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Babolhavaegi H R, Afshar Y, Malekahmadi R, Hosseini M S, dehdashti B, Norouzi H A, et al . Determination of Aflatoxin M1 in Pasteurized and Traditional Milk in Hamadan Province, Iran
. J Environ Health Sustain Dev 2018; 3 (2) :504-508
URL: http://jehsd.ssu.ac.ir/article-1-117-en.html
URL: http://jehsd.ssu.ac.ir/article-1-117-en.html
Hamid Reza Babolhavaegi 
, Yosof Afshar , Roya Malekahmadi , Motahareh Sadat Hosseini , Bahare Dehdashti , Hossein Ali Norouzi , Ali Reza Zafar Mirmohamadi , Afshin Holaki , Lida Rafati *
, Yosof Afshar , Roya Malekahmadi , Motahareh Sadat Hosseini , Bahare Dehdashti , Hossein Ali Norouzi , Ali Reza Zafar Mirmohamadi , Afshin Holaki , Lida Rafati *
Deputy of Health, Hamadan University of Medical Sciences, Hamadan, Iran.
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Determination of Aflatoxin M1 in Pasteurized and Traditional Milk in Hamadan Province, Iran
Hamid Reza Babolhavaegi 1, Yosof Afshar 1, Roya Malekahmadi 2, Motahareh Sadat Hosseini 3, Bahare dehdashti 4, Hossein Ali Norouzi 1, Ali Reza Zafar Mirmohamadi 1, Afshin Holaki 1, Lida Rafati 1*
1 Deputy of Health, Hamadan University of Medical Sciences, Hamadan, Iran.
2 Environmental Science and Technology Research Center, Department of Environmental Health Engineering, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3 Department of Food Hygiene and Safety, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
4 Department of Environmental Health Engineering, School of Health, Isfahan University of Medical Sciences, Isfahan, Iran.
Hamid Reza Babolhavaegi 1, Yosof Afshar 1, Roya Malekahmadi 2, Motahareh Sadat Hosseini 3, Bahare dehdashti 4, Hossein Ali Norouzi 1, Ali Reza Zafar Mirmohamadi 1, Afshin Holaki 1, Lida Rafati 1*
1 Deputy of Health, Hamadan University of Medical Sciences, Hamadan, Iran.
2 Environmental Science and Technology Research Center, Department of Environmental Health Engineering, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3 Department of Food Hygiene and Safety, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
4 Department of Environmental Health Engineering, School of Health, Isfahan University of Medical Sciences, Isfahan, Iran.
| A R T I C L E I N F O | ABSTRACT | |
| ORIGINAL ARTICLE | Introduction: Milk is one of the most complete food products that is effective in reducing blood pressure and increasing its beneficial fats, preventing colon cancer and osteoporosis, and providing many nutrients, such as protein and calcium. Therefore, the contamination of this valuable foodstuff and its products is considered as a serious risk to the public health of the community. Aflatoxin is a dangerous fungal toxin that is produced in the presence of moisture and heat as well as lack of proper storage conditions; moreover, it is considered as a hazardous substance in human health. The aim of this study was to evaluate the level of Aflatoxin M1 (AFM1) in raw milk, pasteurized and sterilized milk in food distribution centers of Hamadan province in 2016. Materials and Methods: In the present study, 586 traditional and pasteurized milk samples (446 pasteurized milk samples and 140 traditional milk samples) produced in Hamadan province in summer of 2016 were investigated for AFM1 using Quick AFM1 Strip Test Code ASTM1/96 kit. Results: AFM1 was not observed in 2% of traditional samples and 6.7% of pasteurized specimens. In this study, 37.85% of traditional milk, 56.3% of pasteurized samples had AFM1 less than 50 ppt. Moreover, 12.5% of traditional milk and 1.5% of pasteurized samples had AFM1 more than 50 ppt, which was higher than Iran standard limitation. Conclusion: The results of this study indicate the presence of AFM1 toxin contamination in traditional and pasteurized milks of Hamedan province. Further investigation and monitoring is needed in Hamedan province. |
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| Article History: Received: 2 January 2018 Accepted: 20 April 2018 |
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| *Corresponding Author: Lida Rafati Email: l.rafati@yahoo.com Tel: +989183334018 |
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| Keywords: Aflatoxin M1, Hamadan City, Pasteurized Milk, Traditional Milk. |
Citation: Babolhavaegi HR, Afshar Y, Malekahmadi R. Determination of Aflatoxin M1 in Pasteurized and Traditional Milk in Hamadan Province, Iran. J Environ Health Sustain Dev. 2018; 3(2): 504-8.
Introduction
Aflatoxins are natural fungal toxins and are mainly produced by special strains of Aspergillus flavus and Aspergillus parasiticus 1, 2. These fungi are toxinogenic and contaminate food products at different stages of production, especially in appropriate moisture and heat conditions. Aflatoxins have several types, such as G2, G1 B2, and B1. Aspergillus flavus produces only aflatoxins B 3, while other species produce both aflatoxin B and G1. These toxins are found in numerous human and animal foods. Aflatoxicosis occurs in industrialized and advanced countries, and in addition to being dependent on environmental, social and economic conditions, it depends on climatic conditions such as humidity and heat that are suitable for fungal growth 4. Aflatoxin M1 (AFM1) and Aflatoxin M2 (AFM2) are oxidative metabolites of Aflatoxin B1 (AFB1) and Aflatoxin B2 (AFB2), which are produced by the action of microsomal liver enzymes and are usually found in milk, urine and faeces of livestock, and some of the mammalian species that have been fed with aflatoxin contaminated food 4, 5. AFM1 is bound to the protein of milk that is casein 6. Aflatoxin is a combination of acute toxicity, immunosuppressive, mutagenic, teratogenic, and carcinogenic 7. The World Health Organization (WHO) International Agency for Research on Cancer has identified AFB1 and AFM1 as the primary and secondary metabolites of carcinogens, respectively 8. The target organ for toxicity and carcinogenicity is the liver. Although AFM1 is mutagenic and cassinogenic lower than AFB1, it has a higher genotoxic activity 9. AFM1 resists the heat of pasteurization, autoclaving, and other stages of food process, and they are ineffective in reducing it 10, 11. The European Codex Food and Drug Administration have set maximum levels of AFM1 in raw milk, powder, heat-treated milk and processed milk products at 50 ng/kg, which should not be exceeded 12. According to the US regulations, the level of AFM1 should not exceed 500 ng/kg 13. The Iranian Institute for Standardization has declared the limit of AFM1 in raw milk 0.5 μg/L 14. In Australia and Switzerland, this figure has dropped to 10 ng/kg in children food 15. The AFB1 contamination limit in the livestock feed is 5 μg/kg. Other mycotoxins may even exist in small amounts in milk and dairy products 16. Since milk is an important nutrient in daily human diet, the aim of this study was to compare the AFM1 level in traditional and pasteurized milk in Hamadan province in the summer of 2016.
Materials and Methods
This cross-sectional study was performed on 586 traditional and pasteurized milk samples (446 and 140 traditional milk samples) with different consumption dates collected from supermarkets of Hamedan province during summer of 2016, in order to evaluate Aflatoxin. For testing AFM1, the Quick Afla M1 Strip Test Code ASTM1/96 kit, a fast and commercial test, was used, which its result is visible and readable by eyes. This kit is used to determine the amount of Aflatoxin remaining at 50-100 ppt. To carry out the test, first 200 μL samples from the milk sample were poured into a clear plastic reaction microwell. Then the lyophilized reagents were re-suspended to a uniform pink color in the bottom of the microwell for each sample. The milk containing pot was left 5 minutes so that the anti-Aflatoxin M1 is bonded with gold particles.
If the antibody linked to the gold particles has engaged with the AFM1 presented in the milk sample, the gold particles will flow past the T-Line and reach the Control line (C-Line). Figure 1 shows the visual interpretation of the test results.
Aflatoxins are natural fungal toxins and are mainly produced by special strains of Aspergillus flavus and Aspergillus parasiticus 1, 2. These fungi are toxinogenic and contaminate food products at different stages of production, especially in appropriate moisture and heat conditions. Aflatoxins have several types, such as G2, G1 B2, and B1. Aspergillus flavus produces only aflatoxins B 3, while other species produce both aflatoxin B and G1. These toxins are found in numerous human and animal foods. Aflatoxicosis occurs in industrialized and advanced countries, and in addition to being dependent on environmental, social and economic conditions, it depends on climatic conditions such as humidity and heat that are suitable for fungal growth 4. Aflatoxin M1 (AFM1) and Aflatoxin M2 (AFM2) are oxidative metabolites of Aflatoxin B1 (AFB1) and Aflatoxin B2 (AFB2), which are produced by the action of microsomal liver enzymes and are usually found in milk, urine and faeces of livestock, and some of the mammalian species that have been fed with aflatoxin contaminated food 4, 5. AFM1 is bound to the protein of milk that is casein 6. Aflatoxin is a combination of acute toxicity, immunosuppressive, mutagenic, teratogenic, and carcinogenic 7. The World Health Organization (WHO) International Agency for Research on Cancer has identified AFB1 and AFM1 as the primary and secondary metabolites of carcinogens, respectively 8. The target organ for toxicity and carcinogenicity is the liver. Although AFM1 is mutagenic and cassinogenic lower than AFB1, it has a higher genotoxic activity 9. AFM1 resists the heat of pasteurization, autoclaving, and other stages of food process, and they are ineffective in reducing it 10, 11. The European Codex Food and Drug Administration have set maximum levels of AFM1 in raw milk, powder, heat-treated milk and processed milk products at 50 ng/kg, which should not be exceeded 12. According to the US regulations, the level of AFM1 should not exceed 500 ng/kg 13. The Iranian Institute for Standardization has declared the limit of AFM1 in raw milk 0.5 μg/L 14. In Australia and Switzerland, this figure has dropped to 10 ng/kg in children food 15. The AFB1 contamination limit in the livestock feed is 5 μg/kg. Other mycotoxins may even exist in small amounts in milk and dairy products 16. Since milk is an important nutrient in daily human diet, the aim of this study was to compare the AFM1 level in traditional and pasteurized milk in Hamadan province in the summer of 2016.
Materials and Methods
This cross-sectional study was performed on 586 traditional and pasteurized milk samples (446 and 140 traditional milk samples) with different consumption dates collected from supermarkets of Hamedan province during summer of 2016, in order to evaluate Aflatoxin. For testing AFM1, the Quick Afla M1 Strip Test Code ASTM1/96 kit, a fast and commercial test, was used, which its result is visible and readable by eyes. This kit is used to determine the amount of Aflatoxin remaining at 50-100 ppt. To carry out the test, first 200 μL samples from the milk sample were poured into a clear plastic reaction microwell. Then the lyophilized reagents were re-suspended to a uniform pink color in the bottom of the microwell for each sample. The milk containing pot was left 5 minutes so that the anti-Aflatoxin M1 is bonded with gold particles.
If the antibody linked to the gold particles has engaged with the AFM1 presented in the milk sample, the gold particles will flow past the T-Line and reach the Control line (C-Line). Figure 1 shows the visual interpretation of the test results.
Figure 1: Interpretation of the results
According to Figure 1 if the T-Line signal intensity is stronger than the signal at the C-Line it means that the result is lower than 50 ppt. If the signal at the T-Line is less intense compared to the C-Line it means that the concentration of AFM1 in the milk sample is higher than 50 ppt. If the two lines have equal signal intensities and it is not possible to distinguish which one is more intense by eye, then the test result is about 50 ppt. When only the C-Line is visible and
the T-Line cannot be seen, the test result is strongly positive (equal or higher than 300-400 ppt).
Results
The contamination level of AFM1 pasteurized and traditional milk is presented in Table 1. Based on the results, AFM1 was not observed in 2% of the traditional samples and 6.7% of the pasteurized samples. In 47% of traditional milk and 35.5% of pasteurized samples, AFM1 was 50 ppt Moreover, in 12.5% of traditional milk and 1.5% of pasteurized samples AFM1 was higher than ppt 50 and in 37.85% of traditional milk and 56.3% of pasteurized samples AFM1was less than 50 ppt. Furthermore, the results showed that 95.7% of samples were according to Iran standards (Table 1, Figure 2).
the T-Line cannot be seen, the test result is strongly positive (equal or higher than 300-400 ppt).
Results
The contamination level of AFM1 pasteurized and traditional milk is presented in Table 1. Based on the results, AFM1 was not observed in 2% of the traditional samples and 6.7% of the pasteurized samples. In 47% of traditional milk and 35.5% of pasteurized samples, AFM1 was 50 ppt Moreover, in 12.5% of traditional milk and 1.5% of pasteurized samples AFM1 was higher than ppt 50 and in 37.85% of traditional milk and 56.3% of pasteurized samples AFM1was less than 50 ppt. Furthermore, the results showed that 95.7% of samples were according to Iran standards (Table 1, Figure 2).
Table 1: AFM1 levels in traditional and pasteurized milk in Hamadan province separated by cities
| City | ND* | 50 ppt | More than50 ppt | Less than 50 ppt | |
| Bahar | Pasteurized | 20 | 36 | 4 | 75 |
| Traditional | 1 | 6 | 6 | 2 | |
| Hamedan | Pasteurized | 5 | 33 | 3 | 108 |
| Traditional | 0 | 11 | 0 | 33 | |
| Nahavand | Pasteurized | 0 | 37 | 0 | 19 |
| Traditional | 0 | 26 | 2 | 4 | |
| Malayer | Pasteurized | 0 | 19 | 0 | 36 |
| Traditional | 0 | 16 | 8 | 12 | |
| Toyserkan | Pasteurized | 5 | 33 | 0 | 13 |
| Traditional | 2 | 7 | 2 | 2 | |
| Total | Both | 33 | 224 | 25 | 304 |
ND* = Not detected
Figure 2: Characteristics of AFM1 in Traditional and Pasteurized Milk in Hamadan Province
Discussion
In this study, milk samples were collected and evaluated. The results showed that 95.7% of samples were in accordance with Iran standards. In general, it can be concluded that AFM1 infection in Hamadan province milk is not a serious health problem. Due to the fact that thermal processes such as sterilization and pasteurization have no effect on reducing the amount of milk aflatoxin 16, if the products in the province are provided
from less polluted centers, they will be more consistent with international standards 16. In a study conducted by Kim Yeak et al. in Korea, the prevalence of AFM1 in pasteurized milk and dairy products was investigated. Out of 180 samples collected from the capital of Seoul, 76% of pasteurized milk samples were contaminated with toxin with an average of 18 pg/g 17. In the study of Golipour et al., which was performed on Mazandaran pasteurized milk, it was found that 96% of samples had Aflatoxin, and in 67.62% of the samples Aflatoxin concentration was higher than the limit established by European Codex Committee for Food and Diet 18.
In a study of 44 lactating dairy products in Taiwan in 2002, 90.9% of samples were contaminated with AFM1 19- 20. In a review study conducted by Sadeghi et al. in 2012, the results showed that Aflatoxin was higher than the standard in different milk and contamination was more in warm seasons than in cold seasons in most cases 16. In the study conducted in Shiraz, the infection rate was reported in 100% of samples 21. Although the necessity of supplementary studies in the province of Hamedan and provinces with contamination is recommended, preventive measures should be taken to prevent the entry of this toxin. Therefore, it is possible to reduce and control the amount of toxin by using the superior technology by experts and the application of rules and regulations in the promotion of the milk quality received by the community and animal feed.
Conclusion
The results of this study raise the issue of AFM1 in local and pasteurized milk, which showed a higher percentage of contamination in local sample than the pasteurized one. Although Aflatoxin is caused by poorly fed livestock by mildew breads and residues, milk of livestock should be tested and investigated for drug residues, vegetable pests and mycotoxins. Finally, it is suggested that the officials of the Agricultural Jihad and other organizations provide appropriate solutions to reduce the amount of contamination in the milk collection centers, such as control of animal feed and storage and keeping of forage and livestock feed. Therefore, careful and continuous monitoring of the control and maintenance of forage and animal feed in contaminated areas is necessary.
Acknowledgement
Thanks are owed to Deputy of Health, Hamadan University of Medical Sciences, for financial and technical supports during this study.
Funding
In this study, milk samples were collected and evaluated. The results showed that 95.7% of samples were in accordance with Iran standards. In general, it can be concluded that AFM1 infection in Hamadan province milk is not a serious health problem. Due to the fact that thermal processes such as sterilization and pasteurization have no effect on reducing the amount of milk aflatoxin 16, if the products in the province are provided
from less polluted centers, they will be more consistent with international standards 16. In a study conducted by Kim Yeak et al. in Korea, the prevalence of AFM1 in pasteurized milk and dairy products was investigated. Out of 180 samples collected from the capital of Seoul, 76% of pasteurized milk samples were contaminated with toxin with an average of 18 pg/g 17. In the study of Golipour et al., which was performed on Mazandaran pasteurized milk, it was found that 96% of samples had Aflatoxin, and in 67.62% of the samples Aflatoxin concentration was higher than the limit established by European Codex Committee for Food and Diet 18.
In a study of 44 lactating dairy products in Taiwan in 2002, 90.9% of samples were contaminated with AFM1 19- 20. In a review study conducted by Sadeghi et al. in 2012, the results showed that Aflatoxin was higher than the standard in different milk and contamination was more in warm seasons than in cold seasons in most cases 16. In the study conducted in Shiraz, the infection rate was reported in 100% of samples 21. Although the necessity of supplementary studies in the province of Hamedan and provinces with contamination is recommended, preventive measures should be taken to prevent the entry of this toxin. Therefore, it is possible to reduce and control the amount of toxin by using the superior technology by experts and the application of rules and regulations in the promotion of the milk quality received by the community and animal feed.
Conclusion
The results of this study raise the issue of AFM1 in local and pasteurized milk, which showed a higher percentage of contamination in local sample than the pasteurized one. Although Aflatoxin is caused by poorly fed livestock by mildew breads and residues, milk of livestock should be tested and investigated for drug residues, vegetable pests and mycotoxins. Finally, it is suggested that the officials of the Agricultural Jihad and other organizations provide appropriate solutions to reduce the amount of contamination in the milk collection centers, such as control of animal feed and storage and keeping of forage and livestock feed. Therefore, careful and continuous monitoring of the control and maintenance of forage and animal feed in contaminated areas is necessary.
Acknowledgement
Thanks are owed to Deputy of Health, Hamadan University of Medical Sciences, for financial and technical supports during this study.
Funding
This study was funded by Deputy of Health, Hamadan University of Medical Sciences.
Conflict of interest
The authors declare that there is no conflict of interest.
This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work for commercial use.
References
Conflict of interest
The authors declare that there is no conflict of interest.
This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work for commercial use.
References
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10.Deshpande SS. Fungal toxins. In: SS. Deshpande, Editor. Handbook of food toxicology. New York: Marcel Decker; 2002.
11. Park DL. Effect of processing on aflatoxin. Adv Exp Med Biol. 2002; 504: 173–9.
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13.Stoloff L, Van Egmond HP, Park DL. Rationales for the establishment of limits and regulations for mycotoxins. Food Addit Contam. 1991; 8(2): 213-21.
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15. Margolles E, Escobar A, Acosta A. Aflatoxin B1 residuality determination directly in milk by ELISA. Revista De Saude Alimentation. 1992; 12: 35–8.
16. Sadeghi E, Mohammadi M, Sadeghi M, et al. Systematic review study of Aflatoxin M1 level
in raw, pasteurized and UHT milk in Iran. Iranian Journal of Nutrition Sciences & Food Technology. 2013; 7(5): 599-612.
17. Kim EK, Shon DH, Ryu D, et al. Occurrence of aflatoxin M1 in Korean dairy products determined by ELISA and HPLC. Food Addit Contam, 2000; 17(1): 59-64
18. Gholipour M, Karimzadeh L, Ali Nia F, et al. Determination of aflatoxin M1 in milk processed in Mazandaran dairy factories. Journal of Mazandaran University of Medical Sciences. 2012; 22(93): 39-46. [In Persian]
19. Zinedine A, Gonzalez Osnaya L, Soriano JM, et al. Presence of aflatoxin M1 in pasteurized milk from Morocco. International Journal of food Microbiology. 2007; 114(1): 25-9.
20.Lin LC, Liu FM, FU YM, et al. Survey of aflatoxin M1 contamination of dairy products in Taiwan. J Food Drug Analy. 2004; 12(2): 154-60.
21.Alborzi S, pourabbas B, Rashidi M, et al. Aflatoxin M1 contamination in pasteurized milk in Shiraz (south of Iran). Food Control. 2005; 17: 582-4.
3. Aycicek H, Aksoy A, Saygi S. Determination of aflatoxin levels in some dairy and food products which consumed in Ankara, Turkey. Food Control. 2005; 16(3): 263-6.
4. Creppy EE. Update of survey, regulation and toxic effects of mycotoxins in Europe. Toxico Lett. 2002; 127(1-3): 19-28.
5. Khodadadi M, Khosravi R, Allahresani A, et al. Occurrence of aflatoxin M1 in pasteurized and traditional cheese marketed in southern Khorasan, Iran. Journal of Food Quality and Hazards Control. 2014; 1: 77-80.
6. Brackett RE, Marth EH. Fate of aflatoxin M1 in Parmesan and Mozzarella cheese. J Food Prot. 1982; 45: 597-600.
7 .Ricordy R, Coacci R, Augusti OF. Aflatoxin B1 and cell cycle perturbation. Food and Nutrition Toxicity. 2004; 4: 213-33.
8. Dragacci S, Gleizes E, Fremi JM, et al. Use of immunoaffinity chromatography as a purification step for the determination of aflatoxin M1 in cheeses. Food Add Contam. 1995; 12(1): 59–65.
9.Kocabas CD, Sekerel BE. Does systemic exposure to aflatoxin B1 cause allergic sensitization?. Allergy. 2003; 58: 347-52.
10.Deshpande SS. Fungal toxins. In: SS. Deshpande, Editor. Handbook of food toxicology. New York: Marcel Decker; 2002.
11. Park DL. Effect of processing on aflatoxin. Adv Exp Med Biol. 2002; 504: 173–9.
12. Food and Agriculture Organization, Worldwide regulations for mycotoxins in food and feed in 2003, Food and Agriculture Organization. Rome 2004; FAO Food and Nutrition; pp: 81.
13.Stoloff L, Van Egmond HP, Park DL. Rationales for the establishment of limits and regulations for mycotoxins. Food Addit Contam. 1991; 8(2): 213-21.
14. Institute of Standards and Industrial Research of Iran. Maximum validity Maycotoxins in human food. ISIRI no 5925. 1st edition, Karaj: ISIRI; 2001. [In Persian]
15. Margolles E, Escobar A, Acosta A. Aflatoxin B1 residuality determination directly in milk by ELISA. Revista De Saude Alimentation. 1992; 12: 35–8.
16. Sadeghi E, Mohammadi M, Sadeghi M, et al. Systematic review study of Aflatoxin M1 level
in raw, pasteurized and UHT milk in Iran. Iranian Journal of Nutrition Sciences & Food Technology. 2013; 7(5): 599-612.
17. Kim EK, Shon DH, Ryu D, et al. Occurrence of aflatoxin M1 in Korean dairy products determined by ELISA and HPLC. Food Addit Contam, 2000; 17(1): 59-64
18. Gholipour M, Karimzadeh L, Ali Nia F, et al. Determination of aflatoxin M1 in milk processed in Mazandaran dairy factories. Journal of Mazandaran University of Medical Sciences. 2012; 22(93): 39-46. [In Persian]
19. Zinedine A, Gonzalez Osnaya L, Soriano JM, et al. Presence of aflatoxin M1 in pasteurized milk from Morocco. International Journal of food Microbiology. 2007; 114(1): 25-9.
20.Lin LC, Liu FM, FU YM, et al. Survey of aflatoxin M1 contamination of dairy products in Taiwan. J Food Drug Analy. 2004; 12(2): 154-60.
21.Alborzi S, pourabbas B, Rashidi M, et al. Aflatoxin M1 contamination in pasteurized milk in Shiraz (south of Iran). Food Control. 2005; 17: 582-4.
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Received: 2018/01/2 | Accepted: 2018/04/20 | Published: 2018/06/13
Received: 2018/01/2 | Accepted: 2018/04/20 | Published: 2018/06/13
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