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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
Seasonal patterns of aflatoxin M1 contamination in commercial pasteurised milk from different areas in Thailand a
Witaya Suriyasathaporn & Watinee Nakprasert
a
a
Department of Food Animal Clinics, Faculty of Veterinary Medicine , Chiang Mai University , Mae Hia , Chiang Mai Province, 50100 , Thailand Accepted author version posted online: 30 Mar 2012.Published online: 10 May 2012.
To cite this article: Witaya Suriyasathaporn & Watinee Nakprasert (2012) Seasonal patterns of aflatoxin M1 contamination in commercial pasteurised milk from different areas in Thailand, Food Additives & Contaminants: Part B: Surveillance, 5:2, 145-149, DOI: 10.1080/19393210.2012.681072 To link to this article: http://dx.doi.org/10.1080/19393210.2012.681072
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 and Contaminants: Part B Vol. 5, No. 2, June 2012, 145–149
Seasonal patterns of aflatoxin M1 contamination in commercial pasteurised milk from different areas in Thailand Witaya Suriyasathaporn* and Watinee Nakprasert Department of Food Animal Clinics, Faculty of Veterinary Medicine, Chiang Mai University, Mae Hia, Chiang Mai Province, 50100, Thailand
Downloaded by [University of West Florida] at 14:19 06 October 2014
(Received 4 May 2011; final version received 27 March 2012) Aflatoxin M1 (AFM1) levels were determined in pasteurised milk from five commercial trademarks produced in different areas in Thailand. One hundred and twenty milk samples were collected from local markets in Chiang Mai province, Thailand, to evaluate AFM1 concentrations using immunoaffinity columns and high-performance liquid chromatography with fluorescence detection. The overall median AFM1 level was 0.023 mg L1 ranging from 0.004 to 0.293 mg L1. All trademarks had average AFM1 concentrations lower than 0.05 mg L1, with those in Trademarks 3 to 5 being higher than Trademarks 1 and 2 (P 5 0.01). All trademarks had different seasonal patterns of AFM1, even though operating in the same area. However, only Trademark 3 showed significant differences of AFM1 levels between seasons. The results suggested that farm management factors, rather than environment factors, were likely to be the main cause of AFM1 contamination in dairy products. Keywords: pasteurised milk; aflatoxin M1; seasons; Thailand
Introduction Aflatoxins, a group of mycotoxins that causes health damage to humans and animals with adverse economic effects nationally and internationally on food supplies and food markets, are produced by certain strains of Aspergillus flavus and Aspergillus parasiticus (Prandini et al. 2007). The aflatoxin B1 (AFB1), the most acutely toxic, is a potential mutagen, teratogen, hepatotoxin, hepatocarcinogen and DNA-damaging agent (Kotsonis et al. 1996; Aflatoxins in Foods 2001). When consuming rations with AFB1, dairy cattle consequently metabolise the toxin and excrete aflatoxin M1 (AFM1) in their milk (Yiannikouris and Jouany 2002). On the demonstrated toxic and carcinogenic effects of AFM1, the toxin initially classified by IARC as a Group 2B human carcinogen (IARC 1993) has now moved to Group 1 (IARC 2002). To control AFM1 contamination in milk, strict regulatory limits for these compounds are currently in force in the European Community, where the prescribed maximum level of AFM1 in liquid milk is 0.05 mg L1 (European Commission 2006). In Thailand, the school milk program provides an outlet for locally produced milk and is a vital part of the Thai dairy market, accounting for more than 30% of the total liquid milk market (Suwanabol 2005). Chiang Mai province, a popular tourist city in northern Thailand and the second largest city in the country, *Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online ß 2012 Taylor & Francis http://dx.doi.org/10.1080/19393210.2012.681072 http://www.tandfonline.com
has a high demand for milk. However, production in this northern area is limited, accounting for only 8% of the national total, whereas the central area is the main area for raw milk production in Thailand, accounting for about 70% (Ohmomo et al. 2002; OAE 2003). Therefore, most of the locally produced raw milk goes into the school milk program, whereas the liquid milk products in the department stores, supermarkets and convenience stores mostly originate from the central part of Thailand. Concentrations of AFM1 in raw milk vary seasonally, with the highest levels in winter and the lowest in summer (Kamkar 2005; Hussain and Anwar 2008). Using samples collected from a collecting centre in the central part of Thailand, the average concentration of AFM1 of milk collected in the winter was significantly higher than those found in rainy and summer seasons in either school milk samples (Ruangwises and Ruangwises 2009) or raw milk samples (Ruangwises and Ruangwises 2010). In Thailand, differences in weather and farm management styles between various areas might result in differences of AFM1 concentration patterns among seasons. Therefore, the objectives of this study were to determine the differences of AFM1 levels of five trademarks of commercial pasteurised milk from companies located in different areas in Thailand and to determine seasonal AFM1 contamination patterns of each trademark by
146
W. Suriyasathaporn and W. Nakprasert
comparisons of AFM1 concentration between summer, rainy and cool seasons.
Materials and methods
Downloaded by [University of West Florida] at 14:19 06 October 2014
Materials Milk samples One hundred and twenty commercial pasteurised milk samples were purchased from different local markets in multiple areas of Chiang Mai province, Thailand, from February to October 2009. The sources included five dairy trademarks including one trademark producing for school milk in Chiang Mai province (Trademark 1) and four trademarks of large dairy companies all operating in the central area of Thailand (Trademarks 2 to 5) whose milk sources were from dairy farms surrounding the companies (Figure 1). At least one sample from each trademark was randomly collected monthly. Immediately after purchase, milk samples were transported to the laboratory in a cooler box at about 4–7 C and stored at 20 C until analysis. Chemicals and standard Methanol (HPLC grade) was purchased from Fisher Scientific (Lougborough, Leicestershire, UK). The immunoaffinity columns (AflaTestÕ ) were obtained from VICAM (Watertown, MA, USA). The water used during analysis was ultrapure water (Milli Q, Millipore, Ligand Scientific Co., Ltd., Thailand). Standard of AFM1 (10 mg L1 in acetonitrile) was purchased from Supelco (Bellifonte, PA, USA) and stored at 20 C. Working standard solution of 0.1 mg mL1 was prepared from stock standard solution and was stored in a tightly stoppered vial below 4 C.
Figure 1. Locations of milk companies including one trademark producing for school milk in Chiang Mai province (Tm1) and four trademarks of large dairy companies all operating in the central area of Thailand (Tm2–Tm5) whose milk sources were from dairy farms surrounding the companies.
Methods Determination of aflatoxin M1. The AOAC official method as reported by Suprasert and Arkanurak (1998) was applied, with some adaptations as proposed by the manufacturer of the immunoaffinity columns (VICAM 2000). Extraction and cleanup. Forty millilitres of milk samples, warmed at 37 C, with 1 g NaCl added and thoroughly mixed, were centrifuged at 2000 g. The skim portion in the bottom layer of the milk was carefully collected for analysis using a syringe needle without disturbing the fat layer on the top, and then was filtered through glass microfibre filter paper. Ten millilitres of filtered milk sample were passed through immunoaffinity columns (IACs) at a flow rate of about 1–2 drops per second. After completely passing through the column, the IAC was transferred to a clean syringe barrel and 10 mL of 10% methanol–90%
water solution was passed through the IAC twice at a 2 drops per second flow rate, until all liquid was passed. The IAC was eluted at a flow rate of about 1–2 drops per second with 1.0 mL of 80% methanol–20% water. The eluted sample was then collected in a clean glass cuvette. HPLC Determination with fluorescence detection. Identification and quantification of AFM1 was achieved by high-performance liquid chromatography (HPLC), using a Shimadzu (Kyoto, Japan) liquid chromatograph with fluorescence detector (excitation ¼ 360 nm, emission ¼ 440 nm). A Whatman Partisphere RTF C-18 4.6 mm 150 mm, 5 mm column was used (Whatman Asia Pacific Pte. Ltd., Singapore Science Park 1, Singapore). Calibration curves of AFM1 were prepared using standard solutions of AFM1, at concentrations of 0.5, 1.0, 2.0, 3.0 and 4.0 mg L1. Fifty microlitres of sample extracts
Downloaded by [University of West Florida] at 14:19 06 October 2014
Food Additives and Contaminants: Part B
147
Figure 2. Geometric means with standard error of means of AFM1 of various trademarks of commercial pasteurised milk purchased from the local market in Chiang Mai province, Thailand. Different letters (a, b, c) denote differences between the means at P50.05. The bold line indicates the LOD of the method at 0.004 mg L1.
were injected into HPLC for determination of AFM1. The isocratic mobile phase consisted of 45% methanol and 55% water with a flow rate 0.8 mL min1. Under these conditions, the retention time for AFM1 was approximately 1.8 minutes.
Statistical analysis On the basis of the manufacturing date, seasons were separated into summer (March to May), rainy (June to October) and cool (November to February). The AFM1 concentrations, transferred with natural logarithm because the AFM1 data were not normally distributed, were statistically analysed by repeated one-way analysis of variance to compare AFM1 concentrations among milk companies and seasons within milk sources. Least-squares differences multiple comparison tests were applied to obtain significance levels between sampling groups.
Results and discussion The typical calibration curve using AFM1 standard was in the range 0.5–4.0 mg L1 and the correlation coefficient (R2) of this curve was 0.9998, indicating good linearity. The chromatograms of AFM1 at the retention time of approximately 1.8 minutes of standard solution and naturally contaminated pasteurised milk samples did not contain interfering peaks. The limit of detection (LOD ¼ 3 S/N) and the limit of quantification (LOQ ¼ 10 S/N) were 0.004 mg L1 and 0.01 mg L1, respectively. Recovery was studied by spiking milk samples with AFM1 standard (10 mg mL1) at the levels 0.5, 1.0 and 2.0 mg L1. The
recoveries were found to be 83%, 87% and 91%, respectively. The overall median AFM1 level was 0.023 mg L1 (n ¼ 120), within a range from 0.004 to 0.293 mg L1. Among the contaminated milk samples, it was observed that 27.5% of all samples were above the EU maximum limit of 0.05 mg L1 (European Commission 2006). The levels found in this study were similar to those obtained by recent studies in Thailand (Ruangwises and Ruangwises 2009, 2010). Figure 2 shows geometric means of AFM1 found in various commercial pasteurised milk samples. All trademarks had average concentrations of AFM1 lower than 0.05 mg L1. The mean AFM1 levels of Trademarks 1 and 2 were lower than LOD levels, which might indicate no incidence of AFM1 contamination. According to statistical analysis, AFM1 levels in Trademarks 3 to 5 were higher than Trademarks 1 and 2 (P 5 0.01). The difference in AFM1 might be caused by the differences in farm management and environment in Thailand. Figure 3 shows the geometric means of AFM1 among trademarks separated into seasons. All trademarks had different seasonal patterns of AFM1, even when operating in the same area. For example, Trademarks 2 to 5 were companies located in central Thailand, but only Trademark 3 had significantly different AFM1 levels between seasons (P 5 0.01). In contrast, the averages of AFM1 concentration of Trademark 5 were at high levels in all seasons ranging between 0.036 and 0.040 mg L1. For Trademark 3, the cool season had higher AFM1 concentration than summer and rainy seasons (P 5 0.01). This pattern is similar to the pattern reported in recent studies using samples from the central part of Thailand (Ruangwises and Ruangwises 2009, 2010). This is also consistent
Downloaded by [University of West Florida] at 14:19 06 October 2014
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Figure 3. Geometric means with standard error of means of AFM1 of various trademarks of commercial pasteurised milk separated into seasons including summer (March–May), rainy (June–October) and cool (November–February). Different letters (a, b) denote differences between the means at P50.05 in comparison within the trademark milk samples. The bold line indicates the LOD of the method at 0.004 mg L1.
with some previous studies in tropical countries, such as Iran (Fallah 2010), Pakistan (Hussain and Anwar 2008) and Turkey (Unusan 2006), showing that AFM1 levels were highest in winter and lowest in summer. Our findings show stronger relationships between contamination of AFM1 and farm management than with environment and climate. In seasons when agricultural products were available, fresh roughages are used for dairy cows. For consuming in the dry season, dairy farms in some central areas of Thailand store and prepare corn silage during the rainy season (Ohmomo et al. 2002), whereas rice straw is used as roughage in the north (Suriyasathaporn 2011). Corn silage has high moisture, with a greater tendency to produce fungal growth and aflatoxins than straw. In the central area of Thailand, rations used in dairy farms had the highest AFB1 in winter compared to rainy and summer seasons (Mahosotanand 2002).
Conclusion The results of the present study provide information on AFM1 contamination of commercial pasteurised milk marketed in Chiang Mai province, Thailand. They reveal that seasonal variations significantly influence AFM1 level in only one trademark of a company located in the central area of Thailand. In contrast, other trademarks, either in the central or north, had no significant seasonal variations related to AFM1 concentrations. Also, the present study showed that the AFM1 level of commercial milk samples, especially in Trademark 5, is a serious problem for public health during the whole year. This indicated that farm management factors, rather than environmental factors, are the main cause of AFM1 contamination in dairy products. Reducing the levels of AFB1 in animal
feed ration by improvement of storage and processing practices can be the initial approach to deal with this problem. In addition, the variation in feedstuff management practices in small-holder dairy farms in Thailand that are associated with AFM1 in bulk tank milk and AFB1 in ration is an important issue for the inspection and control of AFM1 in dairy products.
Acknowledgements This study was supported by a grant from National Research Council of Thailand. The authors would like to thank Dr. Wasana Chaisri for her technical support.
References Aflatoxins in Foods: Risk Assessment Studies. 2001. Report No. 5. Food and Environmental Hygiene Department. The Government of the Hong Kong Special Administrative Region. European Commission. 2006. Regulation 2006/1881/EC of 19 December 2006 replacing Regulation (EC) 466/2001 setting maximum levels for certain contaminants in foodstuffs. Offic J Eur Union. L364:5–24. Fallah AA. 2010. Assessment of aflatoxins M1 contamination in pasteurized and UHT milk marketed in central part of Iran. Food Chem Toxicol. 48:988–991. Hussain I, Anwar J. 2008. A study on contamination of aflatoxin M1 in raw milk in the Punjab province of Pakistan. Food Control. 19:393–395. International Agency for Research on Cancer [IARC]. 1993. Aflatoxins. Some naturally occurring substances: food items and constituents, heterocyclic aromatic amines and mycotoxins. IARC monograph on the evaluation of carcinogenic risks to humans. Vol. 56. IARC, Lyon (France): World Health Organization. p. 245–395.
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Food Additives and Contaminants: Part B International Agency for Research on Cancer [IARC]. 2002. Aflatoxins. IARC monograph on the evaluation of carcinogenic risks to humans. Vol. 82. IARC, Lyon (France): World Health Organization. p. 171. Kamkar U. 2005. A study on the occurrence of aflatoxins M1 in raw milk produced in Sarab City of Iran. Food Control. 19:593–599. Kotsonis FN, Burdock GA, Flamm WG. 1996. Food toxicology. In: Cassarett and Doull’s toxicology: The basic science of poisons. New York: McGraw-Hill. p. 938–939. Mahosotanand B. 2002. Relationship between aflatoxin M1 in milk and aflatoxin B1 in feedstuffs. Proceedings of evaluation and management of integrated problems solving of aflatoxins in food and feedstuffs, Thai Industrial Standards Institute. Bangkok: Ministry of Industry. p. 67–69. Office of Agricultural Economics [OAE]. 2003. Agricultural statistics of Thailand 2003. Office of Agricultural Economics, Ministry of Agriculture and Co-operatives, Bangkok. Ohmomo S, Nitisinprasert S, Hiranpradit S. 2002. Silagemaking and recent trend of dairy farming in Thailand. Jpn Agric Res Q. 36:227–234.
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Prandini A, Tansini G, Sigolo S, Filippi L, Laporta M, Piva G. 2007. On the occurrence of aflatoxin M1 in milk and dairy products. Food Chem Toxicol. 47:984–991. Ruangwises S, Ruangwises N. 2009. Occurrence of aflatoxin M1 in pasteurized milk of the school milk project in Thailand. J Food Prot. 72:1761–1763. Ruangwises N, Ruangwises S. 2010. Aflatoxin M1 contamination in raw milk within the central region of Thailand. Bull Environ Contam Toxicol. 85:195–198. Suprasert D, Arkanurak N. 1998. Comparison of AflaTest-P and Afla-M1 column for aflatoxin M1 analysis in milk. Kasetsart J. 32:292–298. Suriyasathaporn W. 2011. Epidemiology of subclinical mastitis and their antibacterial susceptibility in small holder dairy farms, Chiang Mai province, Thailand. J Anim Vet Adv. 10:316–321. Suwanabol I. 2005. School milk programme in Thailand. Paper presented at: The 3rd International School Milk Conference; April 11–14; Kunming, China. Unusan N. 2006. Occurrence of aflatoxin M1 in UHT milk in Turkey. Food Chem Toxicol. 44:1897–1900. VICAM. 2000. AflaTestÕ Instruction Manual. Procedure 5.3. Watertown, MA, USA, p. 69–70. Yiannikouris A, Jouany JP. 2002. Mycotoxins in feed and their fate in animals: a review. Anim Res. 51:81–89.
Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Seasonal patterns of aflatoxin M1 contamination in commercial pasteurised milk from different areas in Thailand a
Witaya Suriyasathaporn & Watinee Nakprasert
a
a
Department of Food Animal Clinics, Faculty of Veterinary Medicine , Chiang Mai University , Mae Hia , Chiang Mai Province, 50100 , Thailand Accepted author version posted online: 30 Mar 2012.Published online: 10 May 2012.
To cite this article: Witaya Suriyasathaporn & Watinee Nakprasert (2012) Seasonal patterns of aflatoxin M1 contamination in commercial pasteurised milk from different areas in Thailand, Food Additives & Contaminants: Part B: Surveillance, 5:2, 145-149, DOI: 10.1080/19393210.2012.681072 To link to this article: http://dx.doi.org/10.1080/19393210.2012.681072
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 and Contaminants: Part B Vol. 5, No. 2, June 2012, 145–149
Seasonal patterns of aflatoxin M1 contamination in commercial pasteurised milk from different areas in Thailand Witaya Suriyasathaporn* and Watinee Nakprasert Department of Food Animal Clinics, Faculty of Veterinary Medicine, Chiang Mai University, Mae Hia, Chiang Mai Province, 50100, Thailand
Downloaded by [University of West Florida] at 14:19 06 October 2014
(Received 4 May 2011; final version received 27 March 2012) Aflatoxin M1 (AFM1) levels were determined in pasteurised milk from five commercial trademarks produced in different areas in Thailand. One hundred and twenty milk samples were collected from local markets in Chiang Mai province, Thailand, to evaluate AFM1 concentrations using immunoaffinity columns and high-performance liquid chromatography with fluorescence detection. The overall median AFM1 level was 0.023 mg L1 ranging from 0.004 to 0.293 mg L1. All trademarks had average AFM1 concentrations lower than 0.05 mg L1, with those in Trademarks 3 to 5 being higher than Trademarks 1 and 2 (P 5 0.01). All trademarks had different seasonal patterns of AFM1, even though operating in the same area. However, only Trademark 3 showed significant differences of AFM1 levels between seasons. The results suggested that farm management factors, rather than environment factors, were likely to be the main cause of AFM1 contamination in dairy products. Keywords: pasteurised milk; aflatoxin M1; seasons; Thailand
Introduction Aflatoxins, a group of mycotoxins that causes health damage to humans and animals with adverse economic effects nationally and internationally on food supplies and food markets, are produced by certain strains of Aspergillus flavus and Aspergillus parasiticus (Prandini et al. 2007). The aflatoxin B1 (AFB1), the most acutely toxic, is a potential mutagen, teratogen, hepatotoxin, hepatocarcinogen and DNA-damaging agent (Kotsonis et al. 1996; Aflatoxins in Foods 2001). When consuming rations with AFB1, dairy cattle consequently metabolise the toxin and excrete aflatoxin M1 (AFM1) in their milk (Yiannikouris and Jouany 2002). On the demonstrated toxic and carcinogenic effects of AFM1, the toxin initially classified by IARC as a Group 2B human carcinogen (IARC 1993) has now moved to Group 1 (IARC 2002). To control AFM1 contamination in milk, strict regulatory limits for these compounds are currently in force in the European Community, where the prescribed maximum level of AFM1 in liquid milk is 0.05 mg L1 (European Commission 2006). In Thailand, the school milk program provides an outlet for locally produced milk and is a vital part of the Thai dairy market, accounting for more than 30% of the total liquid milk market (Suwanabol 2005). Chiang Mai province, a popular tourist city in northern Thailand and the second largest city in the country, *Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online ß 2012 Taylor & Francis http://dx.doi.org/10.1080/19393210.2012.681072 http://www.tandfonline.com
has a high demand for milk. However, production in this northern area is limited, accounting for only 8% of the national total, whereas the central area is the main area for raw milk production in Thailand, accounting for about 70% (Ohmomo et al. 2002; OAE 2003). Therefore, most of the locally produced raw milk goes into the school milk program, whereas the liquid milk products in the department stores, supermarkets and convenience stores mostly originate from the central part of Thailand. Concentrations of AFM1 in raw milk vary seasonally, with the highest levels in winter and the lowest in summer (Kamkar 2005; Hussain and Anwar 2008). Using samples collected from a collecting centre in the central part of Thailand, the average concentration of AFM1 of milk collected in the winter was significantly higher than those found in rainy and summer seasons in either school milk samples (Ruangwises and Ruangwises 2009) or raw milk samples (Ruangwises and Ruangwises 2010). In Thailand, differences in weather and farm management styles between various areas might result in differences of AFM1 concentration patterns among seasons. Therefore, the objectives of this study were to determine the differences of AFM1 levels of five trademarks of commercial pasteurised milk from companies located in different areas in Thailand and to determine seasonal AFM1 contamination patterns of each trademark by
146
W. Suriyasathaporn and W. Nakprasert
comparisons of AFM1 concentration between summer, rainy and cool seasons.
Materials and methods
Downloaded by [University of West Florida] at 14:19 06 October 2014
Materials Milk samples One hundred and twenty commercial pasteurised milk samples were purchased from different local markets in multiple areas of Chiang Mai province, Thailand, from February to October 2009. The sources included five dairy trademarks including one trademark producing for school milk in Chiang Mai province (Trademark 1) and four trademarks of large dairy companies all operating in the central area of Thailand (Trademarks 2 to 5) whose milk sources were from dairy farms surrounding the companies (Figure 1). At least one sample from each trademark was randomly collected monthly. Immediately after purchase, milk samples were transported to the laboratory in a cooler box at about 4–7 C and stored at 20 C until analysis. Chemicals and standard Methanol (HPLC grade) was purchased from Fisher Scientific (Lougborough, Leicestershire, UK). The immunoaffinity columns (AflaTestÕ ) were obtained from VICAM (Watertown, MA, USA). The water used during analysis was ultrapure water (Milli Q, Millipore, Ligand Scientific Co., Ltd., Thailand). Standard of AFM1 (10 mg L1 in acetonitrile) was purchased from Supelco (Bellifonte, PA, USA) and stored at 20 C. Working standard solution of 0.1 mg mL1 was prepared from stock standard solution and was stored in a tightly stoppered vial below 4 C.
Figure 1. Locations of milk companies including one trademark producing for school milk in Chiang Mai province (Tm1) and four trademarks of large dairy companies all operating in the central area of Thailand (Tm2–Tm5) whose milk sources were from dairy farms surrounding the companies.
Methods Determination of aflatoxin M1. The AOAC official method as reported by Suprasert and Arkanurak (1998) was applied, with some adaptations as proposed by the manufacturer of the immunoaffinity columns (VICAM 2000). Extraction and cleanup. Forty millilitres of milk samples, warmed at 37 C, with 1 g NaCl added and thoroughly mixed, were centrifuged at 2000 g. The skim portion in the bottom layer of the milk was carefully collected for analysis using a syringe needle without disturbing the fat layer on the top, and then was filtered through glass microfibre filter paper. Ten millilitres of filtered milk sample were passed through immunoaffinity columns (IACs) at a flow rate of about 1–2 drops per second. After completely passing through the column, the IAC was transferred to a clean syringe barrel and 10 mL of 10% methanol–90%
water solution was passed through the IAC twice at a 2 drops per second flow rate, until all liquid was passed. The IAC was eluted at a flow rate of about 1–2 drops per second with 1.0 mL of 80% methanol–20% water. The eluted sample was then collected in a clean glass cuvette. HPLC Determination with fluorescence detection. Identification and quantification of AFM1 was achieved by high-performance liquid chromatography (HPLC), using a Shimadzu (Kyoto, Japan) liquid chromatograph with fluorescence detector (excitation ¼ 360 nm, emission ¼ 440 nm). A Whatman Partisphere RTF C-18 4.6 mm 150 mm, 5 mm column was used (Whatman Asia Pacific Pte. Ltd., Singapore Science Park 1, Singapore). Calibration curves of AFM1 were prepared using standard solutions of AFM1, at concentrations of 0.5, 1.0, 2.0, 3.0 and 4.0 mg L1. Fifty microlitres of sample extracts
Downloaded by [University of West Florida] at 14:19 06 October 2014
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Figure 2. Geometric means with standard error of means of AFM1 of various trademarks of commercial pasteurised milk purchased from the local market in Chiang Mai province, Thailand. Different letters (a, b, c) denote differences between the means at P50.05. The bold line indicates the LOD of the method at 0.004 mg L1.
were injected into HPLC for determination of AFM1. The isocratic mobile phase consisted of 45% methanol and 55% water with a flow rate 0.8 mL min1. Under these conditions, the retention time for AFM1 was approximately 1.8 minutes.
Statistical analysis On the basis of the manufacturing date, seasons were separated into summer (March to May), rainy (June to October) and cool (November to February). The AFM1 concentrations, transferred with natural logarithm because the AFM1 data were not normally distributed, were statistically analysed by repeated one-way analysis of variance to compare AFM1 concentrations among milk companies and seasons within milk sources. Least-squares differences multiple comparison tests were applied to obtain significance levels between sampling groups.
Results and discussion The typical calibration curve using AFM1 standard was in the range 0.5–4.0 mg L1 and the correlation coefficient (R2) of this curve was 0.9998, indicating good linearity. The chromatograms of AFM1 at the retention time of approximately 1.8 minutes of standard solution and naturally contaminated pasteurised milk samples did not contain interfering peaks. The limit of detection (LOD ¼ 3 S/N) and the limit of quantification (LOQ ¼ 10 S/N) were 0.004 mg L1 and 0.01 mg L1, respectively. Recovery was studied by spiking milk samples with AFM1 standard (10 mg mL1) at the levels 0.5, 1.0 and 2.0 mg L1. The
recoveries were found to be 83%, 87% and 91%, respectively. The overall median AFM1 level was 0.023 mg L1 (n ¼ 120), within a range from 0.004 to 0.293 mg L1. Among the contaminated milk samples, it was observed that 27.5% of all samples were above the EU maximum limit of 0.05 mg L1 (European Commission 2006). The levels found in this study were similar to those obtained by recent studies in Thailand (Ruangwises and Ruangwises 2009, 2010). Figure 2 shows geometric means of AFM1 found in various commercial pasteurised milk samples. All trademarks had average concentrations of AFM1 lower than 0.05 mg L1. The mean AFM1 levels of Trademarks 1 and 2 were lower than LOD levels, which might indicate no incidence of AFM1 contamination. According to statistical analysis, AFM1 levels in Trademarks 3 to 5 were higher than Trademarks 1 and 2 (P 5 0.01). The difference in AFM1 might be caused by the differences in farm management and environment in Thailand. Figure 3 shows the geometric means of AFM1 among trademarks separated into seasons. All trademarks had different seasonal patterns of AFM1, even when operating in the same area. For example, Trademarks 2 to 5 were companies located in central Thailand, but only Trademark 3 had significantly different AFM1 levels between seasons (P 5 0.01). In contrast, the averages of AFM1 concentration of Trademark 5 were at high levels in all seasons ranging between 0.036 and 0.040 mg L1. For Trademark 3, the cool season had higher AFM1 concentration than summer and rainy seasons (P 5 0.01). This pattern is similar to the pattern reported in recent studies using samples from the central part of Thailand (Ruangwises and Ruangwises 2009, 2010). This is also consistent
Downloaded by [University of West Florida] at 14:19 06 October 2014
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Figure 3. Geometric means with standard error of means of AFM1 of various trademarks of commercial pasteurised milk separated into seasons including summer (March–May), rainy (June–October) and cool (November–February). Different letters (a, b) denote differences between the means at P50.05 in comparison within the trademark milk samples. The bold line indicates the LOD of the method at 0.004 mg L1.
with some previous studies in tropical countries, such as Iran (Fallah 2010), Pakistan (Hussain and Anwar 2008) and Turkey (Unusan 2006), showing that AFM1 levels were highest in winter and lowest in summer. Our findings show stronger relationships between contamination of AFM1 and farm management than with environment and climate. In seasons when agricultural products were available, fresh roughages are used for dairy cows. For consuming in the dry season, dairy farms in some central areas of Thailand store and prepare corn silage during the rainy season (Ohmomo et al. 2002), whereas rice straw is used as roughage in the north (Suriyasathaporn 2011). Corn silage has high moisture, with a greater tendency to produce fungal growth and aflatoxins than straw. In the central area of Thailand, rations used in dairy farms had the highest AFB1 in winter compared to rainy and summer seasons (Mahosotanand 2002).
Conclusion The results of the present study provide information on AFM1 contamination of commercial pasteurised milk marketed in Chiang Mai province, Thailand. They reveal that seasonal variations significantly influence AFM1 level in only one trademark of a company located in the central area of Thailand. In contrast, other trademarks, either in the central or north, had no significant seasonal variations related to AFM1 concentrations. Also, the present study showed that the AFM1 level of commercial milk samples, especially in Trademark 5, is a serious problem for public health during the whole year. This indicated that farm management factors, rather than environmental factors, are the main cause of AFM1 contamination in dairy products. Reducing the levels of AFB1 in animal
feed ration by improvement of storage and processing practices can be the initial approach to deal with this problem. In addition, the variation in feedstuff management practices in small-holder dairy farms in Thailand that are associated with AFM1 in bulk tank milk and AFB1 in ration is an important issue for the inspection and control of AFM1 in dairy products.
Acknowledgements This study was supported by a grant from National Research Council of Thailand. The authors would like to thank Dr. Wasana Chaisri for her technical support.
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