OCCURRENCE OF AFLATOXIN M1 IN DAIRYPRODUCTS CIRCULATED IN JORDAN MARKET USING ELISA

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1 OCCURRENCE OF AFLATOXIN IN DAIRYPRODUCTS CIRCULATED IN JORDAN MARKET USING ELISA Nazieh I. Alkhalaileh Department of Nutrition and Food Science, Faculty of Agriculture, Mutah University, Karak, Jordan ABSTRACT A total of 150 samples, representing 75 samples of dairy product that produced in farms and dairy factories local and 75imported samples, All samples were collected from different supermarket in Amman city of Jordan, and analyzed for aflatoxins (AF) using Enzyme-Linked Immunosorbent Assay (ELISA) The results showed that the percentage 14% of raw milk samples, 20% of full cream milk UHT, 10% of whole milk powdered and 30% skimmed milk UHT were contaminated with AF, with an average of 5.82 ±0.72 ppt, ± 1.08ppt, 6.89 ± 1.05ppt and 39.5 ± 1.45ppt, respectively % of imported samples (11 samples) and 12% (9 samples) of Samples that locally produced were contaminated with AF. Evaporated milk, low fat milk UHT (1.5%) pasteurized milk and yogurt Zabadi were free of Aflatoxin. Keywords: aflatoxins (AF), dairy product, ELISA. INTRODUCTION Mold are live microorganisms widespread in nature, can spread of through direct contact, or it can be airborne (Kubenaet al., 1992). Certain strains of mold have been implicated in food borne intoxication, such as Aspergillus flavus and Aspergillus. parasiticus that can occur in foods and feeds and produces mycotoxins at optimal temperature and humidity such as Aflatoxins (Edds, 1979; Moss, 1996; Haschek et al., 2002; Khayoon et al., 2010). The major aflatoxins are B1, B2, G1, and G2. Aflatoxins cause aflatoxicosis, it was reported to cause death of tissue, cirrhosis, and carcinoma of the liver (Bohm et al., 2005; Wild and Turner, 2002). Aflatoxin (AF) is a major hydroxylated metabolite of aflatoxin B1 (AFB1), which is formed when animals eat feed contaminated with aflatoxin B1. The AFB1, once ingested by the animal, is rapidly absorbed by the gastrointestinal tract and is transformed into the metabolite (Van Egmond and Dragacci, 2001). AF, which appears in the blood after 15 minutes and is then secreted in the milk by the mammary gland (Iqbal and Asi, 2013; Battacone, et al., 2003). AF has been found in a range of animal products, including milk and milk products, eggs, meat and meat products (Iqbal and Asi, 2013; Qureshi et al., 1998). The amount of AF which is found in milk linked to a number of factors, such as animal breed, lactation period and mammary infections. It has, been demonstrated that up to 6% of the ingested AFB1 is secreted into the milk as aflatoxin (Van Egmond and Dragacci, 2001). Handling and storage of dairy products slightly effect has an AF content in milk and milk products because AFis resistant to temperatures used in cooking (JECFA and W HO, 2001; Iha et al., 2013; Josephs et al., 2005). Aflatoxin is cytotoxic and its acute toxicity is similar to that of aflatoxin B1 (Iqbal and Asi, 2013). Identified AF by Several chromatographic methods in different food that include on ELISA, flow injection immunoassay system and HPLC methods (Sarimehmetoglu et al., 2004; Badea et al., 2004; Wang et al, 2012; Herzallah, 2009). The aims of this study were to detect the existence of aflatoxin in dairy product that produced by various farms and dairy factories in Jordan. MATERIALS AND METHODS SAMPLE COLLECTION A total 150 samples of dairy product from local farms and dairy factories and sample imported, representing 65 samples of raw milk, 30 samples of full cream milk UHT (3% fat), 10 samples of skimmed milk UHT, 5 samples of low fat milk UHT (1.5%), 20 samples of whole milk powdered, 10 samples of evaporated milk, 5 samples of pasteurized milk and 5 samples of yogurt Zabadi. All collected samples were stored under refrigeration and kept away from direct light exposure until analysis. Sample Preparation for ELISA Analysis 35.0 g from liquid samples has been centrifuge at 10 C for minutes for degreasing, after that centrifugation, remove upper cream layer completely by aspirating through Pasteur Pipette - use the skimmed milk directly in the test, The prepared samples were using microplate enzyme-linked immunosorbent assay (ELISA) using a quantitative test kits (Ridascreen, r-biofarm, Germany) and ELISA reader (Expert plus, Switzerland).). The samples were analyzed using the Aflatoxin (AF) test procedure (Art. No.; R1121), as described by r-biofarm test, procedure provided with the kit for enzyme immunoassay of quantitative analysis of aflatoxin in milk and milk powder (RIDASCREEN, 2000). 14

2 Aflatoxins Working Standards Six working standard dilutions of 0, 5, 10, 20, 40 and 80 ng//l(kg) were provided with ELISA kit. Aflatoxin was used and immunoassayed in duplicate. A zero standard used was methanol diluted in sample buffer (1+9) and assayed in the presence of the enzyme conjugate. Blank wells were same as zero standards, but assayed without the enzyme conjugate. (RIDASCREEN, 2000). Test Protocol of Aflatoxins by ELISA 100 μl standard or prepared sample to separate duplicate wells. Mix gently by shaking and incubate for 30 min at room temperature (20-25 C in the dark. fill all the wells with 250 μl wash buffer (phosphate buffer solution, ph 7.2), then Add 100 μl of substrate/chromogene to each well. Mix gently by shaking and incubate for 15 min at room temperature (20-25 C in the dark. Then Add 100 μl of the stop solution to each well. Mix gently by shaking, measure the absorbance at 450 nm. Read within 15 min after addition of stop solution, (RIDASCREEN, 2000) Statistical Analysis A statistical analysis conducted of the data obtained with ELISA using the arithmetic means, standard deviations and significance level (p< 0.05) was evaluated using the SPSS package. RESULTS AND DISCUSSIONS The results of aflatoxins contamination of different dairy product are presented in Table-1, the data showed that the percentage of 13% (20 samples) of all samples were contaminated with aflatoxins also the results showed that 9 of the 65 samples raw milk, 6 samples of full cream milk UHT (3% fat) of 30 samples, 3samples of skimmed milk UHT of 10 samples and 2 samples of whole milk powdered of 20 samples were contaminated with aflatoxins. The average of AF 5.82 ±0.72 ppt, ± 1.08ppt, 39.5 ± 1.45ppt, and 6.89 ± 1.05ppt, for raw milk, UHT (3% fat), UHT skim milk, and whole milk powdered respectively. The results showed that all samples of evaporated milk, low fat milk UHT (1.5% fat) pasteurized milk and yogurt Zabadi were free of Aflatoxin. The ingestion of feed contaminated by AFB1 is secreted into the milk as AF. The storage conditions at a non-appropriate temperature and relative humidity, encourages the growth of molds that produce the aflatoxin B1 (Van Egmond and Dragacci, 2001; Alshawabkeh et al., 2015). Dairy product Table-1. Aflatoxin content of selected dairy products. (ppt (ppt Raw Milk ± Full Cream Milk UHT ± (3% fat), Whole Milk powdered ± Skimmed Milk UHT ± Evaporated Milk <3.7 <3.7 <3.7 Low Fat Milk UHT(1.5%) Pasteurized Milk Yogurt Zabadi Total *Values are means (± SD) of A duplicate The AFcontamination for local samples of different dairy product are presented in (Table-2) the results showed that 9 samples of raw milk (14%) of samples, were found to be contaminated with aflatoxins, The an average of 5.82 ±0.72 ppt, with the minimum and maximum observed were 4.26 to 8.74 ppt, AF is relatively considered heat resistant throughout handling and processing of milk, thus limited heat effect on AF content can be caused over heat exposure in dairy products (Galvano et al.,1996; Martins, and Martins, 2000;Tekinsen, and Eken, 2008). 15

3 Dairy product Table-2. AF content of different milk types locally produce. Raw Milk ± Low Fat Milk UHT (1.5%) Pasteurized Milk Total *Values are means (± SD) of a duplicate The results of aflatoxins contamination for imported samples of different dairy product are presented in (Table- 3). The results showed that 6 of full cream milk UHT (3% fat) samples, which represents 20% of the samples, were found contaminated with aflatoxins of an average ± 1.08 ppt. the minimum and maximum observed were 6.41 and 29.8 ppt, Results showed that 3 of skimmed milk UHT samples which represent 30% of the sample were contaminated with aflatoxins with an average of 39.5 ± 1.45ppt and the minimum and maximum observed were 37.6 and 41.4ppt. Two samples of whole milk powdered which represents 10% s, were contaminated with aflatoxins with an average of 6.89 ± 1.05 ppt. The minimum and maximum observed were 6.09 and 7.69 ppt. The presence of the AFin dairy products could be due to the contamination of feed with AFB1as well as the heat resistance of AFused during processing. Therefore, the AF almost retained in pasteurized and powdered milk, and infant formula with only a limited decrease of AF content in UHT milk (Galvano et al. 1996; Martins et al. 2000; Tekinsen et al. 2008). The results found in this study showed that imported dairy products from neighbouring countries contain the highest s of AF compared to locally produced dairy products. This could be attributed to the use of green pastures for animal feeding in Jordan. All these levels less than the maximum admissible levels established by the EU, the maximum admissible levels of AF in milk, 50 ng/kg established by the EU, (European Commission, EC, 2003a), some other countries in Africa, Asia and Latin America also apply the limit of 50 ng/kg. (FAO, 2011). Dairy product Full Cream Milk UHT (3% fat), Whole Milk Powdered Skimmed Milk UHT Evaporated Milk Yogurt Zabadi Table-3. AF of imported milk produce circulated in local market. (ppt/kg) (ppt/kg) (ppt /kg) ± ± ± <3.7 <3.7 <3.7 Total *Values are means (± SD) of A duplicate 16

4 CONCLUSIONS Results of the present study showed that some samples of raw milk, full cream milk UHT (3% fat), skimmed milk UHT and whole milk powdered were contaminated with aflatoxins in different but these levels less than the maximum admissible levels established by the EU, the maximum admissible levels of AF in milk, 50 ng/kg established by the EU, (European Commission, EC, 2003a). REFERENCES Alshawabkeh K., N.I. Alkhalaileh, A. Abdelqader, A. Fataftah and S.M. Herzallah Occurrence of Aflatoxin B1 in Poultry Feedand FeedIngredients in Jordan Using ELISA and HPLC. American-Eurasian Journal of Toxicological Sciences. 7(4): Badea M., L. Micheli and M. C. Messia Aflatoxin Dtermination in Raw Milk Using a Flow-Injection Immunoassay System. Analytica Chimica Acta : Battacone G., A. Nudda, A. Cannas, Cappio, A. Borlino, G. Bomboi and G. Pulina Excretion of Aflatoxin in Milk of Dairy Ewes Treated with Different Doses of Aflatoxin B1 Journal of Dairy Science. 86(8): Bohm J. and E Razzai-Fazeli Effects of Mycotoxins on Domestic Pet Species. In The mycotoxin Blue Book, Ed. D. Diaz, Nottingham University Press, Nottingham, U.K. pp Edds G.T Aflatoxins in W. Shimoda (ed). Conference On Mycotoxins In Animal Feeds And Grains Related To Animal Health. Food and Drug Administration. Report No. FDA/BVM-79/139. pp FAO. Corporate Document Repository. April Worldwide Regulations for Mycotoxins in Food and Feed Available From Galvano F., V. Galofaro and G. Galvano Occurrence and Stability of Aflatoxin in Milk and Milk Products. A Worldwide Review. Journal of Food Protection. 59: Haschek W.M., K.A. Voss and V.R. Beasley Selected Mycotoxins Affecting Animal And Human Health. In Handbook of Toxicologic Pathology, In: W.M. Haschek, C.G. Roussex and M.A. Wallig, (Eds.), 2 ed. Academic Press, New York, NY Herzallah S.M Determination of Aflatoxins in Eggs, Meat and Products Using HPLC fluorescent and UV detectors. Food Chemistry. 114: Iha M. H., C. B. Barbosa, I. A. Okada and M. W. Trucksess Aflatoxin in Milk and Distribution and Stability of Aflatoxin During Production and Storage of Yoghurt and Cheese. Food Control. 29(1): 1-6. Iqbal S. Z. and M. R. Asi Assessment of Aflatoxin in Milk and Milk Products from Punjab, Pakistan Food Control. 30(1): JECFA, World Health Organization Safety evaluation of certainmycotoxins in food. in Proceedings of the 56th Meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), WHO Food Additives Series No. 47, International Programme on Chemical Safety, Geneva, Switzerland. Josephs R. D., F. Ulberth, H. P. Van Egmond and H. Emons Aflatoxin in Milk Powders: Processing, Homogeneity and Stability Testing Of Certified Reference Materials. Food Additives and Contaminants. 22(9): Kubena L.F., R.B. Harvey, W.E. Huff, D.E. Corrier, T.D. Phillips and G.E. Rottinghaus Efficacy of Hydrated Sodium Calcium Aluminosilicate to Reduce the Toxicity Of T-2 Toxin. Poultry Science. 69: Khayoon W.S., B. Saad, C.B. Yan, N.H. Hashim, A.S. M. Ali, M.I. Salleh and B. Salleh The Determination Of aflatoxins In Animal Feedsby HPLC with Multifunctional Column Clean-up. Food Chemistry. 119: Martins M.L. and H.M. Martins Aflatoxin in Raw and UltraHigh Temperature-Treated milk commercialized in Portugal. Food Additives & Contaminants. Part A. 17 (10) Moss M.O Mycotoxic Fungi. In: Microbial Food Poisoning, 2edition; A.R. Elcy, Ed. Chapman and Hall; New York: Qureshi M. A., J. Brake, P. B. Hamilton, W. M. Hagler and S. Nesheim Dietary Exposure Of Broiler Breeders To Aflatoxin Results In Immune Dysfunction In Progeny Chicks. Poultry Science. 77(6): RIDASCREEN Aflatoxin , Enzyme immunoassay for the quantitative analysis of aflatoxin in milk and milk powder. R-Biopharm, A der neuen Bergstra ße Darmstadt, Germany. Sarimehmetoglu B., O. Kuplulu and T. H. Celik Detection of Aflatoxin in Cheese Samples by ELISA. Food Control. 15(1): Tekinsen K.K. and H.S. Eken Aflatoxin Levels In UHT Milk and Kashar Cheese Consumed In Turkey. Food and Chemical Toxicology. 46:

5 Van Egmond, H.P. and S. Dragacci Liquid Chromatographic Method for Aflatoxin in Milk. In: Methods in Molecular Biology. Mycotoxin Protocols II. M.W. Trucksess; A.E. Poland. Vol Wang Y., X. Liu and C. Xiaoet HPLC Determination of AflatoxinInLiquid Milk and Milk Powder Using Solid Phase Extraction on OASISHLB. Food Control. 28(1): Wild C.P. and P.C. Turner The Toxicology of Aaflatoxins as Basis for Public Health decisions Mutagenesis. 17:

Presence of Aflatoxin M1 in Raw Milk for Human Consumption in Palestine

Article Presence of Aflatoxin M1 in Raw Milk for Human Consumption in Palestine Ibrahim Mahmoud AL ZUHEIR 1 and Jamal Abo OMAR 2 1 Faculty of Veterinary Medicine, An Najah National University, Nablus,