Aflatoxin M1 contamination in cow and buffalo milk samples from the ...

Food Additives and Contaminants: Part B Vol. 4, No. 4, December 2011, 282–288

Aflatoxin M1 contamination in cow and buffalo milk samples from the North West Frontier Province (NWFP) and Punjab provinces of Pakistan S.Z. Iqbala*, M.R. Asib and A. Arinõc a Department of Applied Chemistry, GC University Faisalabad, 38000, Faisalabad, Pakistan; bToxicology and Food Chemistry Lab, Nuclear Institute for Agriculture and Biology, Jhang Road Faisalabad, Pakistan; cDepartment of Animal Production and Food Science, University of Zaragoza, Zaragoza, Spain

(Received 13 June 2011; final version received 21 October 2011) A total of 178 milk samples (94 of buffalo and 84 of cow) were randomly taken from Punjab and the North West Frontier Province (NWFP) of Pakistan (n ¼ 89 in each province) and analysed for the presence of aflatoxin M1 (AFM1) by HPLC-FLD. From Punjab about 46% of buffalo’s and 49% of cow’s milks were contaminated with AFM1 as compared with 52% and 51% for milk samples from NWFP, respectively. Overall, the mean AFM1 concentration was 0.046 mg kg1 with a maximum of 0.350 mg kg1. All samples complied with the Codex Alimentarius limit of 0.50 mg kg1 for AFM1 in milk, but 16.3% of samples exceeded the European Union maximum level of 0.05 mg kg1. Another set of 415 buffalo’s and cow’s milk samples (213 morning milks and 202 evening milks) were analysed. Statistical analysis revealed significant differences (p 5 0.05) between mean AFM1 concentrations in milk during the morning (0.043 mg kg1) and the evening (0.028 mg kg1) lactation times. Keywords: aflatoxins; milk

Introduction Pakistan is the fourth largest milk-producing country in the world behind India, China and the United States, with 45 billion litres of milk production per annum. About 55 million people in Pakistan are directly dependent on livestock for their livelihood (Anon 2010). Buffalo is the major milk-producing animal in Pakistan, and this country stands second in world’s buffalo milk production (Hussain, Ghafoor, et al. 2010). Pakistan’s dairy industry is facing a number of problems such as lack of dairy-related education and lack of financial and infrastructure facilities, but the quality check is the most neglected aspect of the whole system. There is no test at any stage along the marketing chain (Jalil et al. 2009). The European Union has established specific hygiene rules to avoid or minimise the possibility of microbiological and chemical hazards appearing in milk, as well as residues and contaminants such as aflatoxins (European Commission 2006b). These mycotoxins are a group of secondary metabolites mainly produced by the filamentous fungi Aspergillus flavus, Aspergillus parasiticus, and rarely by Aspergillus nomius (Iqbal et al. 2010a, 2010b, 2010c, 2011a, 2011b), which can contaminate vegetable foods, notably peanuts and cattle feeds (Garrido et al. 2003).

*Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online 2011 Taylor & Francis http://dx.doi.org/10.1080/19393210.2011.637237 http://www.tandfonline.com

Aflatoxin B1 (AFB1) is considered to be the most potent hepatocarcinogen of this group of mycotoxins. Aflatoxin M1 (AFM1) is produced by hydroxylation of the fourth carbon in the AFB1 molecule and it is secreted in the milk of lactating animals consuming contaminated feed (Ruangwises and Ruangwises 2010). The International Agency for Research on Cancer (IARC) (2002) has classified AFB1 as class 1 (carcinogenic to humans). It has been observed that AFM1 has approximately ten times less carcinogenicity than its parent compound AFB1, but it has high genotoxic activity (Nuryono et al. 2009). Aflatoxins are responsible for acute poisoning (aflatoxicosis), for hepatocellular carcinoma, for growth impairment in children, and immunosuppression. Indeed, due to the fundamental role of milk in the human diet, especially as infant nourishment, the finding of AFM1 in dairy products is regarded as a significant hazard for food safety and public health (Cucci et al. 2007). Strict regulatory limits for AFM1 are currently in force in the European Union. Commission Regulation (EC) 1881/ 2006 (European Commission 2006b) sets a maximum level (ML) for AFM1 in milk intended for adults at 0.050 mg kg1, whereas the level for milk intended for infants or for baby-food production was set at 0.025 mg kg1. However, the Codex Alimentarius has

Food Additives and Contaminants: Part B established a maximum limit of 0.5 mg kg1 for aflatoxin M1 in milk, and the action level for aflatoxin M1 in milk in the United States is also 0.5 mg kg1. Similarly, several Asian countries accept a maximum level of 0.5 mg kg1 aflatoxin M1 in milk, which is also the harmonised MERCOSUR limit applied in Latin America (European Food Safety Authority (EFSA) 2004). There are thus differences in the maximum permissible limit of AFM1 in various countries, and many including Pakistan have no legal limit for AFM1 in milk and dairy products (Hussain and Anwar 2008). Worldwide several studies have been undertaken to determine the presence of AFM1 in milk and dairy products. A high presence of AFM1 in milk has been reported from Iran (Alborzi et al. 2006; Tajkarimi et al. 2008; Ghazani 2009; Fallah 2010; Heshmati and Milani 2010; Nemati et al. 2010; Rahimi et al. 2010; Sani et al. 2010), Kuwait (Dashti et al. 2009; Srivastava et al. 2001), France (Boudra et al. 2007), Nigeria (Atanda et al. 2007), Italy (Cavaliere et al. 2006), Croatia (Bilandzˇic´ et al. 2010), Syria (Ghanem and Orfi 2009), Turkey (Tekins en and Eken 2008), India (Rastogi et al. 2004), China (Pei et al. 2009), Sudan (Elzupir and Elhussein 2010), and Korea (Lee et al. 2009). However, only few reports of the presence of AFM1 were reported from Pakistan (Hussain and Anwar 2008; Hussain et al. 2008; Hussain, Anwar et al. 2010; Asi et al. 2011). Furthermore, most of the previous research worldwide has been focused on cow’s milk, but monitoring activity towards aflatoxin M1 contamination of milk should be intensified and expanded to consumable milk from other animal species. In the present study AFM1 variation levels in buffalo’s and cow’s milk from Punjab and NWFP provinces were documented as well as the excretion of AFM1 during morning and evening lactation times in both dairy species.

Materials and methods Sampling A total of 593 samples of raw buffalo’s and cow’s milk were randomly collected during November 2009–April 2010 from milking sites and farmhouses from major cities of Punjab and NWFP (now known as Khyber Pakhtunkhwa) of Pakistan. The share of milk production of Punjab was 62%, while it was 12% for NWFP. Some samples were used for occurrence analysis (n ¼ 178) and separate samples (n ¼ 415) were used for morning and evening milk comparison. The size of each milk sample was at least 1 litre. During transportation the milk samples were kept in ice packets in an icebox. The milk samples were either analysed immediately or stored in a freezer in case of delayed analysis.

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Chemicals and reagents A standard curve from AFM1 standard (10 mg ml1 in acetonitrile) was prepared with concentrations of 0.05, 0.1, 0.5, 0.10, 0.50 and 10.0 mg l1 solutions and stored in tightly stoppered vials in a refrigerator at 4 C until further analysis. Acetonitrile of HPLC grade (Sigma Aldrich, Steinheim, Germany) and immunoaffinity columns (IAC) of AflaM1TM (VICAM, Watertown, MA, USA) were purchased. During the analysis double-distilled water was used and all other chemicals and reagents were at least of analytical grade.

Extraction and clean-up The extraction of AFM1 from milk samples was carried out by a previously validated method of the authors’ laboratory (Hussain et al. 2008) with some modifications. Liquid milk samples were warmed at 37 C in a water bath and then centrifuged at 2500 rpm to separate the fat layer. After centrifugation they were filtered through Whatman No. 5 filter paper. About 50 ml of filtrate were transferred into a syringe barrel attached to an IAC and passed at 2 ml min1 using a solid-phase extraction manifold. The column was washed with 20 ml double-distilled water to eliminate impurities and AFM1 was eluted with 4 ml pure acetonitrile, for approximately 60 s, to be in contact with the column. Finally, the eluate was evaporated to dryness using a gentle stream of nitrogen at 40 C and diluted with the mobile phase at the time of HPLC determination.

HPLC conditions The HPLC equipment used for AFM1 analysis was a Shimazdzu LC-10A series (Japan), equipped with fluorescence detector (FLD) with excitation and emission wavelengths of 365 and 435 nm, respectively. A Discovery C18 column (4.6 250 mm, 5 mm) of Supelco, USA was used. Acetonitrile in a ratio of 25% with 75% water was used as the mobile phase. The flow rate was 1.0 ml min1. A calibration curve was determined using a series of calibration solutions of AFM1 in acetonitrile with concentrations of 0.05, 0.1, 0.5, 1.0, 5.0 and 10.0 mg l1. The response was linear (R2 ¼ 0.9989). The LOD was 0.004 mg kg1, determined by a signal-to-noise ratio of 3.

Statistical analysis The results of AFM1 concentration were statistically analysed and presented as mean and range. The significant difference (p 5 0.05) between provinces and lactation times (morning and evening) were analysed by one-way analysis of variance (ANOVA) using SPSS software (IBM, PASW Statistics 19, USA).

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S.Z. Iqbal et al. analysed randomly from Punjab and NWFP provinces; occurrence, mean contamination level and range are shown in Table 2. Overall, 88 out of 178 milk samples (49.4%) tested positive for AFM1, and the mean AFM1 concentration was 0.046 mg kg1 with a maximum of 0.350 mg kg1. All samples complied with the Codex Alimentarius maximum limit for AFM1 in milk of 0.50 mg kg1, but 16.3% of samples exceeded the European Union maximum level of 0.05 mg kg1. Regarding the comparison between provinces, about 46% (22 out of 48) of buffalo’s and 49% (20 out of 41) of cow’s milk samples from Punjab were contaminated with AFM1 at detectable level, compared with 52% (24 out of 46) of buffalo’s and 51% (22 out of 43) of cow’s milk samples from NWFP, respectively. From Punjab, about 13% of buffalo’s and 15% of cow’s milks exceeded the European Union permissible limit, compared with 22% of buffalo’s and 16% of cow’s milk from NWFP. Statistical analysis (Table 2) showed that the mean levels of AFM1 in buffalo’s and cow’s milk samples from NWFP province (0.066 and 0.045 mg kg1, respectively) were significantly higher (p 5 0.05) than those from Punjab province (0.040 and 0.030 mg kg1, respectively). When significant quantities of AFB1 are consumed, AFM1 appears in milk at least 12 h post feeding – a time often corresponding to the normal milking time followed after feeding. The excreted amount of aflatoxin M1 in the milk can vary in individual animals,

Analytical quality assurance Precision and recovery analysis were determined according to protocol 2002/657/EC (European Commission 2002) by performing tests on three sets of blank raw milk samples (six replicates each) fortified with AFM1 at concentrations of 0.5, 1.0 and 1.5 times the European Union maximum level (0.050 mg kg1). Recoveries of AFM1 ranged from 86% to 92% (Table 1) and repeatability varied between 9.6% and 11.0% (RSDr), meeting the criteria for methods of analysis of mycotoxins in foodstuffs established in Regulation EC 401/2006 (European Commission 2006a). The good performance characteristics at the low statutory limits indicate that the method based on IAC in combination with liquid chromatography can be used to generate reliable surveillance data. Certified reference material (CRM) is available to the authors’ laboratory and it regularly takes part in proficiency testing. The laboratory is well established, and foodprocessing factories and exporters send their samples for aflatoxin analysis before export to foreign markets. To ensure the results, blank milk samples were run three times before, during and after each series of analytical samples.

Results and discussion In the present study the incidence of AFM1 contamination in milk samples of buffalo and cow were

Table 1. Recovery percentage and precision (relative standard deviation [RSD] at repeatability conditions) studies of aflatoxin M1 in milk. Spiking level (mg kg1)

Parameter

Day 1

Day 2

Day 3

Overall

0.025 (0.5 ML)

Mean (mg kg1) RSD (%)

0.024 7.6

0.023 9.9

0.022 15.5

0.023 (92.0%) 11.0

0.050 (1 ML)


0.045 9.7

0.043 8.7

0.042 14.2

0.043 (86.0%) 10.9

0.075 (1.5 ML)


0.070 9.1

0.067 9.4

0.067 10.1

0.068 (90.7%) 9.6

Notes: ML ¼ maximum level (0.050 mg kg1). RSD was calculated from six replicates at each spiking level.

Table 2. Occurrence, range and contamination level of aflatoxin M1 in buffalo’s and cow’s milk from the provinces of Punjab and NWFP, including the number of samples exceeding the European Union maximum limit. Province

Species

Punjab NWFP Punjab NWFP

Buffalo Buffalo Cow Cow

Total

Buffalo þ cow

n

n 4 LOD

n 4 0.05 mg kg1

Mean (mg kg1)

48 46 41 43

22 24 20 22

6 10 6 7

0.040a 0.066b 0.030a 0.045b

LOD–0.137 LOD–0.350 LOD–0.062 LOD–0.084

178

88

29

0.046

LOD–0.350

Notes: LOD, limit of detection (0.004 mg kg1). a,b Within a species values with different letters in a column are statistically significant (p 5 0.05).

Range (mg kg1)

Food Additives and Contaminants: Part B

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the current European Union statutory limit for AFM1 in milk. The presence of AFM1 concentration in milk samples in the present study was comparable with previously reported studies (Tekins en and Eken 2008; Ghazani 2009; Manetta et al. 2009; Fallah 2010; Rahimi et al. 2010; Assem et al. 2011), as shown in Table 4. However, our reported concentration of AFM1 in milk was lower than that of other studies (Rastogi et al. 2004; Razza 2006; Unusan 2006; Ghanem and Orfi 2009; Nuryono et al. 2009; Elzupir and Elhussein 2010; Sani et al. 2010). Our finding of AFM1 in milk samples was higher when compared with the studies conducted in European Union countries such as Italy (Capei and Neri 2002), Greece (Roussi et al. 2002) and Portugal (Martins et al. 2005). This may be attributed to the adoption of good agricultural and storage practices to control the risk of toxigenic fungi and aflatoxin contamination along the feed supply chain, and also by setting stringent

from day to day and from one milking to the next as it is influenced by various factors, including the feeding regime, health status and finally by actual milk production (EFSA 2004). To understand the variation level of AFM1 during lactation times, 213 morning milks and 202 evening milks were sampled from Punjab and NWFP provinces; their range and concentration are presented in Table 3. In Punjab province, about 32% of samples of buffalo’s and 33% of cow’s morning milks were contaminated by AFM1 compared with 25% and 32% evening milk samples, respectively. In NWFP province, about 42% of buffalo’s and 36% of cow’s morning milks were contaminated by AFM1 compared with 39% and 37% evening milk samples, respectively. Statistical analysis revealed significant differences (p 5 0.05) between mean AFM1 concentrations in milk during morning (0.043 mg kg1) and evening (0.028 mg kg1) lactation times. Again, no samples exceeded the Codex Alimentarius maximum level, but between 7% and 26% of samples were above

Table 3. Aflatoxin M1 contamination levels in morning and evening lactations of buffalo’s and cow’s milk samples. Mean (mg kg1)

n 4 LOD Province

Species

Range (mg kg1)

n

Morning

Evening

Morning

Evening

Morning

Evening

Punjab

Buffalo Cow

124 101

59 (19) 54 (18)

65 (16) 47 (15)

0.052a 0.032a

0.036b 0.024b

LOD–0.186 LOD–0.096

LOD–0.196 LOD–0.057

NWFP

Buffalo Cow

102 88

53 (22) 47 (17)

49 (19) 41 (15)

0.054a 0.032a

0.027b 0.022b

LOD–0.147 LOD–0.072

LOD–0.079 LOD–0.063

Total

Buffalo plus cow

415

213 (76)

202 (65)

0.043a

0.028b

LOD–0.186

LOD–0.196

1

Notes: LOD, limit of detection (0.004 mg kg ). a,b Morning and evening values with different letters in a row are statistically significant (p 5 0.05).

Table 4. Aflatoxin M1 contamination in milk from previous studies from different countries.

Category Milk Pasteurised milk Pasteurised milk Cow’s milk Cow’s raw milk Cow’s milk Water buffalo’s milk UHT milk Milk UHT milk Fresh milk Raw cow’s milk Milk samples Cattle’s milk

Number of samples 27 50 91 25 38 75 75 100 196 80 90 74 87 44

Number of n 4 European positive Union samples limita 16 50 66 25 28 59 29 67 196 9 30 70 76 42

(59.3%) (100%) (72.5) (100%) (73.7%) (78.7%) (38.7%) (67%) (100%) (11.3%) (33.3%) (95%) (87%) (95%)

1 31 33 11 17 27 6 31 158 6 23 41 75 42

(3.7%) (62%) (36.2%) (44%) (44.7%) (36%) (8%) (31%) (80.6%) (7.5%) (25.5%) (55.4%) (86.2%) (95%)

Note: aEuropean Union maximum level (0.05 mg kg1).

Range (mg kg1)

Mean (mg kg1)

50.010–0.051 0–0.259 0.013–0.250 0.030–0.098 0.003–0.126 – – 0.010–0.630 0.019–0.126 0.029–0.103 0.039–0.262 0.020–0.690 0.028–1.01 0.22–6.90

0.022 0.051 0.052 0.056 0.060 0.060 0.032 0.067 0.078 0.073 0.146 0.143 0.299 2.07

Reference and country Gu¨rbay et al. (2006), Turkey Ghazani (2009), Iran Fallah (2010), Iran Manetta et al. (2009), Italy Assem et al. (2011), Lebanon Rahimi et al. (2010), Iran Tekins en and Eken (2008), Turkey Sani et al. (2010), Iran Razza (2006), Pakistan Ghanem and Orfi (2009), Syria Rastogi et al. (2004), India Elzupir and Elhussein (2010), Sudan

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S.Z. Iqbal et al.

regulatory limits for aflatoxins in feed and milk in European Union countries. Maximum levels of AFM1 in milk should be set at a strict level which is reasonably achievable by following good agricultural, storage and feeding practices and by taking into account the risk related to the consumption of the food. The Codex Alimentarius has established a maximum limit for AFM1 in milk (0.50 mg kg1) which is based on a quantitative risk assessment. In turn, the European Union has set maximum concentrations (0.05 mg kg1) at a level that is as low as reasonably achievable (ALARA principle), based on the fact that aflatoxins are genotoxic carcinogens. For the present study it is more suitable to compare results with the Codex Alimentarius limit, which takes into account all relevant factors pertaining to the risk assessment in Pakistan. However, it is also appropriate for the health protection of vulnerable groups such as infants and young children who are milk consumers to compare then also with stricter European Union maximum limits. Therefore, both Codex and European Union approaches should ensure that dairy operators apply measures to prevent and reduce the contamination as far as possible in order to protect public health. It is documented that contamination of AFM1 in milk is a result of exposure of dairy cattle to feedstuffs contaminated with AFB1 (Unusan 2006). The wide variations in AFM1 levels among studies could be related to geographic and climatic differences, but the most important is due to differences in feeding systems and farm management practices (Ghazani 2009). In the present study a relatively high presence of AFM1 was detected in milk samples from NWFP compared with Punjab province. This may be explained by the fact that the availability of fresh fodder is not easy in NWFP because the irrigation system is not as effective and efficient as in Punjab province. Consequently, NWFP farmers use more concentrated feed, cottonseed cake, corn, soybean, threshed wheat straw, paddy straw and wheat bran. All these commodities are vulnerable to attack by moulds and there is a high possibility of the presence of AFB1 in these feed materials (Dutton and Kinsey 1996; Sassahara et al. 2005; Hussain et al. 2008; Pei et al. 2009). The higher concentration of AFM1 in morning milk samples compared with the evening samples may be attributed to the fact that AFM1 is a secondary metabolite of AFB1 and appears in milk approximately 12 h after ingestion of AFB1-contaminated feed (Frobish et al. 1986; van Egmond 1991; Wood 1991; Lopez et al. 2003; Unusan 2006; Boudra et al. 2007). Furthermore, AFM1 concentration in milk decreases to an undetectable level within 72 h (Sassahara et al. 2005; Fallah 2010). As a final conclusion, it was found that milk samples from Punjab were less contaminated by AFM1

than those from NWFP province of Pakistan, and the excretion rate of AFM1 was higher in the morning than in the evening milk samples. All milk samples complied with the Codex Alimentarius maximum limit, but the level of contamination with AFM1 was found to exceed European Union maximum contents in many instances. To achieve a low level of AFM1 in milk, there should be a strict selection of the raw materials and feed samples must be evaluated routinely for aflatoxins, and those with excess contamination should be removed from the ration immediately and new feeds replaced in the diet. However, breeding animals, dry dairy cows or beef cattle can be fed with feed materials with low levels of aflatoxins, especially when a dietary chemisorbent (i.e. clays) is added to the diet at recommended levels. Cows whose milk has exceeded maximum levels for aflatoxin should be fed for several days on aflatoxin-free feed for milk concentrations to fall below tolerance levels. It is recommended that records should be maintained for all feeds, feeding practices, milk contamination, and animal health and performance for all cases of aflatoxin contamination of milk.

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