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Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Aflatoxin M1 in milk from urban and rural farmhouses of Punjab, Pakistan Shahzad Zafar Iqbal
a b
c
, Muhammad Rafique Asi & Jinap Selamat
b
a
Department of Applied Chemistry , Government College University Faisalabad , Faisalabad , Pakistan b
Faculty of Food Science and Technology , Food Safety Research Centre (FOSREC) , Universiti Putra Malaysia , Serdang , Selangor , Malaysia c
Food Toxicology Lab, Plant Protection Division , Nuclear Institute for Agriculture and Biology , Faisalabad , Pakistan Accepted author version posted online: 24 Jul 2013.
To cite this article: Food Additives & Contaminants: Part B (2013): Aflatoxin M1 in milk from urban and rural farmhouses of Punjab, Pakistan, Food Additives & Contaminants: Part B: Surveillance, DOI: 10.1080/19393210.2013.828322 To link to this article: http://dx.doi.org/10.1080/19393210.2013.828322
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Aflatoxin M1 in milk from urban and rural farmhouses of Punjab, Pakistan Shahzad Zafar Iqbal
a,b
, Muhammad Rafique Asic,
a
Department of Applied Chemistry, Government College University Faisalabad, 38000, Pakistan Food Safety Research Centre (FOSREC); Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia c Food Toxicology Lab, Plant Protection Division, Nuclear Institute for Agriculture and Biology, Faisalabad, Pakistan
rip t
an us c
Corresponding Author Email: [email protected] (Shahzad Z. Iqbal) Cell: 0060-12-6861121
M
Abstract
Aflatoxin M1 (AFM1) was determined in 107 milk samples collected from urban and rural
d
farmhouses from Punjab, Pakistan, by HPLC with fluorescence detection. An incidence rate of
pt e
64% (38/59) in milk samples from urban farmhouses was found with a mean concentration of 0.064 ± 0.023 μg L-1. In rural samples about 52% (25/48) milk samples were contaminated with AFM1 with a mean of 0.04 ± 0.034 μg L-1. 42% milk samples from urban and 27% from rural
ce
farmhouses were well above the limit permitted by the EU. However, only 15 and 8% milk samples from urban and rural farmhouses respectively, exceeded the limit of USDA/Codex regulations. The results showed that the contamination of milk with AFM1 from Punjab, Pakistan, when compared with Codex limit are present and needs continuous monitoring. The
Ac
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b
awareness and education among dairy farmers on the potential health risks associated with aflatoxins should be communicated.
Keywords: Milk; AFM1; Urban and Rural farmhouses; Punjab; Pakistan
Introduction Mycotoxins are natural and unavoidable toxic secondary metabolites produced by fungi, which contaminate agricultural staples in the field or during harvest, transport and storage (Iqbal et al. 2010). It has been estimated by the Food and Agriculture Organization that approximately 25% of world’s food crops are affected each year by mycotoxins (Ono et al. 2011). Aflatoxins
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(AFs) are the most toxic class of mycotoxins produced mainly by Aspergillus flavus and A. 2011c). Hydroxylation of AFB1 produces AFM1 which is secreted in the milk of lactating
animals consuming contaminated feed (Ruangwises and Ruangwises 2010; Asi et al. 2012). The
an us c
International Agency for Research on Cancer (2002) has classified AFB1 as class 1 carcinogenic to humans, however the carcinogenicity of AFM1 is approximately ten times less as compared to
AFB1 (Nuryono et al. 2009). It has been evident that AFs are responsible for acute poisoning (aflatoxicosis), for hepatocellular carcinoma, for growth impairment in children and immunosuppresion (Cassel et al. 2001; Sidhu et al. 2009; Fallah 2010). Considering their risk to human health the incidence and presence of AFM1 in dairy products is regarded as a significant
M
hazard for food safety and public health (Cucci et al. 2007).
To minimize the health risk of AFM1 contamination in milk and dairy products most
d
countries implemented strict regulations, however in developing countries like Pakistan an adequate regulation about admissible limits of AFM1 is still lacking. The policy applied by the
pt e
European Union and the USA to ensure food safety is based on the assumption that strict controls on feed and milk would prevent contamination of dairy products (Creppy 2002). The EU maximum level (ML) for AFM1 in milk intended for adults is 0.050 μg kg-1, whereas the
ce
limit intended for infants or for baby-food production was set at 0.025 μg kg-1 (European Commission 2006b). However, the Codex Alimentarius has established a maximum level of 0.5 μg kg-1 for AFM1 in milk (Codex Alimentarius, 1995).
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parasiticus (Iqbal et al. 2011a and b), the most toxic form being aflatoxin B1 (AFB1) (Iqbal et al.
Referring to scientific literature, very limited data has been published on the occurrence
of AFM1 in milk from Pakistan. Previously (Iqbal et al. 2011a; Asi et al. 2012) and other studies (Hussain and Anwar 2008; Hussain et al. 2010; Hussain et al. 2008) showed high percentages of
AFM1 contaminated milk samples. In the present study milk samples have been collected from rural and urban farmhouses to evaluate the feeding practices and their effect on the incidence of AFM1. The main objectives were to evaluate the incidence and contamination level of AFM1 in
milk samples and to compare samples that exceed the European Union (EU) and Codex limits. The results drawn from the present study will be helpful to implement strict regulation and
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Materials and Methods Sampling A total of 107 samples of raw milk from 43 farmhouses, 48 from rural farmhouses and 59
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samples from urban farmhouses, were collected during May 2011 to August 2011 from major districts of Punjab, Pakistan. The selection of urban and rural farmhouses was based on the fact that in most of the urban farmhouses the animal feed consists of more concentrated feed like cottonseed cake, corn, soybean, threshed wheat straw, paddy straw, wheat bran and dry breads
while in rural farmhouses mostly green fodder is used with less or even rare concentrated feed. The size of milk samples was at least 1 liter. During sample collection and transportation, the
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samples were kept in ice packets in an icebox. The samples were either analyzed immediately or stored in a freezer at -4 ºC in case of delayed analysis.
d
Chemicals and Regents The standard of AFM1 (10 μg ml-1 in acetonitrile) and acetonitrile of HPLC grade were
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purchased from Sigma Aldrich (Steinheim, Germany). Immunoaffinity columns (IAC) AflaM1TM were purchased from VICAM (Watertown, MA, USA). To evaluate the concentration of AFM1, a standard curve was prepared with concentrations of 0.05, 0.1, 0.5, 0.10, 0.50 and 10.0 μg L-1
ce
solutions and stored in capped vials in a refrigerator at -4ºC until further analysis. The water used was double-distilled and all other chemicals and reagents were at least of analytical grade. Extraction Milk samples were heated until 37 ºC in a water bath and then centrifuged at 2500 rpm for 5
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examination on utilization of concentrated animal feed and regulate limits for these mycotoxins.
minutes to separate the fat layer. After centrifugation samples were filtered through Whatman No. 5 filter paper and 50 ml filtrate was transferred into a syringe barrel attached to an IAC and passed at a rate 2 ml/min using a solid phase extraction manifold. The column was washed with
20 ml double distilled water and AFM1 was eluted with 4 ml pure acetonitrile passing the IAC in approximately 60 s. Finally the eluate was evaporated to dryness using a gentle stream of N2 at 40 ºC and the residue was dissolved in 1ml mobile phase at the time of HPLC analysis.
HPLC Conditions The HPLC equipment used was a Shimazdzu LC-10A series (Kyoto, Japan) with fluorescence detector FLD (RF-530) with excitation and emission wavelength of 365 and 435 nm, respectively, equipped with a Discovery C18 column (4.6 x 250 mm, 5 μm) of Supelco (Bellefonte, USA). Acetonitrile/water (25: 75 v/v) was used as mobile phase at a flow rate of 1.0 ml min-1. The response of the calibration curve was linear (R2 = 0.9991). The limit of detection
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Validation
Precision and recovery were carried out by performing tests on raw milk samples (four replicates
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each) fortified with AFM1 at 0.02, 0.05, 0.07 and 0.1 μg L-1. The recoveries in these spiked milk samples were 83, 92, 92 and 90%, with RSD from 8, 9, 12, and 4%, respectively. Statistical Analysis
All data were presented as mean ± standard deviation. Correlation and regression analyses were applied to calculate R2. Significant differences of mean between urban and rural milk samples
Results and Discussion
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were determined using Student paired t-test (SPSS, IBM PASW statistics 19, USA).
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All analytical results are presented in table 1. AFM1 was found above the measurable level (0.004 μg L-1) in 64 % (38/59) and 52% (25/48) of milk samples from urban and rural farmhouses, respectively. Considering the European Commission limit, 25 (26.7%) and 13
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(17.4%) milk samples from urban and rural farmhouses had AFM1 concentrations in excess of the maximum limit (0.05 μg L-1), respectively. Less numbers, 9 samples (15%) from urban and 4 (8%) milk samples from rural farmhouses were above the Codex limit of 0.5 μg L-1 (Table 2).
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(LOD) and limit of quantification (LOQ) were 0.004 μg L-1 and 0.012 μg L-1, respectively.
The results show statistically significant difference of AFM1 contamination in milk samples from
urban farmhouses when compared to rural farmhouses by applying student paired t test at (p < 0.05).
The findings of the present study on the occurrence level of AFM1 contamination in milk samples were comparable with a previous study (Iqbal et al. 2011a) in which 88 samples out of 178 were contaminated, with a mean level of AFM1 0.046 μg L-1 and a range from LOD to 0.35
μg/l. Results were comparable with reports from Korea (Kim et al. 2000), Iran (Heshmati and Milani 2010) and India (Rastogi et al. 2004), as presented in Table 3. However, some studies reported lower levels of AFM1 contamination in milk samples: Nigeria (Atanda et al. 2007), Italy (Nachtmann et al. 2007), Morocco (Zinedine et al. 2007) and Turkey (Unusan, 2006). Occurrence of AFM1 in raw milk samples from 14 districts of the Punjab Province, Pakistan has
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been reported by Hussain and Anwar (2008). All samples analyzed were contaminated with
previous studies. Martins and Martins (2000) from Portugal reported AFM1 in 81 raw milk and
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84% UHT milk samples. Similar results were reported by Galvano et al. (2001), who found 78% of liquid milk and 53% of dry milk samples to be contaminated with AFM1, of which 15% from urban and 8% from rural farmhouses were above the codex or USDA limit.
The present findings demonstrate that AFM1 contamination in samples from urban farmhouses is higher when compared with samples collected from rural farmhouses. This could be explained because in urban farmhouses animal feed mostly consists of concentrated feed i.e.
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cottonseed cake, corn, soybean, threshed wheat straw, paddy straw, wheat bran and dry bread. All these commodities are vulnerable to mould attack producing AFs (Dutton and Kinsey 1996; Sassahara et al. 2005; Hussain et al. 2008; Pei et al. 2009). However, in rural farmhouses the
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animal feed mostly consists of green fodder mixed with dry crushed wheat straws. From
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previous studies, it was well documented that AFM1 contamination of milk is a result of the exposure of AFB1 contaminated feedstuffs to dairy animals (Unusan 2006). Therefore, whereas variations of AFM1 levels among different studies could be related to geographic and climatic
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differences, the most important factor is the difference in feeding systems and farm management practices (Ghazani 2009). To achieve and maintain low levels of AFM1 in milk, feed samples must be evaluated
routinely for aflatoxins and those with excess contamination should be discarded. Records
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AFM1 and 99.4% exceeded the European Union limit of 0.05 μg L-1, which was comparable with
should be maintained for all feed, feeding practices, milk contamination and animal health on all cases on aflatoxin contamination of milk. Owners of urban farmhouses should take measures to examine the quality of animal feed. Furthermore government agencies should monitor animal feed and milk samples routinely and enforce food and feed laws in order to minimize health hazards for human and animal health due to these toxins.
Acknowledgements The authors appreciate the funding provided by the Higher Education Commission, Islamabad,
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Pakistan for this study.
Asi MR, Iqbal SZ, Ariño A, Hussain A. 2012. Effect of seasonal variations and lactation times on 25 (1): 34-38.
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aflatoxin M1 contamination in milk of different species from Punjab, Pakistan. Food Contr. Atanda O, Oguntubo A, Adejumo O, Ikeorah J, Akpan I. 2007. Aflatoxin M1 contamination of milk and ice cream in Abeokuta and Odeda local governments of Ogun State, Nigeria. Chemosphere 68: 1455–1458.
Cassel EK, Campbell B, Draper M, Epperson B. 2001. Aflatoxins: Hazards in grain/aflatoxicosis
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and livestock. South Dakota State University. Brookings FS 907.
Codex Alimentarius, 1995. Codex General Standard for contaminants and toxns in food and feed, Codex Standard 193-1995, Adopted 1995. Revised 1997, 2006, 2008, 2009.
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Amended 2010, 2012.
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Creppy EE. 2002. Update of survey, regulation and toxic effects of mycotoxins in Europe. Toxicol. Let. 127:19–28.
Cucci C, Mignani AG, Dall’Asta C, Pela R, Dossena A. 2007. A portable fluorometer for the
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rapid screening of M1 aflatoxin. Sensor Actuator B. 126:467–472. Dutton MF, Kinsey A. 1996. A note on the occurrence of mycotoxins in cereals and animal feedstuffs in Kwazulu Natal, South Africa 1984–1993. S. Afr. J. Anim. Sci. 26:53–57
Fallah AA. 2010. Assessment of aflatoxin M1 contamination in pasteurized and UHT milk
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Heshmati A, Milani JM. 2010. Contamination of UHT milk by aflatoxin M1 in Iran. Food Contr. 21(1):19–22. Hussain I, Anwar J, Asi MR, Munawar MA, Kashif M. 2010. Aflatoxin M1 contamination in milk from five dairy species in Pakistan. Food Contr. 21:122-124. Hussain I, Anwar J, Munawar MA, Asi MR. 2008. Variation of levels of aflatoxin M1 in raw
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Iqbal SZ, Asi MR, Ariño A. 2011a. Comparison of aflatoxin M1 contamination level in milk samples from NWFP and Punjab provinces of Pakistan. Food Addit. Contam. B 4 (4): 282288.
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Iqbal SZ, Paterson RRM, Bhatti IA, Asi MR. 2011c. Comparing aflatoxins contamination in 80.
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chilies from Punjab, Pakistan, produced in summer and winter. Mycotoxin Res. 27:75– Kim EK, Shon DH, Ryu D, Park JW, Hwand HJ, Kim YB. 2000. Occurrence of Aflatoxin M 1 in
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Korean dairy products determined by ELISA and HPLC. Food Addit. Contam. 17: 59– 64.
Martins ML, Martins HM. 2000. Aflatoxin M1 in raw and ultra high temperature-treated milk commercialized in Portugal. Food Addit. Contam. 17: 871–874.
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milk from different localities in the central areas of Punjab, Pakistan. Food Contr. 19:
Nachtmann C, Gallina S, Rastelli M, Ferro GL, Decastelli L. 2007. Regional monitoring plan regarding the presence of aflatoxin M1 in pasteurized and UHT milk in Italy. Food Contr. 18: 623–629. Nuryono N, Agus A, Wedhastri S, Maryudani YB, Setyabudi FMCS, Bohm J, Razzazi-Fazeli E. 2009. A limited survey of aflatoxin M1 in milk from Indonesia by ELISA. Food Contr. 20:721–724.
Ono EYS, Hirooka EY, Rossi CN, Ono MA. 2011. Chapter 13; Mycotoxins in Seeds and Nuts, Book title; Nuts and Seeds in Health and Disease Prevention edited by Victor R. Preedy, Ronald Ross Watson and Vinood B. Patel, Elsevier Inc. ISBN: 978-0-12-375688-6 p.p (121-127). Pei SC, Zhang YY, Eremin SA, Lee WJ. 2009. Detection of aflatoxin M1 in milk products from
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Sassahara M, Pontes ND, Yanaka EK. 2005. Aflatoxin occurrence in foodstuff supplied to dairy cattle and aflatoxin M1 in raw milk in the North of Parana state. Food Chem. Toxicol. 43: 981-984.
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Sidhu OP, Chandra H, Behl HM. 2009. Occurrence of aflatoxins in mahua (Madhuca indica Gmel.) seeds: synergistic effect of plant extracts on inhibition of Aspergillus flavus growth and aflatoxin production. Food Chem. Toxicol. 47: 774-777.
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Unusan N. 2006. Occurrence of aflatoxin M1 in UHT milk in Turkey. Food Chem. Toxicol. Zinedine A, González-Osnaya L, Soriano JM, Moltó JC, Idrissi L, Mañes J. 2007. Presence of
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Rastogi S, Dwivedi PD, Khanna SK, Das M. 2004. Detection of aflatoxin M1 contamination in
Table 1: AFM1 contamination level in milk samples from urban and rural farmhouses Mean (
μg L-1)
Positive samples
Urban Samples
59
38 (64)
< 0.004 to 0.98
0.064 a ± 0.02
Rural samples
48
25 (52)
< 0.004 to 0.71
0.045 b ± 0.03
Total
107
63 (59)
< 0.004 to 0.98
0.055 ± 0.03
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Range (
μg L-1)
n
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Mean values with different letters are statistically different at (p < 0.05) applying Student paired t-test
Sample type
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farmhouses
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Table 2: Incidence of AFM1 contamination in milk samples from urban and rural Positive
Incidence
n > 0.05 μg L-1
n > 0.05 μg L-1
n > 0.5c μg L-1
n > EU
38 (64)
13 (22%)
16 (27%)
9 (15%)
25 (42%)
Milk samples RFb 25 (52)
12 (25%)
9 (19%)
4 (8%)
13 (27%)
a
ce
Milk samples UF
UF = Urban farmhouse RF= Rural farmhouse c Codex/USDA limit b
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Sample type
Positive
n > 0.05 µg/kg
Mean (μg L- Country 1 )
Reference
178 180 210 87 12 10 316 54 129 360 120
49% 79% 55% 87% 100% 100% 2.2% 89% 58.1% 42.5% 52.5%
16% 58% 33% 99% 100% 100% 0.6% 7.4% 47% 52.5% 57.5%
0.046 0.057 0.087 0.299 2.04 4.00 0.027 0.019 0.11 0.027 (buffalo) 0.044
Pakistan Korea Iran India
Iqbal et al. (2011) Kim et al. (2000) Heshmati and Milani (2010) Rastogi et al. (2004)
Nigeria
Atanda et al. (2007)
Italy Morocco Turkey
Nachtmann et al. (2007) Zinedine et al. (2007) Unusan (2006
Pakistan
Hussain and Anwar (2008)
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d pt e ce
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Samples
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Table 3: Occurrence of AFM1 contamination in milk samples from previous studies
Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Aflatoxin M1 in milk from urban and rural farmhouses of Punjab, Pakistan Shahzad Zafar Iqbal
a b
c
, Muhammad Rafique Asi & Jinap Selamat
b
a
Department of Applied Chemistry , Government College University Faisalabad , Faisalabad , Pakistan b
Faculty of Food Science and Technology , Food Safety Research Centre (FOSREC) , Universiti Putra Malaysia , Serdang , Selangor , Malaysia c
Food Toxicology Lab, Plant Protection Division , Nuclear Institute for Agriculture and Biology , Faisalabad , Pakistan Accepted author version posted online: 24 Jul 2013.
To cite this article: Food Additives & Contaminants: Part B (2013): Aflatoxin M1 in milk from urban and rural farmhouses of Punjab, Pakistan, Food Additives & Contaminants: Part B: Surveillance, DOI: 10.1080/19393210.2013.828322 To link to this article: http://dx.doi.org/10.1080/19393210.2013.828322
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Aflatoxin M1 in milk from urban and rural farmhouses of Punjab, Pakistan Shahzad Zafar Iqbal
a,b
, Muhammad Rafique Asic,
a
Department of Applied Chemistry, Government College University Faisalabad, 38000, Pakistan Food Safety Research Centre (FOSREC); Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia c Food Toxicology Lab, Plant Protection Division, Nuclear Institute for Agriculture and Biology, Faisalabad, Pakistan
rip t
an us c
Corresponding Author Email: [email protected] (Shahzad Z. Iqbal) Cell: 0060-12-6861121
M
Abstract
Aflatoxin M1 (AFM1) was determined in 107 milk samples collected from urban and rural
d
farmhouses from Punjab, Pakistan, by HPLC with fluorescence detection. An incidence rate of
pt e
64% (38/59) in milk samples from urban farmhouses was found with a mean concentration of 0.064 ± 0.023 μg L-1. In rural samples about 52% (25/48) milk samples were contaminated with AFM1 with a mean of 0.04 ± 0.034 μg L-1. 42% milk samples from urban and 27% from rural
ce
farmhouses were well above the limit permitted by the EU. However, only 15 and 8% milk samples from urban and rural farmhouses respectively, exceeded the limit of USDA/Codex regulations. The results showed that the contamination of milk with AFM1 from Punjab, Pakistan, when compared with Codex limit are present and needs continuous monitoring. The
Ac
Downloaded by [University of Minnesota Libraries, Twin Cities] at 04:04 25 July 2013
b
awareness and education among dairy farmers on the potential health risks associated with aflatoxins should be communicated.
Keywords: Milk; AFM1; Urban and Rural farmhouses; Punjab; Pakistan
Introduction Mycotoxins are natural and unavoidable toxic secondary metabolites produced by fungi, which contaminate agricultural staples in the field or during harvest, transport and storage (Iqbal et al. 2010). It has been estimated by the Food and Agriculture Organization that approximately 25% of world’s food crops are affected each year by mycotoxins (Ono et al. 2011). Aflatoxins
rip t
(AFs) are the most toxic class of mycotoxins produced mainly by Aspergillus flavus and A. 2011c). Hydroxylation of AFB1 produces AFM1 which is secreted in the milk of lactating
animals consuming contaminated feed (Ruangwises and Ruangwises 2010; Asi et al. 2012). The
an us c
International Agency for Research on Cancer (2002) has classified AFB1 as class 1 carcinogenic to humans, however the carcinogenicity of AFM1 is approximately ten times less as compared to
AFB1 (Nuryono et al. 2009). It has been evident that AFs are responsible for acute poisoning (aflatoxicosis), for hepatocellular carcinoma, for growth impairment in children and immunosuppresion (Cassel et al. 2001; Sidhu et al. 2009; Fallah 2010). Considering their risk to human health the incidence and presence of AFM1 in dairy products is regarded as a significant
M
hazard for food safety and public health (Cucci et al. 2007).
To minimize the health risk of AFM1 contamination in milk and dairy products most
d
countries implemented strict regulations, however in developing countries like Pakistan an adequate regulation about admissible limits of AFM1 is still lacking. The policy applied by the
pt e
European Union and the USA to ensure food safety is based on the assumption that strict controls on feed and milk would prevent contamination of dairy products (Creppy 2002). The EU maximum level (ML) for AFM1 in milk intended for adults is 0.050 μg kg-1, whereas the
ce
limit intended for infants or for baby-food production was set at 0.025 μg kg-1 (European Commission 2006b). However, the Codex Alimentarius has established a maximum level of 0.5 μg kg-1 for AFM1 in milk (Codex Alimentarius, 1995).
Ac
Downloaded by [University of Minnesota Libraries, Twin Cities] at 04:04 25 July 2013
parasiticus (Iqbal et al. 2011a and b), the most toxic form being aflatoxin B1 (AFB1) (Iqbal et al.
Referring to scientific literature, very limited data has been published on the occurrence
of AFM1 in milk from Pakistan. Previously (Iqbal et al. 2011a; Asi et al. 2012) and other studies (Hussain and Anwar 2008; Hussain et al. 2010; Hussain et al. 2008) showed high percentages of
AFM1 contaminated milk samples. In the present study milk samples have been collected from rural and urban farmhouses to evaluate the feeding practices and their effect on the incidence of AFM1. The main objectives were to evaluate the incidence and contamination level of AFM1 in
milk samples and to compare samples that exceed the European Union (EU) and Codex limits. The results drawn from the present study will be helpful to implement strict regulation and
rip t
Materials and Methods Sampling A total of 107 samples of raw milk from 43 farmhouses, 48 from rural farmhouses and 59
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samples from urban farmhouses, were collected during May 2011 to August 2011 from major districts of Punjab, Pakistan. The selection of urban and rural farmhouses was based on the fact that in most of the urban farmhouses the animal feed consists of more concentrated feed like cottonseed cake, corn, soybean, threshed wheat straw, paddy straw, wheat bran and dry breads
while in rural farmhouses mostly green fodder is used with less or even rare concentrated feed. The size of milk samples was at least 1 liter. During sample collection and transportation, the
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samples were kept in ice packets in an icebox. The samples were either analyzed immediately or stored in a freezer at -4 ºC in case of delayed analysis.
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Chemicals and Regents The standard of AFM1 (10 μg ml-1 in acetonitrile) and acetonitrile of HPLC grade were
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purchased from Sigma Aldrich (Steinheim, Germany). Immunoaffinity columns (IAC) AflaM1TM were purchased from VICAM (Watertown, MA, USA). To evaluate the concentration of AFM1, a standard curve was prepared with concentrations of 0.05, 0.1, 0.5, 0.10, 0.50 and 10.0 μg L-1
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solutions and stored in capped vials in a refrigerator at -4ºC until further analysis. The water used was double-distilled and all other chemicals and reagents were at least of analytical grade. Extraction Milk samples were heated until 37 ºC in a water bath and then centrifuged at 2500 rpm for 5
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examination on utilization of concentrated animal feed and regulate limits for these mycotoxins.
minutes to separate the fat layer. After centrifugation samples were filtered through Whatman No. 5 filter paper and 50 ml filtrate was transferred into a syringe barrel attached to an IAC and passed at a rate 2 ml/min using a solid phase extraction manifold. The column was washed with
20 ml double distilled water and AFM1 was eluted with 4 ml pure acetonitrile passing the IAC in approximately 60 s. Finally the eluate was evaporated to dryness using a gentle stream of N2 at 40 ºC and the residue was dissolved in 1ml mobile phase at the time of HPLC analysis.
HPLC Conditions The HPLC equipment used was a Shimazdzu LC-10A series (Kyoto, Japan) with fluorescence detector FLD (RF-530) with excitation and emission wavelength of 365 and 435 nm, respectively, equipped with a Discovery C18 column (4.6 x 250 mm, 5 μm) of Supelco (Bellefonte, USA). Acetonitrile/water (25: 75 v/v) was used as mobile phase at a flow rate of 1.0 ml min-1. The response of the calibration curve was linear (R2 = 0.9991). The limit of detection
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Validation
Precision and recovery were carried out by performing tests on raw milk samples (four replicates
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each) fortified with AFM1 at 0.02, 0.05, 0.07 and 0.1 μg L-1. The recoveries in these spiked milk samples were 83, 92, 92 and 90%, with RSD from 8, 9, 12, and 4%, respectively. Statistical Analysis
All data were presented as mean ± standard deviation. Correlation and regression analyses were applied to calculate R2. Significant differences of mean between urban and rural milk samples
Results and Discussion
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were determined using Student paired t-test (SPSS, IBM PASW statistics 19, USA).
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All analytical results are presented in table 1. AFM1 was found above the measurable level (0.004 μg L-1) in 64 % (38/59) and 52% (25/48) of milk samples from urban and rural farmhouses, respectively. Considering the European Commission limit, 25 (26.7%) and 13
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(17.4%) milk samples from urban and rural farmhouses had AFM1 concentrations in excess of the maximum limit (0.05 μg L-1), respectively. Less numbers, 9 samples (15%) from urban and 4 (8%) milk samples from rural farmhouses were above the Codex limit of 0.5 μg L-1 (Table 2).
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(LOD) and limit of quantification (LOQ) were 0.004 μg L-1 and 0.012 μg L-1, respectively.
The results show statistically significant difference of AFM1 contamination in milk samples from
urban farmhouses when compared to rural farmhouses by applying student paired t test at (p < 0.05).
The findings of the present study on the occurrence level of AFM1 contamination in milk samples were comparable with a previous study (Iqbal et al. 2011a) in which 88 samples out of 178 were contaminated, with a mean level of AFM1 0.046 μg L-1 and a range from LOD to 0.35
μg/l. Results were comparable with reports from Korea (Kim et al. 2000), Iran (Heshmati and Milani 2010) and India (Rastogi et al. 2004), as presented in Table 3. However, some studies reported lower levels of AFM1 contamination in milk samples: Nigeria (Atanda et al. 2007), Italy (Nachtmann et al. 2007), Morocco (Zinedine et al. 2007) and Turkey (Unusan, 2006). Occurrence of AFM1 in raw milk samples from 14 districts of the Punjab Province, Pakistan has
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been reported by Hussain and Anwar (2008). All samples analyzed were contaminated with
previous studies. Martins and Martins (2000) from Portugal reported AFM1 in 81 raw milk and
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84% UHT milk samples. Similar results were reported by Galvano et al. (2001), who found 78% of liquid milk and 53% of dry milk samples to be contaminated with AFM1, of which 15% from urban and 8% from rural farmhouses were above the codex or USDA limit.
The present findings demonstrate that AFM1 contamination in samples from urban farmhouses is higher when compared with samples collected from rural farmhouses. This could be explained because in urban farmhouses animal feed mostly consists of concentrated feed i.e.
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cottonseed cake, corn, soybean, threshed wheat straw, paddy straw, wheat bran and dry bread. All these commodities are vulnerable to mould attack producing AFs (Dutton and Kinsey 1996; Sassahara et al. 2005; Hussain et al. 2008; Pei et al. 2009). However, in rural farmhouses the
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animal feed mostly consists of green fodder mixed with dry crushed wheat straws. From
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previous studies, it was well documented that AFM1 contamination of milk is a result of the exposure of AFB1 contaminated feedstuffs to dairy animals (Unusan 2006). Therefore, whereas variations of AFM1 levels among different studies could be related to geographic and climatic
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differences, the most important factor is the difference in feeding systems and farm management practices (Ghazani 2009). To achieve and maintain low levels of AFM1 in milk, feed samples must be evaluated
routinely for aflatoxins and those with excess contamination should be discarded. Records
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AFM1 and 99.4% exceeded the European Union limit of 0.05 μg L-1, which was comparable with
should be maintained for all feed, feeding practices, milk contamination and animal health on all cases on aflatoxin contamination of milk. Owners of urban farmhouses should take measures to examine the quality of animal feed. Furthermore government agencies should monitor animal feed and milk samples routinely and enforce food and feed laws in order to minimize health hazards for human and animal health due to these toxins.
Acknowledgements The authors appreciate the funding provided by the Higher Education Commission, Islamabad,
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Pakistan for this study.
Asi MR, Iqbal SZ, Ariño A, Hussain A. 2012. Effect of seasonal variations and lactation times on 25 (1): 34-38.
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aflatoxin M1 contamination in milk of different species from Punjab, Pakistan. Food Contr. Atanda O, Oguntubo A, Adejumo O, Ikeorah J, Akpan I. 2007. Aflatoxin M1 contamination of milk and ice cream in Abeokuta and Odeda local governments of Ogun State, Nigeria. Chemosphere 68: 1455–1458.
Cassel EK, Campbell B, Draper M, Epperson B. 2001. Aflatoxins: Hazards in grain/aflatoxicosis
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Codex Alimentarius, 1995. Codex General Standard for contaminants and toxns in food and feed, Codex Standard 193-1995, Adopted 1995. Revised 1997, 2006, 2008, 2009.
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Creppy EE. 2002. Update of survey, regulation and toxic effects of mycotoxins in Europe. Toxicol. Let. 127:19–28.
Cucci C, Mignani AG, Dall’Asta C, Pela R, Dossena A. 2007. A portable fluorometer for the
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Table 1: AFM1 contamination level in milk samples from urban and rural farmhouses Mean (
μg L-1)
Positive samples
Urban Samples
59
38 (64)
< 0.004 to 0.98
0.064 a ± 0.02
Rural samples
48
25 (52)
< 0.004 to 0.71
0.045 b ± 0.03
Total
107
63 (59)
< 0.004 to 0.98
0.055 ± 0.03
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Range (
μg L-1)
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Mean values with different letters are statistically different at (p < 0.05) applying Student paired t-test
Sample type
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farmhouses
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Table 2: Incidence of AFM1 contamination in milk samples from urban and rural Positive
Incidence
n > 0.05 μg L-1
n > 0.05 μg L-1
n > 0.5c μg L-1
n > EU
38 (64)
13 (22%)
16 (27%)
9 (15%)
25 (42%)
Milk samples RFb 25 (52)
12 (25%)
9 (19%)
4 (8%)
13 (27%)
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Milk samples UF
UF = Urban farmhouse RF= Rural farmhouse c Codex/USDA limit b
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Sample type
Positive
n > 0.05 µg/kg
Mean (μg L- Country 1 )
Reference
178 180 210 87 12 10 316 54 129 360 120
49% 79% 55% 87% 100% 100% 2.2% 89% 58.1% 42.5% 52.5%
16% 58% 33% 99% 100% 100% 0.6% 7.4% 47% 52.5% 57.5%
0.046 0.057 0.087 0.299 2.04 4.00 0.027 0.019 0.11 0.027 (buffalo) 0.044
Pakistan Korea Iran India
Iqbal et al. (2011) Kim et al. (2000) Heshmati and Milani (2010) Rastogi et al. (2004)
Nigeria
Atanda et al. (2007)
Italy Morocco Turkey
Nachtmann et al. (2007) Zinedine et al. (2007) Unusan (2006
Pakistan
Hussain and Anwar (2008)
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Samples
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Table 3: Occurrence of AFM1 contamination in milk samples from previous studies
