Survey of aflatoxin M1 in cows’ milk from free-grazing cows in Abeokuta, Nigeria
F. Oluwafemi, A. O. Badmos, S. O. Kareem, O. Ademuyiwa & A. L. Kolapo Mycotoxin Research ISSN 0178-7888 Volume 30 Number 4 Mycotoxin Res (2014) 30:207-211 DOI 10.1007/s12550-014-0204-4
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Author's personal copy Mycotoxin Res (2014) 30:207–211 DOI 10.1007/s12550-014-0204-4
ORIGINAL PAPER
Survey of aflatoxin M1 in cows’ milk from free-grazing cows in Abeokuta, Nigeria F. Oluwafemi & A. O. Badmos & S. O. Kareem & O. Ademuyiwa & A. L. Kolapo
Received: 14 March 2014 / Revised: 18 June 2014 / Accepted: 19 June 2014 / Published online: 18 July 2014 # Society for Mycotoxin Research and Springer-Verlag Berlin Heidelberg 2014
Abstract Aflatoxin M1 (AFM1) in milk from 100 different herds of free-grazing cows in Abeokuta, Nigeria, was analysed by immunoaffinity column cleanup and HPLC with fluorescence detection. AFM1 was found in 75 % of the samples, the toxin levels in positive samples ranged from 9.0 to 456.0 ng/l. The mean AFM1 level in positive samples was 108.15 ng/l, exceeding, for example, the European Union maximum level by a factor of two. These results indicated that there is an urgent need to more closely control the milk of freegrazing cows for AFM1 in order to protect the health of humans consuming milk and milk products. Keywords Free-grazing animals . Cow’s milk . HPLC . Aflatoxin M1 . Public health . Nigeria
Introduction Cow’s milk is an important component of the human diets and plays an important role in nutrition, growth, development and immunity (Keira and Mao 2004). Because of the carry-over rate of aflatoxin B1 from cows’ feed as aflatoxin M1 (AFM1) F. Oluwafemi (*) : A. O. Badmos : S. O. Kareem Department of Microbiology, Federal University of Agriculture, Abeokuta, Nigeria e-mail:
[email protected] A. O. Badmos Mycotoxin Laboratory, National Agency for Food, Drug and Administration, Lagos, Nigeria O. Ademuyiwa Department of Biochemistry, Federal University of Agriculture, Abeokuta, Nigeria A. L. Kolapo Department of Biology, The Polytechnic, Ibadan, Nigeria
into milk, milk and milk products may present a risk for the consumer, in particular if there is no efficient aflatoxin control in feeds (Galvano et al. 2001; Henry et al. 2001; Creppy 2002; Gürbay et al. 2006; Oliveira and Farraz 2007). Carry-over in dairy cows milked two times daily was usually 1–2 % of the ingested aflatoxin B1 (AFB1) for low-yielding cows and up to approximately 6 % for high-yielding cows (Britzi et al. 2013). Aflatoxins are fungal metabolites that contaminate the food supply in certain areas of the world (Gourama and Bullerman 1995; Smela et al. 2001). The contamination of food and feeds with aflatoxins is more serious in tropical countries where relative humidity is high and the temperatures conducive to the growth and production of aflatoxin by moulds. Over 90 % of the ruminant livestock in Nigeria lies in the hands of herders who keep them under extensive and semi-intensive management systems, whereby the animals only rely on natural pasture and crop residues for survival (Lawal-Adebowale 2012). On the understanding that hay, which contains a large complement of cereal grain infested in the field, could be a source of appreciable aflatoxins (Lizárraga-Paulín et al. 2011), it is possible that animal products, in addition to staple foods, might be contributing an appreciable quantum to dietary aflatoxin exposure in Nigeria. The fact that aflatoxins are a general problem in Nigeria is highlighted by the fact that AFM1 is also present in the milk of nursing women who eat food containing AFB1 (Oluwafemi 2012). The occurrence of AFM1 in milk is transitory in nature, usually reaching a peak within 2 days after the ingestion of the contaminated commodity and disappears within 4–5 days after the withdrawal from a contaminated food source (Henry et al. 2001; Degen et al. 2013). The major concerns with aflatoxins are their potent carcinogenic, mutagenic and teratogenic effects in humans (Battacone et al. 2003; Kocabas and Sekerel 2003). Although AFM1 is less carcinogenic and mutagenic than AFB1, it exhibits a high level of genotoxic activity and certainly
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Fig. 1 HPLC scan of standard aflatoxin M1 chromatogram equivalent to 2.0 ng/l
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represents a health risk due to its possible accumulation and linkage to DNA (Shundo and Sabino 2006). Avoidance of contaminated food/feed is rarely possible, and feeds that contain relatively low concentrations of AFB1 may still have deleterious effects on sensitive species such as poultry (Doerr et al. 1983; Giambrone et al. 2005; Rauber et al. 2007). Herds of free-ranging and free-grazing dairy cattle are a difficult task in aspects of feed monitoring. Incidentally, a large portion of dairy cattle in many developing countries such as Nigeria are raised under free-ranging conditions. There is dearth of information of aflatoxin M1 in milk of free-grazing lactating cows. The aim of the present study was to provide data on AFM1 at the individual herd level for one region in Nigeria, to obtain information about whether or not there may be a contamination problem and to indicate the dimensions of the contamination.
Materials and methods Sampling location Abeokuta is one of the most important and populous cities in southwest Nigeria. As of 2012, it has a metropolitan population of 1.117 million people. It is located in the tropical rain forest zone with high humidity and temperature which promotes mould growth and toxin production.
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Cultivation of crops in its suburbs is transforming the vegetation into a wooded savanna, which incidentally favours the herding of cattle by the Fulani pastoralists. In the surrounding villages, the herded cattle are fed with forage and left-over harvest of cultivated crops such as maize. The lactating cows of each herd are usually milked by the wives of the pastoralists, the milk is used to make cheese and other dairy products for public consumption. Sample collection A total of 100 raw milk samples (100 ml each) from freeranging cows were obtained from Fulani herdsmen in Abeokuta, Ogun state of Nigeria. The samples were collected into sterile bottles and brought to the laboratory in an ice box where they were stored at 4 °C until the time of analysis. To avoid separation of the refrigerated milk into two layers, the milk samples were warmed to 37 °C in a water bath and gently stirred with a magnetic stirrer to homogenize the fat layer. This was then centrifuged at 3,000×g, and the thin upper fat layer was discarded. The resulting fluid was then filtered twice through a paper filter. Analytical procedure Solvents such as acetonitrile, methylene chloride, and methanol were of HPLC grade. The standard AFM1 was purchased from Chromogen (New Delhi, Delhi, India). Immunoaffinity
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Fig. 2 HPLC scan of aflatoxin M1 chromatogram showing spiked peak milk equivalent to 2.5 μg/l
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column was Aflastar M1 R (lot: AF 1011 1012), supplied by Roma Labs Diagnostic Technopark 13430, Tulln, Austria. Milk sample extracts were prepared for analysis by a modification of the method of Smith et al. (1994). Aliquots of the milk (50 ml) were passed through an immunoaffinity column. The column contains specific antibodies bound to the solid support materials. As the sample passes through the column, the antibodies selectively bind any aflatoxin M1 (antigen) contained in the sample matrix. The column was prewashed with 10 ml of acetonitrile and 10 mL of distilled water, using a disposable 20-ml syringe. The milk sample was loaded onto the cartridge and eluted at a flow rate of 10 ml/min after which the eluate was discarded. The cartridge (which contained the bound aflatoxin M1) was then washed with 10 ml of basic 10 % acetonitrile, followed by 10 ml of acidic 10 % acetonitrile at a flow rate of 10 ml/min. Five millilitres of acidic 30 % acetonitrile was subsequently added to the cartridge, and the eluate was collected into a 20-ml test tube, to which 2 ml of methylene chloride was added. Subsequent vortexing and extraction were performed as described earlier by Smith et al. (1994). The extracted milk were pooled and concentrated to approximately 0.5 ml under a gentle stream of nitrogen gas. The extract was completely evaporated under nitrogen and then dissolved in 200 μl of the acidic 30 % acetonitrile solution. A volume of 150 μl was injected into the HPLC system and analysed for AFM1. The HPLC system consisted of a Waters 6000A solvent delivery system and a WISP 710B sample processor for sample injections (Waters Associates). The samples were eluted isocratically on a radically compressed 10-μm octadecylsilane cartridge (Waters 100
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Associates) with a mobile phase of acetonitrile/methanol/water (15:15:70) at a flow rate of 2 mL/min. A prefilter was placed between the injector and the cartridge. AFM1 was detected fluorometrically (exCitation wavelength, 365 nm; emission wavelength, 425 nm) with a fluorescence detector (model 420C, Waters Associates). The HPLC chromatograms were recorded on a Waters Data Module (Waters Associates) at a chart speed of 1.0 cm/min. The concentration of AFM1 in the milk samples was determined from the peak area in comparison with samples containing known concentrations of AFM1 (Smith et al. 1994).
Results and discussion Validation of AFM1 measurement was done by imported condensed (‘Peak’, Friesland Campina) milk bought from a supermarket in Lagos, Nigeria. Recoveries at an AFM1 spiking level of 2.5 ng/ml ranged between 99.7 and 100.13 %. The calibration curves showed good linearity. Figures show chromatograms of AFM1 standard solution (Fig. 1), spiked milk sample (Fig. 2) and two naturally contaminated milk samples at AFM1 levels of 27 ng/ml (Fig. 3) and at 55 ng/ml (Fig. 4). Table 1 shows the frequency distribution of AFM1 in milk. The AFM1 levels in positive samples ranged between 9 and 456 ng/l. Although the milk yield of the sampled cows was not recorded, a survey carried out by Shittu et al. (2008) in Sokoto state, north-western Nigeria reported an average yield of 24 l 100
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Fig. 4 HPLC Chromatogram of quantified AFM1 in sample equivalent to 55 ng/l
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Fig. 3 HPLC chromatogram of quantified AFM1 in sample two equivalent to 27 ng/l
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Table 1 AFM1 (ng/l) distribution in milk samples (n=100) Frequency distribution
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