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Occurrence of aflatoxin M1 in UHT milk in Turkey
Food and Chemical Toxicology 44 (2006) 1897–1900 www.elsevier.com/locate/foodchemtox
Occurrence of aflatoxin M1 in UHT milk in Turkey Nurhan Unusan
*
Selcuk University, Education Faculty, 42090 Konya, Turkey Received 7 June 2005; accepted 8 June 2006
Abstract Aflatoxin M1 (AFM1) appears in milk as a direct result of the ingestion of food contaminated with aflatoxin B1 by cattle. The role of milk in human nutrition is well-known. The purpose of the study was to determine the levels of AFM1 in UHT milk samples in Central Anatolia, Turkey. The occurrence of AFM1 contamination in UHT milk samples was investigated by ELISA (Enzyme Linked Immunosorbent Assay) technique. A total of 129 samples of commercial UHT whole milk were analysed. The mean value was 108.17 ng/L. There was a high incidence rate of AFM1, with 75 (58.1%) milk samples being contaminated. Although 68 (53%) were below the limit, the remaining 61 (47%) were well above the limit permitted by the EU. Four of the samples exceeded the prescribed limit of US regulations. It can be concluded that AFM1 levels in the samples purchased in Central Anatolia Region, appear to be a serious public health problem at the moment. Dairy farmers must be educated by the government authorities on potential health consequences of aflatoxins. 2006 Elsevier Ltd. All rights reserved. Keywords: Aflatoxin M1; UHT milk; ELISA
1. Introduction Aflatoxins may be produced by three species of Aspergillus – A. flavus, A. parasiticus, and rare A. nomius-that contaminate plants and its products. A. flavus produces only B aflatoxins, while the others produce both B and G aflatoxins. Aflatoxins M1 and M2 are the hydroxilated metabolites of aflatoxin B1 and B2 and may be found in milk products obtained from livestock that have ingested contaminated feed. The main sources of aflatoxins in feeds are peanut, meal maize and cottonseed meal. Aflatoxin B1 (AFB1) is the most potent hepatocarcinogen known in mammals, the risk assessment of which is very well known. The formation of AFM1 occurs in liver and it is secreted into the milk (Cathey et al., 1994). There was a linear relationship between the amount of AFM1 in milk and AFB1 in feed consumed by the animals (Dragacci et al., 1995). About 0.3–6.2% of AFB1 in animal feed is transformed to aflatoxin M1 in milk (Creppy, 2002). When
*
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lactating cows, sheep, and goats are fed with feedstuffs containing aflatoxin B1 (AFB1), this metabolite can be converted to aflatoxin M1 (AFM1), which is cytotoxic and genotoxic (Cole and Cox, 1981; Jackson and Groopman, 1999). International Agency for Research and Cancer (IARC) of WHO included AFB1 as primary and AFM1 as secondary groups of carcinogenic compounds (Cathey et al., 1994; Dragacci et al., 1995). The tolerance limits for aflatoxins in milk are established by competent national authorities. The legal limits vary significantly both from country to country and by mycotoxin type and matrix (Gilbert and Anklam, 2002). For example, The European Community and Codex Alimentarius prescribe that the maximum level of AFM1 in liquid milk should not exceed 50 ng/L (Codex Alimentarius Commissions, 2001). This limit has been established following the ALARA (As Low As Reasonable Achievable) principle. However, according to US regulations the level of AFM1 in milk should not be higher than 500 ng/L. Milk and milk products are a good source of many nutrients such as proteins and calcium. Therefore, humans are potentially exposed to these metabolites and it is generally assumed that neither storage nor processing provides a
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N. Unusan / Food and Chemical Toxicology 44 (2006) 1897–1900
reduction of AFM1 content (Galvano et al., 1996). The weighted mean concentration of AFM1 in milk is 0.023 lg/kg in the European-type diet, 0.022 lg/kg in the Latin American diet, 0.36 lg/kg in the Far Eastern diet, 0.005 lg/kg in the in the Middle Eastern diet, and 0.002 lg/kg in the in the African diet (Creppy, 2002). Taking into account that UHT technique does not affect AFM1 concentration because of its heat stability (Galvano et al., 1996). Many researchers from different countries have carried out studies about the incidence of AFM1in milk. Kim et al. (2000) showed that, the incidence of AFM1 in milk was 76%, with a mean concentration of 18 pg/g in Korea. In a study in Brazil, Garrido et al. (2003) found that in 20.9% of the milk samples in the range 50–240 ng/L exceeded the concentration of the limit permitted by the European Union. Rastogi et al. (2004)showed that the range of contamination of AFM1 was of the magnitude of 28–164 ng/L in Indian markets. Therefore 4 (33%) out of 12 samples were contaminated. Kamkar (2005) in Iran found that in 85 (76.6%) of the 111 samples, the presence of AFM1 was detected in range between 0.015–0.28 lg/L. Forty percent of positive samples were higher than the tolerance limit accepted by European countries. There is little information about the occurrence of AFM1 in milk in Turkey. Bakirci (2001) found AFM1 in 79 (87.7%) of the milk samples. Oruc and Sonal (2001) determined 10 milk samples by ELISA, and in 10% of milk samples AFM1 was detected. Gu¨rbay et al. (2006) worked with 27 milk samples and AFM1 was detected in 59.3% of samples sold in Turkey by HPLC. The aim of the present study was to investigate the occurrence of AFM1 in UHT milk samples sold in Central Anatolia Region in Turkey by ELISA, and to compare the obtained results with maximum AFM1 tolerance limits accepted by The European Community. 2. Materials and methods 2.1. Samples A total of 129 samples of commercial UHT whole milk (250 ml milk packet) were purchased from supermarkets in Central Anatolia (Konya, Ankara, Sivas, Kayseri, Nigde) between January and February 2005. All samples were analysed before their expiry date.
toxin M1 standard solutions (1.3 ml each 0 ppt, 250 ppt, 500 ppt, 1000 ppt, 2000 ppt), peroxidase conjugated aflatoxin, anti-aflatoxin M1 antibody, substrate/chromogen stained in red, and stop solution contains 1 N sulphuric acid. Methanol and acetonitrile used were of analytical grade and provided by Merck.
2.4. Preparation of the milk samples Preparation of samples was conducted according to the instructions of the RIDASCREEN test kit. 10 ml of milk samples were chilled to 10 C and then centrifuged for 10 min at 3500 rpm. An aliquot (50 ll per well) of the skimmed milk was used directly in the test.
2.5. Test procedure AFM1 standards or the prepared sample solutions were added to wells. 50 ll of enzyme conjugate and 50 ll of anti-aflatoxin M1 antibody solution added to each well. Mixed gently by rocking the plate manually and incubated for 10 min at room temperature in the dark, the antibody binding sites are occupied proportionally to the AFM1 concentration. The liquid was then removed completely from the wells, which were washed twice with distilled water. In the next step, 100 ll of substrate/chromogen added (white dropper) to each well. Mixed manually by rocking the plate and incubated for 5 min at room temperature in the dark. 100 ll of stop solution (yellow dropper) added to each well, and led colour change from blue to yellow. Mixed manually again gently by rocking the plate, and measured the absorbance at 450 nm against an air blank. Read within 10 min.
2.6. Evaluation of AFM1 The absorbance values obtained for the standards and the samples were divided by the absorbance value of the first standard (0 standards) and multiplied by 100. Therefore, the zero standard is thus made equal to 100% and the absorbance values are quoted in percentages. The absorption is inversely proportional to the AFM1. According to the test preparation record, the lower detection limit is 0.01 lg/L for milk. Also according to the instructions for use of the RIDASCREEN kit, the recovery rate in spiked milk (10–80 pg/ml) is 95% with a mean coefficient of variation of 15%. For evaluation of the ELISA kits, software for the RIDASCREEN tests has been developed by R-Biopharm was used.
2.7. Calculation of extrapolated values of AFB1 concentration in cattle feeding stuffs It has been suggested that only 1.6% of ingested AFB1 is converted to AFM1 by the dairy cattle. Therefore, the values of AFB1 contamination in feeding stuffs were back calculated by the formula given below (Forbish et al., 1986; Price et al., 1985; Rastogi et al., 2004): AFB1 (lg/L) = AFM1 (ng/L) · 100/(1.6 · 1000).
3. Results 2.2. Method Determination of AFM1 was based on Enzyme Linked Immunoassay (ELISA) using the RIDASCREEN test kit (R-Biopharm, Germany). This method is quick, reliable, and cost effective for estimation of AFM1 and has been included in the official collection of test procedures by the German Federal Board of Health. The test shows cross-reaction to AFB1 (12.4%) but this is not relevant when analysing AFM1, considering that AFB1 is not to be found in milk.
2.3. Reagents Most of the reagents used were contained in the RIDASCREEN test kit; which included microtiter plate coated with capture antibodies, afla-
The standard curve for AFM1 detection by competitive ELISA is given in Fig. 1. The absorption is inversely proportional to the AFM1 concentration in the sample. As can be seen from the Figure, the calibration curve was found virtually linear in the 250–2000 ppt range. The detection limit was found to be 5 ppt. The recovery score in spiked UHT milk (250–2000 ng/L) was found to be 93% with a mean coefficient of variation of 15%; the results are consistent with Kaniou-Grigoriadou et al. (2005). The occurrence of AFM1 in UHT milk samples is shown in Table 1. The mean value was 108.17 ng/L. There
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4. Discussion
Fig. 1. Calibration curve of AFM1.
Table 1 Occurrence of AFM1 in UHT milk samples Range of AFM1 concentration (ng/L)
UHT milk, n (%)
<10 10–49 50–499 Exceeding EC/CODEX regulations (50 ng/L) P500 Exceeding US regulations (500 ng/L) Range
58 (45) 10 (7.9) 57 (43.9) 4 (3.2) 0–543.64
was a high incidence rate of AFM1, with 75 (58.1%) milk samples being contaminated. Although 68 (53%) were below the limit, the remaining 61 (47%) were well above the limit permitted by the EU. Four of the samples exceeded the prescribed limit of US regulations. These results suggest that although US regulations prescribe 10 fold higher limits of AFM1 even then 3.2% UHT milk samples exceed the maximum limits. It is known that contamination of AFM1 in milk is a result of exposure of AFB1 to dairy cattle through feedstuffs. Further researchers suggested that on average 1.6% of AFB1 fed to the milking cattle is excreted in milk as AFM1 (Forbish et al., 1986; Price et al., 1985) using the factor, the content of AFB1 in the dairy cattle feed was extrapolated from AFM1 contamination in milk samples (Table 2). It can be seen from the results that the contamination of feed with AFB1 in cattle feed may range of 0–33.98, with an average of 6.76 lg/L. Moreover, 51% of the samples exceeded the limits recommended by EC regulations. Thus, it is quite likely that cattle feed in Turkey contains AFB1 in much higher concentration than that prescribed by EC regulations.
Table 2 Extrapolated AFB1 concentration in cattle feedstuffs based on AFM1 contamination in UHT milk samples Range of AFB1 concentration (lg/L)
n (%)
64.99 P5 Exceeding EC/CODEX regulations (5 lg/L) Range
78 (60.9) 51 (39.1) 0–33.98
Forty-seven percent of the sample was over the permissible level of 50 ng/L as accepted by EU. Of the milk samples, 53% were in the range of 0–43.8 ng/L. This contamination range was found higher than other researches in UHT milk samples done in Turkey (Bakirci, 2001; Oruc and Sonal, 2001; Gu¨rbay et al., 2006). The higher incidence of AFM1 found in UHT milk samples may due to the number of samples analysed than other researches done in Turkey. Kamkar (2005) reported that in spring and summer, samples mean concentration of AFM1 were significantly lower than that of samples measured in winter and autumn. The researcher showed that 40% of the samples exceeded the EU limits, while none of the samples exceeded US limits in Iran. He pointed out the importance of seasonal factors in AFM1 levels. Also other researchers reported a higher incidence of AFM1 contamination during cold seasons than hot ones (Bachner et al., 1998; Blanco et al., 1988; Garrido et al., 2003; Lopez et al., 2003). Due to the seasonal conditions in winter, dairy cows are fed with animal feed that exceed the allowed AFB1 content. Thus far, a relationship between AFM1 occurrence level in milk and AFB1 content in feed was reported (Price et al., 1985; Van Egmond, 1991; Wood, 1991). The most important factor on the amount of AFB1 was temperature and moisture, since some moulds like A. flavus, and A. parasiticus can easily grow in feeds having moisture between 13–18%, and environmental moisture between 50–60% (Jay, 1992). It can be concluded that AFM1 levels in the samples purchased in Central Anatolia Region, appear to be a serious public health problem at the moment. Also it should be kept in mind that, total daily aflatoxin intake from other foods could be an important risk factor for people as well. The magnitude of contamination of AFM1 in UHT milk samples in Turkey was comparable to European Countries as Italy (Galvano et al., 1998), Portugal (Markaki and Melissari, 1997), and Greece (Martin and Martin, 2000), but contamination range was higher (543.64 ng/L). A higher range of AFM1 than this research was reported by Rastogi et al. (2004) in Indian milk samples (28– 1012 ng/L). 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, 1997, 1998, 1999). It is concluded that the number of samples analysed must be increased, dairy farmers must be educated by the government authorities on potential health consequences of aflatoxins, and researches must be conducted over an extended period of time to minimise the health hazard risk in population. Acknowledgement This study was supported financially by the Selcuk University Scientific Research Coordination Center (BAP).
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References Bachner, U., Martlbauer, E., Terblan, G., 1998. Detecting aflatoxin M1 in milk from selected parts of Bavaria by using ELISA. German Federal Republic. On Dairy Science Abstract, 1990 52, 901. Bakirci, I., 2001. A study on the occurrence of aflatoxin M1 in milk and milk products produced in Van province of Turkey. Food Control 12 (1), 47–51. Blanco, J.L., Dominguezs, L., Gomez-Lucia, E., Garayzabal, J.F.F., Garcia, J.A., Suarez, G., 1988. Presence of aflatoxin M1 in commercial UHt treated milk. Applied Environmental Microbiology 56, 1622– 1623. Cathey, C.G., Nuang, Z.G., Sarr, A.B., Clement, B.A., Phililips, T.D., 1994. Development and evaluation of a minicolumn assay fro the detection of aflatoxin M1 in milk. Journal of Dairy Science 77, 1223– 1231. Codex Alimentarius Commissions. 2001. Comments submitted on the draft miximum level for aflatoxin M1 in milk. Codex committee on food additives and contaminants, 33rd sessions, Hauge, The Netherlands. Cole, R.J., Cox, R.H., 1981. Handbook of Toxic Fungal Metabolites. Academic Press, New York. Creppy, E.E., 2002. Update of survey, regulation and toxic effects of mycotoxins in Europe. Toxicology Letters 127, 19–28. Dragacci, S., Gleizes, E., Fremi, J.M., Candlish, A.A.G., 1995. Use of immunoaffinity chromatography as a purification step for the determination of aflatoxin M1 in cheeses. Food Additives and Contaminants 12 (1), 59–65. Forbish, R.A., Bradley, B.D., Wagner, D.D., Long-Bradley, P.E., Hariston, H., 1986. Aflatoxin residues in milk of dairy cows after ingestion of naturally contaminated grain. Journal of Food Protection 49, 781–785. Galvano, F., Galofaro, V., Galvano, G., 1996. Occurrence and stability of aflatoxin M1 in milk and milk products: a worldwide review. Journal of Food Protection 59, 1079–1090. Galvano, F., Galofaro, V., De Angelis, A., Galvano, M., Bognano, M., Galvano, G., 1998. Survey of occurrence of aflatoxin M1 in dairy products marketed in Italy. Journal of Food Protection 49, 781– 785. Garrido, N.S., Iha, M.H., Santos Ortolani, M.R., Duarte Favaro, R.M. 2003. Occurrence of aflatoxins M1 and M2 in milk commercialized in Ribeirao Preto-SP, Brazil. Gilbert, J., Anklam, E., 2002. Validation of analytical methods for determining mycotoxins in foodstuffs. Trends in Analytical Chemistry 21 (6–7), 468–486.
Gu¨rbay, A., Aydın, S., Girgin, G., Engin, A.B., S ß ahin, G., 2006. Assessment of aflatoxin M1 levels in milk in Ankara, Turkey. Food Control 17 (1), 1–4. Jackson, P.E., Groopman, J.D., 1999. Aflatoxin and liver cancer. Baillieres Best Practice and Research in Clinical Gastroenterology 13 (4), 545–555. Jay, J.M., 1992. Modern Food Microbiology. 4th ed. Chapman & Hall, New York. Kamkar, A., 2005. A study on the occurrence of aflatoxin M1 in raw milk produced in Sarab city of Iran. Food Control 16, 593–599. Kaniou-Grigoriadou, I., Eleftheriadou, A., Mouratidou, T., Katikou, P., 2005. determination of aflatoxin M1 in ewe’s milk samples and the produced curd and Feta cheese. Food Control 16, 257–261. Kim, E.K., Shon, D.H., Ryu, D., Park, J.W., Hwang, H.J., Kim, Y.B., 2000. Occurrence of aflatoxin M1 in Korean dairy products determined by ELISA and HPLC. Food Additives and Contaminants 17 (1), 59–64. Lopez, C.E., Ramos, L.L., Ramadan, S.S., Bulacio, L.C., 2003. Presence of aflatoxin M1 in milk for human consumption in Argentina. Food Control 14 (1), 31–34. Markaki, P., Melissari, E., 1997. Occurrence of aflatoxin M1 in commercialised milk determined with ELISA and HPLC. Food Additives and Contaminants 14, 451–456. Martin, M.L., Martin, H.M., 2000. Aflatoxin M1 in raw and UHT treated milk commercialised in Portugal. Food Additives and Contaminants 17, 871–874. Oruc, H.H., Sonal, S., 2001. Determination of aflatoxin M1 levels in cheese and milk consumed in Bursa, Turkey. Veterinary Human Toxicology 43 (5), 292–293. Price, R.L., Paulson, J.H., Lough, O.G., Ginng, C., Kurtz, A.G., 1985. Aflatoxin conversion by dairy cattle consuming naturally contaminated whole cottonseed. Journal of Food Protection 48, 11–15. Rastogi, S., Dwivedi, P.D., Khanna, S.K., Das, M., 2004. detection of aflatoxin M1 contamination in milk and infant milk products from Indian markets by ELISA. Food Control 15, 287–290. Trucksess, M.W., 1997. Mycotoxins. Journal of AOAC International 80, 119–126. Trucksess, M.W., 1998. Mycotoxins. Journal of AOAC International 81, 128–137. Trucksess, M.W., 1999. Mycotoxins. Journal of AOAC International 82, 488–495. Van Egmond, H.P., 1991. Mycotoxin International Dairy Federation Special Issue 9101, 131–145. Wood, G.E., 1991. Aflatoxin M1. In: Sharma, R.P., Salunkhe, D.k. (Eds.), Mycotoxins and Phytoalexins. CRC, London, pp. 145–163.
Occurrence of aflatoxin M1 in UHT milk in Turkey Nurhan Unusan
*
Selcuk University, Education Faculty, 42090 Konya, Turkey Received 7 June 2005; accepted 8 June 2006
Abstract Aflatoxin M1 (AFM1) appears in milk as a direct result of the ingestion of food contaminated with aflatoxin B1 by cattle. The role of milk in human nutrition is well-known. The purpose of the study was to determine the levels of AFM1 in UHT milk samples in Central Anatolia, Turkey. The occurrence of AFM1 contamination in UHT milk samples was investigated by ELISA (Enzyme Linked Immunosorbent Assay) technique. A total of 129 samples of commercial UHT whole milk were analysed. The mean value was 108.17 ng/L. There was a high incidence rate of AFM1, with 75 (58.1%) milk samples being contaminated. Although 68 (53%) were below the limit, the remaining 61 (47%) were well above the limit permitted by the EU. Four of the samples exceeded the prescribed limit of US regulations. It can be concluded that AFM1 levels in the samples purchased in Central Anatolia Region, appear to be a serious public health problem at the moment. Dairy farmers must be educated by the government authorities on potential health consequences of aflatoxins. 2006 Elsevier Ltd. All rights reserved. Keywords: Aflatoxin M1; UHT milk; ELISA
1. Introduction Aflatoxins may be produced by three species of Aspergillus – A. flavus, A. parasiticus, and rare A. nomius-that contaminate plants and its products. A. flavus produces only B aflatoxins, while the others produce both B and G aflatoxins. Aflatoxins M1 and M2 are the hydroxilated metabolites of aflatoxin B1 and B2 and may be found in milk products obtained from livestock that have ingested contaminated feed. The main sources of aflatoxins in feeds are peanut, meal maize and cottonseed meal. Aflatoxin B1 (AFB1) is the most potent hepatocarcinogen known in mammals, the risk assessment of which is very well known. The formation of AFM1 occurs in liver and it is secreted into the milk (Cathey et al., 1994). There was a linear relationship between the amount of AFM1 in milk and AFB1 in feed consumed by the animals (Dragacci et al., 1995). About 0.3–6.2% of AFB1 in animal feed is transformed to aflatoxin M1 in milk (Creppy, 2002). When
*
Tel.: +90 532 393 27 72; fax: +90 332 325 21 32. E-mail address: [email protected]
0278-6915/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2006.06.010
lactating cows, sheep, and goats are fed with feedstuffs containing aflatoxin B1 (AFB1), this metabolite can be converted to aflatoxin M1 (AFM1), which is cytotoxic and genotoxic (Cole and Cox, 1981; Jackson and Groopman, 1999). International Agency for Research and Cancer (IARC) of WHO included AFB1 as primary and AFM1 as secondary groups of carcinogenic compounds (Cathey et al., 1994; Dragacci et al., 1995). The tolerance limits for aflatoxins in milk are established by competent national authorities. The legal limits vary significantly both from country to country and by mycotoxin type and matrix (Gilbert and Anklam, 2002). For example, The European Community and Codex Alimentarius prescribe that the maximum level of AFM1 in liquid milk should not exceed 50 ng/L (Codex Alimentarius Commissions, 2001). This limit has been established following the ALARA (As Low As Reasonable Achievable) principle. However, according to US regulations the level of AFM1 in milk should not be higher than 500 ng/L. Milk and milk products are a good source of many nutrients such as proteins and calcium. Therefore, humans are potentially exposed to these metabolites and it is generally assumed that neither storage nor processing provides a
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N. Unusan / Food and Chemical Toxicology 44 (2006) 1897–1900
reduction of AFM1 content (Galvano et al., 1996). The weighted mean concentration of AFM1 in milk is 0.023 lg/kg in the European-type diet, 0.022 lg/kg in the Latin American diet, 0.36 lg/kg in the Far Eastern diet, 0.005 lg/kg in the in the Middle Eastern diet, and 0.002 lg/kg in the in the African diet (Creppy, 2002). Taking into account that UHT technique does not affect AFM1 concentration because of its heat stability (Galvano et al., 1996). Many researchers from different countries have carried out studies about the incidence of AFM1in milk. Kim et al. (2000) showed that, the incidence of AFM1 in milk was 76%, with a mean concentration of 18 pg/g in Korea. In a study in Brazil, Garrido et al. (2003) found that in 20.9% of the milk samples in the range 50–240 ng/L exceeded the concentration of the limit permitted by the European Union. Rastogi et al. (2004)showed that the range of contamination of AFM1 was of the magnitude of 28–164 ng/L in Indian markets. Therefore 4 (33%) out of 12 samples were contaminated. Kamkar (2005) in Iran found that in 85 (76.6%) of the 111 samples, the presence of AFM1 was detected in range between 0.015–0.28 lg/L. Forty percent of positive samples were higher than the tolerance limit accepted by European countries. There is little information about the occurrence of AFM1 in milk in Turkey. Bakirci (2001) found AFM1 in 79 (87.7%) of the milk samples. Oruc and Sonal (2001) determined 10 milk samples by ELISA, and in 10% of milk samples AFM1 was detected. Gu¨rbay et al. (2006) worked with 27 milk samples and AFM1 was detected in 59.3% of samples sold in Turkey by HPLC. The aim of the present study was to investigate the occurrence of AFM1 in UHT milk samples sold in Central Anatolia Region in Turkey by ELISA, and to compare the obtained results with maximum AFM1 tolerance limits accepted by The European Community. 2. Materials and methods 2.1. Samples A total of 129 samples of commercial UHT whole milk (250 ml milk packet) were purchased from supermarkets in Central Anatolia (Konya, Ankara, Sivas, Kayseri, Nigde) between January and February 2005. All samples were analysed before their expiry date.
toxin M1 standard solutions (1.3 ml each 0 ppt, 250 ppt, 500 ppt, 1000 ppt, 2000 ppt), peroxidase conjugated aflatoxin, anti-aflatoxin M1 antibody, substrate/chromogen stained in red, and stop solution contains 1 N sulphuric acid. Methanol and acetonitrile used were of analytical grade and provided by Merck.
2.4. Preparation of the milk samples Preparation of samples was conducted according to the instructions of the RIDASCREEN test kit. 10 ml of milk samples were chilled to 10 C and then centrifuged for 10 min at 3500 rpm. An aliquot (50 ll per well) of the skimmed milk was used directly in the test.
2.5. Test procedure AFM1 standards or the prepared sample solutions were added to wells. 50 ll of enzyme conjugate and 50 ll of anti-aflatoxin M1 antibody solution added to each well. Mixed gently by rocking the plate manually and incubated for 10 min at room temperature in the dark, the antibody binding sites are occupied proportionally to the AFM1 concentration. The liquid was then removed completely from the wells, which were washed twice with distilled water. In the next step, 100 ll of substrate/chromogen added (white dropper) to each well. Mixed manually by rocking the plate and incubated for 5 min at room temperature in the dark. 100 ll of stop solution (yellow dropper) added to each well, and led colour change from blue to yellow. Mixed manually again gently by rocking the plate, and measured the absorbance at 450 nm against an air blank. Read within 10 min.
2.6. Evaluation of AFM1 The absorbance values obtained for the standards and the samples were divided by the absorbance value of the first standard (0 standards) and multiplied by 100. Therefore, the zero standard is thus made equal to 100% and the absorbance values are quoted in percentages. The absorption is inversely proportional to the AFM1. According to the test preparation record, the lower detection limit is 0.01 lg/L for milk. Also according to the instructions for use of the RIDASCREEN kit, the recovery rate in spiked milk (10–80 pg/ml) is 95% with a mean coefficient of variation of 15%. For evaluation of the ELISA kits, software for the RIDASCREEN tests has been developed by R-Biopharm was used.
2.7. Calculation of extrapolated values of AFB1 concentration in cattle feeding stuffs It has been suggested that only 1.6% of ingested AFB1 is converted to AFM1 by the dairy cattle. Therefore, the values of AFB1 contamination in feeding stuffs were back calculated by the formula given below (Forbish et al., 1986; Price et al., 1985; Rastogi et al., 2004): AFB1 (lg/L) = AFM1 (ng/L) · 100/(1.6 · 1000).
3. Results 2.2. Method Determination of AFM1 was based on Enzyme Linked Immunoassay (ELISA) using the RIDASCREEN test kit (R-Biopharm, Germany). This method is quick, reliable, and cost effective for estimation of AFM1 and has been included in the official collection of test procedures by the German Federal Board of Health. The test shows cross-reaction to AFB1 (12.4%) but this is not relevant when analysing AFM1, considering that AFB1 is not to be found in milk.
2.3. Reagents Most of the reagents used were contained in the RIDASCREEN test kit; which included microtiter plate coated with capture antibodies, afla-
The standard curve for AFM1 detection by competitive ELISA is given in Fig. 1. The absorption is inversely proportional to the AFM1 concentration in the sample. As can be seen from the Figure, the calibration curve was found virtually linear in the 250–2000 ppt range. The detection limit was found to be 5 ppt. The recovery score in spiked UHT milk (250–2000 ng/L) was found to be 93% with a mean coefficient of variation of 15%; the results are consistent with Kaniou-Grigoriadou et al. (2005). The occurrence of AFM1 in UHT milk samples is shown in Table 1. The mean value was 108.17 ng/L. There
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4. Discussion
Fig. 1. Calibration curve of AFM1.
Table 1 Occurrence of AFM1 in UHT milk samples Range of AFM1 concentration (ng/L)
UHT milk, n (%)
<10 10–49 50–499 Exceeding EC/CODEX regulations (50 ng/L) P500 Exceeding US regulations (500 ng/L) Range
58 (45) 10 (7.9) 57 (43.9) 4 (3.2) 0–543.64
was a high incidence rate of AFM1, with 75 (58.1%) milk samples being contaminated. Although 68 (53%) were below the limit, the remaining 61 (47%) were well above the limit permitted by the EU. Four of the samples exceeded the prescribed limit of US regulations. These results suggest that although US regulations prescribe 10 fold higher limits of AFM1 even then 3.2% UHT milk samples exceed the maximum limits. It is known that contamination of AFM1 in milk is a result of exposure of AFB1 to dairy cattle through feedstuffs. Further researchers suggested that on average 1.6% of AFB1 fed to the milking cattle is excreted in milk as AFM1 (Forbish et al., 1986; Price et al., 1985) using the factor, the content of AFB1 in the dairy cattle feed was extrapolated from AFM1 contamination in milk samples (Table 2). It can be seen from the results that the contamination of feed with AFB1 in cattle feed may range of 0–33.98, with an average of 6.76 lg/L. Moreover, 51% of the samples exceeded the limits recommended by EC regulations. Thus, it is quite likely that cattle feed in Turkey contains AFB1 in much higher concentration than that prescribed by EC regulations.
Table 2 Extrapolated AFB1 concentration in cattle feedstuffs based on AFM1 contamination in UHT milk samples Range of AFB1 concentration (lg/L)
n (%)
64.99 P5 Exceeding EC/CODEX regulations (5 lg/L) Range
78 (60.9) 51 (39.1) 0–33.98
Forty-seven percent of the sample was over the permissible level of 50 ng/L as accepted by EU. Of the milk samples, 53% were in the range of 0–43.8 ng/L. This contamination range was found higher than other researches in UHT milk samples done in Turkey (Bakirci, 2001; Oruc and Sonal, 2001; Gu¨rbay et al., 2006). The higher incidence of AFM1 found in UHT milk samples may due to the number of samples analysed than other researches done in Turkey. Kamkar (2005) reported that in spring and summer, samples mean concentration of AFM1 were significantly lower than that of samples measured in winter and autumn. The researcher showed that 40% of the samples exceeded the EU limits, while none of the samples exceeded US limits in Iran. He pointed out the importance of seasonal factors in AFM1 levels. Also other researchers reported a higher incidence of AFM1 contamination during cold seasons than hot ones (Bachner et al., 1998; Blanco et al., 1988; Garrido et al., 2003; Lopez et al., 2003). Due to the seasonal conditions in winter, dairy cows are fed with animal feed that exceed the allowed AFB1 content. Thus far, a relationship between AFM1 occurrence level in milk and AFB1 content in feed was reported (Price et al., 1985; Van Egmond, 1991; Wood, 1991). The most important factor on the amount of AFB1 was temperature and moisture, since some moulds like A. flavus, and A. parasiticus can easily grow in feeds having moisture between 13–18%, and environmental moisture between 50–60% (Jay, 1992). It can be concluded that AFM1 levels in the samples purchased in Central Anatolia Region, appear to be a serious public health problem at the moment. Also it should be kept in mind that, total daily aflatoxin intake from other foods could be an important risk factor for people as well. The magnitude of contamination of AFM1 in UHT milk samples in Turkey was comparable to European Countries as Italy (Galvano et al., 1998), Portugal (Markaki and Melissari, 1997), and Greece (Martin and Martin, 2000), but contamination range was higher (543.64 ng/L). A higher range of AFM1 than this research was reported by Rastogi et al. (2004) in Indian milk samples (28– 1012 ng/L). 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, 1997, 1998, 1999). It is concluded that the number of samples analysed must be increased, dairy farmers must be educated by the government authorities on potential health consequences of aflatoxins, and researches must be conducted over an extended period of time to minimise the health hazard risk in population. Acknowledgement This study was supported financially by the Selcuk University Scientific Research Coordination Center (BAP).
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