This article was downloaded by: [New York University] On: 04 June 2015, At: 12:13 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
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 milk in Qazvin province, Iran: a seasonal study ab
c
d
Aziz A. Fallah , Afshin Barani & Zeinab Nasiri a
Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord 34141, Iran b
Research Institute of Zoonotic Diseases, Shahrekord University, Shahrekord 34141, Iran
c
Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran d
Click for updates
Graduate from Department of Chemistry, Karaj Branch, Islamic Azad University, Karaj, Iran Accepted author version posted online: 22 May 2015.
To cite this article: Aziz A. Fallah, Afshin Barani & Zeinab Nasiri (2015): Aflatoxin M1 in raw milk in Qazvin province, Iran: a seasonal study, Food Additives & Contaminants: Part B: Surveillance, DOI: 10.1080/19393210.2015.1046193 To link to this article: http://dx.doi.org/10.1080/19393210.2015.1046193
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
Publisher: Taylor & Francis Journal: Food Additives & Contaminants: Part B DOI: 10.1080/19393210.2015.1046193
an us cr ip t
Aflatoxin M1 in raw milk in Qazvin province, Iran: a seasonal study Aziz A. Fallaha,b, Afshin Baranic,*, Zeinab Nasirid
Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Shahrekord
University, Shahrekord 34141, Iran b c
Research Institute of Zoonotic Diseases, Shahrekord University, Shahrekord 34141, Iran
Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Urmia
University, Urmia, Iran
Graduate from Department of Chemistry, Karaj Branch, Islamic Azad University, Karaj, Iran
M
d
*Corresponding author. Tel./Fax: +98 381 4424427
ce
Abstract
pt
ed
E-mail address: [email protected] (A. Barani)
Occurrence of aflatoxin M1 (AFM1) was determined in 254 samples of raw milk obtained from dairy cow farms of Qazvin province, Iran. Aflatoxin M1 analysis was
Ac
Downloaded by [New York University] at 12:13 04 June 2015
a
carried out by using the competitive enzyme-linked immunosorbent assay (ELISA) technique for screening and high performance liquid chromatography with fluorescence detection (HPLC-FLD) for confirmatory purposes. The limit of detection (LOD) and quantification (LOQ) of the confirmatory method were 0.003 and 0.01 µg/l, respectively. Aflatoxin M1 was detected in 204 analyzed samples (80.3%), ranging from 0.011 to 0.321 μg/l and 144 samples (56.7%) had levels above the Iranian national
standard limit of 0.050 μg/l. Considering the seasonal variability, the occurrence and levels of AFM1 in samples obtained in winter were significantly higher (P < 0.05) than those obtained in summer. The results of this survey indicate the usefulness of a
an us cr ip t
monitoring program to supervise food safety for consumers.
Introduction
Aflatoxins are secondary toxic metabolites of fungi that are produced by species
M
such as Aspergillus flavus and Aspergillus parasiticus. Aflatoxin B1 is the most toxic
ed
and carcinogenic compound that can contaminate animal feed (Sweeney & Dobson 1998; Fallah et al. 2014). In the liver of animals consuming aflatoxin B1 contaminated
pt
feed, cytochrome P450 associated enzymes hydroxylate it into aflatoxin M1 and thus contaminate milk products (Murphy et al. 2006; Prandini et al. 2009). There is a linear
ce
relationship between the concentration of AFM1 in milk and of AFB1 in feed for dairy cattle. It has been estimated that about 0.3–6.2% of AFB1 consumed by dairy cattle is
Ac
Downloaded by [New York University] at 12:13 04 June 2015
Keywords: Aflatoxin M1, dairy farms, ELISA, HPLC-FLD, seasonal influence.
metabolized to AFM1 and excreted into milk. The toxin can be detected in milk 12–24 h after consuming AFB1. When consumption of contaminated feed is stopped the
concentration of AFM1 in milk decreases to an undetectable level within 4–5 days (Fink-Gremmels 2008; Fallah 2010a). Although the mutagenic and carcinogenic potency of aflatoxin M1 is less when compared to its parent compound AFB1, the carcinogenic damage caused by aflatoxin M1 convinced the International Agency for
Research on Cancer (IARC 2002) to classify it in class 1 human carcinogenic compounds. Many countries have established a maximum limit (ML) for aflatoxin M1 in milk and milk products to protect consumer’s health. Depending on the economic
an us cr ip t
considerations, the regulations vary from one country to another (Stoloff et al. 1991; Fallah et al. 2009). In Codex regulations (Codex Alimentarius, 1995) the maximum
the Institute of Standards and Industrial Research of Iran (ISIRI 2002) have set a ML of 0.050 µg/kg and 0.050 µg/l for AFM1 in raw milk, respectively.
Due to the fact that milk and dairy products are good sources of essential nutrients for health and growth of children, several surveys have been conducted on the
M
occurrence of AFM1 in different types of milk and dairy products around the world
ed
(Bilandžić et al. 2010; El-Tras et al. 2011; Zheng et al. 2013; Iqbal et al. 2014; Kanungo & Bhand 2014). Also, several studies have been performed in Iran about this subject
pt
(Kamkar et al. 2014a). In the current study, occurrence and levels of AFM1 were determined in raw milk samples from dairy farms in the Qazvin province, supplying the
ce
raw milk of the central part of Iran.
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level for AFM1 in raw milk is 0.500 µg/l. The European Commission (EC 2006) and
Materials and methods Sample collection During the year 2014, 127 dairy farms were randomly selected for sampling among the farms in Qazvin province, Iran by using a random number table. All farms were
authorized by the Iran Ministry of Agriculture. The samples (1500 ml each) were taken from the bulk tank milk of each farm during winter (January to March) and Summer (July to September), transported to the laboratory in an icebox and stored at – 18 ºC until analyses.
an us cr ip t
ELISA analysis
For screening purposes AFM1 was determined by competitive enzyme-linked
Biopharm, Darmstadt, Germany), with a limit of detection (LOD) of 5 ng/l for milk. Sample extraction and clean-up
To confirm the presence of AFM1 and also for quantification, milk samples revealing levels above ELISA kit’s LOD were extracted and cleaned-up again with the official
M
HPLC-FLD reference method described by the Institute of Standards and Industrial
ed
Research of Iran (ISIRI 2010). The milk samples were centrifuged at 2000g for 10 min and the upper fat layer was discarded. The skimmed milk (20 ml) was passed through an
pt
immunoaffinity column previously conditioned with 10 ml of phosphate buffer (pH 7.4) at a flow rate of 3–4 ml/min. The column was washed twice with 10 ml distilled water
ce
and AFM1 was eluted with 2.5 ml acetonitrile into a clean glass tube. The solvent was evaporated to dryness under a gentle stream of nitrogen at 50 °C. The residue was
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immunosorbent assay (ELISA) using a RIDASCREEN Aflatoxin M1 test kit (R-
dissolved in 1 ml of mobile phase and filtered through a 0.45 µm syringe filter before HPLC analysis.
High-performance liquid chromatography analysis HPLC analyses were carried out with a Waters 2695 chromatograph (Waters Corporation, Milford, MA, USA), with a quaternary pump, an auto sampler, a vacuum degasser and a fluorescence detector. Chromatographic separation was performed on a
Discovery® C18 HPLC column (5 µm particle size, 250 mm × 4.6 mm i.d.) protected with a Discovery® C18 Supelguard column (5 µm particle size, 20 mm × 4.6 mm i.d.), both from Supelco (Bellefonte, USA). Water, acetonitrile and methanol (60:20:20, v/v/v) was used as mobile phase at a flow rate of 1.0 ml/min. The fluorescence detector
an us cr ip t
was set at wavelengths of 360 and 430 nm for excitation and emission, respectively. The injection volume of the sample or standard solutions was 100 µl.
The validation parameters were determined to ensure method performance quality: limit of detection (LOD), limit of quantification (LOQ), recovery, linearity, repeatability and
within-laboratory reproducibility. LOD and LOQ were based on signal to noise ratios of 3:1 and 10:1, respectively. Accuracy of the method was assessed by analyzing blank
M
milk samples spiked with AFM1 at levels of 0.5 × ML, 1 × ML and 1.5 × ML
ed
(European Commission 2002). Spiking was carried out 6 times for each level. The analyses were performed on 3 different days with the same instruments, but with
pt
different batches of reagents and different operators. The recovery was calculated by the following formula: (measured content/fortified level) × 100%. Precision (repeatability
ce
and within-laboratory reproducibility) was expressed as the relative standard deviation (RSD) of the recovery. A six-point calibration curve was prepared at 0.05, 0.1, 0.25,
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Method validation
0.5, 1 and 2 ng/ml to check linearity and for quantification of AFM1 in the samples.
Statistical analyses Statistical analyses were performed using chi-square test and t-test of the SPSS software
version 20 for windows (SPSS Inc., Chicago, IL, USA) to compare detection rates and AFM1 concentrations between the seasons, respectively. The differences were considered significant at P < 0.05.
Results and discussion Mean recoveries were 79.7–89.9% and the relative standard deviations were less than
an us cr ip t
7.1% for repeatability and within-laboratory reproducibility. LOD and LOQ of the
method were 0.003 and 0.01 μg/l, respectively (Table 1). AFM1 occurrence and levels
Table 2. It was detected in 204 out of 254 samples (80.3%), ranging from 0.011 to 0.321 μg/l and 144 samples (56.7%) had levels above the ISIRI (2002) limit of 0.050 μg/l.
None of the samples exceeded the Codex Alimentarius Commission (1995) limit of 0.50
µg/kg for AFM1 in milk. In previous surveys conducted in different parts of Iran
M
incidences of AFM1 contamination were found in raw milk which are comparable to the
ed
results of this study (Ghiasian et al. 2007; Nemati et al. 2010; Fallah et al. 2011; Rohani et al. 2011; Vagef & Mahmoudi 2013; Kamkar et al. 2014b). Comparing our results
pt
with some European countries, the occurrence and levels of AFM1 are higher than those found in Croatia (Bilandžić et al. 2010), Portugal (Martins & Martins 2000), Turkey
ce
(Kara & Ince 2014), France (Boudra et al. 2007) and Italy (Santini et al. 2013). Considering seasonal variability, this study showed that AFM1 incidence and
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in raw milk samples obtained from dairy farms of Qazvin province, Iran are shown in
levels in raw milk samples obtained in winter were significantly higher (P < 0.05) than those obtained in summer. Moreover, occurrence of samples exceeding the ISIRI limit (0.050 μg/l) was 3.5 times higher in winter compared to summer (112 vs. 32) samples; Table 2). These findings are consistent with previous studies indicating seasonal trend in AFM1 contamination with higher occurrence and levels in cold seasons (Ghiasian et al. 2007; Fallah 2010b; Nemati et al. 2010; Suriyasathaporn & Nakprasert 2012; Vagef
& Mahmoudi 2013). This is due to the fact that in cold seasons dairy animals are fed with higher amounts of concentrated feed that might be stored under inappropriate
an us cr ip t
conditions and thus be contaminated with toxigenic fungi producing AFB1.
Conclusions
obtained from dairy farms in Qazvin, Iran. Moreover, there is a seasonal trend, with a
higher occurrence and levels of this mycotoxin in winter than in summer. It could be a
serious hazard for health of consumers, especially children. Monitoring AFM1 contamination in milk and various dairy products should be performed regularly to
M
provide information for better risk assessment. Products with AFM1 levels higher than
pt
ed
the legal limits must be prevented from human consumption.
Acknowledgment
ce
This study was financially supported by the Vice Chancellor for Research, Shahrekord University, Iran.
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This study showed incidence and levels of AFM1 contamination in raw milk samples
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Boudra H, Barnouin J, Dragacci S, Morgavi DP. 2007. Aflatoxin M1 and Ochratoxin A in raw bulk milk from French dairy herds. J Dairy Sci. 90:3197–3201. Codex Alimentarius, 1995. Codex General Standard for contaminants and toxins in food and feed, Codex Standard 193-1995, Adopted 1995. Revised 1997, 2006, 2008, 2009.
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mycotoxins: maximum tolerated level. 1st ed. Standard No. 5925. Tehran: ISIRI. Institute of Standards and Industrial Research of Iran (ISIRI). 2010. Milk and Milk
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aflatoxin M1 in raw milk during the summer and winter seasons in Hamedan, Iran. J
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treated milk commercialized in Portugal. Food Addit Contam. 17:871–874.
(Italy). Food Addit Contam Part B. 6:181–186.
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Sweeney MJ, Dobson ADW. 1998. Mycotoxin production by Aspergillus, Fusarium and penicillium species. Int J Food Microbiol. 43:141–158. Vagef R, Mahmoudi R. 2013. Occurrence of aflatoxin M1 in raw and pasteurized milk produced in west region of Iran (during summer and winter). Int Food Res J. 20:1421–
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in raw milk in the five provinces of China. Food Addit Contam Part B. 6:110–115.
Day 2 repeatability (n = 6)
RSD (%)
Mean recovery (%)
RSD (%)
Mean recovery (%)
6.63
83.9
6.53
84.9
3.43
87.0
6.17
84.2
2.74
82.3
5.34
80.9
6.94
84.5
5.53
83.5
4.16
89.9
Day 1 repeatability (n = 6) RSD (%)
Spiked level (µg/l)
Linearity (r2)
LOQ (µg/l)
LOD (µg/l)
0.998
0.01
0.003
P
Mean recovery (%)
7.06
79.7
0.025
4.16
89.5
0.050
5.69
80.0
0.075
M
an
us
P
t
Day 3 repeatability (n = 6)
cr ip
Within-laboratory reproducibility (n = 18) RSD (%) Mean recovery (%)
Table 2 The occurrence of aflatoxin M1 (AFM1) in raw milk in Qazvin province, Iran. Exceed regulationb, Distribution of positive samples, n (%) n (%) > 0.20 0.10–0.20 0.050–0.10 LOQa–0.050 µg/l µg/l µg/l µg/l P
Concentration (µg/l) Range Mean ± SE
pt ed
P
P
P
Positive samples, n (%)
Samples, n
Season
127
Summer
2 (1.57)
20 (15.7)
10 (7.87)
45 (35.4)
0.011–0.210
0.037 ± 0.005x
77 (60.6)x
112 (88.2)
8 (6.30)
40 (31.5)
64 (50.4)
15 (11.8)
0.032–0.321
0.097 ± 0.004y
127 (100.0)y
127
Winter
144 (56.7)
10 (3.94)
60 (23.6)
74 (29.1)
60 (23.6)
0.011–0.321
0.066 ± 0.003
204 (80.3)
254
Total
a P
P
P
P
P
ISIRI limit for AFM1 in milk is 0.050 µg/l.
x,y P
P
Limit of quantification (0.01 µg/l). P
b P
ce
32 (25.2)
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Table 1 Method performance parameters for aflatoxin M1 (AFM1) in spiked samples of raw milk.
P
Contamination percentages and mean levels ± SD in the same column with different superscript letters are significantly different between the seasons (P < 0.05).
12
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 milk in Qazvin province, Iran: a seasonal study ab
c
d
Aziz A. Fallah , Afshin Barani & Zeinab Nasiri a
Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord 34141, Iran b
Research Institute of Zoonotic Diseases, Shahrekord University, Shahrekord 34141, Iran
c
Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran d
Click for updates
Graduate from Department of Chemistry, Karaj Branch, Islamic Azad University, Karaj, Iran Accepted author version posted online: 22 May 2015.
To cite this article: Aziz A. Fallah, Afshin Barani & Zeinab Nasiri (2015): Aflatoxin M1 in raw milk in Qazvin province, Iran: a seasonal study, Food Additives & Contaminants: Part B: Surveillance, DOI: 10.1080/19393210.2015.1046193 To link to this article: http://dx.doi.org/10.1080/19393210.2015.1046193
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
Publisher: Taylor & Francis Journal: Food Additives & Contaminants: Part B DOI: 10.1080/19393210.2015.1046193
an us cr ip t
Aflatoxin M1 in raw milk in Qazvin province, Iran: a seasonal study Aziz A. Fallaha,b, Afshin Baranic,*, Zeinab Nasirid
Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Shahrekord
University, Shahrekord 34141, Iran b c
Research Institute of Zoonotic Diseases, Shahrekord University, Shahrekord 34141, Iran
Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Urmia
University, Urmia, Iran
Graduate from Department of Chemistry, Karaj Branch, Islamic Azad University, Karaj, Iran
M
d
*Corresponding author. Tel./Fax: +98 381 4424427
ce
Abstract
pt
ed
E-mail address: [email protected] (A. Barani)
Occurrence of aflatoxin M1 (AFM1) was determined in 254 samples of raw milk obtained from dairy cow farms of Qazvin province, Iran. Aflatoxin M1 analysis was
Ac
Downloaded by [New York University] at 12:13 04 June 2015
a
carried out by using the competitive enzyme-linked immunosorbent assay (ELISA) technique for screening and high performance liquid chromatography with fluorescence detection (HPLC-FLD) for confirmatory purposes. The limit of detection (LOD) and quantification (LOQ) of the confirmatory method were 0.003 and 0.01 µg/l, respectively. Aflatoxin M1 was detected in 204 analyzed samples (80.3%), ranging from 0.011 to 0.321 μg/l and 144 samples (56.7%) had levels above the Iranian national
standard limit of 0.050 μg/l. Considering the seasonal variability, the occurrence and levels of AFM1 in samples obtained in winter were significantly higher (P < 0.05) than those obtained in summer. The results of this survey indicate the usefulness of a
an us cr ip t
monitoring program to supervise food safety for consumers.
Introduction
Aflatoxins are secondary toxic metabolites of fungi that are produced by species
M
such as Aspergillus flavus and Aspergillus parasiticus. Aflatoxin B1 is the most toxic
ed
and carcinogenic compound that can contaminate animal feed (Sweeney & Dobson 1998; Fallah et al. 2014). In the liver of animals consuming aflatoxin B1 contaminated
pt
feed, cytochrome P450 associated enzymes hydroxylate it into aflatoxin M1 and thus contaminate milk products (Murphy et al. 2006; Prandini et al. 2009). There is a linear
ce
relationship between the concentration of AFM1 in milk and of AFB1 in feed for dairy cattle. It has been estimated that about 0.3–6.2% of AFB1 consumed by dairy cattle is
Ac
Downloaded by [New York University] at 12:13 04 June 2015
Keywords: Aflatoxin M1, dairy farms, ELISA, HPLC-FLD, seasonal influence.
metabolized to AFM1 and excreted into milk. The toxin can be detected in milk 12–24 h after consuming AFB1. When consumption of contaminated feed is stopped the
concentration of AFM1 in milk decreases to an undetectable level within 4–5 days (Fink-Gremmels 2008; Fallah 2010a). Although the mutagenic and carcinogenic potency of aflatoxin M1 is less when compared to its parent compound AFB1, the carcinogenic damage caused by aflatoxin M1 convinced the International Agency for
Research on Cancer (IARC 2002) to classify it in class 1 human carcinogenic compounds. Many countries have established a maximum limit (ML) for aflatoxin M1 in milk and milk products to protect consumer’s health. Depending on the economic
an us cr ip t
considerations, the regulations vary from one country to another (Stoloff et al. 1991; Fallah et al. 2009). In Codex regulations (Codex Alimentarius, 1995) the maximum
the Institute of Standards and Industrial Research of Iran (ISIRI 2002) have set a ML of 0.050 µg/kg and 0.050 µg/l for AFM1 in raw milk, respectively.
Due to the fact that milk and dairy products are good sources of essential nutrients for health and growth of children, several surveys have been conducted on the
M
occurrence of AFM1 in different types of milk and dairy products around the world
ed
(Bilandžić et al. 2010; El-Tras et al. 2011; Zheng et al. 2013; Iqbal et al. 2014; Kanungo & Bhand 2014). Also, several studies have been performed in Iran about this subject
pt
(Kamkar et al. 2014a). In the current study, occurrence and levels of AFM1 were determined in raw milk samples from dairy farms in the Qazvin province, supplying the
ce
raw milk of the central part of Iran.
Ac
Downloaded by [New York University] at 12:13 04 June 2015
level for AFM1 in raw milk is 0.500 µg/l. The European Commission (EC 2006) and
Materials and methods Sample collection During the year 2014, 127 dairy farms were randomly selected for sampling among the farms in Qazvin province, Iran by using a random number table. All farms were
authorized by the Iran Ministry of Agriculture. The samples (1500 ml each) were taken from the bulk tank milk of each farm during winter (January to March) and Summer (July to September), transported to the laboratory in an icebox and stored at – 18 ºC until analyses.
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ELISA analysis
For screening purposes AFM1 was determined by competitive enzyme-linked
Biopharm, Darmstadt, Germany), with a limit of detection (LOD) of 5 ng/l for milk. Sample extraction and clean-up
To confirm the presence of AFM1 and also for quantification, milk samples revealing levels above ELISA kit’s LOD were extracted and cleaned-up again with the official
M
HPLC-FLD reference method described by the Institute of Standards and Industrial
ed
Research of Iran (ISIRI 2010). The milk samples were centrifuged at 2000g for 10 min and the upper fat layer was discarded. The skimmed milk (20 ml) was passed through an
pt
immunoaffinity column previously conditioned with 10 ml of phosphate buffer (pH 7.4) at a flow rate of 3–4 ml/min. The column was washed twice with 10 ml distilled water
ce
and AFM1 was eluted with 2.5 ml acetonitrile into a clean glass tube. The solvent was evaporated to dryness under a gentle stream of nitrogen at 50 °C. The residue was
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immunosorbent assay (ELISA) using a RIDASCREEN Aflatoxin M1 test kit (R-
dissolved in 1 ml of mobile phase and filtered through a 0.45 µm syringe filter before HPLC analysis.
High-performance liquid chromatography analysis HPLC analyses were carried out with a Waters 2695 chromatograph (Waters Corporation, Milford, MA, USA), with a quaternary pump, an auto sampler, a vacuum degasser and a fluorescence detector. Chromatographic separation was performed on a
Discovery® C18 HPLC column (5 µm particle size, 250 mm × 4.6 mm i.d.) protected with a Discovery® C18 Supelguard column (5 µm particle size, 20 mm × 4.6 mm i.d.), both from Supelco (Bellefonte, USA). Water, acetonitrile and methanol (60:20:20, v/v/v) was used as mobile phase at a flow rate of 1.0 ml/min. The fluorescence detector
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was set at wavelengths of 360 and 430 nm for excitation and emission, respectively. The injection volume of the sample or standard solutions was 100 µl.
The validation parameters were determined to ensure method performance quality: limit of detection (LOD), limit of quantification (LOQ), recovery, linearity, repeatability and
within-laboratory reproducibility. LOD and LOQ were based on signal to noise ratios of 3:1 and 10:1, respectively. Accuracy of the method was assessed by analyzing blank
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milk samples spiked with AFM1 at levels of 0.5 × ML, 1 × ML and 1.5 × ML
ed
(European Commission 2002). Spiking was carried out 6 times for each level. The analyses were performed on 3 different days with the same instruments, but with
pt
different batches of reagents and different operators. The recovery was calculated by the following formula: (measured content/fortified level) × 100%. Precision (repeatability
ce
and within-laboratory reproducibility) was expressed as the relative standard deviation (RSD) of the recovery. A six-point calibration curve was prepared at 0.05, 0.1, 0.25,
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Method validation
0.5, 1 and 2 ng/ml to check linearity and for quantification of AFM1 in the samples.
Statistical analyses Statistical analyses were performed using chi-square test and t-test of the SPSS software
version 20 for windows (SPSS Inc., Chicago, IL, USA) to compare detection rates and AFM1 concentrations between the seasons, respectively. The differences were considered significant at P < 0.05.
Results and discussion Mean recoveries were 79.7–89.9% and the relative standard deviations were less than
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7.1% for repeatability and within-laboratory reproducibility. LOD and LOQ of the
method were 0.003 and 0.01 μg/l, respectively (Table 1). AFM1 occurrence and levels
Table 2. It was detected in 204 out of 254 samples (80.3%), ranging from 0.011 to 0.321 μg/l and 144 samples (56.7%) had levels above the ISIRI (2002) limit of 0.050 μg/l.
None of the samples exceeded the Codex Alimentarius Commission (1995) limit of 0.50
µg/kg for AFM1 in milk. In previous surveys conducted in different parts of Iran
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incidences of AFM1 contamination were found in raw milk which are comparable to the
ed
results of this study (Ghiasian et al. 2007; Nemati et al. 2010; Fallah et al. 2011; Rohani et al. 2011; Vagef & Mahmoudi 2013; Kamkar et al. 2014b). Comparing our results
pt
with some European countries, the occurrence and levels of AFM1 are higher than those found in Croatia (Bilandžić et al. 2010), Portugal (Martins & Martins 2000), Turkey
ce
(Kara & Ince 2014), France (Boudra et al. 2007) and Italy (Santini et al. 2013). Considering seasonal variability, this study showed that AFM1 incidence and
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in raw milk samples obtained from dairy farms of Qazvin province, Iran are shown in
levels in raw milk samples obtained in winter were significantly higher (P < 0.05) than those obtained in summer. Moreover, occurrence of samples exceeding the ISIRI limit (0.050 μg/l) was 3.5 times higher in winter compared to summer (112 vs. 32) samples; Table 2). These findings are consistent with previous studies indicating seasonal trend in AFM1 contamination with higher occurrence and levels in cold seasons (Ghiasian et al. 2007; Fallah 2010b; Nemati et al. 2010; Suriyasathaporn & Nakprasert 2012; Vagef
& Mahmoudi 2013). This is due to the fact that in cold seasons dairy animals are fed with higher amounts of concentrated feed that might be stored under inappropriate
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conditions and thus be contaminated with toxigenic fungi producing AFB1.
Conclusions
obtained from dairy farms in Qazvin, Iran. Moreover, there is a seasonal trend, with a
higher occurrence and levels of this mycotoxin in winter than in summer. It could be a
serious hazard for health of consumers, especially children. Monitoring AFM1 contamination in milk and various dairy products should be performed regularly to
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provide information for better risk assessment. Products with AFM1 levels higher than
pt
ed
the legal limits must be prevented from human consumption.
Acknowledgment
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This study was financially supported by the Vice Chancellor for Research, Shahrekord University, Iran.
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This study showed incidence and levels of AFM1 contamination in raw milk samples
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Day 2 repeatability (n = 6)
RSD (%)
Mean recovery (%)
RSD (%)
Mean recovery (%)
6.63
83.9
6.53
84.9
3.43
87.0
6.17
84.2
2.74
82.3
5.34
80.9
6.94
84.5
5.53
83.5
4.16
89.9
Day 1 repeatability (n = 6) RSD (%)
Spiked level (µg/l)
Linearity (r2)
LOQ (µg/l)
LOD (µg/l)
0.998
0.01
0.003
P
Mean recovery (%)
7.06
79.7
0.025
4.16
89.5
0.050
5.69
80.0
0.075
M
an
us
P
t
Day 3 repeatability (n = 6)
cr ip
Within-laboratory reproducibility (n = 18) RSD (%) Mean recovery (%)
Table 2 The occurrence of aflatoxin M1 (AFM1) in raw milk in Qazvin province, Iran. Exceed regulationb, Distribution of positive samples, n (%) n (%) > 0.20 0.10–0.20 0.050–0.10 LOQa–0.050 µg/l µg/l µg/l µg/l P
Concentration (µg/l) Range Mean ± SE
pt ed
P
P
P
Positive samples, n (%)
Samples, n
Season
127
Summer
2 (1.57)
20 (15.7)
10 (7.87)
45 (35.4)
0.011–0.210
0.037 ± 0.005x
77 (60.6)x
112 (88.2)
8 (6.30)
40 (31.5)
64 (50.4)
15 (11.8)
0.032–0.321
0.097 ± 0.004y
127 (100.0)y
127
Winter
144 (56.7)
10 (3.94)
60 (23.6)
74 (29.1)
60 (23.6)
0.011–0.321
0.066 ± 0.003
204 (80.3)
254
Total
a P
P
P
P
P
ISIRI limit for AFM1 in milk is 0.050 µg/l.
x,y P
P
Limit of quantification (0.01 µg/l). P
b P
ce
32 (25.2)
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Table 1 Method performance parameters for aflatoxin M1 (AFM1) in spiked samples of raw milk.
P
Contamination percentages and mean levels ± SD in the same column with different superscript letters are significantly different between the seasons (P < 0.05).
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