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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 raw cow and buffalo milk in Shush city of Iran a
b
b
Abolfazl Kamkar , Samira Yazdankhah , Abdolreza Mohammadi Nafchi & Amir Sasan Mozaffari Nejad
c d
a
Department of Food Hygiene , Faculty of Veterinary Medicine , , University of Tehran , Tehran , Iran b
Department of Food Science and Technology , Islamic Azad University , Damghan Branch, Damghan , Iran c
Institute of Science and Technology, Center for Biotechnology , Jawaharlal Nehru Technological University , Hyderabad , Andhra Pradesh , India d
Member of Young Researchers and Elite Club , Garmsar Branch, Islamic Azad University , Garmsar , Iran Accepted author version posted online: 05 Aug 2013.
To cite this article: Food Additives & Contaminants: Part B (2013): Aflatoxin M1 in raw cow and buffalo milk in Shush city of Iran, Food Additives & Contaminants: Part B: Surveillance, DOI: 10.1080/19393210.2013.830277 To link to this article: http://dx.doi.org/10.1080/19393210.2013.830277
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
1
Aflatoxin M1 in raw cow and buffalo milk in Shush city of Iran
2 3
Abolfazl Kamkara, Samira Yazdankhahb, Abdolreza Mohammadi Nafchib, Amir Sasan
4
Mozaffari Nejadc,d,*
t a
7
Tehran, Iran
8
b
9
Damghan, Iran
cr ip
6
us
Department of Food Hygiene, Faculty of Veterinary Medicine, University of Tehran,
an
Department of Food Science and Technology, Islamic Azad University, Damghan Branch,
10
c
11
Technological University, Hyderabad, Andhra Pradesh, India
12
d
13
Garmsar, Iran
15
M
Member of Young Researchers and Elite Club, Garmsar Branch, Islamic Azad University,
ep te d
14
Center for Biotechnology, Institute of Science and Technology, Jawaharlal Nehru
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16 17
* Corresponding author:
18
Telephone: +98 21 61117042.
19
Fax: +98 21 66933222
20
E-mail addresses: [email protected] and [email protected] 1
Abstract Aflatoxin M1 (AFM1) contamination is evaluated in 120 samples of raw milk from cow
23
and buffalo (60 each), collected randomly in the Shush (southwest Iran). Enzyme-linked
24
immunoabsorbent assay (ELISA) was applied to analyze AFM1 in the samples. Aflatoxin M1
25
was detected in 44 (69%) raw cow milk samples with a mean of 55 ng/l at a range of 3.6-419
26
ng/l) and in 46 (79%) raw buffalo milk samples with a mean of 116 ng/l at a range of 13-423
27
ng/l. In all samples the AFM1 concentration was lower than the Iranian national standard and
28
FDA limit of 500 ng/l. According to the European Union and Codex Alimentarius
29
Commission 18 (28%) and 32 (52%) of cow and buffalo raw milk samples is above the 50
30
ng/l limit, respectively. Results showed that AFM1 contamination of raw milk could pose a
31
problem for public health, since all age groups, including infants and children consume this
32
product.
33
Keywords: Aflatoxin M1; Raw milk; Cow; Buffalo; ELISA; Iran.
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Introduction
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Aflatoxins (AFs) are the best known and most intensively researched mycotoxins world-
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wide, which are produced by different Aspergillus (A) species, like A. flavus, A. parasiticus,
40
A. bombycis, A. ochraceoroseus, A. nomius and A. pseudotamarii. It contaminates
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agricultural commodities and thus food and feed, particularly in critical temperature and
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humidity conditions before or during harvest, or because of inappropriate storage (Lee et al.,
43
2009; Firdous et al., 2012. The main types of aflatoxins are B1, B2, G1 and G2 which are 2
45
products, as aflatoxin M1 and M2 (Tavakoli et al., 2012; Tavakoli et al., 2013). AFM1 is the
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monohydroxylated metabolite of AFB1 formed in liver by means of microsomial cytochrome
47
P450- associated enzymes and excreted through body fluids such as milk, urine, faeces and
48
blood (Ardic et al., 2009a; Ghanem and Orfi, 2009).
t
currently produced in plant product. Biotransformed aflatoxins may occur in milk and dairy
There is a linear relationship between the amount of AFM1 in milk and AFB1 in the feed
50
which is consumed by animals (Bakirci, 2001; Abbes et al., 2012). Monitoring studies
51
reported that approximately 0.3–6.2% of aflatoxin B1 ingested by livestock is transformed to
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aflatoxin M1 in milk. However, transmission rate varies from animal to animal, day to day
53
and one milking process to the next. Aflatoxin M1 could be detected in milk 12-24 h after the
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first ingestion of aflatoxin B1. When the intake of aflatoxin B1 is stopped, the aflatoxin M1
55
concentration in the milk decreases to an undetectable level after 72 hours (Van Egmond,
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1989; Ardic, 2009b, Fallah, 2010a). The International Agency for Research on Cancer
57
(IARC) of the World Health Organization (WHO) included AFB1 as primary and AFM1 as
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secondary group of carcinogenic compounds (Tavakoli et al., 2012). Several studies showed
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that AFM1 is relatively stable to heat and high temperature with pasteurization, sterilization
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(UHT techniques), autoclaving, freezing, fermenting, preparation and cold-storage of various
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dairy products and also the level of aflatoxin M1 does not change in contaminated milk
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(Bakici, 2001; Abbes et al., 2012; Tavakoli et al., 2013). Most of the developed countries
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have set or proposed legal regulations for AFM1 levels in milk and dairy products to reduce
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this hazard. These regulations vary from one country to another country and are dependent on
65
economic considerations (Fallah, 2010b; Rahimi et al., 2010).
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The Khuzestan province of Iran is a major source for abundant milk production, obtained
67
from cow, buffalo, goat, sheep and camel. Therefore, this study was carried out with the
3
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objective of investigating the presence of Aflatoxin M1 in raw milk from cow and buffalo in
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southwest Iran.
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t Materials and Methods
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Samples
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A total of 120 samples of raw milk, cow (n= 60) and buffalo (n= 60), were taken randomly
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from different parts of Shush in Khuzestan province of Iran during 6 months (February to
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July 2012). These months were divided into three seasons: winter, spring and summer. All
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samples were transported to the laboratory in an icebox at 2-4°C and kept at -20°C until
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analysis.
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Sample preparation
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Raw milk samples were centrifuged at 2000 × g for 5 min. After centrifugation the upper
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fat layer was removed by aspirating with a Pasteur pipette and used for AFM1 analysis.
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Analysis
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Quantitative analysis of aflatoxin M1 in the raw milk samples was performed by
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competitive ELISA using an AFM1 kit (Euro Clone, EuroClone S.p.A., Pero (Milano) Italy).
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200 microliters of the AFM1 standard solutions and test samples in duplicate were added to
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the wells of micro-titer plates pre-coated with antibodies for AFM1 and incubated for 30 min
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at room temperature (20-25°C) in the dark. The liquid was poured out and the wells were
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filled with approx. 300 µl washing buffer and poured out the liquid again. This washing step
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was repeated twice. In the next stage 200 µl of enzyme conjugate was added to occupy the 4
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enzyme conjugates. Then 50 µl of enzyme substrate and 50 µl of chromogen were added to
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the wells and incubated for 10 min at room temperature in the dark. The reaction was stopped
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by adding 50 µl stop solution to each well and absorbance was measured at 450 nm in a
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spectrophotometer ELISA plate reader. Results were expressed as the means of two analyses.
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The absorbances obtained for standards and samples were divided by the absorbance of the
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first standard (the zero standard) and multiplied by 100. Therefore, the zero standards are
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considered 100% and the absorbance values are expressed in percentage.
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Statistical analysis
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remaining free binding sites and in the washing step 250 µl of buffer washed any unbound
Data were analysed by Statistics 19 SPSS IBM software. To evaluate differences in the
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means between months and two groups of samples, t-test and ANOVA for independent
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samples were applied. Differences between values were considered significant at P ≤ 0.05.
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Results
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The standard curve was linear from 50 to 100 ng aflatoxin M1/l. The correlation
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coefficient R2 was 0.9998. The limit of detection (LOD) was defined as the minimum level at
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which the analyte could be detected and was determined to be 3 ng/l. The incidence and
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levels of aflatoxin M1 in raw cow and buffalo milk samples are presented in Tables 1 and 2
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respectively. The concentration of AFM1 in all samples was lower than the Institute of
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Standards and Industrial Research of Iran and FDA limit of 500 ng/l (US FDA, 1996; ISIRI,
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2005). But 28% (18/60) of cow milk and 52% (32/60) of buffalo milk samples contained
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91
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higher levels than the maximum limit of 50 ng/l of the European Union and Codex
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Alimentarius Commission (Codex Alimentarius Commission, 2001; European Commission,
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2006). Regarding seasonal effect influences, mean concentration of aflatoxin M1 in the
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buffalo samples collected in the three seasons was significantly (P < 0.05) higher than in cow
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samples.
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Discussion
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Aflatoxin M1 contamination of milk and milk products is the result of feeding cows
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material containing aflatoxin B1. The concentration of aflatoxin B1 in animal feed is
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influenced by feed type, time and method of harvesting, temperature and moisture content.
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A. flavus, and A.parasiticus can easily grow in feed having a moisture content between 13-
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18% and an environmental humidity between 50-60% (Bakirci, 2001; Ardic, 2009). Our
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findings revealed a high incidence of aflatoxin M1 in raw milk samples. Also, statistical
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analysis of the data shows that the percentage of aflatoxin M1 contamination in buffalo milk
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is more than in cow milk, which was not similar to a study conducted by Rahimi et al. (2010).
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In another study, Bilandzic et al. (2010) reported less AFM1 (1.6%) at detectable levels in
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raw milk samples, but in a recent study Davoudi et al. (2011) determined AFM1 in 100% of
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the investigated raw milk samples. In previous surveys conducted in Iran by TLC methods,
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Kamkar (2005) reported that 77% (85 of 111) raw milk samples were contaminated with
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AFM1 in Sarab and 40% had levels above the European limit. A survey from Morocco (El
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Marnissi et al. 2012) reported aflatoxin M1 in raw milk in 13 out of 48 samples (27%),
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ranging 10-100 ng/kg and within the positive samples 4 (~8% of the total) were above the EU 6
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contained AFM1 and 61% of the positive samples were higher than the EU limit (Assem et
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al., 2011). Similar results were documented from Tunisia by Abbes et al. (2012), who found
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by an ELISA method that 61% of 112 raw milk samples included aflatoxin M1. An earlier
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study by Lee et al (2009) from South Korea reported 48 (48%) of 100 raw milk samples
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contained AFM1. In Punjab, Pakistan, Hussain et al. (2008) analysed 480 raw milk samples,
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360 of buffalo and 120 of cow. Percentages of AFM1 contamination in buffalo and cow milk
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were 42.5 and 52.5%, respectively. Several studies by other researchers are shown in Table 3.
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limit. A study on raw milk samples in Lebanon showed that 28 (74%) of 38 raw milk samples
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Conclusions
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Aflatoxin M1 is determined in a high percentage of milk samples, which can justify to
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perform a monitoring program for the total milk chain, as especially infants and children
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consume these products. Control could start from the farm, by regular control of aflatoxin B1
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contamination of animal feed and control of its storage conditions. The use of a food control
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system, like the HACCP system in the food industries is suggested as an efficient means of
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limiting mycotoxin contamination, especially for aflatoxin in Iranian food.
156 157 158 159
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Ardic M. 2009b. Occurrence of aflatoxin M1 in raw ewe’s milk produced in Sanliurfa,
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produced in Van province of Turkey. Food Control. 12: 47-51. Bilandzic N, Varenina I, Solomun B. 2010. Aflatoxin M1 in raw milk in Croatia. Food Control. 21: 1279-1281.
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Codex Alimentarius Commission. 2001. Comments submitted on the draft maximum level
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for Aflatoxin M1 in milk. Codex committee on food additives and contaminants 33rd
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Ardic M, Karakaya Y, Atasever M, Adiguzel G. 2009a. Aflatoxin M1 levels of Turkish white
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El Marnissia B, Belkhoua R, Morgavic DP, Bennanib L, Boudra H. 2012. Occurrence of
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aflatoxin M1 in raw milk collected from traditional dairies in Morocco. Food Chem
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setting maximum levels of certain contaminants in foods. Official Journal of the European
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Hussain I, Anwar J, Munawar MA, Asi MR. 2008. Variation of levels of aflatoxin M1 in raw
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milk from different localities in the central areas of Punjab, Pakistan. Food Control. 19:
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Lee JE, Kwak B-M, Ahn J-H, Jeon T-H. 2009. Occurrence of aflatoxin M1 in raw milk in
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milk of five dairy species in Ahvaz, Iran. Food Chem Toxicol. 48: 129-131. Rastogi S, Dwivedi PD, Khanna SK, Das M. 2004. Detection of aflatoxin M1 contamination
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Tajkarimi M, Shojaee Aliabadi F, Salah Nejad M, Poursoltani H, Motallebi AA, Mahdavi H.
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Tavakoli HR, Riazipour M, Kamkar A, Rafati H, Mozaffari Nejad AS. 2012. Occurrence of aflatoxin M1 in cheese samples from Tehran, Iran. Food Control. 23: 293-295.
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Rahimi E, Bonyadian M, Rafei M, Kazemeini HR. 2010. Occurrence of aflatoxin M1 in raw
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Aflatoxin M1 (cpg 7106.210), FDA Compliance Policy Guides, Washington, DC, FDA, p.
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219.
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Van Egmond HP. 1989. Introduction. In: van Egmond, H.P. (Ed.), Mycotoxins in dairy products. Elsevier Applied Science, London and New York, pp. 1-10.
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226 227 228 229
4 (40%) 4.5 14.1 6.1a ± 2.7 20 (67%) 3.6 100.9 32.6a ± 8.4 20 23.2 419.5 114.9b ± (100%) 36.3 Total 60 16 (31%) 44 (69%) 3.6 419.5 55.5 a 230 Values in parentheses indicate % negative and positive samples. b 231 Mean ± SE with different letters is significantly different. c 232 The EU limit for AFM1 is 50 ng/l for raw milk. 233 234 235 236 237 238 239 Table 2 240 Distribution by season of aflatoxin M1 (ng/l) in 60 raw buffalo milk samples. Tested Negativea Positivea Minimum Maximum Mean ± SEb Season
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6 (60%) 10 (33%) 0 (0%)
18(28)
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10 30 20
93.2a ± 47.1 109.1a ± 40.5 Summer 20 2 (10%) 18 (90%) 12.7 367.3 138.4a ± 40.9 Total 60 14 (21%) 46 (79%) 12.7 422.7 116.2 ± 25.1 a 241 Values in parentheses indicate % negative and positive samples. b 242 Mean ± SE with different letters is significantly different. c 243 The EU limit for AFM1 is 50 ng/l for raw milk. 244 245 246 247 248 249 250 251 252 253 254 Winter Spring
10 30
2 (20%) 10 (33%)
8 (80%) 20 (67%)
19.1 19.5
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Winter Spring Summer
Exceeding regulation,c n (%) 0(0) 4(13) 14(70)
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Table 1 Distribution by season of aflatoxin M1 (ng/l) in 60 raw cow milk samples. Tested Negativea Positivea Minimum Maximum Mean ± SEb Season
11
209.5 422.7
Exceeding regulation,c n (%) 4 (40) 14 (47) 14 (70) 32 (52)
255 256
Table 3 Occurrence and levels of aflatoxin M1 in raw milks reported in previous studies. Country Samples Positive (%) Range Method Reference 12
33%
28-164
ELISA
Rastogi et al. (2004)
Iran
111
76.6
15-280
TLC
Kamkar (2005)
Iran
98
100
3-329
HPLC
Tajkarimi et al. (2007)
Turkey
90
88.9
17-232
ELISA
Ardic (2009)
Turkey
45
91.1
75
ELISA
Kart et al. (2009)
Syrian
108
83.3
6-690
ELISA
Iran
311
42.1
50
ELISA
Croatia
61
1.6
0.6-58.7
ELISA
Iran
100
100
30-630
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TLC
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India
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(ng/l)
Ghanem and Orfi (2009) Rahimi et al. (2010)
Bilandzic et al. (2010) Davoudi et al. (2011)
Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Aflatoxin M1 in raw cow and buffalo milk in Shush city of Iran a
b
b
Abolfazl Kamkar , Samira Yazdankhah , Abdolreza Mohammadi Nafchi & Amir Sasan Mozaffari Nejad
c d
a
Department of Food Hygiene , Faculty of Veterinary Medicine , , University of Tehran , Tehran , Iran b
Department of Food Science and Technology , Islamic Azad University , Damghan Branch, Damghan , Iran c
Institute of Science and Technology, Center for Biotechnology , Jawaharlal Nehru Technological University , Hyderabad , Andhra Pradesh , India d
Member of Young Researchers and Elite Club , Garmsar Branch, Islamic Azad University , Garmsar , Iran Accepted author version posted online: 05 Aug 2013.
To cite this article: Food Additives & Contaminants: Part B (2013): Aflatoxin M1 in raw cow and buffalo milk in Shush city of Iran, Food Additives & Contaminants: Part B: Surveillance, DOI: 10.1080/19393210.2013.830277 To link to this article: http://dx.doi.org/10.1080/19393210.2013.830277
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
1
Aflatoxin M1 in raw cow and buffalo milk in Shush city of Iran
2 3
Abolfazl Kamkara, Samira Yazdankhahb, Abdolreza Mohammadi Nafchib, Amir Sasan
4
Mozaffari Nejadc,d,*
t a
7
Tehran, Iran
8
b
9
Damghan, Iran
cr ip
6
us
Department of Food Hygiene, Faculty of Veterinary Medicine, University of Tehran,
an
Department of Food Science and Technology, Islamic Azad University, Damghan Branch,
10
c
11
Technological University, Hyderabad, Andhra Pradesh, India
12
d
13
Garmsar, Iran
15
M
Member of Young Researchers and Elite Club, Garmsar Branch, Islamic Azad University,
ep te d
14
Center for Biotechnology, Institute of Science and Technology, Jawaharlal Nehru
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5
16 17
* Corresponding author:
18
Telephone: +98 21 61117042.
19
Fax: +98 21 66933222
20
E-mail addresses: [email protected] and [email protected] 1
Abstract Aflatoxin M1 (AFM1) contamination is evaluated in 120 samples of raw milk from cow
23
and buffalo (60 each), collected randomly in the Shush (southwest Iran). Enzyme-linked
24
immunoabsorbent assay (ELISA) was applied to analyze AFM1 in the samples. Aflatoxin M1
25
was detected in 44 (69%) raw cow milk samples with a mean of 55 ng/l at a range of 3.6-419
26
ng/l) and in 46 (79%) raw buffalo milk samples with a mean of 116 ng/l at a range of 13-423
27
ng/l. In all samples the AFM1 concentration was lower than the Iranian national standard and
28
FDA limit of 500 ng/l. According to the European Union and Codex Alimentarius
29
Commission 18 (28%) and 32 (52%) of cow and buffalo raw milk samples is above the 50
30
ng/l limit, respectively. Results showed that AFM1 contamination of raw milk could pose a
31
problem for public health, since all age groups, including infants and children consume this
32
product.
33
Keywords: Aflatoxin M1; Raw milk; Cow; Buffalo; ELISA; Iran.
35 36
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Introduction
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Aflatoxins (AFs) are the best known and most intensively researched mycotoxins world-
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wide, which are produced by different Aspergillus (A) species, like A. flavus, A. parasiticus,
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A. bombycis, A. ochraceoroseus, A. nomius and A. pseudotamarii. It contaminates
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agricultural commodities and thus food and feed, particularly in critical temperature and
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humidity conditions before or during harvest, or because of inappropriate storage (Lee et al.,
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2009; Firdous et al., 2012. The main types of aflatoxins are B1, B2, G1 and G2 which are 2
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products, as aflatoxin M1 and M2 (Tavakoli et al., 2012; Tavakoli et al., 2013). AFM1 is the
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monohydroxylated metabolite of AFB1 formed in liver by means of microsomial cytochrome
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P450- associated enzymes and excreted through body fluids such as milk, urine, faeces and
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blood (Ardic et al., 2009a; Ghanem and Orfi, 2009).
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currently produced in plant product. Biotransformed aflatoxins may occur in milk and dairy
There is a linear relationship between the amount of AFM1 in milk and AFB1 in the feed
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which is consumed by animals (Bakirci, 2001; Abbes et al., 2012). Monitoring studies
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reported that approximately 0.3–6.2% of aflatoxin B1 ingested by livestock is transformed to
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aflatoxin M1 in milk. However, transmission rate varies from animal to animal, day to day
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and one milking process to the next. Aflatoxin M1 could be detected in milk 12-24 h after the
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first ingestion of aflatoxin B1. When the intake of aflatoxin B1 is stopped, the aflatoxin M1
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concentration in the milk decreases to an undetectable level after 72 hours (Van Egmond,
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1989; Ardic, 2009b, Fallah, 2010a). The International Agency for Research on Cancer
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(IARC) of the World Health Organization (WHO) included AFB1 as primary and AFM1 as
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secondary group of carcinogenic compounds (Tavakoli et al., 2012). Several studies showed
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that AFM1 is relatively stable to heat and high temperature with pasteurization, sterilization
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(UHT techniques), autoclaving, freezing, fermenting, preparation and cold-storage of various
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dairy products and also the level of aflatoxin M1 does not change in contaminated milk
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(Bakici, 2001; Abbes et al., 2012; Tavakoli et al., 2013). Most of the developed countries
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have set or proposed legal regulations for AFM1 levels in milk and dairy products to reduce
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this hazard. These regulations vary from one country to another country and are dependent on
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economic considerations (Fallah, 2010b; Rahimi et al., 2010).
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The Khuzestan province of Iran is a major source for abundant milk production, obtained
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from cow, buffalo, goat, sheep and camel. Therefore, this study was carried out with the
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objective of investigating the presence of Aflatoxin M1 in raw milk from cow and buffalo in
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southwest Iran.
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Samples
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A total of 120 samples of raw milk, cow (n= 60) and buffalo (n= 60), were taken randomly
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from different parts of Shush in Khuzestan province of Iran during 6 months (February to
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July 2012). These months were divided into three seasons: winter, spring and summer. All
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samples were transported to the laboratory in an icebox at 2-4°C and kept at -20°C until
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analysis.
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Sample preparation
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Raw milk samples were centrifuged at 2000 × g for 5 min. After centrifugation the upper
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fat layer was removed by aspirating with a Pasteur pipette and used for AFM1 analysis.
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Analysis
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Quantitative analysis of aflatoxin M1 in the raw milk samples was performed by
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competitive ELISA using an AFM1 kit (Euro Clone, EuroClone S.p.A., Pero (Milano) Italy).
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200 microliters of the AFM1 standard solutions and test samples in duplicate were added to
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the wells of micro-titer plates pre-coated with antibodies for AFM1 and incubated for 30 min
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at room temperature (20-25°C) in the dark. The liquid was poured out and the wells were
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filled with approx. 300 µl washing buffer and poured out the liquid again. This washing step
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was repeated twice. In the next stage 200 µl of enzyme conjugate was added to occupy the 4
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enzyme conjugates. Then 50 µl of enzyme substrate and 50 µl of chromogen were added to
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the wells and incubated for 10 min at room temperature in the dark. The reaction was stopped
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by adding 50 µl stop solution to each well and absorbance was measured at 450 nm in a
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spectrophotometer ELISA plate reader. Results were expressed as the means of two analyses.
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The absorbances obtained for standards and samples were divided by the absorbance of the
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first standard (the zero standard) and multiplied by 100. Therefore, the zero standards are
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considered 100% and the absorbance values are expressed in percentage.
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Statistical analysis
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remaining free binding sites and in the washing step 250 µl of buffer washed any unbound
Data were analysed by Statistics 19 SPSS IBM software. To evaluate differences in the
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means between months and two groups of samples, t-test and ANOVA for independent
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samples were applied. Differences between values were considered significant at P ≤ 0.05.
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Results
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The standard curve was linear from 50 to 100 ng aflatoxin M1/l. The correlation
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coefficient R2 was 0.9998. The limit of detection (LOD) was defined as the minimum level at
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which the analyte could be detected and was determined to be 3 ng/l. The incidence and
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levels of aflatoxin M1 in raw cow and buffalo milk samples are presented in Tables 1 and 2
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respectively. The concentration of AFM1 in all samples was lower than the Institute of
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Standards and Industrial Research of Iran and FDA limit of 500 ng/l (US FDA, 1996; ISIRI,
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2005). But 28% (18/60) of cow milk and 52% (32/60) of buffalo milk samples contained
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higher levels than the maximum limit of 50 ng/l of the European Union and Codex
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Alimentarius Commission (Codex Alimentarius Commission, 2001; European Commission,
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2006). Regarding seasonal effect influences, mean concentration of aflatoxin M1 in the
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buffalo samples collected in the three seasons was significantly (P < 0.05) higher than in cow
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samples.
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Discussion
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Aflatoxin M1 contamination of milk and milk products is the result of feeding cows
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material containing aflatoxin B1. The concentration of aflatoxin B1 in animal feed is
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influenced by feed type, time and method of harvesting, temperature and moisture content.
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A. flavus, and A.parasiticus can easily grow in feed having a moisture content between 13-
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18% and an environmental humidity between 50-60% (Bakirci, 2001; Ardic, 2009). Our
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findings revealed a high incidence of aflatoxin M1 in raw milk samples. Also, statistical
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analysis of the data shows that the percentage of aflatoxin M1 contamination in buffalo milk
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is more than in cow milk, which was not similar to a study conducted by Rahimi et al. (2010).
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In another study, Bilandzic et al. (2010) reported less AFM1 (1.6%) at detectable levels in
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raw milk samples, but in a recent study Davoudi et al. (2011) determined AFM1 in 100% of
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the investigated raw milk samples. In previous surveys conducted in Iran by TLC methods,
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Kamkar (2005) reported that 77% (85 of 111) raw milk samples were contaminated with
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AFM1 in Sarab and 40% had levels above the European limit. A survey from Morocco (El
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Marnissi et al. 2012) reported aflatoxin M1 in raw milk in 13 out of 48 samples (27%),
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ranging 10-100 ng/kg and within the positive samples 4 (~8% of the total) were above the EU 6
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contained AFM1 and 61% of the positive samples were higher than the EU limit (Assem et
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al., 2011). Similar results were documented from Tunisia by Abbes et al. (2012), who found
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by an ELISA method that 61% of 112 raw milk samples included aflatoxin M1. An earlier
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study by Lee et al (2009) from South Korea reported 48 (48%) of 100 raw milk samples
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contained AFM1. In Punjab, Pakistan, Hussain et al. (2008) analysed 480 raw milk samples,
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360 of buffalo and 120 of cow. Percentages of AFM1 contamination in buffalo and cow milk
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were 42.5 and 52.5%, respectively. Several studies by other researchers are shown in Table 3.
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limit. A study on raw milk samples in Lebanon showed that 28 (74%) of 38 raw milk samples
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Conclusions
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Aflatoxin M1 is determined in a high percentage of milk samples, which can justify to
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perform a monitoring program for the total milk chain, as especially infants and children
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consume these products. Control could start from the farm, by regular control of aflatoxin B1
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contamination of animal feed and control of its storage conditions. The use of a food control
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system, like the HACCP system in the food industries is suggested as an efficient means of
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limiting mycotoxin contamination, especially for aflatoxin in Iranian food.
156 157 158 159
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References
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occurrence of aflatoxins (B1 and M1) in feed, plasma and raw milk of lactating dairy cows
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Assem E, Mohamad A, Oula EA. 2011. A survey on the occurrence of aflatoxin M1 in raw and processed milk samples marketed in Lebanon. Food Control. 22: 1856-1858.
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Ardic M. 2009b. Occurrence of aflatoxin M1 in raw ewe’s milk produced in Sanliurfa,
Bakirci I. 2001. A study on the occurrence of aflatoxin M1 in milk and milk products
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brined cheese. Food Control. 20: 196-199.
produced in Van province of Turkey. Food Control. 12: 47-51. Bilandzic N, Varenina I, Solomun B. 2010. Aflatoxin M1 in raw milk in Croatia. Food Control. 21: 1279-1281.
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Codex Alimentarius Commission. 2001. Comments submitted on the draft maximum level
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for Aflatoxin M1 in milk. Codex committee on food additives and contaminants 33rd
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Ardic M, Karakaya Y, Atasever M, Adiguzel G. 2009a. Aflatoxin M1 levels of Turkish white
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Davoudi Y, Garedaghi Y, Nazeri M. 2011. Survey on contaminated raw milks with aflatoxin M1 in the Sarab region, Iran. Aust J Basic and Appl Sci. 5: 97-100.
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El Marnissia B, Belkhoua R, Morgavic DP, Bennanib L, Boudra H. 2012. Occurrence of
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Toxicol. 50: 2819-2821.
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setting maximum levels of certain contaminants in foods. Official Journal of the European
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Fallah AA. 2010b. Assessment of aflatoxin M1 contamination in pasteurized and UHT milk
Ghanem I, Orfi M. 2009. Aflatoxin M1 in raw, pasteurized and powdered milk available in Syrian market. Food Control. 20: 603- 605.
Hussain I, Anwar J, Munawar MA, Asi MR. 2008. Variation of levels of aflatoxin M1 in raw
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milk from different localities in the central areas of Punjab, Pakistan. Food Control. 19:
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ISIRI, 2005. Milk and Milk Products-raw Milk-specifications and Test Methods. Iranian
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winter and summer. Food Control. 21: 1478-1481.
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Fallah AA. 2010a. Aflatoxin M1 contamination in dairy products marketed in Iran during
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Kamkar A. 2005. A study on the occurrence of aflatoxin M1 in raw milk produced in Sarab city of Iran. Food Control. 16: 593-599.
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Kart A, Elmali M, Yapar K, Yaman H. 2009. Occurrence of aflatoxin M1 determined by
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ELISA in UHT (Sterilized) and raw milk samples produced in Turkey. Asian J Chem. 21:
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Lee JE, Kwak B-M, Ahn J-H, Jeon T-H. 2009. Occurrence of aflatoxin M1 in raw milk in
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milk of five dairy species in Ahvaz, Iran. Food Chem Toxicol. 48: 129-131. Rastogi S, Dwivedi PD, Khanna SK, Das M. 2004. Detection of aflatoxin M1 contamination
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in milk and infant milk products from Indian markets by ELISA. Food Control. 15: 287-
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Tajkarimi M, Shojaee Aliabadi F, Salah Nejad M, Poursoltani H, Motallebi AA, Mahdavi H.
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2007. Seasonal study of aflatoxin M1 contamination in milk in five regions in Iran. Int J
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Food Microbiol. 116: 345-349.
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Tavakoli HR, Riazipour M, Kamkar A, Rafati H, Mozaffari Nejad AS. 2012. Occurrence of aflatoxin M1 in cheese samples from Tehran, Iran. Food Control. 23: 293-295.
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Tavakoli HR, Kamkar A, Riazipour M, Mozaffari Nejad AS, Rafati H. 2013. Assessment of
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Rahimi E, Bonyadian M, Rafei M, Kazemeini HR. 2010. Occurrence of aflatoxin M1 in raw
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219.
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Van Egmond HP. 1989. Introduction. In: van Egmond, H.P. (Ed.), Mycotoxins in dairy products. Elsevier Applied Science, London and New York, pp. 1-10.
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226 227 228 229
4 (40%) 4.5 14.1 6.1a ± 2.7 20 (67%) 3.6 100.9 32.6a ± 8.4 20 23.2 419.5 114.9b ± (100%) 36.3 Total 60 16 (31%) 44 (69%) 3.6 419.5 55.5 a 230 Values in parentheses indicate % negative and positive samples. b 231 Mean ± SE with different letters is significantly different. c 232 The EU limit for AFM1 is 50 ng/l for raw milk. 233 234 235 236 237 238 239 Table 2 240 Distribution by season of aflatoxin M1 (ng/l) in 60 raw buffalo milk samples. Tested Negativea Positivea Minimum Maximum Mean ± SEb Season
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18(28)
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93.2a ± 47.1 109.1a ± 40.5 Summer 20 2 (10%) 18 (90%) 12.7 367.3 138.4a ± 40.9 Total 60 14 (21%) 46 (79%) 12.7 422.7 116.2 ± 25.1 a 241 Values in parentheses indicate % negative and positive samples. b 242 Mean ± SE with different letters is significantly different. c 243 The EU limit for AFM1 is 50 ng/l for raw milk. 244 245 246 247 248 249 250 251 252 253 254 Winter Spring
10 30
2 (20%) 10 (33%)
8 (80%) 20 (67%)
19.1 19.5
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Winter Spring Summer
Exceeding regulation,c n (%) 0(0) 4(13) 14(70)
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Table 1 Distribution by season of aflatoxin M1 (ng/l) in 60 raw cow milk samples. Tested Negativea Positivea Minimum Maximum Mean ± SEb Season
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209.5 422.7
Exceeding regulation,c n (%) 4 (40) 14 (47) 14 (70) 32 (52)
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Table 3 Occurrence and levels of aflatoxin M1 in raw milks reported in previous studies. Country Samples Positive (%) Range Method Reference 12
33%
28-164
ELISA
Rastogi et al. (2004)
Iran
111
76.6
15-280
TLC
Kamkar (2005)
Iran
98
100
3-329
HPLC
Tajkarimi et al. (2007)
Turkey
90
88.9
17-232
ELISA
Ardic (2009)
Turkey
45
91.1
75
ELISA
Kart et al. (2009)
Syrian
108
83.3
6-690
ELISA
Iran
311
42.1
50
ELISA
Croatia
61
1.6
0.6-58.7
ELISA
Iran
100
100
30-630
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TLC
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India
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(ng/l)
Ghanem and Orfi (2009) Rahimi et al. (2010)
Bilandzic et al. (2010) Davoudi et al. (2011)
