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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
Survey of aflatoxin M1 in raw milk in the five provinces of China Nan Zheng & Peng Sun
a b c
, Jia-Qi Wang
a b c
, Rong-Wei Han
a b c d
, Yun-Peng Zhen
b c
, Xiao-Min Xu
b c
a
a
State Key Laboratory of Animal Nutrition, Institute of Animal Science , Chinese Academy of Agricultural Sciences , Beijing , P.R. China b
Milk and Dairy Product Inspection Center (Beijing), Ministry of Agriculture , Beijing , P.R. China c
Laboratory of Quality & Safety Risk Assessment for Dairy Products (Beijing), Ministry of Agriculture , Beijing , P.R. China d
College of Food Science and Engineering , Qingdao Agricultural University , Qingdao , P.R. China Accepted author version posted online: 07 Jan 2013.Published online: 18 Feb 2013.
To cite this article: Nan Zheng , Jia-Qi Wang , Rong-Wei Han , Yun-Peng Zhen , Xiao-Min Xu & Peng Sun (2013): Survey of aflatoxin M1 in raw milk in the five provinces of China, Food Additives & Contaminants: Part B: Surveillance, 6:2, 110-115 To link to this article: http://dx.doi.org/10.1080/19393210.2012.763191
PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions 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. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
Food Additives & Contaminants: Part B, 2013 Vol. 6, No. 2, 110–115, http://dx.doi.org/10.1080/19393210.2012.763191
Survey of aflatoxin M1 in raw milk in the five provinces of China Nan Zhenga,b,c, Jia-Qi Wanga,b,c*, Rong-Wei Hana,b,c,d, Yun-Peng Zhenb,c, Xiao-Min Xub,c and Peng Suna a
State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, P.R. China; Milk and Dairy Product Inspection Center (Beijing), Ministry of Agriculture, Beijing, P.R. China; cLaboratory of Quality & Safety Risk Assessment for Dairy Products (Beijing), Ministry of Agriculture, Beijing, P.R. China; dCollege of Food Science and Engineering, Qingdao Agricultural University, Qingdao, P.R. China b
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(Received 4 April 2012; final version received 29 December 2012) Aflatoxin M1 (AFM1) is the only mycotoxin that has a legal limit in milk all over the world. In the present study, 360 raw milk samples were collected from Beijing, Hebei, Shanxi, Shanghai and Guangdong provinces in China in September 2010, and their AFM1 levels were determined by using enzyme-linked immunosorbent assay (ELISA). More than three-fourths (78.1%) of the 360 raw milk samples contained AFM1 at concentrations of 5–123 ng L−1. AFM1 contents in all positive samples were far below the Chinese and US legal limit of 500 ng L−1, but 10% of the raw milk samples exceeded the EU legal limit of 50 ng L−1. Moreover, both incidence and content of AFM1 in milk collected from the southern provinces, including Shanghai and Guangdong, were higher than those collected from the northern provinces, including Beijing, Hebei and Shanxi. Keywords: aflatoxin M1; raw milk; ELISA; China
Introduction Aflatoxins are toxic secondary metabolites of moulds Aspergillus flavus and A. parasiticus and are the main mycotoxin which could be detected in milk (Boudra et al. 2007). Eighteen aflatoxins have been identified up to now, and B1, B2, G1, G2 and M1 are the aflatoxins of most concern (Decastelli et al. 2007). Moreover, aflatoxin M1 (AFM1) is the only mycotoxin that has been set a legal limit in milk by many national governments and international organisations, such as the Codex Alimentarius Commission (Food and Agriculture Organisation 2004; Sugiyama et al. 2008). Aflatoxin B1 (AFB1) is the most toxic aflatoxin and has been designated as a primary carcinogenic compound by the International Agency for Research on Cancer (IARC) of the World Health Organisation (WHO) (International Agency for Research on Cancer 1993). Dairy cows consuming feeds contaminated with AFB1 can excrete a hydroxylated metabolite of AFB1 as AFM1 in milk (Frobish et al. 1986; Chopra et al. 1999; Kuilman et al. 2000). AFM1 has been classified as a second-class possible human carcinogen by the IARC, and its carcinogenicity was estimated to be 1/10th of that of AFB1 by the Joint Expert Committee on Contamination and Food Additives in 2001 (International Agency for Research on Cancer 1993; Sugiyama et al. 2008). Furthermore, AFM1 is very stable, and it is assumed that neither storage nor processing, such as pasteurisation, autoclaving, fermentation or other methods commonly used to process milk products, destroys its toxin (Tajkarimi et al. 2008). *Corresponding author. Email: [email protected] Nan Zheng and Rong-Wei Han contributed equally. © 2013 Taylor & Francis
A legal limit for AFM1 in milk existed in 60 countries at the end of 2003. Among them, 50 ng L−1 set by the EU and 500 ng L−1 by the United States are two peak limits (Food and Agriculture Organisation 2004). Further, many countries such as New Zealand, United Kingdom, Brazil and Iran have conducted national surveys to assess AFM1 contamination in raw milk in order to ensure safety of milk products (UKFSA 2001; Sassahara et al. 2005; Tajkarimi et al. 2008; NZFSA 2012). The Chinese government has established 500 ng L−1 as the national legal limit of AFM1 in milk. Pei et al. (2009) measured the AFM1 content in 12 raw milk samples collected from the market in northeastern China in 2008. AFM1 was detected in all samples with contents in the range of 160–500 μg L−1, and 75% of samples had AFM1 concentrations in the range 320–500 µg L−1, which were much closer to the Chinese national legal limit. The findings suggested that the contamination of AFM1 in raw milk in the northeast of China is serious and stressed the need for continued assessment of AFM1. In the present study, AFM1 contamination in 360 raw milk samples collected from five provinces, covering the north and south of China, was assessed using ELISA. Material and methods Sampling During September 2010, raw milk samples were collected from 360 milk stations in Beijing (80), Hebei (80), Shanxi (80), Shanghai (60) and Guangdong (60) provinces in
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Food Additives & Contaminants: Part B China. Geographically, Beijing, Hebei and Shanxi provinces are located in the north, whereas Shanghai and Guangdong provinces are located in the south. They represent two typical climates of China, and the mean temperature and precipitation in the five provinces in September 2010 is listed in Table 1. The 360 milk stations can be classified into three types according to their ownership, owned by large-scale farms (181), milk processing plants (66) and small farm cooperatives (113), respectively. The raw milk was collected directly from the milk holding tanks at the milk stations in dairy farms. After stirring of milk holding tanks, 200 mL of milk sample was taken from the upper third of the tank, 200 mL from the middle third and 200 mL from the lower third. Thus, 600 mL of milk from each tank was mixed, and 100-mL samples were taken and stored at −20°C until analysis was performed. Milk sample analysis The quantitative analysis of AFM1 in milk samples was determined using RIDASCREEN® Aflatoxin M1 test kit (R1111, R-Biopharm AG, Darmstadt, Germany), which is a competitive enzyme immunoassay kit. All reagents required for the enzyme immunoassay, including standards (0, 5, 10, 20, 40 and 80 ng L−1), are contained in the test kit. The test was conducted according to the instruction manual supplied with the kit. Sample preparation Eight millilitres of milk sample was defatted by centrifugation at 3500 g and 10°C for 10 min. The upper cream layer was removed and the defatted supernatant was subjected to ELISA for AFM1 detection.
111
CA, USA). Next, 100 μL of the diluted enzyme conjugate was added to the wells and incubated for 15 min at room temperature in the dark. The washing procedure was repeated twice again. Hundred microlitres of chromogen was added to each well and incubated for 15 min at room temperature in the dark. Lastly, 100 μL of the stop solution was added to each well and the absorbance was measured at 450 nm by using a microplate reader (Infinite 200, Tecan Austria GmbH, Groedig, Austria). Quality control Every 20th sample of milk was fortified with 20 ng L−1 AFM1 to verify the reliability of the analysis. Calculation The content of AFM1 in samples was calculated by using a special software, the RIDA®SOFT Win (Z9999, RBiopharm AG, Darmstadt, Germany). According to the instructions, the detection limit of the present method is 5 ng L−1 for milk. So the AFM1 concentration in the 5– 80 ng L−1 range can be read from the calibration curve. Statistical analysis All samples were run in duplicate. Differences in AFM1 concentration were statistically analysed by using a nonparametric test, followed by Mann-Whitney or KruskalWallis comparisons, using SPSS version 11.5 (SPSS, Inc., Chicago, IL). The level of confidence required for significance was set at P < 0.05. Results
Test procedure Hundred microlitres of the standard solutions or defatted samples was added to the wells and incubated for 30 min at room temperature (20–25°C) in the dark. The liquid then was sucked out of the wells and they were filled with 250 μL washing buffer, which again was sucked out. The washing procedure was repeated twice using immuno wash (Model 1875, Bio-Rad Laboratories, Inc., Hercules,
AFM1 recovery from the 20 ng L−1 fortified raw milk samples (n = 36) was 92.5–115.0%. For the 360 raw milk samples, AFM1 was detected in 281 samples, with an incidence of 78.1%. Among the positive samples, 62.2% contained AFM1 at levels of 5.0–29.9 ng L−1, 5.8% at levels of 30.0–49.9 ng L−1, 9.7% at levels of 50.0– 99.9 ng L−1, and 0.3% at levels of 100.0–129.9 ng L−1 (Table 2).
Table 1. Temperature and precipitation in September 2010 in five provinces of China. Province Beijing Hebei Shanxi Shanghai Guangdong Northa Southb
Temperature (ºC)
Precipitation (mm)
20.4 18.8 17.7 26.2 27.4 19.0 26.8
75.6 90.7 84.6 125.7 351.6 83.6 238.7
Notes: aNorth part includes Beijing, Hebei and Shanxi provinces. b South part includes Shanghai and Guangdong provinces.
39 20 1 60 360
Total
Total
60
Total
Large-scale farms Milk processing plants Small farm cooperatives
50 10 0
80
Total
Large-scale farms Milk processing plants Small farm cooperatives
15 13 52
Large-scale farms Milk processing plants Small farm cooperatives
80
Total
−1
79 (21.9)
5 (8.3)
2 (5.1) 3 (15.0) 0
5 (8.3)
4 (8.0) 1 (10.0) –
37 (46.3)
9 (60.0) 8 (61.5) 20 (38.5)
20 (25.0)
12 (24.5) – 8 (25.8)
12 (15.0)
2 (7.1) 5 (21.7) 5 (17.2)
Negative samples,a no. (%)
Notes: Negative samples had an AFM1 level lower than the detection limit of 5 ng L . b Positive samples had an AFM1 level greater than the detection limit of 5 ng L−1.
a
Guangdong
Shanghai
Shanxi
49 0 31
Large-scale farms Milk processing plants Small farm cooperatives
80
Total
Hebei
28 23 29
Large-scale farms Milk processing plants Small farm cooperatives
Beijing
No. of samples
Milk station type
Level of aflatoxin M1 (AFM1) in raw milk in five provinces of China.
Province
Table 2.
224 (62.2)
41 (68.3)
30 (76.9) 11 (55.0) 0
38 (63.3)
32 (64.0) 6 (60.0) –
39 (48.8)
6 (40.0) 5 (38.5) 28 (53.8)
51 (63.8)
33 (67.3) – 18 (58.1)
55 (68.8)
22 (78.6) 12 (52.2) 21 (72.4)
5.0–29.9 ng L−1
21 (5.8)
6 (10.0)
5 (12.8) 1 (5.0) 0
6 (10.0)
6 (12.0) 0 –
2 (2.5)
0 0 2 (3.8)
3 (3.8)
1 (2.0) – 2 (6.5)
4 (5.0)
1 (3.6) 1 (4.3) 2 (6.9)
30.0–49.9 ng L−1
35 (9.7)
8 (13.3)
2 (5.1) 5 (25.0) 1 (100)
11 (18.3)
8 (16.0) 3 (30.0) –
2 (2.5)
0 0 2 (3.8)
5 (6.3)
3 (6.1) – 2 (6.5)
9 (11.3)
3 (10.7) 5 (21.7) 1 (3.4)
50.0–99.9 ng L−1
Positive samples,b no. (%)
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1 (0.3)
0
0
0
0
0 0 –
0
0 0 0
1 (1.3)
0 – 1 (3.2)
0
0 0 0
100.0–129.9 ng L−1
112 N. Zheng et al.
Food Additives & Contaminants: Part B
113
Table 3. Comparison of aflatoxin M1 (AFM1) levels in raw milk collected from northa and southb China.
No. of samples
Negative sample,c no. (%)
Positive sample,d no. (%)
Samples exceeding EU legal limit,e no. (%)
Samples exceeding CN legal limit,f no. (%)
North South
240 120
69 (28.8%) 10 (8.3%)
171 (71.3%) 110 (91.7%)
17 (7.1%) 19 (15.8%)
Total
360
79 (21.9%)
281 (78.1%)
36 (10.0%)
Location
AFM1 content (ng L–1) Minimum
Maximum
Mean ± SD
0 0
5.0 5.5
123.0 97.5
18.9 ± 20.8A 25.9 ± 22.4B
0
5.0
123.0
21.6 ± 21.6
The incidence of AFM1 in the milk samples collected from the north was 71.3%, with AFM1 content ranging from 5.0 to 123.0 ng L−1. And the incidence of AFM1 in milk samples collected from the south was 91.7%, with AFM1 content from 5.5 to 97.5 ng L−1(Table 3). According to our statistical analysis, the mean of AFM1 content in milk samples collected from the north (18.9 ng L−1) was significantly lower (P < 0.05) than that collected from the south (25.9 ng L−1) (Table 3). The incidence of AFM1 in raw milk samples collected from the five provinces was different, which in increasing order was Shanxi (53.7%), Hebei (75.0%), Beijing (85.0%), Guangdong (91.7%) and Shanghai (91.7%) (Table 2). Moreover, the AFM1 content was significantly different (P < 0.05) in samples collected from the five provinces, and the ascendant succession was Shanxi (15.3 ng L−1), Beijing (19.2 ng L−1), Hebei (21.2 ng L−1), Guangdong (23.7 ng L−1) and Shanghai (28.1 ng L−1) (Figure 1). In each of the five provinces, the incidence of AFM1 in milk was different in milk stations owned by large-scale farms, milk processing plants, and small farm cooperatives. However, there was no consistent trend in three types of milk stations in the five provinces (Table 2). Overall, the incidence of AFM1 was lowest in samples collected from
milk stations owned by small farm cooperatives (70.8%), medium in those from milk stations owned by milk processing plants (74.2%), and highest in those from milk stations owned by large-scale farms (84.0%). But the AFM1 content in milk samples collected from the three types of milk stations had no significant (P > 0.05) difference (Table 4). Discussion Although most of the milk samples were contaminated by AFM1, the AFM1 content in all positive samples was within the Chinese and US legal limit of 500 ng L−1. And the minimum and maximum AFM1 content was 5 and 123 ng L−1, respectively. Both the incidence and level of AFM1 in the present study was lower than in an earlier report by Pei et al. (2009). Moulds can easily grow and produce toxins in high temperature and a wet environment, so temperature and moisture were the most important impacts on the amount of AFB1 occurrence in feed (Bakirci 2001). Furthermore, AFB1 content in dairy cow feed determines AFM1 content in raw milk, so higher levels of temperature and moisture also led to higher concentrations of AFM1 in raw milk. Of the five provinces, Shanghai and
30.0 25.0 AFM1 content (ng/L)
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Notes: Different superscript capital letters A and B denote that the mean difference in samples between north and south is significant at P < 0.05. a North part includes Beijing, Hebei and Shanxi. b South part includes Shanghai and Guangdong. c Negative samples had an AFM1 level lower than the detection limit of 5 ng L−1. d Positive samples had an AFM1 level greater than the detection limit of 5 ng L−1. e EU legal limit for AFM1 in milk is 50 ng L−1. f CN legal limit for AFM1 in milk is 500 ng L−1.
20.0 15.0 10.0 5.0 0.0
Beijing
Hebei
Shanxi
Shanghai
Guangdong
Province
Figure 1.
Comparison of aflatoxin M1 (AFM1) content in raw milk in five provinces of China.
114
N. Zheng et al.
Table 4. Comparison of aflatoxin M1 (AFM1) level in raw milk collected from three types of milk stations.
Milk station type
Samples exceeding EU legal limit,c no. (%)
No. of samples
Negative sample,a no. (%)
Positive sample,b no. (%)
181 66 113 360
29 (16.0%) 17 (25.8%) 33 (29.2%) 79 (21.9%)
152 (84.0%) 49 (74.2%) 80 (70.8%) 281 (78.1%)
Large-scale farms Milk processing plants Small farm cooperatives Total
16 13 7 36
Samples exceeding CN legal limit,d no. (%)
(8.8%) (19.7%) (6.2%) (10.0%)
AFM1 content (ng L−1) Minimum
Maximum
5.0 5.6 5.1 5.0
98.7 80.9 123.0 123.0
0 0 0 0
Mean ± SD 21.1 27.5 20.0 21.6
± ± ± ±
19.8A 22.4A 22.6A 21.6
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Notes: Superscript capital letter A denotes that the mean difference in samples from three type milk stations is not significant at P > 0.05. a Negative samples had an AFM1 level lower than the detection limit of 5 ng L−1. b Positive samples had an AFM1 level greater than the detection limit of 5 ng L−1. c The EU legal limit for AFM1 in milk is 50 ng L−1. d The CN legal limit for AFM1 in milk is 500 ng L−1.
Guangdong are located in the south of China whereas Beijing, Hebei and Shanxi are located in the north of China. The climate in the south has higher temperature and moisture than in the north (Table 1). It leads to the higher incidence and content of AFM1 in raw milk in the south than in the north (Table 3). Contamination of AFM1 in raw milk in different countries is shown in Table 5. With regard to levels of AFM1, samples from China (78.1%) were higher than those from New Zealand (0), United Kingdom (3%), Brazil (23.8%), Iran (54%) and Indonesia (58.2%) but lower than samples in Portugal (80.6%) and Thailand (98.5%). However, as for the level of AFM1 in Brazil, Indonesia and Thailand, 7%, 21% and 25.4% of samples, respectively, contained AFM1 above the Chinese and US legal limit of 500 ng L−1. And 10% and 23% of samples in
Table 5.
China and Iran, respectively, contained AFM1 above the EU legal limit of 50 ng L−1, but within the Chinese and US legal limit. On the other hand, in New Zealand, United Kingdom, Japan and Portugal, no samples contained AFM1 above the EU legal limit (Kriengsag 1997; Martins & Martins 2000; UKFSA 2001; Sassahara et al. 2005; Sugiyama et al. 2008; Tajkarimi et al. 2008; NZFSA 2012). It seems that AFM1 contamination in raw milk samples in China was worse than that in New Zealand, United Kingdom and Japan, but better than samples in Brazil, Indonesia and Thailand. Conclusion The incidence of AFM1 (78.1%) in raw milk was high, but AFM1 content in all positive samples was
Analysis of aflatoxin M1 (AFM1) in the raw milk in different countries.
No. of samples
Positive samples, no. (%)
Samples exceeding EU legal limit,a no. (%)
Samples exceeding CN legal limit,b no. (%)
AFM1 concentration, ng L−1
Analysis method
Reference
Country
Period
New Zealand
July 2011– Jun 2012 2001
303
0
0
0
–
HPLC
NZFSA (2012)
100
3 (3%)
0
0
10–21
HPLC
UKFSA (2001)
Jan 2004
101
–
0
0
11 ± 3.5
HPLC
31
25 (80.6%)
0
0
5–50
HPLC
319 42
172 (54%) 10 (23.8%)
73 (23%) –
0 3 (7%)
10 ± 119 295–1975
HPLC ELISA
Sugiyama et al. (2008) Martins and Martins (2000) Tajkarimi et al. (2008) Sassahara et al. (2005)
342
199 (58.2%) 66 (98.5%)
–
73 (21%)
310–5400
HPLC
Tajkarimi et al. (2008)
–
17 (25.4%)
–
ELISA
Kriengsag (1997)
United Kingdom Japan Portugal
Jun–Sep, 1999
Iran Brazil
2004 Jul 2001– Nov 2002 1990–1993, 1999 –
Indonesia Thailand
67
Notes: “–”: not mentioned in the reference. a The EU legal limit for AFM1 in milk is 50 ng L−1. b The CN legal limit for AFM1 in milk is 500 ng L−1.
Food Additives & Contaminants: Part B far below the Chinese and U.S. legal limit of 500 ng L−1. In addition, AFM1 contamination in raw milk in the southern provinces was more serious than that in the northern provinces, shown by both AFM1 incidence and content. Acknowledgement This research was funded by the China Agricultural Ministry (2013-Z10) and Institute of Animal Science, Chinese Academy of Agricultural Sciences (2013ywf-yb-3 and 2011cj-3).
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References Bakirci I. 2001. A study on the occurrence of aflatoxin M1 in milk and milk products produced in Van province of Turkey. Food Contr. 12:47–51. 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. Chopra RC, Chhabra A, Prasad KSN, Dudhe A, Murthy TN, Prasad T. 1999. Carry-over of aflatoxin M1 in milk of cows fed aflatoxin B1 contaminated ration. Ind J Anim Nutr. 16:103–106. Decastelli L, Lai J, Gramaglia M, Monaco A, Nachtmann C, Oldano F, Ru YM, Sezian A, Bandirola C. 2007. Aflatoxins occurrence in milk and feed in Northern Italy during 20042005. Food Contr. 18:1263–1266. Food and Agriculture Organisation. 2004. Worldwide regulations for mycotoxins in food and feed in 2003. Food and Nutrition Paper No. 81. Rome (Italy): FAO. Frobish RA, Bradley BD, Wagner DD, Long-Bradley PE, Hairston H. 1986. Aflatoxin residues in milk of dairy cows after ingestion of naturally contaminated grain. J Food Prot. 49:781–785.
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International Agency for Research on Cancer. 1993. Some naturally occurring substances: food items and constituents, heterocyclic aromatic amines and mycotoxins. Lyon (France): World Health Organisation, International Agency for Research on Cancer. Report No. 56. Kriengsag S. 1997. Incidence of aflatoxin M1 in Thai milk products. J Food Protect. 60:1010–1012. Kuilman ME, Maas RF, Fink-Gremmels J. 2000. Cytochrome P450-mediated metabolism and cytotoxicity of aflatoxin B1 in bovine hepatocytes. Toxicol In Vitro. 14:321–327. Martins ML, Martins HM. 2000. Aflatoxin M1 in raw and ultra high temperature-treated milk commercialized in Portugal. Food Addit Contam A. 17:871–874. [NZFSA] New Zealand Food Safety Authority. 2012. Dairy National Chemical Contaminants Programme 2009/10 full year results [Internet]. [cited 2011 Nov 25]. Available from: www.foodsafety.govt.nz/elibrary/industry/ Pei SC, Zhang YY, Eremin SA, Lee WJ. 2009. Detection of aflatoxin M1 in milk products from China by ELISA using monoclonal antibodies. Food Contr. 20:1080–1085. Sassahara M, Pontes ND, Yanak EK. 2005. Aflatoxin occurrence in foodstuff supplied to dairy cattle and aflatoxin M1 in raw milk in the North of Parana state. Food Chem Toxicol. 43:981–984. Sugiyama K, Hiraoka H, Sugita-Konishi Y. 2008. Aflatoxin M1 contamination in raw bulk milk and the presence of aflatoxin B1 in corn supplied to dairy cattle in Japan. J Food Hyg Soc Jpn. 49:352–355. Tajkarimi M, Aliabadi-Sh F, Salah NA, Poursoltani H, Motallebi AA, Mahdavi H. 2008. Aflatoxin M1 contamination in winter and summer milk in 14 states in Iran. Food Contr. 19:1033–1036. [UKFSA] United Kingdom Food Standards Agency. 2001. Survey of milk for mycotoxins (Number 17/01) [Internet] [cited 2001 Sep 14]. Available from: http://www.food.gov. uk/science/surveillance/fsis2001/milk-myco
Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Survey of aflatoxin M1 in raw milk in the five provinces of China Nan Zheng & Peng Sun
a b c
, Jia-Qi Wang
a b c
, Rong-Wei Han
a b c d
, Yun-Peng Zhen
b c
, Xiao-Min Xu
b c
a
a
State Key Laboratory of Animal Nutrition, Institute of Animal Science , Chinese Academy of Agricultural Sciences , Beijing , P.R. China b
Milk and Dairy Product Inspection Center (Beijing), Ministry of Agriculture , Beijing , P.R. China c
Laboratory of Quality & Safety Risk Assessment for Dairy Products (Beijing), Ministry of Agriculture , Beijing , P.R. China d
College of Food Science and Engineering , Qingdao Agricultural University , Qingdao , P.R. China Accepted author version posted online: 07 Jan 2013.Published online: 18 Feb 2013.
To cite this article: Nan Zheng , Jia-Qi Wang , Rong-Wei Han , Yun-Peng Zhen , Xiao-Min Xu & Peng Sun (2013): Survey of aflatoxin M1 in raw milk in the five provinces of China, Food Additives & Contaminants: Part B: Surveillance, 6:2, 110-115 To link to this article: http://dx.doi.org/10.1080/19393210.2012.763191
PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions 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. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
Food Additives & Contaminants: Part B, 2013 Vol. 6, No. 2, 110–115, http://dx.doi.org/10.1080/19393210.2012.763191
Survey of aflatoxin M1 in raw milk in the five provinces of China Nan Zhenga,b,c, Jia-Qi Wanga,b,c*, Rong-Wei Hana,b,c,d, Yun-Peng Zhenb,c, Xiao-Min Xub,c and Peng Suna a
State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, P.R. China; Milk and Dairy Product Inspection Center (Beijing), Ministry of Agriculture, Beijing, P.R. China; cLaboratory of Quality & Safety Risk Assessment for Dairy Products (Beijing), Ministry of Agriculture, Beijing, P.R. China; dCollege of Food Science and Engineering, Qingdao Agricultural University, Qingdao, P.R. China b
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(Received 4 April 2012; final version received 29 December 2012) Aflatoxin M1 (AFM1) is the only mycotoxin that has a legal limit in milk all over the world. In the present study, 360 raw milk samples were collected from Beijing, Hebei, Shanxi, Shanghai and Guangdong provinces in China in September 2010, and their AFM1 levels were determined by using enzyme-linked immunosorbent assay (ELISA). More than three-fourths (78.1%) of the 360 raw milk samples contained AFM1 at concentrations of 5–123 ng L−1. AFM1 contents in all positive samples were far below the Chinese and US legal limit of 500 ng L−1, but 10% of the raw milk samples exceeded the EU legal limit of 50 ng L−1. Moreover, both incidence and content of AFM1 in milk collected from the southern provinces, including Shanghai and Guangdong, were higher than those collected from the northern provinces, including Beijing, Hebei and Shanxi. Keywords: aflatoxin M1; raw milk; ELISA; China
Introduction Aflatoxins are toxic secondary metabolites of moulds Aspergillus flavus and A. parasiticus and are the main mycotoxin which could be detected in milk (Boudra et al. 2007). Eighteen aflatoxins have been identified up to now, and B1, B2, G1, G2 and M1 are the aflatoxins of most concern (Decastelli et al. 2007). Moreover, aflatoxin M1 (AFM1) is the only mycotoxin that has been set a legal limit in milk by many national governments and international organisations, such as the Codex Alimentarius Commission (Food and Agriculture Organisation 2004; Sugiyama et al. 2008). Aflatoxin B1 (AFB1) is the most toxic aflatoxin and has been designated as a primary carcinogenic compound by the International Agency for Research on Cancer (IARC) of the World Health Organisation (WHO) (International Agency for Research on Cancer 1993). Dairy cows consuming feeds contaminated with AFB1 can excrete a hydroxylated metabolite of AFB1 as AFM1 in milk (Frobish et al. 1986; Chopra et al. 1999; Kuilman et al. 2000). AFM1 has been classified as a second-class possible human carcinogen by the IARC, and its carcinogenicity was estimated to be 1/10th of that of AFB1 by the Joint Expert Committee on Contamination and Food Additives in 2001 (International Agency for Research on Cancer 1993; Sugiyama et al. 2008). Furthermore, AFM1 is very stable, and it is assumed that neither storage nor processing, such as pasteurisation, autoclaving, fermentation or other methods commonly used to process milk products, destroys its toxin (Tajkarimi et al. 2008). *Corresponding author. Email: [email protected] Nan Zheng and Rong-Wei Han contributed equally. © 2013 Taylor & Francis
A legal limit for AFM1 in milk existed in 60 countries at the end of 2003. Among them, 50 ng L−1 set by the EU and 500 ng L−1 by the United States are two peak limits (Food and Agriculture Organisation 2004). Further, many countries such as New Zealand, United Kingdom, Brazil and Iran have conducted national surveys to assess AFM1 contamination in raw milk in order to ensure safety of milk products (UKFSA 2001; Sassahara et al. 2005; Tajkarimi et al. 2008; NZFSA 2012). The Chinese government has established 500 ng L−1 as the national legal limit of AFM1 in milk. Pei et al. (2009) measured the AFM1 content in 12 raw milk samples collected from the market in northeastern China in 2008. AFM1 was detected in all samples with contents in the range of 160–500 μg L−1, and 75% of samples had AFM1 concentrations in the range 320–500 µg L−1, which were much closer to the Chinese national legal limit. The findings suggested that the contamination of AFM1 in raw milk in the northeast of China is serious and stressed the need for continued assessment of AFM1. In the present study, AFM1 contamination in 360 raw milk samples collected from five provinces, covering the north and south of China, was assessed using ELISA. Material and methods Sampling During September 2010, raw milk samples were collected from 360 milk stations in Beijing (80), Hebei (80), Shanxi (80), Shanghai (60) and Guangdong (60) provinces in
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Food Additives & Contaminants: Part B China. Geographically, Beijing, Hebei and Shanxi provinces are located in the north, whereas Shanghai and Guangdong provinces are located in the south. They represent two typical climates of China, and the mean temperature and precipitation in the five provinces in September 2010 is listed in Table 1. The 360 milk stations can be classified into three types according to their ownership, owned by large-scale farms (181), milk processing plants (66) and small farm cooperatives (113), respectively. The raw milk was collected directly from the milk holding tanks at the milk stations in dairy farms. After stirring of milk holding tanks, 200 mL of milk sample was taken from the upper third of the tank, 200 mL from the middle third and 200 mL from the lower third. Thus, 600 mL of milk from each tank was mixed, and 100-mL samples were taken and stored at −20°C until analysis was performed. Milk sample analysis The quantitative analysis of AFM1 in milk samples was determined using RIDASCREEN® Aflatoxin M1 test kit (R1111, R-Biopharm AG, Darmstadt, Germany), which is a competitive enzyme immunoassay kit. All reagents required for the enzyme immunoassay, including standards (0, 5, 10, 20, 40 and 80 ng L−1), are contained in the test kit. The test was conducted according to the instruction manual supplied with the kit. Sample preparation Eight millilitres of milk sample was defatted by centrifugation at 3500 g and 10°C for 10 min. The upper cream layer was removed and the defatted supernatant was subjected to ELISA for AFM1 detection.
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CA, USA). Next, 100 μL of the diluted enzyme conjugate was added to the wells and incubated for 15 min at room temperature in the dark. The washing procedure was repeated twice again. Hundred microlitres of chromogen was added to each well and incubated for 15 min at room temperature in the dark. Lastly, 100 μL of the stop solution was added to each well and the absorbance was measured at 450 nm by using a microplate reader (Infinite 200, Tecan Austria GmbH, Groedig, Austria). Quality control Every 20th sample of milk was fortified with 20 ng L−1 AFM1 to verify the reliability of the analysis. Calculation The content of AFM1 in samples was calculated by using a special software, the RIDA®SOFT Win (Z9999, RBiopharm AG, Darmstadt, Germany). According to the instructions, the detection limit of the present method is 5 ng L−1 for milk. So the AFM1 concentration in the 5– 80 ng L−1 range can be read from the calibration curve. Statistical analysis All samples were run in duplicate. Differences in AFM1 concentration were statistically analysed by using a nonparametric test, followed by Mann-Whitney or KruskalWallis comparisons, using SPSS version 11.5 (SPSS, Inc., Chicago, IL). The level of confidence required for significance was set at P < 0.05. Results
Test procedure Hundred microlitres of the standard solutions or defatted samples was added to the wells and incubated for 30 min at room temperature (20–25°C) in the dark. The liquid then was sucked out of the wells and they were filled with 250 μL washing buffer, which again was sucked out. The washing procedure was repeated twice using immuno wash (Model 1875, Bio-Rad Laboratories, Inc., Hercules,
AFM1 recovery from the 20 ng L−1 fortified raw milk samples (n = 36) was 92.5–115.0%. For the 360 raw milk samples, AFM1 was detected in 281 samples, with an incidence of 78.1%. Among the positive samples, 62.2% contained AFM1 at levels of 5.0–29.9 ng L−1, 5.8% at levels of 30.0–49.9 ng L−1, 9.7% at levels of 50.0– 99.9 ng L−1, and 0.3% at levels of 100.0–129.9 ng L−1 (Table 2).
Table 1. Temperature and precipitation in September 2010 in five provinces of China. Province Beijing Hebei Shanxi Shanghai Guangdong Northa Southb
Temperature (ºC)
Precipitation (mm)
20.4 18.8 17.7 26.2 27.4 19.0 26.8
75.6 90.7 84.6 125.7 351.6 83.6 238.7
Notes: aNorth part includes Beijing, Hebei and Shanxi provinces. b South part includes Shanghai and Guangdong provinces.
39 20 1 60 360
Total
Total
60
Total
Large-scale farms Milk processing plants Small farm cooperatives
50 10 0
80
Total
Large-scale farms Milk processing plants Small farm cooperatives
15 13 52
Large-scale farms Milk processing plants Small farm cooperatives
80
Total
−1
79 (21.9)
5 (8.3)
2 (5.1) 3 (15.0) 0
5 (8.3)
4 (8.0) 1 (10.0) –
37 (46.3)
9 (60.0) 8 (61.5) 20 (38.5)
20 (25.0)
12 (24.5) – 8 (25.8)
12 (15.0)
2 (7.1) 5 (21.7) 5 (17.2)
Negative samples,a no. (%)
Notes: Negative samples had an AFM1 level lower than the detection limit of 5 ng L . b Positive samples had an AFM1 level greater than the detection limit of 5 ng L−1.
a
Guangdong
Shanghai
Shanxi
49 0 31
Large-scale farms Milk processing plants Small farm cooperatives
80
Total
Hebei
28 23 29
Large-scale farms Milk processing plants Small farm cooperatives
Beijing
No. of samples
Milk station type
Level of aflatoxin M1 (AFM1) in raw milk in five provinces of China.
Province
Table 2.
224 (62.2)
41 (68.3)
30 (76.9) 11 (55.0) 0
38 (63.3)
32 (64.0) 6 (60.0) –
39 (48.8)
6 (40.0) 5 (38.5) 28 (53.8)
51 (63.8)
33 (67.3) – 18 (58.1)
55 (68.8)
22 (78.6) 12 (52.2) 21 (72.4)
5.0–29.9 ng L−1
21 (5.8)
6 (10.0)
5 (12.8) 1 (5.0) 0
6 (10.0)
6 (12.0) 0 –
2 (2.5)
0 0 2 (3.8)
3 (3.8)
1 (2.0) – 2 (6.5)
4 (5.0)
1 (3.6) 1 (4.3) 2 (6.9)
30.0–49.9 ng L−1
35 (9.7)
8 (13.3)
2 (5.1) 5 (25.0) 1 (100)
11 (18.3)
8 (16.0) 3 (30.0) –
2 (2.5)
0 0 2 (3.8)
5 (6.3)
3 (6.1) – 2 (6.5)
9 (11.3)
3 (10.7) 5 (21.7) 1 (3.4)
50.0–99.9 ng L−1
Positive samples,b no. (%)
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1 (0.3)
0
0
0
0
0 0 –
0
0 0 0
1 (1.3)
0 – 1 (3.2)
0
0 0 0
100.0–129.9 ng L−1
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Table 3. Comparison of aflatoxin M1 (AFM1) levels in raw milk collected from northa and southb China.
No. of samples
Negative sample,c no. (%)
Positive sample,d no. (%)
Samples exceeding EU legal limit,e no. (%)
Samples exceeding CN legal limit,f no. (%)
North South
240 120
69 (28.8%) 10 (8.3%)
171 (71.3%) 110 (91.7%)
17 (7.1%) 19 (15.8%)
Total
360
79 (21.9%)
281 (78.1%)
36 (10.0%)
Location
AFM1 content (ng L–1) Minimum
Maximum
Mean ± SD
0 0
5.0 5.5
123.0 97.5
18.9 ± 20.8A 25.9 ± 22.4B
0
5.0
123.0
21.6 ± 21.6
The incidence of AFM1 in the milk samples collected from the north was 71.3%, with AFM1 content ranging from 5.0 to 123.0 ng L−1. And the incidence of AFM1 in milk samples collected from the south was 91.7%, with AFM1 content from 5.5 to 97.5 ng L−1(Table 3). According to our statistical analysis, the mean of AFM1 content in milk samples collected from the north (18.9 ng L−1) was significantly lower (P < 0.05) than that collected from the south (25.9 ng L−1) (Table 3). The incidence of AFM1 in raw milk samples collected from the five provinces was different, which in increasing order was Shanxi (53.7%), Hebei (75.0%), Beijing (85.0%), Guangdong (91.7%) and Shanghai (91.7%) (Table 2). Moreover, the AFM1 content was significantly different (P < 0.05) in samples collected from the five provinces, and the ascendant succession was Shanxi (15.3 ng L−1), Beijing (19.2 ng L−1), Hebei (21.2 ng L−1), Guangdong (23.7 ng L−1) and Shanghai (28.1 ng L−1) (Figure 1). In each of the five provinces, the incidence of AFM1 in milk was different in milk stations owned by large-scale farms, milk processing plants, and small farm cooperatives. However, there was no consistent trend in three types of milk stations in the five provinces (Table 2). Overall, the incidence of AFM1 was lowest in samples collected from
milk stations owned by small farm cooperatives (70.8%), medium in those from milk stations owned by milk processing plants (74.2%), and highest in those from milk stations owned by large-scale farms (84.0%). But the AFM1 content in milk samples collected from the three types of milk stations had no significant (P > 0.05) difference (Table 4). Discussion Although most of the milk samples were contaminated by AFM1, the AFM1 content in all positive samples was within the Chinese and US legal limit of 500 ng L−1. And the minimum and maximum AFM1 content was 5 and 123 ng L−1, respectively. Both the incidence and level of AFM1 in the present study was lower than in an earlier report by Pei et al. (2009). Moulds can easily grow and produce toxins in high temperature and a wet environment, so temperature and moisture were the most important impacts on the amount of AFB1 occurrence in feed (Bakirci 2001). Furthermore, AFB1 content in dairy cow feed determines AFM1 content in raw milk, so higher levels of temperature and moisture also led to higher concentrations of AFM1 in raw milk. Of the five provinces, Shanghai and
30.0 25.0 AFM1 content (ng/L)
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Notes: Different superscript capital letters A and B denote that the mean difference in samples between north and south is significant at P < 0.05. a North part includes Beijing, Hebei and Shanxi. b South part includes Shanghai and Guangdong. c Negative samples had an AFM1 level lower than the detection limit of 5 ng L−1. d Positive samples had an AFM1 level greater than the detection limit of 5 ng L−1. e EU legal limit for AFM1 in milk is 50 ng L−1. f CN legal limit for AFM1 in milk is 500 ng L−1.
20.0 15.0 10.0 5.0 0.0
Beijing
Hebei
Shanxi
Shanghai
Guangdong
Province
Figure 1.
Comparison of aflatoxin M1 (AFM1) content in raw milk in five provinces of China.
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Table 4. Comparison of aflatoxin M1 (AFM1) level in raw milk collected from three types of milk stations.
Milk station type
Samples exceeding EU legal limit,c no. (%)
No. of samples
Negative sample,a no. (%)
Positive sample,b no. (%)
181 66 113 360
29 (16.0%) 17 (25.8%) 33 (29.2%) 79 (21.9%)
152 (84.0%) 49 (74.2%) 80 (70.8%) 281 (78.1%)
Large-scale farms Milk processing plants Small farm cooperatives Total
16 13 7 36
Samples exceeding CN legal limit,d no. (%)
(8.8%) (19.7%) (6.2%) (10.0%)
AFM1 content (ng L−1) Minimum
Maximum
5.0 5.6 5.1 5.0
98.7 80.9 123.0 123.0
0 0 0 0
Mean ± SD 21.1 27.5 20.0 21.6
± ± ± ±
19.8A 22.4A 22.6A 21.6
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Notes: Superscript capital letter A denotes that the mean difference in samples from three type milk stations is not significant at P > 0.05. a Negative samples had an AFM1 level lower than the detection limit of 5 ng L−1. b Positive samples had an AFM1 level greater than the detection limit of 5 ng L−1. c The EU legal limit for AFM1 in milk is 50 ng L−1. d The CN legal limit for AFM1 in milk is 500 ng L−1.
Guangdong are located in the south of China whereas Beijing, Hebei and Shanxi are located in the north of China. The climate in the south has higher temperature and moisture than in the north (Table 1). It leads to the higher incidence and content of AFM1 in raw milk in the south than in the north (Table 3). Contamination of AFM1 in raw milk in different countries is shown in Table 5. With regard to levels of AFM1, samples from China (78.1%) were higher than those from New Zealand (0), United Kingdom (3%), Brazil (23.8%), Iran (54%) and Indonesia (58.2%) but lower than samples in Portugal (80.6%) and Thailand (98.5%). However, as for the level of AFM1 in Brazil, Indonesia and Thailand, 7%, 21% and 25.4% of samples, respectively, contained AFM1 above the Chinese and US legal limit of 500 ng L−1. And 10% and 23% of samples in
Table 5.
China and Iran, respectively, contained AFM1 above the EU legal limit of 50 ng L−1, but within the Chinese and US legal limit. On the other hand, in New Zealand, United Kingdom, Japan and Portugal, no samples contained AFM1 above the EU legal limit (Kriengsag 1997; Martins & Martins 2000; UKFSA 2001; Sassahara et al. 2005; Sugiyama et al. 2008; Tajkarimi et al. 2008; NZFSA 2012). It seems that AFM1 contamination in raw milk samples in China was worse than that in New Zealand, United Kingdom and Japan, but better than samples in Brazil, Indonesia and Thailand. Conclusion The incidence of AFM1 (78.1%) in raw milk was high, but AFM1 content in all positive samples was
Analysis of aflatoxin M1 (AFM1) in the raw milk in different countries.
No. of samples
Positive samples, no. (%)
Samples exceeding EU legal limit,a no. (%)
Samples exceeding CN legal limit,b no. (%)
AFM1 concentration, ng L−1
Analysis method
Reference
Country
Period
New Zealand
July 2011– Jun 2012 2001
303
0
0
0
–
HPLC
NZFSA (2012)
100
3 (3%)
0
0
10–21
HPLC
UKFSA (2001)
Jan 2004
101
–
0
0
11 ± 3.5
HPLC
31
25 (80.6%)
0
0
5–50
HPLC
319 42
172 (54%) 10 (23.8%)
73 (23%) –
0 3 (7%)
10 ± 119 295–1975
HPLC ELISA
Sugiyama et al. (2008) Martins and Martins (2000) Tajkarimi et al. (2008) Sassahara et al. (2005)
342
199 (58.2%) 66 (98.5%)
–
73 (21%)
310–5400
HPLC
Tajkarimi et al. (2008)
–
17 (25.4%)
–
ELISA
Kriengsag (1997)
United Kingdom Japan Portugal
Jun–Sep, 1999
Iran Brazil
2004 Jul 2001– Nov 2002 1990–1993, 1999 –
Indonesia Thailand
67
Notes: “–”: not mentioned in the reference. a The EU legal limit for AFM1 in milk is 50 ng L−1. b The CN legal limit for AFM1 in milk is 500 ng L−1.
Food Additives & Contaminants: Part B far below the Chinese and U.S. legal limit of 500 ng L−1. In addition, AFM1 contamination in raw milk in the southern provinces was more serious than that in the northern provinces, shown by both AFM1 incidence and content. Acknowledgement This research was funded by the China Agricultural Ministry (2013-Z10) and Institute of Animal Science, Chinese Academy of Agricultural Sciences (2013ywf-yb-3 and 2011cj-3).
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