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Food Control 34 (2013) 714e718

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Food Control

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Variation of aflatoxin M1 contamination in milk and milk productscollected during winter and summer seasons

Shahzad Zafar Iqbal a,b,*, Muhammad Rafique Asi c, S. Jinap b,d

aDepartment of Applied Chemistry, Government College University, Faisalabad 38000, Pakistanb Food Safety Research Centre (FOSREC), Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysiac Food Toxicology Lab, Plant Protection Division, Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad, Pakistand Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

a r t i c l e i n f o

Article history:Received 10 March 2013Received in revised form2 June 2013Accepted 8 June 2013

Keywords:Aflatoxin M1

Milk and milk productsHPLCSeasonal variations

* Corresponding author. Food Safety Research CenScience and Technology, Universiti Putra MalaysiaMalaysia. Tel.: þ60 126861121 (Cell), þ60 389468393

E-mail address: shahzad10542005@yahoo.com (S.

0956-7135/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodcont.2013.06.009

a b s t r a c t

Total 221 samples of milk and milk products were collected during winter (November 2011eFebruary2012) and 212 samples were collected during summer (MayeAugust 2012) from central areas of Punjab,Pakistan. The samples were analyzed for the presence of aflatoxin M1 (AFM1) with a validated HPLCmethod equipped with florescence detector. The results revealed that from winter season almost 45%samples of milk and milk products were found to be contaminated with AFM1 i.e. 40% of raw milk, 51% ofUHT milk, 37% of yogurt, 60% of butter and 43% of ice cream samples and 27, 24, 25, 34 and 17% ofsamples were found above the recommended limit for AFM1, respectively. However, from summerseason 32% samples of milk and milk products were found to be contaminated i.e. 36% of raw milk, 31% ofUHT milk, 29% of yogurt, 40% of butter and 24% of ice cream and 23, 23, 18, 20 and 5% of samples werefound above the permissible limit for AFM1, respectively. The levels of contamination in winter milk andmilk product samples were significantly higher (a � 0.05) than in summer season. The occurrence ofAFM1 in milk and milk products were higher, demanding to implement strict regulations and also urgedthe need for continuous monitoring of milk and milk products in order to minimize the health hazards.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Now a days with the advancement in analytical facility and withthe increasing international trade in food, especially when themanufacturing country provide their materials to other manufac-turers or finished goods to consumers living in importing countrieswill emphasize the need to adapt harmonization in food safetycontrol protocols (Stringer, 2005). It has been reported that almost25% of food and food products are affected annually by mycotoxins(Iqbal, Asi, Zuber, Akhtar, & Saif, 2013). The fungi can attack cropsduring pre-harvest or post harvest conditions, which resulted forthe production of mycotoxins (Iqbal, Asi, Zuber, Akram, & Batool,2013). Mycotoxin represents naturally occurring secondary me-tabolites which are mainly produced by the filamentous fungi(Iqbal & Asi, 2013). Aflatoxins (AFs) are the most toxic and carci-nogenic class of mycotoxins and capable to contaminate cereals,

tre (FOSREC), Faculty of Food, 43400 Serdang, Selangor,(Office).

Z. Iqbal).

All rights reserved.

spices, fruits, and vegetables (Asi, Iqbal, Ariño, & Hussain, 2012).Among AFs, aflatoxin B1 (AFB1) is the most toxic group (Iqbal,Paterson, Bhatti, & Asi, 2011) and aflatoxin M1 (AFM1) is the hy-droxylatedmetabolite of AFB1 (Asi et al., 2012), which is excreted inmilk in the mammary glands of both human and lactating animalsas a result of feeding AFs contaminated feed (Fallah, Jafari, Fallah, &Rahnama, 2009; Gürbay et al., 2010).

It was concluded that AFM1 is 2e10% of less toxic than AFB1(Creppy, 2002) but International Agency for Research on Cancer hasplaced it along with AFB1 as Group 1 carcinogen (IARC, 2002).Previous studies have shown that approximately 0.3e6.2% of AFB1ingested by livestock is metabolized into AFM1 and excreted inmilkhowever; it mainly depends on the genetics of animals, seasonalvariation, milking process and on the environmental conditions(Unusan, 2006). It has been observed that AFM1 is very stable athigh temperatures like other forms of AFs and the concentration ofAFM1 in milk is not affected significantly with the application ofthermal processes i.e. pasteurization and ultra-high-temperature(UHT) treatments used in dairy industry (Prandini et al., 2009).Therefore, to prevent or minimize the health risks associated fromthese toxins, most of the countries have implemented regulation(Iqbal et al., 2013). The European Union (EU) has implement the

S.Z. Iqbal et al. / Food Control 34 (2013) 714e718 715

maximum AFM1 level in liquid milk and dried or processed milkproducts intended for adults i.e. 0.050 mg/L and 0.025 mg/L for milkintended for infants (European Commission Regulation, 2004).However, the US Food and Drug Administration stated themaximum permissible level of 0.5 mg/kg in milk (Kav, Col, &Tekinsen, 2011). The Codex Alimentarius legal limit for AFM1 inbutter is 0.05 mg/kg (Codex Alimentarius Commission, 2001). Thereare thus differences in the maximum tolerance limit for AFM1 invarious countries and Pakistan has no legal limit for AFM1 inmilk ordairy products (Iqbal, Asi, & Ariño, 2011).

The presence of AFM1 in milk and milk products is a seriousissue, since these products are regularly consumed by each agegroup in their daily diet (Fallah et al., 2009). The situation is evenworse in developing countries where lack of awareness, constraintsin resources and the use of conventional analytical facilities makesthe situation alarming. Pakistan stands forth in milk producingcountries with production of 47.5 million tons per annum (Iqbal &Asi, 2013). There are few reports for the contamination of AFM1 inmilk and milk products from Pakistan (Asi et al., 2012; Hussain &Anwar, 2008; Hussain, Anwar, Asi, Munawar, & Kashif, 2010;Hussain, Anwar, Munawar, & Asi, 2008; Iqbal & Asi, 2013; Iqbal,Asi, et al., 2011; Sadia et al., 2012). However, very limited data isavailable on the effect of seasonal variations for AFM1 in milk andmilk products. The climatic conditions in Pakistan are conducive forthe growth of toxigenic fungi (Iqbal, Asi, et al., 2011) and therefore,there should be regularmonitoring of food products especially milkand milk products to prevent health hazards. The contamination ofAFM1 in UHT milk and ice cream samples has never been reportedfrom Pakistan. The main objectives of the present study are (i) toassess the natural presence of AFM1 in raw milk, UHT milk, yogurt,butter and ice cream samples from central areas of Punjab,Pakistan, (ii) to investigate the seasonal effect on AFM1 level in milkproducts, (iii) to compare the samples which exceeds the permis-sible level of EU and (iv) to construct a database to help policymaker in local governments, law enforcement agencies and tradersto focus urgent attention to prevent or minimize the health risksassociated from these toxin.

2. Materials and methods

2.1. Sampling

A total of 433 samples of raw milk and milk products of which221 samples from winter season (raw milk (48), UHT milk (45),yogurt (51), butter (35) and ice cream (42)) and 212 samples fromsummer season (56, 39, 45, 35, and 37, respectively) were randomlycollected during November 2011eSeptember 2012 from the maindistricts of Punjab, Pakistan. The samples of milk andmilk productswere collected frommilking sites, small and large dairies and dairyfarmhouses. The size of milk samples were maintained at least of1 L, while yogurt, butter and ice cream samples were maintained at

Table 1The fortified level of AFM1 in milk, UHT milk, yogurt, butter and ice cream.

AFM1 fortified(mg/L)

Milk UHT milk Yo

aMean(mg/L)

RSD(%)

aMean(mg/L)

RSD(%)

aM(mg

0.025 0.021 (84) 10 0.022 (87) 9 0.00.050 0.044 (89) 11 0.045 (89) 5 0.00.075 0.067 (90) 7 0.066 (88) 11 0.00.100 0.091 (91) 5 0.090 (90) 3 0.00.200 0.160 (78) 21 0.170 (87) 7 0.1

The data in parenthesis represents the % age recovery.a Mean represents the 5 replicate data of each fortified concentration.

least of 200e500 g. The samples were preserved in plastic bags andduring transportation kept in an icebox. The samples were stored ina freezer at �4 �C until further analysis.

2.2. Chemicals

Aflatoxin M1 standard (10 mg/L in acetonitrile, SigmaeAldrich,St. Louis, Mo., USA) was provided by Food Toxicology Lab, NuclearInstitute of Agriculture and Biology, Faisalabad, Pakistan. The sol-vent acetonitrile (HPLC grade) was purchased from SigmaeAldrich,Steinheim, Germany and immunoaffinity columns (IAC) fromVICAM, Watertown, MA, USA. A standard curve for AFM1 wasprepared by diluting the standard with acetonitrile into differentconcentrations of 0.05, 0.1, 0.5, 1.0, 5.0 and 10.0 mg/L solutions andstored in caped vials in a refrigerator at�4 �C until further analysis.All other reagents were at least of analytical grade.

2.3. Extraction

The analysis of AFM1 in milk, yogurt, butter and ice creamsamples were performed according to our previously validatedmethod (Iqbal & Asi, 2013).

2.4. HPLC conditions

For the detection of AFM1 in milk and milk products, HPLC ofShimadzu LC-10A series (Kyoto, Japan) was used, equipped withfluorescence detector FLD (RF-530) with excitation and emissionwavelengths at 365 and 435 nm, respectively. The column was aC18 (4.6 � 250 mm, 5 mm) Discovery of Supelco, Bellefonte, USA.The mobile phase acetonitrile and water (25:75 v/v) was used witha flow rate of 1.0 ml/min isocratically. The method has shown linearresponse (R2 ¼ 0.9992) and limit of detection (LOD) 0.004 mg/L.

2.5. Recovery analysis

Precision and recovery assays were carried out by performingtests on each of the blank raw milk, yogurt, butter and ice creamsamples (five replicates each) fortified with AFM1 at concentrationsof 0.025, 0.050, 0.075, 0.1 and 0.2 mg/L, as shown in Table 1. Inpresent study the recovery percentages of AFM1 in spiked milksamples were found 78e96%with relative standard deviation (RSD)from 4 to 21%. The method has shown good recoveries in milk andmilk products.

2.6. Statistical analysis

The data was presented as mean � standard deviation. Corre-lation and regression analyses were carried out to find out thecoefficient of determination (R2). The significant difference of AFM1

gurt Butter Ice cream

ean/L)

RSD(%)

aMean(mg/kg)

RSD(%)

aMean(mg/L)

RSD(%)

22 (88) 10 0.020 (86) 10 0.021 (89) 1144 (88) 8 0.043 (85) 4 0.048 (96) 467 (89) 5 0.069 (88) 12 0.064 (85) 2090 (88) 9 0.091 (89) 7 0.087 (88) 870 (83) 15 0.180 (83) 19 0.150 (78) 19

Table 2The occurrence and mean concentration of AFM1 in milk and milk products samples collected during winter and summer seasons.

Dairy samples Winter Summer

Samplesanalyzedn

Positive samplesn (%)

Mean � SD(mg/kg)

Samples analyzedn

Positive samplesn (%)

Mean � SD(mg/kg)

Raw milk 48 19 (40) 0.073a � 0.006 56 20 (36) 0.028b � 0.002UHT milk 45 23 (51) 0.060a � 0.009 39 12 (31) 0.021b � 0.007Yogurt 51 19 (37) 0.053a � 0.003 45 13 (29) 0.019b � 0.009Butter 35 21 (60) 0.036a � 0.007 35 14 (40) 0.015b � 0.005Ice cream 42 18 (43) 0.021a � 0.004 37 9 (24) 0.012b � 0.006Total samples 221 100 (45) 212 68 (32)

The data in parenthesis represents the parentage of samples to total samples analyzed.The English letter with different words represents the significant difference (a ¼ 0.05).

S.Z. Iqbal et al. / Food Control 34 (2013) 714e718716

contamination among seasons was determined at (a ¼ 0.05) usingSPSS (IBM PASW statistics 19 software, USA).

Table 3The range and samples exceeds the permissible limit of AFM1 inwinter and summersamples of milk and milk products.

Dairy samples Winter Summer

Exceedregulationsa

n (%)

Range(mg/kg, mg/L)

Exceedregulationsa

n (%)

Range(mg/kg, mg/L)

Raw milk 13 (27) LODe0.45 13 (23) LODe0.89UHT milk 11 (24) LODe0.51 9 (23) LODe0.51Yogurt 13 (25) LODe0.44 8 (18) LODe0.88Butter 12 (34) LODe0.57 7 (20) LODe0.78Ice cream 7 (17) LODe0.67 2 (5) LODe0.34Total samples 56 (25) 39 (18)

The data in parenthesis represents the parentage of samples to total samplesanalyzed.

a EU limits (0.05 mg/L) for AFM1 in raw milk, UHT milk, yogurt, and ice cream0.020 mg/kg for butter.

3. Results and discussions

3.1. Raw milk and UHT milk

In present study, the incidence of AFM1 contamination in rawmilk and UHT milk samples were found 40% (mean0.073� 0.006 mg/L) and 51% (mean 0.060� 0.009 mg/L) fromwinterseason as compared to 36% (mean 0.028 � 0.002 mg/L) and 31%(mean 0.021 � 0.007 mg/L) samples respectively from summerseason, as shown in Table 1. The concentration of AFM1 in raw milkand UHT milk in winter season samples were found significant(p < 0.05) as compared to the summer seasons. The samples whichexceed the maximum recommended limit in raw milk and UHTmilk from winter season were 27% and 24% as compared to thesamples found above the recommended limit from summer season23% each respectively, as shown in Table 3. The occurrence andconcentration of AFM1 in milk samples from present finding arelower when compared to the results of our previous study (Iqbal &Asi, 2013), which reported that 71% of milk samples were found tobe contaminated with the average level of 0.15 � 0.001 mg/L and24% of the samples were found to be above the recommended limit.However, the results were comparable to in another study whichreported the mean level of AFM1 in cow milk from winter seasonwas 0.089 � 0.004 mg/L and in summer season 0.022 � 0.006 mg/L(Asi et al., 2012). The results of present finding were comparable toprevious studies from other Countries (Assem, Mohamad, & Oula,2011; Manetta et al., 2009; Rahimi, Bonyadian, Rafei, &Kazemeini, 2010; Sani, Nikpooyan, & Moshiri, 2010; Tekinsen &Eken, 2008). From Turkey, Tekinsen and Eken (2008) have re-ported that 67% of UHT milk samples were found positive withAFM1 with mean level of 0.067 mg/L and 31% of samples were foundto exceed the EU recommended limit. Manetta et al. (2009) fromItaly have reported 100% of cow milk samples were found positivewith mean level of AFM10.056 mg/L and 44% of samples were foundto be above the permissible level. About 73.7% of cow’s milk sam-ples were found to be contaminated with 44.7% of samples werefound to be above the recommended limit with a mean level of0.060 mg/L (Assem et al., 2011). Similar results were reported fromIran by Rahimi et al. (2010) in cow’s milk samples and reported78.7% positive samples and 36% of samples were found to be abovethe recommended limit with mean level of 0.060 mg/L. Sani et al.(2010) from Iran have observed 100% of milk samples positivewith AFM1 and 80.6% of samples exceeds the maximum limit withmean level of 0.078 mg/L.

The variation in AFM1 contamination in milk samples fromwinter and summer seasons could be explained by feeding

practices adopted in Pakistan (Asi et al., 2012). In summer seasonfarmers mostly use fresh animal feed such as pasture, grass, weedsand green fodder but in winter due to shortage of fresh green feed,more concentrate feeding based on corn, cotton seeds and wheatderived products are being used in animal farmhouses. It has alsobeen reported that toxigenic Aspergillus fungi can attack on greenfodder and hay preserved as silage during storage and AFs may beproduced (Herzallah, 2009; Heshmati & Milani, 2010). Seasons alsoaffect the yield of milk because the yield of milk is higher in sum-mer season as compared to winter, therefore AFM1 and othercomponents become more concentrated during winter season (Asiet al., 2012). Our findings are consistent with previous studies thatreported that high contamination level of AFM1 in cold seasonscomparing to hot ones (Ayar, Sert, & Çon, 2007; Hussain & Anwar,2008; Kamkar, 2005; Ruangwises & Ruangwises, 2009).

3.2. Yogurt and butter

Total 96 samples of yogurt (winter¼51, and summer¼45) and35each samples of butter from winter and summer seasons wereanalyzed for the contamination of AFM1. The results are shown inTable 2. The contaminations of AFM1 in winter yogurt and buttersamples were found significant (p � 0.05) compared with the sum-mer samples, respectively. The levels of AFM1 in yogurt and buttersamples from present study were found low as compared to ourpervious study in which 61% of yogurt samples (mean 0.090 � 0.01mg/L) and 45% of butter samples (mean 0.069 � 0.009 mg/kg) werefound contaminated (Iqbal & Asi, 2013). However, the contaminationof AFM1 in yogurt samples from present study were found high ascompared to the level of 0.011e0.013 mg/kg in yogurt samples re-ported by Maqbool, Anwar-Ul-Haq, and Ahmad (2009). SimilarlyFallah (2010) from Iranhas concluded that the level ofAFM1 inyogurt

Fig. 1. The comparison of positive and the samples which exceed the EU permissiblelimit of milk and milk product from summer and winter seasons.

S.Z. Iqbal et al. / Food Control 34 (2013) 714e718 717

and butter samples from winter season was higher compared withsummer samples but reported lower contamination level ascompared to presentfinding. He reported that 66% of yogurt samples(mean 0.032 � 0.004 mg/L) and 25.8% of butter samples (mean0.005�0.002mg/kg)were foundpositivewithAFM1and21%and10%of sampleswere found to be above the regulatory limits, respectively.In present study about 25% of yogurt and 34% of butter sampleswerefound to be above the permissible limit, as shown in Table 3. FromTurkey, AFM1was found in56%of yogurt samples ranging from0.002to 0.078 mg/kg and 14% of yogurt samples has AFM1 level higher thanthe acceptable (Ertas, Gonulalan, Yildirim, & Karadal, 2011).

3.3. Ice cream

Total 79 samples of ice creamwere analyzed for the detection ofAFM1 during winter and summer seasons. The occurrence andmean contamination level is shown in Table 2. The results revealedthat 43% of ice cream samples from winter season were foundpositive (mean 0.021 � 0.004 mg/L) as compared to summer seasonsamples i.e. 24% (mean 0.012 � 0.006 mg/L). The levels of AFM1 inice cream samples in winter season were significant (p � 0.05) ascompared to summer samples. The mean contamination levels ofpresent finding were low as compare to other finding from Nigeriaby Atanda, Oguntubo, Adejumo, Ikeorah, and Akpan (2007), theyhave analyzed 6 ice cream samples and found level of 2.23 mg/L.However, in another study from Iran, higher occurrence and meancontamination of AFM1 was reported by Fallah (2010), comparedwith the results of present finding in ice-cream samples. He re-ported 69.4% of samples were found positive with mean level of0.041 � 0.006 mg/L. The present study revealed that 17% of icecream samples from winter season were exceeded as compared toonly 5% of summer samples to EU permissible limit as shown inTable 2. However, Atanda et al. (2007) have reported that no sampleof ice cream was found to be above the permissible limit for AFM1.The comparison of positive samples and those which exceed thepermissible limits in milk and milk product samples from summerand winter seasons is represented in Fig. 1.

4. Conclusions

The study is basically the continuous part of our previous studies,to regularly monitor the contamination level of AFM1 in milk and

milk products. The results have revealed that about 27% of raw milk,24% of UHT milk, 25% of yogurt, 34% of butter and 17% of ice creamsamples from winter season and 23, 23, 18, 20, 5% of samples,respectively from summer season were found to be above the EUlimits for AFM1. Total 25% of samples of milk and milk products fromwinter season compared with 18% of samples from summer seasonwere found to be above the recommended limit. The recommenda-tions includes that, there should be more studies on AFM1 contam-ination in milk focusing on feeding practices in order to investigatethe main factors that are responsible for high occurrence of AFM1contamination.

Acknowledgments

The funding provided by Higher Education Commission (HEC),Islamabad, Pakistan for present project is highly appreciated. Theauthors acknowledge the analytical facilities provided by NuclearInstitute for Agriculture and Biology (NIAB) Faisalabad, Pakistan.

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