Pakistan Research Repository

Micro-Analysis of Aflatoxin mi in Dairy Products at Trace Levels and its Elimination

Hussain, Imtiaz (2010) Micro-Analysis of Aflatoxin mi in Dairy Products at Trace Levels and its Elimination. PhD thesis, University of the Punjab, Lahore .



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 M1 isexcreted in milk of those lactating animals which have ingested aflatoxin B1 contaminated feed. Aflatoxin B1 (AFB1) is metabolized to aflatoxin M1 in liver and then excreted in milk and urine. Aflatoxin B 1 is a potent carcinogen and aflatoxin M1 (AFM1), being the metabolite of AFB1, has toxic properties similar to AFB1. Several researches have demonstrated the potential toxicity of exposure to AFM1. Aflatoxin M1 is present in milk and milk products. This study includes the determination of contamination of aflatoxin M1 in milk and milk products and contamination of aflatoxin B1 in dairy feed in the Punjab province of Pakistan. The analytical techniques used in the determination of AFM1 were high performance liquid chromatography (HPLC), fluorometry (using Fluorometer), and enzyme linked immunosorbent assay (ELISA). For the determination of AFB1, 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 AFM1 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 AFB1 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 AFM1, however in 96.4% samples the level of contamination was below the US tolerance limit of 0.5 μg/ L. Only 3% samples showed AFM1 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 AFM1 contamination in milk. ANOVA analysis indicated significant difference (p < 0.01) in AFM1 concentration in milk in different seasons.The AFM1 contamination was higher in winter as compared to summer and this was supported by previous studies. During the study of AFM1 contamination in raw milk taken from different localities, variation in levels of AFM1 was found in raw milk from different localities in the central areas of the Punjab, Pakistan.Total 480 milk samples of buffaloes and cows from different localities (urban, semi-urban, and rural) were analyzed by using HPLC with prior clean-up step applying immunoaffinity columns.The percentage of AFM1 contamination in buffalo and cow milk was 42.5% and 52.5% respectively. In both type of milk, level of AFM1 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 AFM1 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 herdsize (6-10 cattle) showed higher AFM1 concentration as compared to large herd-size (more than 10 cattle). Another study was conducted to investigate the AFM1 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 AFM1 contamination in buffalo milk, cow milk, goat milk, and sheep milk was found to be 34.5%, 37.5%, 20%, and 16.7% respectively. AFM1 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 AFM1 concentration is not affected by normal milk processes, AFM1 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 AFM1 contamination was found to be 87.5% and 70% in cheese and yoghurt samples respectively. Because of the possibility of presence of aflatoxin B1, feed plays a major role in the occurrence of aflatoxin M1 in milk.The monitoring of AFB1 contamination in dairy feed is compulsory to ensure safety of milk consumers. The study on the contamination of AFB1 in the dairy feed samples showed high contamination of AFB1 in cotton-seed cake samples and concentrate feed samples. Total 260 samples of different commodities, used as dairy feed, were analyzed for AFB1 contamination by HPLC. The average AFB1 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.The contamination level was high as compared to US tolerance i.e., 20 μg/ Kg. The study revealed that the menace of AFM1 concentration in milk and milk products is present in the area. It is imperative to take measures to control and reduce the AFM1 contamination in milk and milk products in the area. Contamination of AFM1 was found in milk and milk products, but only a small percentage of contaminated samples exceeded the US tolerance limit.This can be controlled by taking precautionary measures. The study conducted for AFB1 contamination determination showed the high concentration of AFB1 in cottonseed cake and concentrate feed. The use of these two commodities must be controlled in the feed regimen of milking animals. Moreover, the proper use of toxin binders can reduce the menace of AFM1 contamination in milk and milk products in the area. The feed, straw, bread and other commodities contaminated with moulds must not be used as a feed for milking animals.Availability of sufficient moisture is the most critical factor in mould growth. A relative humidity of 70% to 90% is suitable for growth of moulds and production of mycotoxins. Long storage of dairy feed and feedstuffs should not be practiced or these should be stored in a proper way, especially at low moisture content, to avoid the growth of moulds and to eliminate the contamination of aflatoxins. Low moisture content and low humidity percentage can be achieved by aeration.The concerned authorities should set a legal limit for AFM1 in dairy products and AFB1 in dairy feed. Furthermore, establishment of sophisticated laboratories for aflatoxins’ analysis and arrangement of surveillance programs for aflatoxin contamination will prove to be of a great help in controlling aflatoxin contamination.

Item Type:Thesis (PhD)
Uncontrolled Keywords:Elimination, Punjab, Analysis, Milk, Products, Samples, Trace, Contamination, Micro, Levels, Aflatoxin, Compared, Stored
Subjects:Physical Sciences (f) > Chemistry(f2)
ID Code:6052
Deposited By:Mr. Javed Memon
Deposited On:25 Apr 2011 10:35
Last Modified:20 Aug 2011 10:14

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