Micro-Analysis of Aflatoxin M 1 in Dairy Products at Trace Levels and Its Elimination

Abstract

Milk is a complex mixture of fat, protein, carbohydrate, and mineral components and it has been a source of human food since the recorded history. Aflatoxin M 1 is excreted in milk of those lactating animals which have ingested aflatoxin B 1 contaminated feed. Aflatoxin B 1 (AFB 1 ) is metabolized to aflatoxin M 1 in liver and then excreted in milk and urine. Aflatoxin B 1 is a potent carcinogen and aflatoxin M 1 (AFM 1 ), being the metabolite of AFB 1, has toxic properties similar to AFB 1 . Several researches have demonstrated the potential toxicity of exposure to AFM 1 . Aflatoxin M 1 is present in milk and milk products. This study includes the determination of contamination of aflatoxin M 1 in milk and milk products and contamination of aflatoxin B 1 in dairy feed in the Punjab province of Pakistan. The analytical techniques used in the determination of AFM 1 were high performance liquid chromatography (HPLC), fluorometry (using Fluorometer), and enzyme linked immunosorbent assay (ELISA). For the determination of AFB 1 , HPLC was used. Immunoaffinity columns were used to accomplish cleanup step during HPLC and fluorometric determination. A total of 977 samples of milk, cheese, and yoghurt were analyzed for AFM 1 contamination. Whereas a total of 260 samples of feed commodities (concentrate feed, cottonseed cake, wheat bran, bread, paddy straw, and wheat straw) were analyzed for AFB 1 contamination. In the first phase of study 168 sample of raw milk from fourteen districts, were analyzed by using immunoaffinity columns and Fluorometer. All the samples were found contaminated with AFM 1 , however in 96.4% samples the level of contamination was below the US tolerance limit of 0.5 μg/ L. Only 3% samples showed AFM 1 contamination higher than the US tolerance limit. While considering EU maximum permissible limit (0.05 μg/ L), 99.4% samples exceeded this limit. Seasonal effect was also studied on the presence of AFM 1 contamination in milk. ANOVA analysis indicated significant difference (p < 0.01) in AFM 1 concentration in milk in different seasons. The AFM 1 contamination was higher in winter as compared to summer and this was supported by previous studies. During the study of AFM 1 contamination in raw milk taken from different localities, variation in levels of AFM 1 was found in raw milk from different localities in the central areas of the Punjab, Pakistan. Total 480 milk samples of buffaloes and cows xviifrom different localities (urban, semi-urban, and rural) were analyzed by using HPLC with prior clean-up step applying immunoaffinity columns. The percentage of AFM 1 contamination in buffalo and cow milk was 42.5% and 52.5% respectively. In both types of milk, level of AFM 1 contamination was higher in milk samples obtained from urban and semi-urban areas and it was minimal in milk samples taken from rural areas. The AFM 1 contamination in buffalo milk was studied statistically with respect to herd-size variation also. The results showed significant variations with respect to herd-size (F= 6.631, p= 0.001). Milk samples in case of small herd-size (1-5 cattle) and medium herd- size (6-10 cattle) showed higher AFM 1 concentration as compared to large herd-size (more than 10 cattle). Another study was conducted to investigate the AFM 1 contamination in the milk of five mammalian species namely buffalo, cow, goat, sheep, and camel from the area of Faisalabad district of the Punjab province, Pakistan. Analysis was made by using HPLC with fluorescence detection. Immunoaffinity columns, which are based on the principle of affinity chromatography, were used for clean-up purposes. Total 169 milk samples were analyzed. The percentage of AFM 1 contamination in buffalo milk, cow milk, goat milk, and sheep milk was found to be 34.5%, 37.5%, 20%, and 16.7% respectively. AFM 1 contamination was not detected in camel milk in this area. Although there is massive use of fresh milk in Pakistan, but still significant consumption occurs after milk has been processed. As AFM 1 concentration is not affected by normal milk processes, AFM 1 is also present in milk products like cheese and yoghurt. The milk product samples including 80 cheese samples and 80 yoghurt samples were analyzed by using ELISA technique. The percentage of AFM 1 contamination was found to be 87.5% and 70% in cheese and yoghurt samples respectively. Because of the possibility of presence of aflatoxin B 1 , feed plays a major role in the occurrence of aflatoxin M 1 in milk. The monitoring of AFB 1 contamination in dairy feed is compulsory to ensure safety of milk consumers. The study on the contamination of AFB 1 in the dairy feed samples showed high contamination of AFB 1 in cotton-seed cake samples and concentrate feed samples. Total 260 samples of different commodities, used as dairy feed, were analyzed for AFB 1 contamination by HPLC. The average AFB 1 contamination levels in cottonseed cake, concentrate feed, wheat bran, bread pieces, and paddy straw were found to be 242, 176, 98, 23, and 37 μg/ Kg respectively. contamination level was high as compared to US tolerance i.e., 20 μg/ Kg.