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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 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.
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