Abstract:
In this study, raw cow milk samples were collected from milk churns at 40 traditional and semi-industrial cattle farms located in Babol (Northern Iran) in winter 2006. In total, 120 raw milk samples were tested for Aflatoxin M1 (AFM1) contamination by competitive ELISA. In 68 out of 120 samples (56.7%) the presence of AFM1 was detected by concentration ranging from 50 to 352.3 ng L-1. Fifty two samples (43.3%) contained AFM1 at levels of 4-50 ng L-1 (The AFM1 contamination levels were between 4-352.3 ng L-1 by the average of 102.73 ng L-1). In general, 56.7% of samples were beyond the limit of European community regulations (50 ng L-1). In other words, the AFM1 contamination levels in raw milk were more than twice over standard levels. The AFM1 contamination levels, (>50 ng L-1), in January, February and March were 40, 65 and 65%, respectively. Estimation of contamination of AFB1 using AFM1 in feed stuff showed that it was about 0.25 to 22 µg kg-1 holding the average of 46.7%, being higher than European community limit (5 μg kg-1).
INTRODUCTION
Aflatoxins are structurally related to a group of toxic compounds, found in most plant products such as peanuts, copra, soya, maize, race and wheat. They are generally produced by special strains of Aspergillus flavus and Aspergillus parasiticus. These toxigenic fungi contaminate food products in different phases of production and processing especially in suitable heat and moist conditions. Aflatoxicosis depends on both environmental, social and economical conditions and the climate (Aycicek et al., 2005). AFM1 and AFM2 are oxidative metabolic AFB1 and AFB2 which are made by live mycosomal enzymes functions. AFM1 and AFM2 are discharged via milk, urinary and feces of cattle and some species of mammals that consume feed stuff contaminated to Aflatoxin (Creppy, 2002; Aycicek et al., 2005).
Their main target organ for toxicity and carcinogenicity is liver. Although mutagenic and carcinogenic level of AFM1 is lower than AFB1, its genotoxic activity is high. Aflatoxin in some human diseases, particularly, liver primary cancer is involved by engaging DNA, mutagen P53 gene (Kocabas and Sekerel, 2003). European community and Codex Alimentary determined the maximum level of AFM1 50 ng kg-1 in raw milk, liquid, powder, heat-treated milk and processed dairy products and it must not exceed the amount.
The permitted contamination limit to AFB1 in feed stuff is 5 μg kg-1. AFM1 appears in milk by cattle`s consuming AFB1 after 12-24 h. Milk and dairy products consumption is high in human particularly among children; therefore, they are exposed to AFM1. On the other hand, milk is consumed not only as liquid but as children formulae, yoghurt, cheese and milk-based sweets such as chocolate and donuts. Therefore, AFM1 recognition in milk and dairy products is crucial. As a result, consumers of various ages are protected from its potential risks (Bakirci, 2001).
Many countries have conducted inspection program and controlling on mycotoxins for several years to promote public health. Some studies have been conducted in some provinces in Iran. No research is observed to be done in the Northern provinces. The aim of the study is to survey the occurrence of AFM1 in raw milk at cattle farms in Babol, Iran. There has been no detection in area.
MATERIALS AND METHODS
Samples: A total of 120 raw cow milk samples randomly collected from milk churns at 40 traditional and semi-industrial cattle farms located in Babol (Northern Iran) in winter 2006. In total, 120 raw milk samples were tested for Aflatoxin M1 (AFM1) contamination by competitive Enzyme Linked Immunosorbent Assay (Tecna, AFLA M1- code MA418). Solid phase in plastic micro wells was coated with anti-Aflatoxin M1 antibodies.
Methods: The samples were stored in a refrigerator (-20°C) and were centrifuged at 2-8°C for 10 min at 3000 g, then fat was completely separated from skimmed milk by pipettepastur with micropipette. We added 100 μL skimmed milk and Aflatoxin M1 standard solutions in each well. In each micro-plate, we appointed 7 wells for standards and then incubated the plate at 20-25°C for 45 min. Each well was washed four times by washing buffer 20X concentration. Then 100 μL conjugated solution (100X) was added to each well. After that, the plate was incubated at 20-25°C for 15 min. Next, the wells were washed. Substrate then was added in the wells. Then we incubated the plate at 20-25°C in a dark place for 15 min. The reaction was stopped by the stop solution. At most after one hour, light absorption was read at 450 nm by ELISA reader. It is suggested that only 1.6% of AFB1 fed by cattle convert to AFM1; therefore, AFB1 level found in foods is calculated by the following equation (Rastogi et al., 2004):
In the study, in total, 120 raw milk samples were tested for aflatoxin M1 (AFM1) contamination by competitive ELISA. In 68 out of 120 samples (56.7%) the presence of AFM1 was detected by concentration ranging from 50 to 352.3 ng L-1 and 52 samples (43.3%) contained AFM1 at levels of 4-50 ng L-1 (Table 1). The AFM1 contamination levels were between 4-352.3 ng L-1 by the average of 102.73 ng L-1. The AFM1 contamination levels (>50 ng L-1) in January, February and March were 40, 65 and 65%, respectively (Table 2). In general, 56.7% of samples were beyond the limit of European community regulations (50 ng L-1). In other words, the AFM1 contamination levels in raw milk were more than twice over standard levels. (Table 3). In 40 cattle farms of the 26 were traditional and 14 were semi-industrial. The AFM1 contamination levels (>50 ng L-1) in traditional and semi-industrial cattle farms were 58.97 and 52.38%, respectively (Table 3). The AFM1 contamination levels (>50 ng L-1) in traditional cattle farms in January, February and March were 23.46, 69.38 and 69.38%, respectively, but in semi-industrial cattle farms this levels were 42.86, 57.14 and 57.14% (Table 3). Estimation of contamination of AFB1 using AFM1 in feed stuff shows that it is about 0.25-22 μg kg-1 which bears the average of 46.7% (Table 4), being higher than European community limit (5 μg kg-1). The lowest contamination was observed in January. The highest contamination was observed in February and March (Table 5). Statistical evaluations show that there is not a significant relationship between AFM1 contamination and different months of winter, the type of the cattle farm and the kind of feed stuff.
To decrease AFM1 in milk to the lowest point, feed stuff ration should be checked regularly and it should be kept away from fungal contamination. AFM1 seasonal contamination with low level in summer and high level in winter is considerable, since cattle feed concentrated materials in winter and in summer they graze in pastures.
To increase milk quality, it is necessary for feed stuff to be without AFB1 contamination (Kim et al., 2000). In northern Iran due to favorable aflatoxigenic moulds growth especially in cold seasons, some measures should be taken in production, processing and storage of feed stuff.
In Greece, AFM1 was measured by ELISA and HPLC methods in pasteurized milk, found at supermarkets. Of 81 milk samples, 32 had 2-2.5 ng L-1, 9 cases above 5 ng L-1, 31 cases 0.5-1 ng L-1 and 9 cases had no AFM1. In India AFM1 was measured in milk and children dairy products at supermarkets by applying competitive ELISA method. Out of 87 samples 87.3% were contaminated. Children
| Table 1: | The AFM1 level distribution in raw milk at Babol`s cattle-farms in different months |
| *: >50 ng L-1, SD = Standard Deviation, SE = Standard Error of Mean, N = Number | |
| Table 2: | The AFM1 level distribution in raw milk in Babol, Iran |
| Table 3: | The level of AFM1 contamination and the type of cattle farms |
| *: >50 ng L-1, SD = Standard Deviation, SE = Standard Error of Mean, N = Number | |
| Table 4: | The AFB1 level distribution in ration in Babol, Iran |
| Table 5: | The AFB1 level distribution in ration at Babol`s cattle-farms in different months |
| *: >50 ng L-1 | |
dairy products (65-1012 ng L-1) were more contaminated than the liquid milk (28-164 ng L-1). Almost permitted by European community Alimentary, whereas 9% of samples had higher level allowed by US standard (500 ng L-1) (Rastogi et al., 2004). In 2001 winter, AFM1 was detected in 207 (99.5%) samples out of 208 (0.001-0.24 μg kg-1) which was found no significant difference in AFM1 contamination in various areas in Japan (Nakajima et al., 2004).
The results of the study show that AFM1 contamination level in milk is high. It is a serious public health problem, because all age groups including babies and children extensively consume the product. As a result, milk and dairy products should be controlled regularly at least twice a year. Beside this, keeping low AFB1 level in feed stuff is of importance. To reach the goal, feed stuff should be kept away from probable contamination.