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Food Control xxx (2011) 1e3

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Food Control journal homepage: www.elsevier.com/locate/foodcont

A survey on the occurrence of aflatoxin M1 in raw and processed milk samples marketed in Lebanon Elkak Assem a, b, *, Abbas Mohamad a, El Atat Oula a a

Laboratoire de Biotechnologie des Substances Naturelles et Produits de Santé (BSNPS), Doctoral School of Sciences and Technology, Lebanese University, Rafic Hariri University Campus, P.O.BOX 14/6573, Hadath, Lebanon b Groupe de Recherche Environnementale Suivi, Impact et Traitement (GRESIT), Doctoral School of Sciences and Technology, Lebanese University, P.O.BOX 14/6573, Hadath, Lebanon

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 February 2011 Received in revised form 15 April 2011 Accepted 30 April 2011

A survey was conducted to determine the occurrence of AFM1 in 77 cow and goat milk samples (38 raw milk, 25 pasteurized milk and 14 powder milk samples); obtained either from local small farms, or markets. The competitive enzyme e linked immunosorbent assay (ELISA) method was applied for this purpose positively detecting AFM1 in 64.9% of all tested milk samples. The revealed rates of AFM1 contamination were 73.6%, 68.0%, 35.7% for the raw, pasteurized and powder milk samples, respectively. The individual values, within each category of milk samples, ranged from 2.63 to 126 ng/l (average ¼ 60 ng/l), 3.27e84.4 ng/l (average ¼ 30.6 ng/l) and 9.18e16.5 ng/l (average ¼ 13.7 ng/l) for the raw, pasteurized and powder milk samples, respectively. Of the positive samples, 29 were still below the permitted limit (50 ng/l) set by the European Commission whereas 21 exceeded the permissible limit. This work represents the data of the first survey on the occurrence of AFM1 in raw and processed milk marketed and consumed in Lebanon. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.

Keywords: AFM1 Milk ELISA Public health

1. Introduction Mycotoxins are small molecules that are naturally produced as secondary metabolites by filamentous fungi which can contaminate feed and food and cause disease and death in human (Gourama & Bullerman, 1995; Hedayati, Pasqualotto, Warn, Bowyer, & Denning, 2007; Peraica, Radi c, Lucic, & Pavlovi c, 1999; Pittet, 1998). More than 200 secondary metabolites of fungi have been identified, but only a few, including aflatoxins, have been shown to bear an important impact on public health. Aflatoxins are among the best known and most widely studied mycotoxins. They are highly toxic, mutagenic and carcinogenic compounds that have been involved as a potential agent in human hepatic carcinogenesis (Wogan, 1999). Aflatoxin M1 is the principle hydroxylated metabolite of aflatoxin B1 which is transformed at the hepatic level by means of cytochrome P450 enzymes and excreted into the milk in the mammary glands of both human and lactating animal after ingestion by the animal of pellets and forage contaminated with aflatoxin B1 (Oveisi, Jannat, Sadeghi, Hajimahmoodi, & Nikzad, 2007; Prandini et al., 2009).

* Corresponding author. Laboratoire de Biotechnologie des Substances Naturelles et Produits de Santé (BSNPS), Doctoral School of Sciences and Technology, Lebanese University, Rafic Hariri University Campus, P.O.BOX 14/6573, Hadath, Lebanon. Tel.: þ961 5 46 33 67; fax: þ961 5 46 33 75. E-mail address: aelkak@ul.edu.lb (E. Assem).

The WHO e International Agency for Research on Cancer (IARC) have classified both aflatoxin B1 and aflatoxin M1 as carcinogenic agents to humans (IARC, 2002). Aflatoxin M1 links to nucleic acid in toxic ways leading to hepatotoxicity and carcinogenicity (Wogan, Hecht, Felton, Conney, & Loeb, 2004). The presence of AFM1 in milk and dairy products can be a potential threat toward babies and young children because they are major consumers of milk and derivatives. Consequently, to protect consumers several countries throughout the world have set regulations limits for aflatoxin M1 to reduce this hazard. The European Commission (EC) has indicated that the maximum residual limit (MRL) of AFM1 in liquid, and dried processed milk products should not exceeded 50 ng/l (European Commission , 2006). ELISA (Enzyme-Linked Immunosorbent Assay) method was used for measurement of AFM1. This method is established as a high throughput assay with low sample volume requirements, and often has less sample clean e up procedures compared to HPLC method (ISO, 2002). In Lebanon little available data, if any, was found about the occurrence of AFM1 in milk and milk derivatives. The aim of the present study was to investigate for the first time, the occurrence of AFM1 in raw, pasteurized and powdered milk available in Lebanon by using a 96-well microtitre plates ELISA test kits for AFM1 determination, and to compare the obtained levels to those set by the European Commission.

0956-7135/$ e see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.04.026

Please cite this article in press as: Assem, E., et al., A survey on the occurrence of aflatoxin M1 in raw and processed milk samples marketed in Lebanon, Food Control (2011), doi:10.1016/j.foodcont.2011.04.026


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E. Assem et al. / Food Control xxx (2011) 1e3

2. Experimental 2.1. Milk samples A total number of 77 samples were collected from different Lebanese regions and markets during 5 months (March to July 2010,spring e summer time) and were then evaluated for the presence of AFM1. Collected raw milk samples were transported to the laboratory in ice box and stored at 20  C while being protected from light until the time for analysis. 2.1.1. Raw milk Twenty one raw milk samples were collected between the 4th of March and 21st of May (spring time), while 17 samples were collected between the 1st of June and 6th of July (summer time). Samples were from different local small farms from various regions of Lebanon. 2.1.2. Pasteurized milk Twenty five samples of commercial pasteurized cow milk were purchased from supermarkets in Beirut. Samples were from Lebanese, Arabian and European milk industries and were analyzed before their expiry date. 2.1.3. Powder milk Fourteen samples of imported cow and goat powder milk were tested. All these samples were purchased from supermarkets in Beirut and were analyzed before their expiry date.

Table 1 Levels of AFM1 in milk consumed in Lebanon. Milk product

AFM1 (ng/l) Samples number (%)

Raw milk Pasteurized milk Powdered milk Total

38 25 14 77

BDL <5 5e25

49 10 32 8 18 9 100 27

2 2 0 4

6 7 5 18

25e50 >50 3 4 0 7

17 4 0 21

BDL: Below detection limit.

100 mL of the stop solution were added into each well and mixed gently by shaking the plate manually. Absorbance was measured at 450 nm using ELISA microplate reader (KC-100. Caretium, Shenzhen, China). 2.2.4. Evaluation of AFM1 The absorbance values obtained for the standards and the samples were divided by the absorbance value of the first standard (zero standards) and multiplied by 100 (percentage maximum absorbance). Therefore, the zero standards is thus made equal to 10 and the registered absorbance are quoted in percentages. The absorption is inversely proportional to the AFM1 concentration in the sample. The detection limit of the method is 5 ng/l and the test was validated with matrices milk and milk powder. The dilution factor was 1. After calculating the percentage of absorbance, The values were implicated in calculation method to evaluate the final concentration in (ng/l) by using a special software, the RIDAÒ SOFT Win.

2.2. Methods 3. Results and discussion 2.2.1. Determination of AFM1 content The quantitative analysis to evaluate the incidence of AFM1 in the different milk samples was performed by competitive enzyme immunoassay using RidascreenÒ AFM1 kits (R-Biopharm, Dermstadt, Germany), which contained microtiter plates coated with specific antibodies to AFM1, AFM1 standard solution of (0, 5, 10, 20, 40, and 80 ng/l), peroxidase conjugated AFM1, together with substrate/chromogen and stop solution. 2.2.2. Preparation of milk samples 2.2.2.1. Milk powder. Ten grams of powder milk was placed in a flask, and 100 mL of deionized water was added. The mixture was stirred for 5 min and then centrifuged at 3500 g for 10 min at 10 C temperature. After centrifugation, the upper fatty layer was removed and 100 mL of the skimmed milk was used for ELISA analysis. 2.2.2.2. Liquid milk. For raw and pasteurized milk, 20 mL of milk were chilled to 10 C and was centrifuged for 10 min at 3500 g. The fatty layer was removed and 100 mL of the defatted milk was applied directly in the ELISA microtiter plate. 2.2.3. Test procedure A sufficient number of microtiter wells were inserted into the microwell holder according to the manufacturer’s instructions. One hundred microliter of the AFM1 standard solutions and of the samples (100 mL/well) were added in duplicates to the wells and incubated for 30 min at room temperature in the dark. The wells were washed three times with 250 mL washing buffer. After the washing steps, 100 mL of the peroxidase conjugated AFM1 were added and incubated for 15 min at room temperature in the dark. After incubation, wells were washed again for three times with 250 mL washing buffer, and one 100 mL of substrate/chromogen were added to each well, gently shaking the plate and incubated for 15 min at room temperature in the dark. At the end of incubation

As far as the measurement of AFM1 is concerned in the different milk samples, we have used (ELISA) method, by Ridascreen test (R-Biopharm AG, Germany); already show to be reliable, simple and could be standardized for routine analysis of the mycotoxins present in food and feeds materials. This has been reported in ISO (International Standards Organization) guidelines (Rosi et al., 2007). Absorbance values obtained for standards measurement allows to calculate the concentration of AFM1 in the different milk samples. According to kit provided by the manufacturer, the recovery rate in milk (10e80 ng/l is approximately 95% with a Coefficient of Variation (CV) of 1. In our study the mean recovery score in milk samples was 89.5% with CV ¼ 3.6. A total of 77 samples consisting of 38 raw cow and goat milk, 25 pasteurized cow milk and 14 powdered cow and goat milk, were analyzed to determine the amount of AFM1. The occurrence and levels of AFM1 in the different tested milk samples are shown in Tables 1 and 2. Aflatoxin M1 was found at detectable level in 73.6% (28/38), 68.0% (17/25) and 35.7% (5/14) of raw, pasteurized and dried powdered milk samples (Table 2). Concerning the pasteurized milk 17 samples out of 25 were found positive with AFM1 levels ranging from 3.27 to 84.4 ng/l (mean value: 30.6 ng/l). The toxin was also detected in 28 of the raw milk samples at levels

Table 2 Occurrence of AFM1 in milk available in Lebanon. Milk product

Samples tested (n) Positive samples (%)

Raw milk 38 Pasteurized milk 25 Milk powder 14

28 17 5

Concentration range of positive samples (ng/l)

Mean 73.6 60.4 68.0 30.6 35.7 13.7

Min Max 2.63 126 3.27 84.4 9.18 16.5

Please cite this article in press as: Assem, E., et al., A survey on the occurrence of aflatoxin M1 in raw and processed milk samples marketed in Lebanon, Food Control (2011), doi:10.1016/j.foodcont.2011.04.026


E. Assem et al. / Food Control xxx (2011) 1e3 Table 3 Occurrence of AFM1 according to the category of milk samples. Dairy milk

Representation of milk types

AFM1 range (ng/l)

Positive sample exceeding EC limit

Raw milk

35(cow milk) 3(goat milk)

2.63e126 BDL

17/28(60.7%) 0

Pasteurized milk

14(local cow milk) 11(imported cow milk)

5.18e55.3 3.27e84.4

1/17(5.88%) 3/17(17.6%)

Powder milk

13(cow milk) 1(goat milk)

9.18e16.5 BDL

0 0

BDL: Below detection limit EC limit: European Community permissible limit of AFM1 in milk ¼ 50 ng/l.

ranging from 2.63 to 126 ng/l (mean value: 60.4 ng/l). For the powdered milk 5 samples were found positive and ranged from 9.18 to 16.5 ng/l (mean value: 13.7 ng/l). Table 1 shows the levels of AFM1 in the different local and imported milk samples. The contamination in some of the local raw milk samples may reach higher levels than the detection limit. On the other hand, 11 raw milk samples of the positive ones showed a low contamination level ranging between 2.63 and 45.7 ng/l, whereas seventeen (60.7%) of them varied in their AFM1 content between 51.8 and 126 ng/l, thus exceeding European Commission limit of 50 ng/l. Concerning the pasteurized milk, 13 positive sample had acceptable detection values ranging between 3.27 and 38.4 ng/l, while only 4 (23.5%) from the positive samples ranged between 55.3 and 84.4 ng/l thus exceeding the EC limit of 50 ng/l (Table 1 and Table 3). The imported powdered formula samples showed contamination levels ranging from 9.18 to 16.5 ng/l. Table 3 shows the occurrence of AFM1 according to their category. The range of contamination varied among the different categories of milk samples. AFM1 levels in raw cow, pasteurized local cow, pasteurized imported cow and powdered cow milk ranged from 2.63 to 126, 5.18 to 55.3, 3.27 to 84.4 and 9.18e16.5 ng/ l respectively. The highest number of AFM1 positive samples was detected in raw cow milk from local small farms. As for raw goat and powdered goat milk, the levels of AFM1 were below the detection limit. Based on the above results, the concentration of AFM1 in liquid raw cow and pasteurized cow milk was higher than in powdered milk. The highest concentration being found in the raw milk (Table 3). In the present study we confirmed the incidence of AFM1 contamination in locally marketed milk samples at equal or comparable values previously observed and reported by (Biland zic, Varenina, & Solomun, 2010; Roussi, Govaris, Varagouli, & Botsoglou, 2002). However, it is noteworthy to mention that such contamination values were lower than those reported by many surveys (Fallah, 2010; Ghanem & Orfi, 2009; Lan-Chi, Fang e Ming, You e Min, & Yang e Chih Shih, 2004; Razza, 2006; Unusan, 2006). An increase in the consumption of milk and dairy products in Lebanon has been noticed. As a result, the occurrence of AFM1 in milk can constitute a potential hazard for Lebanese consumers

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specially for babies and children who are more susceptible than adults. The obtained results indicate that the occurrence of AFM1 in pasteurized and powder milk can be hazardous for consumption in Lebanon. Concerning the raw milk, the study showed it to be a significant risk to public health. This situation clearly shows the urgent need of a wider national survey in which a larger number of milk samples should be collected from all Lebanese regions and during the various seasons of the year. Acknowledgement This study was funded by the GRESIT research group at the Lebanese University. The authors thank Dr. Zeinab Saad, Dean of The Doctoral School Sciences and Technology for her continuous support. References Biland zi c, N., Varenina, I., & Solomun, B. (2010). Aflatoxin M1 in raw milk in Croatia. Food Control, 21, 1279e1281. European Commission. (2006). Commission regulation. 1881/2006 of December 12th setting maximum levels of certain contaminants in foods. Official Journal European Communities, . L364/5. Fallah, A. A. (2010). Assessment of aflatoxin M1 contamination in pasteurized and UHT milk marketed in central part of Iran. Food and Chemical Toxicology, 48, 988e991. Ghanem, I., & Orfi, M. (2009). Aflatoxin M1 in raw, pasteurized and powdered milk available in the Syrian market. Food Control, 20, 603e605. Gourama, H., & Bullerman, L. B. (1995). Aspergillus flavus: aflatoxigenic fungi of concern in foods and feed. Journal of Food Protection, 58, 1395e1404. Hedayati, M. T., Pasqualotto, A. C., Warn, P. A., Bowyer, P., & Denning, D. W. (2007). Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology, 153, 1677e1692. Pittet, A. (1998). Natural occurrence of mycotoxins in foods and feeds. An updated review. Revue de Médecine Vétérinaire, 149, 479e492. ISO. (2002). Milk and milk products. In Guidelines for standardized description of competitive enzyme immunoassays-determination of AFM1 content standard 14675 Geneva, Switzerland: International Standards Organization. Lan-Chi, L., Fang-Ming, L., You-Min, F., & Yang-Chih Shih, D. (2004). Survey of aflatoxin M1 contamination of dairy products in Taiwan. Journal of Food and Drug Analysis, 12(2), 154e160. Oveisi, M. R., Jannat, B., Sadeghi, N., Hajimahmoodi, M., & Nikzad, A. (2007). Presence of aflatoxin M1 in milk and infant milk products in Tehran, Iran. Food Control, 18, 1216e1218. Peraica, M., Radi c, B., Luci c, A., & Pavlovi c, M. (1999). Toxic effects of mycotoxins in humans. Bulletin of the World Health Organization, 77(9), 754e766. Prandini, A., Tansini, G., Sigolo, S., Fillipi, L., La Porta, M., & Piva, G. (2009). On the occurrence of aflatoxin M1 in milk and dairy products. Food Chemistry and Toxicology, 47, 984e991. Razza, R. (2006). Occurrence of aflatoxin M1in the milk marketed in the city of Karachi. Pakistan. Journal of the Chemical Society of Pakistan, 28(2), 155e157. Rosi, P., Borsari, A., Lasi, G., Lodi, S., Galanti, A., Fava, A., et al. (2007). Aflatoxin M1 in milk: reliability of the immunoenzymatic assay. International Dairy Journal, 17, 429e435. Roussi, V., Govaris, A., Varagouli, A., & Botsoglou, N. A. (2002). Occurrence of aflatoxin M1 in raw and market milk commercialized in Greece. Food Additives and Contaminants, 19(9), 863e868. Unusan, N. (2006). Occurrence of aflatoxin M1 in UHT milk in Turkey. Food and Chemical Toxicology, 44, 1897e1900. WHOe; International Agency for Research on Cancer (IARC). (2002). Monograph on the evaluation of carcinogenic risks to humans, vol. 82. WHO. 171e275. Wogan, G. N. (1999). Aflatoxin as a human carcinogen. Hepatology, 30(2), 573e575. Wogan, G. N., Hecht, S. S., Felton, J. S., Conney, A. H., & Loeb, L. A. (2004). Environmental and chemical carcinogenesis. Seminars in Cancer Biology, 14, 473e486.

Please cite this article in press as: Assem, E., et al., A survey on the occurrence of aflatoxin M1 in raw and processed milk samples marketed in Lebanon, Food Control (2011), doi:10.1016/j.foodcont.2011.04.026


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