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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
Aflatoxins in black tea in Iran a
Zohreh Pouretedal & Mansooreh Mazaheri
a
a
Department of Food and Agriculture , Standard Research Institute , Karaj , Iran Accepted author version posted online: 09 Jan 2013.Published online: 25 Feb 2013.
To cite this article: Zohreh Pouretedal & Mansooreh Mazaheri (2013) Aflatoxins in black tea in Iran, Food Additives & Contaminants: Part B: Surveillance, 6:2, 127-129, DOI: 10.1080/19393210.2013.764551 To link to this article: http://dx.doi.org/10.1080/19393210.2013.764551
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
Food Additives & Contaminants: Part B, 2013 Vol. 6, No. 2, 127–129, http://dx.doi.org/10.1080/19393210.2013.764551
Aflatoxins in black tea in Iran Zohreh Pouretedal and Mansooreh Mazaheri* Department of Food and Agriculture, Standard Research Institute, Karaj, Iran
Downloaded by [University of Saskatchewan Library] at 18:18 10 October 2014
(Received 26 May 2012; final version received 6 January 2013) Aflatoxins (AFs) are highly toxic, and carcinogenic secondary fungal metabolites and have been detected in various food commodities. In this regard, 40 black tea samples including domestic and imported black tea were analysed for aflatoxin contamination by high-performance liquid chromatography using a post-column derivatisation procedure (Kobra cell) with fluorescence detection. Samples were randomly collected in 2010 from Tehran markets. The results revealed that 30 among 40 samples were contaminated with aflatoxins (27.5% of the total). Mean AFB1 content was 10.0 ng/g and mean of aflatoxin total was 12.07 ng/g for the 11 contaminated samples. Keywords: aflatoxins; black tea; Iran; HPLC
Introduction Aflatoxins (AFs) are a group of carcinogenic polyketide secondary metabolites which are produced by strains of Aspergillus flavus, Aspergillus parasiticus, Aspergillus nomius and Aspergillus pseudotamarii (Payne 1998). They are the secondary metabolites produced by filamentous fungi in food crops that cause toxic response (mycotoxicosis) when ingested by animals and humans. There are four naturally occurring AFs, designated B1, B2, G1 and G2, with AFB1 being the most common and toxic. The International Agency for Research on Cancer has classified AFB1 as a Group I carcinogen, primarily affecting the liver (International Agency for Research on Cancer 1993). Aflatoxins are found as contaminants in various agricultural commodities such as cereals, tree nuts, groundnut and cottonseed (Pittet 1998). The occurrence of aflatoxins in tropical areas is unavoidable because of the climate conditions for fungal growth and aflatoxin production. Tea is an aromatic beverage prepared by adding cured leaves of the Camellia sinensis plant to hot water. The term also refers to the plant itself. After water, tea is the most widely consumed beverage in the world. Tea is native to East and South Asia and probably originated around the point of confluence of the lands of northeast India, north Burma and southwest China. In Iran, black tea is available and consumed regularly. Despite lots of studies on the mycotoxins in agricultural products, only a few are concerned with tea, which is becoming more and more common in our daily diet and which plays an important role in the economy. Furthermore, previous works show that aflatoxin levels are not reduced by domestic cooking with either microwave or conventional gas oven heating (Midio et al. 2001) *Corresponding author. Email: [email protected] © 2013 Taylor & Francis
and that AFs do not decompose at the temperature of boiling water during the preparation of the drink (Feuell 1996). Determination of aflatoxins in tea and medical plants is difficult because of their complex matrices consisting of various chemical components. The purpose of this study is to provide useful information on aflatoxin levels in black tea in order to achieve some knowledge on the evaluation of the risk regarding the consumption of this product. Therefore, the incidence of aflatoxins in 40 samples (domestic and imported black tea) from Tehran market was investigated and determined using HPLC with fluorescence detection. Materials and methods Chemicals Mixed aflatoxin standards were purchased from Sigma Chemical (Sigma-Aldrich, St. Louis, MO, USA). A stock standard solution of aflatoxins at 1000 μg/ml for B1 and G1 and 200 μg/ml for B2 and G2 in methanol was prepared and kept wrapped in aluminium foil at – 20°C. Aflatoxin working solutions were prepared by dilution in the same solvent and stored in stoppered glass vials at 0°C. All solvents used for the experiments (methanol, acetonitrile, deionised water) were HPLC grade. Deionised water was obtained using a Mili-Q water purification system (Millipore, Bedford, MA, USA). Aflatest immunoaffinity columns (IAC) were purchased from Romer (Romer Labs, Tulln, Austria). Sampling Samples were collected randomly from Tehran markets. In this regard, 40 black tea samples including Iranian tea and
128
Z. Pouretedal and M. Mazaheri were eluted with methanol, which passed through by gravity. The eluate was diluted with water and analysed by HPLC.
imported tea samples were taken in July 2010. The sample size package was about 250 g. The weight of the incremental sample is the weight of the retail packing (250 g), or one retail pack has been considered as one incremental sample. Ten incremental samples were taken randomly from each marketing brand, resulting in an aggregate sample of 2.5 kg. Each sample was mixed and then all samples were transferred to the Mycotoxins Lab in the Institute of Standard and Industrial Research of Iran in Karaj, where aflatoxins were analysed in all samples.
Sample fortification procedure The recovery (percentage of aflatoxin standards added to the sample that is recovered after extraction and clean-up) of the extraction method was determined by sample fortification. Fifty grams of milled tea was fortified 1 hour before extraction with a solution of aflatoxins in methanol at 5 μg/ml (for B1 and G1) and 1 μg/ml (for B2 and G2). The aflatoxin fortification solution was prepared in methanol and used for quantification of analyte recovered after extraction.
To minimise the sub-sampling error in aflatoxin analysis, the whole sample was grinded with a mill and collected in a plastic bag. The mill grinded the samples with a mesh size of 1 mm. The ground tea powder that passed through a 1-mm sieve was separately mixed thoroughly by tumbling at 50 rpm for 15 minutes. The homogeneity of the bulk materials was checked by analysis of subsamples (n = 10) using the Association of Official Analytical Chemists (AOAC) Method (Stroka et al. 2000). All samples were stored in a refrigerator at 4°C until analysis. Before analysis, each sample was mixed again to ensure homogeneity.
Analysis of aflatoxin using HPLC Aflatoxins were determined by reversed-phase HPLC with fluorescence detection after post-column derivatisation involving bromine (Stroka et al. 2000). A Waters HPLC system (pump 1525, florescence detector 2475, analytical column, Nova-pack C18 250 × 4.6 mm: 4 μm, Waters, Milford, MA, USA). Post-column derivatisation was achieved with a Kobra cell and addition of bromide to the mobile phase. After dilution of the aflatoxins eluate with water, 100 μl was injected into the HPLC with a mobile phase of water, methanol and acetonitrile (600:300:200, v/v/v) and 350 μl nitric acid 4 mol/l and 12 mg of potassium bromide with a flow rate of 1 ml/min. The fluorescence detector was operated at an excitation wavelength of 365 nm and emission wavelength of 435 nm. Each working day, a five-point calibration curve was constructed for aflatoxin AFB1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) and checked for linearity and use for quantification of aflatoxins in tea samples. AFG2 eluted first followed by AFG1, AFB2 and AFB1. Calibration points were set at 3.6, 2.8, 2, 1.2 and 0. 5 for AFB1 and G1, and 0.72, 0.54, 0.4, 0.24 and 0.08 for B2 and G2. Good linearity was achieved, as regression coefficients were 0.99. LODs for AFB1, AFB2, AFG1 and AFG2 were 1, 0.2, 1 and 0.2 ng/g, respectively.
Extraction and clean up Samples were analysed using the validated method of the AOAC International (Stroka et al. 2000). To a test portion of 50 g of milled tea, 5 g of sodium chloride was added and then extracted with 300 ml of methanol/water (80 ml/ 20 ml) by shaking for 30 minutes. After filtration, the extract was diluted with phosphate buffer saline (PBS) and filtered through a glass micro-fiber filter. For cleanup of samples, Aflatest IACs were used. First, 10 ml PBS was passed through the IAC. Then, the filtrate was passed through the IAC at a flow rate of ca. 1 drop/s. The column was washed with 20 ml PBS buffer two times and the solution was removed by pressing air through the column and dried by applying little vacuum. Finally, aflatoxins B1 250 Aflatoxin B1 (ng/g)
200 150
B1
100 50
No. of samples
Figure 1.
Concentration of aflatoxin B1 in black tea samples.
40
37
34
31
28
25
22
19
16
13
10
7
4
0 1
Downloaded by [University of Saskatchewan Library] at 18:18 10 October 2014
Sample preparation
Food Additives & Contaminants: Part B
Total aflatoxins (ng/g)
250
Downloaded by [University of Saskatchewan Library] at 18:18 10 October 2014
Total aflatoxins
200 150
Total
100 50 0
Figure 2.
129
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 No. of samples
Concentration of total aflatoxins in black tea samples.
Quality assurance Regarding internal quality control, the accuracy and precision of the methods were verified. In this regard, recoveries of AFB1, AFB2, AFG1 and AFG2 were recorded by analysing a blank tea sample spiked at 5 ng/g for AFB1 and AFG1 and 1 ng/g for AFB2 and AFG2. Aflatoxins levels were corrected for recoveries.
and more data on the distribution and contamination levels of AFs in food like black tea. This study shows that some commercially sold products like black tea can be contaminated with mycotoxins, which indicates it is essential to monitor the presence of mycotoxins in these commodities. It is also necessary to develop and apply strategies to prevent the formation of mycotoxins in these food products in order to ensure they are wholesome and safe for consumers.
Results and discussion Average recoveries for AFB1, AFB2, AFG1 and AFG2 in tea were in the range 75–110%. According to AOAC and Codex guidelines, the recovery range should be 70−110% at a level of 10−100 μg/kg and 60−120% at a level of 1−10 μg/kg, so the obtained recoveries are acceptable. Among 40 samples analysed, 29 samples (72.5%) were not contaminated with AFB1 (
Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Aflatoxins in black tea in Iran a
Zohreh Pouretedal & Mansooreh Mazaheri
a
a
Department of Food and Agriculture , Standard Research Institute , Karaj , Iran Accepted author version posted online: 09 Jan 2013.Published online: 25 Feb 2013.
To cite this article: Zohreh Pouretedal & Mansooreh Mazaheri (2013) Aflatoxins in black tea in Iran, Food Additives & Contaminants: Part B: Surveillance, 6:2, 127-129, DOI: 10.1080/19393210.2013.764551 To link to this article: http://dx.doi.org/10.1080/19393210.2013.764551
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
Food Additives & Contaminants: Part B, 2013 Vol. 6, No. 2, 127–129, http://dx.doi.org/10.1080/19393210.2013.764551
Aflatoxins in black tea in Iran Zohreh Pouretedal and Mansooreh Mazaheri* Department of Food and Agriculture, Standard Research Institute, Karaj, Iran
Downloaded by [University of Saskatchewan Library] at 18:18 10 October 2014
(Received 26 May 2012; final version received 6 January 2013) Aflatoxins (AFs) are highly toxic, and carcinogenic secondary fungal metabolites and have been detected in various food commodities. In this regard, 40 black tea samples including domestic and imported black tea were analysed for aflatoxin contamination by high-performance liquid chromatography using a post-column derivatisation procedure (Kobra cell) with fluorescence detection. Samples were randomly collected in 2010 from Tehran markets. The results revealed that 30 among 40 samples were contaminated with aflatoxins (27.5% of the total). Mean AFB1 content was 10.0 ng/g and mean of aflatoxin total was 12.07 ng/g for the 11 contaminated samples. Keywords: aflatoxins; black tea; Iran; HPLC
Introduction Aflatoxins (AFs) are a group of carcinogenic polyketide secondary metabolites which are produced by strains of Aspergillus flavus, Aspergillus parasiticus, Aspergillus nomius and Aspergillus pseudotamarii (Payne 1998). They are the secondary metabolites produced by filamentous fungi in food crops that cause toxic response (mycotoxicosis) when ingested by animals and humans. There are four naturally occurring AFs, designated B1, B2, G1 and G2, with AFB1 being the most common and toxic. The International Agency for Research on Cancer has classified AFB1 as a Group I carcinogen, primarily affecting the liver (International Agency for Research on Cancer 1993). Aflatoxins are found as contaminants in various agricultural commodities such as cereals, tree nuts, groundnut and cottonseed (Pittet 1998). The occurrence of aflatoxins in tropical areas is unavoidable because of the climate conditions for fungal growth and aflatoxin production. Tea is an aromatic beverage prepared by adding cured leaves of the Camellia sinensis plant to hot water. The term also refers to the plant itself. After water, tea is the most widely consumed beverage in the world. Tea is native to East and South Asia and probably originated around the point of confluence of the lands of northeast India, north Burma and southwest China. In Iran, black tea is available and consumed regularly. Despite lots of studies on the mycotoxins in agricultural products, only a few are concerned with tea, which is becoming more and more common in our daily diet and which plays an important role in the economy. Furthermore, previous works show that aflatoxin levels are not reduced by domestic cooking with either microwave or conventional gas oven heating (Midio et al. 2001) *Corresponding author. Email: [email protected] © 2013 Taylor & Francis
and that AFs do not decompose at the temperature of boiling water during the preparation of the drink (Feuell 1996). Determination of aflatoxins in tea and medical plants is difficult because of their complex matrices consisting of various chemical components. The purpose of this study is to provide useful information on aflatoxin levels in black tea in order to achieve some knowledge on the evaluation of the risk regarding the consumption of this product. Therefore, the incidence of aflatoxins in 40 samples (domestic and imported black tea) from Tehran market was investigated and determined using HPLC with fluorescence detection. Materials and methods Chemicals Mixed aflatoxin standards were purchased from Sigma Chemical (Sigma-Aldrich, St. Louis, MO, USA). A stock standard solution of aflatoxins at 1000 μg/ml for B1 and G1 and 200 μg/ml for B2 and G2 in methanol was prepared and kept wrapped in aluminium foil at – 20°C. Aflatoxin working solutions were prepared by dilution in the same solvent and stored in stoppered glass vials at 0°C. All solvents used for the experiments (methanol, acetonitrile, deionised water) were HPLC grade. Deionised water was obtained using a Mili-Q water purification system (Millipore, Bedford, MA, USA). Aflatest immunoaffinity columns (IAC) were purchased from Romer (Romer Labs, Tulln, Austria). Sampling Samples were collected randomly from Tehran markets. In this regard, 40 black tea samples including Iranian tea and
128
Z. Pouretedal and M. Mazaheri were eluted with methanol, which passed through by gravity. The eluate was diluted with water and analysed by HPLC.
imported tea samples were taken in July 2010. The sample size package was about 250 g. The weight of the incremental sample is the weight of the retail packing (250 g), or one retail pack has been considered as one incremental sample. Ten incremental samples were taken randomly from each marketing brand, resulting in an aggregate sample of 2.5 kg. Each sample was mixed and then all samples were transferred to the Mycotoxins Lab in the Institute of Standard and Industrial Research of Iran in Karaj, where aflatoxins were analysed in all samples.
Sample fortification procedure The recovery (percentage of aflatoxin standards added to the sample that is recovered after extraction and clean-up) of the extraction method was determined by sample fortification. Fifty grams of milled tea was fortified 1 hour before extraction with a solution of aflatoxins in methanol at 5 μg/ml (for B1 and G1) and 1 μg/ml (for B2 and G2). The aflatoxin fortification solution was prepared in methanol and used for quantification of analyte recovered after extraction.
To minimise the sub-sampling error in aflatoxin analysis, the whole sample was grinded with a mill and collected in a plastic bag. The mill grinded the samples with a mesh size of 1 mm. The ground tea powder that passed through a 1-mm sieve was separately mixed thoroughly by tumbling at 50 rpm for 15 minutes. The homogeneity of the bulk materials was checked by analysis of subsamples (n = 10) using the Association of Official Analytical Chemists (AOAC) Method (Stroka et al. 2000). All samples were stored in a refrigerator at 4°C until analysis. Before analysis, each sample was mixed again to ensure homogeneity.
Analysis of aflatoxin using HPLC Aflatoxins were determined by reversed-phase HPLC with fluorescence detection after post-column derivatisation involving bromine (Stroka et al. 2000). A Waters HPLC system (pump 1525, florescence detector 2475, analytical column, Nova-pack C18 250 × 4.6 mm: 4 μm, Waters, Milford, MA, USA). Post-column derivatisation was achieved with a Kobra cell and addition of bromide to the mobile phase. After dilution of the aflatoxins eluate with water, 100 μl was injected into the HPLC with a mobile phase of water, methanol and acetonitrile (600:300:200, v/v/v) and 350 μl nitric acid 4 mol/l and 12 mg of potassium bromide with a flow rate of 1 ml/min. The fluorescence detector was operated at an excitation wavelength of 365 nm and emission wavelength of 435 nm. Each working day, a five-point calibration curve was constructed for aflatoxin AFB1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) and checked for linearity and use for quantification of aflatoxins in tea samples. AFG2 eluted first followed by AFG1, AFB2 and AFB1. Calibration points were set at 3.6, 2.8, 2, 1.2 and 0. 5 for AFB1 and G1, and 0.72, 0.54, 0.4, 0.24 and 0.08 for B2 and G2. Good linearity was achieved, as regression coefficients were 0.99. LODs for AFB1, AFB2, AFG1 and AFG2 were 1, 0.2, 1 and 0.2 ng/g, respectively.
Extraction and clean up Samples were analysed using the validated method of the AOAC International (Stroka et al. 2000). To a test portion of 50 g of milled tea, 5 g of sodium chloride was added and then extracted with 300 ml of methanol/water (80 ml/ 20 ml) by shaking for 30 minutes. After filtration, the extract was diluted with phosphate buffer saline (PBS) and filtered through a glass micro-fiber filter. For cleanup of samples, Aflatest IACs were used. First, 10 ml PBS was passed through the IAC. Then, the filtrate was passed through the IAC at a flow rate of ca. 1 drop/s. The column was washed with 20 ml PBS buffer two times and the solution was removed by pressing air through the column and dried by applying little vacuum. Finally, aflatoxins B1 250 Aflatoxin B1 (ng/g)
200 150
B1
100 50
No. of samples
Figure 1.
Concentration of aflatoxin B1 in black tea samples.
40
37
34
31
28
25
22
19
16
13
10
7
4
0 1
Downloaded by [University of Saskatchewan Library] at 18:18 10 October 2014
Sample preparation
Food Additives & Contaminants: Part B
Total aflatoxins (ng/g)
250
Downloaded by [University of Saskatchewan Library] at 18:18 10 October 2014
Total aflatoxins
200 150
Total
100 50 0
Figure 2.
129
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 No. of samples
Concentration of total aflatoxins in black tea samples.
Quality assurance Regarding internal quality control, the accuracy and precision of the methods were verified. In this regard, recoveries of AFB1, AFB2, AFG1 and AFG2 were recorded by analysing a blank tea sample spiked at 5 ng/g for AFB1 and AFG1 and 1 ng/g for AFB2 and AFG2. Aflatoxins levels were corrected for recoveries.
and more data on the distribution and contamination levels of AFs in food like black tea. This study shows that some commercially sold products like black tea can be contaminated with mycotoxins, which indicates it is essential to monitor the presence of mycotoxins in these commodities. It is also necessary to develop and apply strategies to prevent the formation of mycotoxins in these food products in order to ensure they are wholesome and safe for consumers.
Results and discussion Average recoveries for AFB1, AFB2, AFG1 and AFG2 in tea were in the range 75–110%. According to AOAC and Codex guidelines, the recovery range should be 70−110% at a level of 10−100 μg/kg and 60−120% at a level of 1−10 μg/kg, so the obtained recoveries are acceptable. Among 40 samples analysed, 29 samples (72.5%) were not contaminated with AFB1 (
