DETERMINATION OF AFLATOXIN M1 LEVELS IN BOVINE FARMED MILK WITH SPECIAL REFERENCE TO THE LEVELS OF AFLATOXIN B1 IN THE ANIMAL FEED

Dept. of Food Hygiene, Port-Said Lab.,

Animal Health Research Institute.

Determination of aflatoxin M₁ levels in bovine farmed milk with special reference to the levels of

aflatoxin B₁ in the animal feed

(With 3 Tables)

By

Azza A.H. El-Tabiy and Heba M. Hassan*

* Central Lab. for Vet. Quality Control of Poultry Production, Animal Health Research Institute.

(Received at 14/9/2011)

تحديد مستوى الأفلاتوکسين م1 فى ألبان المزارع البقرية مع الإشارة إلى مستوى الأفلاتو کسين ب1 فى أعلاف الحيوانات

عزة علي حسين التابعي ، هبه محمد حسن

في دراسة لتحديد مستوى الأفلاتوکسين ب 1& م1 تم جمع 40 عينة من أربعة مزارع مختلفة من مدينة بورسعيد بواقع 20 عينة من کل من أعلاف الحيوانات وألبان الأبقار(5عينات ألبان و5 عينات أعلاف من کل مزرعة). ووجد ان متوسط مستوى الأفلاتوکسين ب 1 في عينات أعلاف الحيوانات کان 41,38 ± 9,6 ,2,07 ±1,6 , 7,48 ± 3,5 و1,19 ± 0,9 جزء في البليون, بينما کان متوسط  مستوى الأفلاتوکسين م 1 في عينات الألبان البقرية 51,6 ± 10,8 ,21,48 ±4,5 , 35,72 ± 7,3 و8,63 ± 3,9 جزء في التريليون. وقد أسفرت النتائج عن وجود الأفلاتوکسين ب1 و الأفلاتوکسين م 1 في عينات أعلاف الحيوانات والألبان البقرية بنسبة 80% ( 16) , 65% (13) علي التوالي. وبمقارنة مستوى الأفلاتوکسين ب 1& الأفلاتوکسين م1 في العينات موضع الدراسة وجد ان 25% (5) و20% (4) من کل من أعلاف الحيوانات والألبان البقرية علي التوالي قد تعدت الحد المسموح به طبقا لمنظمة FAO/WHO. وقد تم مقارنة مستوي الأفلاتوکسين ب1 والأفلاتوکسين م 1 في العينات موضع الدراسة وکذلک مناقشة تأثير الأفلاتوکسين م 1 علي صحة المستهلک.

SUMMARY

A total of 40 samples of animal feed and milk (20 of each) were randomly collected from four small dairy farms and analyzed for aflatoxin B₁ and M₁ respectively. The mean average of AFB₁ and AFM₁ were 41.38 ± 9.6, 1.07 ± 0.6, 7.48 ± 3.5 and 1.19 ± 0.9 ppb; 51.6 ± 10.8, 21.48 ± 4.5, 35.72 ± 7.3 and 8.63 ± 3.9 ppt in the examined animal feed and milk samples respectively. The incidence of AFB₁ and AFM₁ was detected in 80% (16) and 65% (13) of the examined animal feed and milk samples respectively. On the other hand, 25% (5) and 20% (4) of the examined animal feed and milk samples were exceeded the limits recommended by FAO/WHO. The relationship between the levels of AFB₁and AFM₁in the examined animal feed and milk were discussed. Also the effect of AFM₁ on the human health was discussed. There is need to create awareness and establish routine monitoring of animal feeds and milk to reduce risk to animal and consequently human response.

Key words: Aflatoxins, Aspergillus spp., milk, animal feed.

Introduction

Aflatoxins (AFs) are secondary metabolites produced by some species of Aspergilus, especially Aspergilus flavus and Aspergilus parasiticus. They are naturally contaminants of food and several feedstuffs, initiated under favorable conditions of temperature, relative humidity/moisture and poor storage conditionswith the long storage under favorable conditions molds produce their metabolites in the form of fungal toxins (IARC, 2002; Strosnider et al., 2006).

Aflatoxins are a group of toxins classified into aflatoxin B₁, B₂, G₁ and G₂. These toxins are fluorescent under the ultraviolet light and don't have flavor and scent. They are resistant to high temperatures up to 320^ºC thus didn't exterminate or fragment by boiling and pasteurization (Early, 2000). The potential danger of aflatoxins group is the transfer of these toxins to human through food chain (Rory and Enda, 2008).

The toxicity of aflatoxin B₁ (AFB₁) in animals is varied due to differences in susceptibility (Lanza et al., 1982). The toxic dose for cattle has been shown to be from 300–700 ppb depending up on the individual animal. The adverse effects include low weight gain and dietary intake causing growth impairment and depress the immune status (Raisuddin et al., 1993).

In human, aflatoxin B₁ is the most toxic compound produced by some Aspergilus species and the most potent hepato-carcinogens. The International Agency for Research on Cancer of WHO cited that aflatoxin B₁ is carcinogenic of "Group 1" for humans (IARC, 2002). The long-term exposure to low levels of AFB₁ in the diet produced carcinogenic, mutagenic, teratogenic, oes-trogenic, neurotoxic and immunotoxic effect (Albert et al., 2006).

In the liver of animal, AFB₁ was transformed biologically by hepatic microsomal cytochrome P450 into aflatoxin M₁ (AFM₁). Aflatoxin M₁ (AFM₁) is the principal hydroxylated AFB₁ metabolite present in milk of cows fed with a diet contaminated with AFB₁ (Battacone et al., 2005) and excreted within 12 hours after administration of contaminated feeds (Battacone et al., 2003; Applebaum et al., 2003). The US Food and Drug Administration (FDA) have indicated that aflatoxin is the only mycotoxin that currently warrants regulation in milk (Wood and Trucksess, 1998).

Milk containing violative levels of AFM₁cannot be marketed. The action level for most feeds is 20 ppb; the action level for human food is also 20 ppb for total aflatoxins, with the exception of milk which has an action level of 0.5 ppb for aflatoxin M₁.(FDA, 2005). the acute toxicity begins and seems to be similar or slightly less than that of aflatoxin B₁ but its carcinogenic potency is probably one or even two orders of magnitude lower than that of aflatoxin B₁(Henry et al., 2001).  

This study was initiated to assess the levels of AFB₁ and AFM₁ in animal feeds and milk of different farms respectively, and the significant differences of parameters analyzed.

Materials and Methods

1- Samples collection:

1-1: Milk:

Milk samples (20) from four smallholder dairy farming households (5 of each) in Port-Said city were collected in sterile 15 milliliters tubes. All the samples were thoroughly identified and transported to the laboratory in cool boxes. The samples were analyzed as soon as possible and any delay, the samples should be frozen. 

1-2: Animal feeds:

From the same smallholder dairy farming households, about 500g of animal feed were taken and thoroughly identified (20 samples) transported to the laboratory and analysed as soon as possible.

2- Sample preparation:

2-1: Milk:

Individual milk sample was centrifuged at 3500 rpm for 10 min. at 10^°C for a process of de-fatting. By using a Pasteur pipette, the upper cream layer was removed completely, stored in cool place and protect against light(A.O.A.C., 2000).

2-2: Cereals and feed:

The cereal and feed sample was mixed and thoroughly ground. In a screw cap bottle 20 g of the mixed and ground sample was mixed with 10 ml of methanol: distilled water (70:30, v/v) by using shaker for 10 minutes at room temperature. The homogenate was filtered through Whatman® No. 1 filter paper. 100µ l of the sample filtrate was diluted with 600µ l of the sample dilution buffer. The diluted sample was stored in cool place and protect against light(Kang'ethe and Lang'a, 2009).

3- Enzyme immunoassay:

3-1: Aflatoxin M₁ (AFM₁) in milk:

In each well, 100 µl of the skimmed milk sample were taken and analyzed directly using an ELISA kit for M₁ following the manufacturer's instructions (Ridascreen® Aflatoxin M₁, purchased from r-biopharm, Germany). Samples were run in duplicates. The sample was diluted and re-tested when OD reading above the reading of 40 parts per trillion (ppt) standards. The kit had a sensitivity of 5ppt.

3-2: Aflatoxin B₁ (AFB₁) in cereals and feed:

50µl per well were employed directly in the assay. Competitive enzyme immunoassay for AFB₁ was done using Aflatoxin kit following the manufacturer's instructions (Ridascreen® Aflatoxin, Commercial License Eliza Kit, obtained from r-biopharm, Germany). Any sample having more than 13.5 ppb was diluted further (sample dilution buffer containing 10% methanol) and re-tested. This concentration was the upper limit of the sensitivity of the standard curve. The kit had a sensitivity limit of 1.8 ppb.

4- Calculation:

Special software, the RIDA®SOFT win (Art. No. Z9999) is available for evaluation of the RIDASCREEN® enzyme immunoassays.

The course of the slandered curve is shown in the quality assurance certificate enclosed in the test kit.

Remark for the calculation without software:

5- Sensitivity:

The mean lower detection limit of the RIDASCREEN ® Aflatoxin total assay is about 50 ng/kg (0.5 ppb) for milk. According to the test preparation record, the detection limit is (1.75ppb) for cereals and feed. 

6- Statistical methods:

            Data were submitted to one-way ANOVA to test differences between treatments, and linear regression analysis was carried out to test AFM₁ and AFB₁(SPSS, 2007).

Results

Table 1: Quantitative estimation of aflatoxins B₁and M₁ in the examined animal feed and milk samples in different farms (n=5).

 ppb = part per billion

 ppt= part per trillion

 N/D=not detectable    

Table 2: Statistical analytical results of aflatoxins B₁ and M₁ in the examined animal feed and milk samples in different farms (n=5).

Table 3: Incidence of aflatoxin B₁ and M₁ in the examined animal feeds and milk samples in different farms. (n=5).

N/D =not detectable    

PMls =Permissible limits

Discussion

A total of 20 animal feeds samples were collected from 4 farms (5 of each). The results tabulatedin Table 1 showed that the highest recorded levels of aflatoxin B₁ and M₁ were 90.20 ppb and 58.08 ppt in the examined feed animal and milk samples of farm 1 respectively, while the lowest levels were 0.038 ppb and 5.78 ppt in feed animal and milk samples of farm 4 respectively. The results of feed animal samples were agree with the results recorded by Karki and Sinha, (1989) but not agreed with theresult of Abdel-Fattah and Yacoub, (2008) who stated that aflatoxin not detected in animal feed. The variation in the levels of aflatoxin B ₁ between the different samples and farms may be attributed to the differences in storages facilities of animals' feeds, temperature, humidity and high insect activity (Strosnider et al., 2006). On the other hand, milk samples results were agree with the results of Battacone       et al. (2003) but disagree with the results recorded by Westlake et al. (1989). Also, Table 1 showed a significant relationship between the levels of aflatoxin B₁ and M₁ in animal feed and milk samples respectively. Thus Aflatoxin M₁used as possible marker of exposure to aflatoxin B₁, as the amount of AFB₁ ingested increased the AFM₁ concentration in milk rose. These results agree with the result of Veldmann et al. (1992); Michael et al. (2002) but not agree with that of Frobish et al. (1986). The significant relationship between the levels of aflatoxin B₁ and M₁ may be regards to that the considerable part of the ingested aflatoxin B₁ is degraded in the rumen and extensively metabolized in the liver, resulting predominantly in aflatoxin M₁. Circulating aflatoxin M₁ can be excreted via the kidneys and appears in milk. The excreted amount of aflatoxin M₁ in the milk of dairy cows was estimated to represent 1-2 % of the ingested aflatoxin B₁. Also the changes in the plasma- milk barrier result in a higher carry over rate of aflatoxin M₁ into milk(Munksgaard et al., 1987; Veldmann et al., 1992).

As shown inTable 2 the mean levels of aflatoxin B₁ and M₁ in the examined animal feed and milk samples were 41.38 ± 9.6, 1.07 ± 0.6, 7.48 ± 3.5 and 1.19 ± 0.9 ppb; 51.6 ± 10.8, 21.48 ± 4.5, 35.72 ± 7.3 and 8.63 ± 3.9 ppt, respectively in the four farms. The variation between the levels of aflatoxin B₁ in different farms and within the same farm is regarded to the variation of the feeds nutrient content of the different farms i.e. feed manufactured from grains is considered the major source for aflatoxin B₁ contamination that find their way to animal feed. Also high temperature, humidity and moisture content induce the mold growth and aflatoxin B₁ production in animal feed (IARC, 2002).

The obtained results recorded in Table 3 showed that 80% (16) and 65% (13) of the examined animal feeds and milk samples were contaminated with aflatoxin B₁ and M₁ respectively. This result is nearly agreed with the results recorded by Rory and Enda (2008), they reported that eighty six percent of the feed samples from farmers were contaminated with aflatoxin B₁. Contamination of animal feeds with AFB₁ was prevalent throughout the farms. 20% (4) of samples were free from AFB₁, Meanwhile, 25% (5) of the contaminated samples had aflatoxin B₁levels above FAO/WHO limits (20 ppb), and also 20% (4) of contaminated milk samples had aflatoxin M₁ levels above FAO/WHO limits0.05µg/Kg (50 ppt). (FAO/WHO, 2002). These results confirm unavoidable contaminants in food and feed, so AFB₁ level must be lowered 20 ppb of total aflatoxin in animal feed to avoid its harmful effects for the animals or human who consume its products. Total rations containing aflatoxin at levels greater than 20 pbb certainly should not be fed to milk cows. In this concern, the Food and Agriculture Organization (1996) reported that 25% of the world's food crops are affected by mycotoxins.

Conclusion

These results suggest that presently the contamination of milk with AFM₁ does not appear to be a serious health problem in Port-said, and may be a direct and immediate correlation between the presence of AFB₁ in feedstuff and the detection of AFM₁ in milk from the same farm. It is necessary to highlight the procedures in place for minimizing potential risks to animals and subsequent risks to consumers from animal derived food products. Aflatoxin M₁ contamination of milk cannot be completely prevented because AFB₁ does occur naturally in grains. It is not practical to completely eliminate AFB₁ from feeds or AFM₁from milk. However, it is possible to control the amount of AFM₁present in milk by limiting the amount of aflatoxin in animal feeds. The FDA has established specific guidelines on acceptable levels of aflatoxins in human food and animal feed by establishing action levels that allow for the removal of violative lots from commerce (Smith, 2005). Nevertheless, a continuous surveillance programme may be warranted to monitor regularly the occurrence of aflatoxin in the animal feeds responsible for current limited contamination and to note rapidly any worsening in the situation that may depend on market changes or on unfavorable climatic developments

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