Iranian Journal of Environmental Health, Science and Engineering Iranian Association of Environmental Health (IAEH) ISSN: 1735-1979 Vol. 8, Num. 2, 2011, pp. 123-128

Iranian Journal of Environmental Health, Science and Engineering, Vol. 8, No. 2, 2011, pp. 123-128

Occurrence Of Aflatoxin M1 In Raw Milk Produced In Ardebil Of Iran

¹A. Kamkar, *²Gh. R. Jahed Khaniki, ³S. A. Alavi

¹Department of Food Hygiene, Faculty of Veterinary Medicine, Tehran University, Tehran, Iran
²Department of Environmental Health Engineering, School of public Health, Tehran University of Medical Sciences, Tehran, Iran
³Veterinary Organization of Iran. Ardebil Veterinary Office, Ardebil, Iran

Received 10 April 2010; revised 11 December 2010; accepted 13 March 2011

Code Number: se11015

ABSTRACT

Contamination of milk and dairy products to aflatoxin M1 is a risk for human and it can be a public heath concern. The aim of this study was to evaluate the presence of aflatoxin M1 in raw milk samples produced in Ardebil City (Iran) by ELISA (Enzyme Linked Immuno Sorbent Assay) technique. 122 samples of raw milk were collected from milk collecting centers and dairy plants in the region and aflatoxin M1 contamination was detected in all of milk samples. The mean concentration of aflatoxin M1 was 40.01ng/L and 14.75 percent of the samples had higher levels than the maximum recommended limits by ISIRI, European Community and Codex Alimentarius. With a view of the fact that milk is used by all the age groups including infants and children in the city of Ardebil, it is necessary to apply an ideal recommended limit to minimize the health hazard from aflatoxin M1 contamination in milk. Application of Good Agricultural Practices and Good Veterinary Practices by agriculture and also the Hazard Analysis and Critical Control Points (HACCP) system as a draft code of practice for preharvest and postharvest control of dairy cow’s feed and in milk and dairy products processing is effective.

Key words: Aflatoxin M1; Raw milk; Enzyme linked immuno sorbent assay; Milk safety

INTRODUCTION

Aflatoxins are generally produced in animal feed by toxigenic fungi such as Aspergillus flavus, Aspergillus parasiticus and the rare Aspergillus nomius. There are fungi spores in the environment and poor storage of commodities after harvest allow the growth of fungi. Some studies have shown that various foods, peanuts and corns can be contaminated by Aspergillus fungi (Khanafari et al., 2007; Rostami et al., 2009). The conditions of high humidity and warm temperatures can give rise to the highest levels of aflatoxins in food (Adams and Moss, 2005). They are toxic, immunosuppressive, mutagenic, teratogenic and carcinogenic compounds to animals and humans (Piva et al., 1995; Peraica et al., 1999; Kocabas and Sekerel, 2003).

Aflatoxin (AFM1) is a hydroxylated metabolite of aflatoxin B1 that may be found in milk and milk products obtained from livestock that have ingested contaminated feed (Creppy, 2002). Contamination of milk and dairy products to AFM1 can lead to increase the risk of hepatocellular carcinoma and liver cancer (Krogh, 1987; Peraica et al., 1999). International Agency for Research on Cancer (IARC) of WHO considers aflatoxin B1 as primary and aflatoxin M1 as secondary groups of carcinogenic compounds (Cathey et al., 1994; Dragacci et al., 1995).

The main feed sources of aflatoxins are peanut meal, maize and cottonseed meal. Many researchers reported that there was a linear relationship between the amount of aflatoxin M1 in milk and aflatoxin B1 in feed consumed by animals (Bakirci, 2001). Milk usually may have the greatest demonstrated potential for introducing aflatoxin residues from food animal origin into the human diet (Stoloff, 1977). Many countries have established regulations to control the levels of aflatoxin B1 in feeds and to have maximum permissible levels of aflatoxin M1 in milk to reduce this hazard (Van Egmond, 1989; Sarimehmetoglo et al., 2004). The European Community and Codex Alimentarius Commission prescribed that the maximum level of aflatoxin M1 in milk and milk products should not exceed 50 ng/L (EC, 1992; CAC, 2001).

Some studies have been done about aflatoxin M1 contamination in raw milk in the world and their results have presented exceeded concentrations regarding the European Community and Codex Alimentarius regulatory limit (Dashti et al., 2009; Ghanem and Orfi, 2009; Hussain et al., 2008). Although, there are some studies about the contamination of aflatoxin M1 in milk and dairy products in different cities of Iran (Oveisi et al., 2007; Ghazani, 2009; Fallah et al., 2009), but there is no published data regarding aflatoxin M1 in raw milk produced in Ardebil City in north west of Iran and this study was carried out to evaluate the occurrence of aflatoxin M1 in raw milk produced in this region.

MATERIALS AND METHODS

Sample collection

A total of 122 samples of raw milk samples were randomly collected with in 6 months for the analysis of aflatoxin M1. The raw milk samples were obtained directly from cooling tanks of the milk collecting centers and dairy plants of Ardebil City before pasteurization. 30 mL milk was gathered and placed in an amber glass flask and kept refrigerated until analysis.

Sample examination and ELISA test procedure

The quantity of aflatoxin M1 was determined according to Enzyme-Linked Immuno Sorbent Assay (ELISA) method by using the Ridascreen^® aflatoxin M1 (R-biopharm, Darmstadt, Germany) test kit which is a competitive enzyme immunoassay based on antigen-antibody reaction (Anonymous, 1999).

The milk samples were centrifuged for 10 minutes with 3500 rpm in 10°C. After centrifugation, the upper creamy layer was completely removed by aspirating through a Pasteur pipette. 100 µL of skimmed milk was used directly in each well for quantitative test. A sufficient number of micro titer wells were inserted into the micro well holder for all standards and prepared samples. Then 100 µL of the standard solutions and prepared samples were added in separate wells and they were incubated for 60 min at room temperature in the dark. The liquid was poured off the wells and the micro well holder was tapped upside down vigorously (three times) against absorbent paper to ensure complete removal of liquid from the wells. All the wells were filled with 250 μL of washing buffer and emptied as described earlier. The washing procedure was repeated twice. After that 100 µL of the enzyme conjugate was added and incubated for 60 min at room temperature in the dark.

The washing sequence was repeated three times. 50 µL of the substrate solution and 50 μL of chromogen solution were added to each well and mixed thoroughly. Then they were incubated for 30 min at room temperature in the dark. After this time, 100 µL of the stop reagent was added to each well and mixed completely. Finally, the measurement of AFM1 was done photometrically at the wavelength of 450 nm against the air blank in ELISA reader. Absorbance percentages were taken to the calibration curve performed with standards at different concentrations.

Statistical analysis

All statistical analyses were performed using the software SPSS, version 11.5. Analysis of variance ANOVA was employed after logarithmic conversion when necessary to detect significant differences among means. A probability level of p<0.05 was considered statistically significant.

RESULTS

In this study, a total of 122 raw milk samples were analyzed with the competitive ELISA technique. The occurrence and levels of aflatoxin M1 in raw milk samples produced in Ardebil City during six months are shown in Tables Table 1 and Table 2. Aflatoxin M1 contamination was detected in all of the examined raw milk samples. The aflatoxin M1 contamination levels were between 4 -112.4 ng/L with the mean of 40.01ng/L. There was a high incidence rate of aflatoxin M1 with 122 (100 %) milk samples being contaminated. The mean of aflatoxin M1 contamination levels in October, November, December, January, February and March were 29.74, 40.69, 45.78, 46.13, 43.35 and 34.38ng/L, respectively. Also, the range of contamination levels varied among six months and they were ranged from 4 - 96 ng/L, 4.2 - 96 ng/L, 8.4 - 112.4 ng/L, 8 - 110 ng/L, 5.6 - 106.5 ng/L and 4.5 - 102.2 ng/L in October, November, December, January, February and March, respectively (Table 1). The lowest contamination was observed in October and the highest contamination was observed in January. The frequency of aflatoxin M1 contamination levels above 50 ng/L in October, November, December, January, February and March were 0.81, 1.64, 4.10, 4.10, 2.46 and 1.64 %, respectively (Table 2). In general, 18 out of 122 raw milk samples (14.75%) were beyond the limit of ISIRI, Codex Alimentarius and European Community regulations. Also, results showed that in 104 samples (85.25%) the aflatoxin M1 concentrations were less than 50 ng/L and in 27 samples (22.13%) aflatoxin M1 was found less than 10ng/l. In 77 samples (67.11%), the concentrations were above 10 ng/L and less than 50 ng/L.

DISCUSSION

According to results obtained in this study, aflatoxin M1 was found in 100% of raw milk samples with 14.75% of the samples were higher than the permissible level of 50 ng/L as accepted by Iranian food standards (ISIRI, 2002; ISIRI, 2005) and European Community. With a view of the fact that milk is used by all the age groups including infants and children in Ardebil City, even the low amount of aflatoxin M1 in milk can be a serious public health problem. Since the commission of the European communities stated that even if aflatoxin M1 is regarded a less dangerous genotoxic carcinogenic substance than Aflatoxin B1, it is necessary to prevent the presence in milk and consequently in milk products, intended for human consumption and for young children in particular (Prandini et al., 2009).

Other researches have previously worked on the prevalence of aflatoxin M1 in milk samples (Table 3). The rate of aflatoxin M1 contamination levels obtained from our study is higher than the aflatoxin M1 levels in examined raw milk samples from Urmia (Tajik et al., 2007) and it is lower than aflatoxin M1 levels in reported raw milk samples from Sarab (Kamkar, 2005) and Tehran (Karim, 1998; Oveisi et al., 2007). It has been indicated that in many countries of Europe showed relatively low levels of aflatoxin M1 have been found in milk and milk products (Trucksess, 1999). The occurrence of aflatoxin M1 at such low levels in European countries may be the result of stringent regulations of aflatoxin B1 in complementary feedstuffs for dairy cattle. In many Asian countries and in Iran no severe control method for aflatoxin M1 in milk and milk products has been applied. In an Indian study of 87 liquid milk samples, incidence of contamination of aflatoxin M1 was of the magnitude of 87.3%. Almost 99% of the contaminated samples exceeded the European Communities and Codex Alimentarius recommended limits (EC, 1992; Shipra et al., 2004). Also, some studies have been done about aflatoxin M1 contamination in milk in other Asian countries like Kuwait, Pakistan and Syria. In a study, Dashti et al. (2009) reported that all fresh milk samples except one from Kuwaiti markets were contaminated with aflatoxin M1 ranging from 4.9 to 68.7 ng/L and eight samples exceeded the EC regulatory limit. In another research, Hussain et al. (2008) reported that all of the examined raw milk samples from fourteen districts of the Punjab province, Pakistan, were contaminated with aflatoxin M1 and 99.4% samples exceeded the European Union limit (0.05 μg/L). Also, Ghanem and Orfi, 2009 showed that 80% of tested raw milk samples collected from the Syrian market were contaminated with various levels of aflatoxin M1 ranging from 20 - 765 ng/L and the percentage of aflatoxin M1-contaminated samples exceeding the European tolerance limits was 52%. In other study, Bakirci (2001) found that 87.77% of the examined raw milk samples from the dairy plant in Turkey were contaminated with aflatoxin M1 and 44.30% of the positive samples were higher than the maximum tolerance limit (0.05 ppb) accepted by Turkey and European Union. The percentage of contaminated raw milk in present study is less than above mentioned studies in other countries.

Bilandzic et al. (2010) reported that the maximum mean concentrations of aflatoxin M1 recorded in winter-spring season were in the range of 35.8–58.6 ng/L and in summer–autumn season in the range of 11.6–14.9 ng/L and examined milk samples in winter–spring season have high levels of aflatoxin M1.

The aflatoxin M1 levels of our study in all months of October, November, December, January, February and March were higher than Iranian and European tolerance limits (50 ng/L), but many contaminated samples with high levels of aflatoxin M1 were found in months of December and January. This variation can be related to dairy feed quality. Because the quality of fodder used for feeding the cattle due to aflatoxin M1 in milk is important and the ingestion of food contaminated with aflatoxin B1 by dairy cattle is the way of exposure to aflatoxin M1 in milk (Lopez et al., 2003).

Many factors may affect the formation of aflatoxins in animal feeds. Geographic and climate changes can affect the farm management practices and feed quality. These effects can lead to the wide variations in AFM1 levels in milk (Ghazani, 2009). The preserved fodder such as silage and hay might have been contaminated by aflatoxin producing fungi and the improper storage led to aflatoxin production. The level of aflatoxin M1 in feed in rainy seasons is more than in dry seasons. It can be also probable to use higher amounts of contaminated concentrates in the cold months. Tajkarimi et al (2008) reported that the high levels of aflatoxin M1 contamination can occur in the cold months. The higher levels of aflatoxin M1 in this study obtained from cold months (e.g. December, January and February), which was reported by previous studies (Tajkarimi et al., 2007; Tajkarimi et al., 2008). Dairy cattle feed stuffs should be kept away from aflatoxin contamination as much as possible. It is important to maintain control and to apply an ideal recommended limit to minimize the health hazard from aflatoxin M1 contamination in milk which it can be used by infants and children. About this, governments have responsibility for making regulations to protect consumers against harm arising from chemical in milk. Government and producer must apply some methods and plans for prevention and control of aflatoxin M1 in milk and dairy products. About this, application of the Good Agricultural Practices (GAP) and Good Veterinary Practices (GVP) by agriculture and also the Hazard Analysis and Critical Control Point (HACCP) system as a draft code of practice for preharvest and postharvest control of dairy cow’s feed and in milk and dairy products processing is effective. Responsibility for aflatoxin M1 control in milk and dairy products lies with all participitants in the production process, from farmers through to consumers.

ACKNOWLEDGEMENTS

The authors would like to thank the laboratory personnel of Ardebil Veterinary Office for kind technical assistance.

REFERENCES

• Adams, M.R., Moss, M.O., (2005). Food Microbiology. New Age International (P) Limited, Publishers, New Dehli., 228-244.
• Anonymous., (1999). Enzyme immunoassay for the quantitative analysis of aflatoxin M1. Ridascreen aflatoxin M1, R-Biopharm Gmbh, Darmstadt, Germany.
• 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.
• Bilandzic, N., Varenina, I., Solomun, B., (2010). Aflatoxin M1 in raw milk in Croatia.   Food Control, 21 (9): 1279-1281.
• Cathey, C.G., Huang, Z.G., Sarr, A.B., Clement, B.A., Philips, T.D., (1994). Development and evaluation of a minicolumn assay for the detection of aflatoxin M1 in milk. Journal of Dairy Science, 77:1223-1231.
• CAC., (2001). Comments submitted on the draft maximum level for aflatoxin M1 in milk. Codex Alimentarius Commissions (CAC). Joint FAO/WHO Food Standards Programme Codex Alimentarius Commission, Report of the 33^rd session of the Codex Committee on Food Additives and Contaminants, The Hague, The Netherlands.,127-133.
• Creppy, E. E., (2002). Update of survey, regulation and toxic effects of mycotoxins in Europe. Toxicology Letters, 127: 19-28.
• Dashti, B., Al-Hamli, S., Alomirah, H., Al-Zenki, S., Bu Abbas, A., Sawaya, W., (2009). Levels of aflatoxin M1 in milk, cheese consumed in Kuwait and occurrence of total aflatoxin in local and imported animal feed. Food Control, 20 (7):686-690.
• Decastelh, D., Lai, J., Grammaglia, M., Monaco, A., Nachtmann, C., Oldano, F., Ruffier, M., Sezian, A., Bandirola, C., (2006). Aflatoxin occurrence in milk and feed in Northern Italy during 2004-2005. Food Control, 18:1263-1266.
• 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: 59-65.
• EC., (1992). Commission Directive 92/95/EEC and Commission Directive 94/14/EC amending the annex of the Seventh Commission Directive 76/372/EEC establishing community methods of analysis for the official control of feeding stuffs. European Community (EC). Official Journal of the European Communities, 15: 4.
• Fallah, A.A., Jafari, T., Fallah, A., Rahnama, M., (2009). Determination of aflatoxin M1 levels in Iranian white and cream cheese. Food and Chemical Toxicology., 47: 1872-1875.
• Ghanem, I., and Orfi, M., (2009). Aflatoxin M1 in raw, pasteurized and powdered milk available in the Syrian market. Food Control, 20 (6): 603-605. Ghazani, M.H.M., (2009). Aflatoxin M1 contamination in pasteurized milk in Tabriz (northwest of Iran). Food and Chemical Toxicology, 47:1624-1625.
• Henry, S.H., Whitaker, T. B., Rabbani, I., Bowers, J., Park, D., Price, W., Bosch, F.X., Pennington, J., Verger, P., Yoshizawa, T., Van Egmond, H., Jonker, M.A., Coker, and R., (2001). Aflatoxin M1, chemical safety information from inter government organizations. Joint FAO/WHO Expert Committee on Food Additives (JFCFA), WHO Food Additives Series 47:45-80.
• Hussain, I., and Anwar, J., (2008). A study on contamination of aflatoxin M1 in raw milk in the Punjab province of Pakistan Food Control, 19(4):393-395.
• ISIRI., (2002). Food and Feed- Mycotoxins: Maximum tolerated level. First edition. Institute of standards and industrial research of Iran (ISIRI), Tehran, Iran., 3-20.
• ISIRI., (2005). Milk and milk products specifications and test methods. Iranian national standard, No.164, 2^nd revision. Institute of standards and industrial research of Iran (ISIRI), Tehran, Iran. ., 4-22.
• Kamkar, A., (2005). A study on the occurrence of aflatoxin M1 in raw milk produced in Sarab city of Iran. Food Control, 16: 593-9.
• Karim, G., (1998). Study on the contamination of milk with aflatoxin in Tehran area using ELISA method. Pajouhesh-va-Sazandegi Journal, 40:163-165.
• Khanafari, A., Soudi, H., Miraboulfathi, M., (2007). Biocontrol of Aspergillus flavus and aflatoxin B1 production in corn. Iran J Environ Health Sci & Eng, 4: 163-168.
• Kocabas, C. N., Sekerel, B.E., (2003). Does systemic exposure to aflatoxin B1 cause allergic sensitization? Allergy, 58: 363.
• Krogh, P., (1987). Mycotoxins in food. Food Science and Technology, A series of Monographs Academic press, Harcourt Brace Jovanovich, Publishers, London.
• Lopez, C.E., Ramos, L.L., Ramadan, S.S., Bulacio, L.C., (2003). Presence of aflatoxin M1 in milk for human consumption Argentina. Food Control, 14: 31–34.
• Markaki, P., and Melissari, E., (1997). Occurrence of aflatoxin M1 in commercial pasteurized milk determined with ELISA and HPLC. Food Additives and Contaminants, 14: 451-456.
• Oveisi, M.R., Jannat, B., Sadeghi, N., Hajimahmoodi, M., Nikzad, A., (2007). Presence of aflatoxin M1 in milk and infant milk product in Tehran. Food Control, 18: 1216-1218.
• Peraica, M., Radic, B., Lucic, A., Pavlovic, M., (1999). Toxic effects of mycotoxins in humans. Bulletin of the World Health Organization, 77: 754-766.
• Piva, G., Galvano, F., Pietri, A., Piva, A., (1995). Detoxification methods of aflatoxin. A review. Nutrition Reaserch, 15: 689-715.
• Prandini, A., Tansini, G., Sigolo, S., Filippi, L., Laporta, M., Piva, G., (2009). Review: On the occurrence of aflatoxin M1 in milk and dairy products. Food and Chemical Toxicology, 47: 984-991.
• Rostami, R., Naddafi, K., Aghamohamadi, A., Najafi saleh, H., Fazlzadeh davil , M., (2009). Survey of peanut fungal contamination and its relashionship with ambient conditions in the bazar of Zanjan. Iran J Environ Health Sci & Eng, 6: 295-300. 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: 863-868.
• Sarimehmetoglo, B., Kuplulu, O., Celik, T.H., (2004). Detection of aflatoxin M1 in cheese samples by ELISA. Food Control, 15: 287-290.
• Sefidgar, S.A.A., Azizi, A.R.G., Kosravi, A.R., Roudbar-Mohammadi, S., (2008). Presence of aflatoxin M1 in raw milk at cattle farmers in Babol, Iran. Pakistan Journal of Biological Sciences, 11: 484-486.
• Shipra, R., Premendra, D.D., Subhash, K.K., Mukul, D., (2004). Detection of Aflatoxin M1 contamination in milk and infant milk products from Indian markets by ELISA. Food control, 15: 287-290.
• Stoloff, L., (1977). Aflatoxins: An overview. Proceedings of a conference on mycotoxins in human and animal health. Park Forest South. Pathotox Publishers Inc. pp. 7-28.
• Tajik, H., Rohani, S.M.R., Moradi, M., (2007). Detection of Aflatoxin M1 in raw and commercial pasteurized milk in Urmia, Iran. Pakistan Journal of Biological Sciences, 10: 4103-4107.
• Tajkarimi, M., Aliabadi, S.F., Salah Nejad, M., Poursoltani, H., Motallebi, A.A., Mahdavi, H., (2007). Seasonal study of aflatoxin M1 contamination in milk in five regions in Iran. International Journal of Food Microbiology, 116: 346–349.
• Tajkarimi, M., Aliabadi, S.F., Salah Nejad, A., Poursoltani, H., Motallebi, A.A., Mahdavi, H., (2008). Aflatoxin M1 contamination in winter and summer milk in 14 states in Iran. Food Control, 19: 1033–1036.
• Trucksess, M. W., (1999). Mycotoxins. Journal of AOAC International, 82: 448-495.
• Van Egmond, H. P., (1989). Current situation on regulations for mycotoxins. Overview of tolerances and status of standard method of sampling and analysis. Food Additives and Contaminants, 6: 139-188.

Copyright © 2011 - Tehran University of Medical Sciences Publications

The following images related to this document are available:

Photo images