analytical methods employed (Sarimehmetoglu et al. 2004).

CONCLUSION

In the study 70 cheese samples from 7 different firms were investigated and in none of the samples AFM₁ levels exceeding the TFC values were determined, thus these cheese samples does not represent a risk for public health. Raw milk samples were obtained from 3 different open air markets and an unacceptable value was not determined but this finding should be supported with more number of samples. However, in pasteurised milk 12.5% of the samples exceeded the TFC standard value and are thought to be an important risk for public health especially for infants that are consuming large amounts of milk.

ACKNOWLEDGEMENT

This study was supported by a grant from Gazi University Research Foundation (Project no: 02/2009-16).