First, aflatoxin introduction
Aflatoxin (aflatoxin, referred to as AF) is the most toxic mycotoxins so far discovered. It can contaminate food and feed in a variety of ways, directly or indirectly into the human food chain, threatening human health and life safety, and causing serious damage to human and animal internal organs, especially the liver. The toxin is the metabolism of toxigenic strains of Aspergillus flavus and Aspergillus parasiticus. The product is ubiquitous in moldy food and food products. Aflatoxin is very heat-resistant and can be completely destroyed by heating to 230 °C, so general cooking is not easy to eliminate.
Second, the harm of aflatoxin to the human body
1. Cause acute and chronic poisoning:
Aflatoxin is a highly toxic substance with a toxicity equivalent to 10 times that of potassium cyanide and 68 times that of arsenic . Aflatoxin is a liver poison. In addition to inhibiting the synthesis of DNA and RNA , it also inhibits the synthesis of liver proteins. Aflatoxin is indexed from human acute poisoning events. There are many reports at home and abroad. The most typical is moldy corn in India. The incident directly killed dozens of people and hundreds of people suffered from different types of liver disease.
2 , carcinogenicity:
Aflatoxin is highly carcinogenic, and long-term intake of aflatoxin induces liver cancer. Its ability to induce liver cancer is 75 times greater than that of dimethyl nitrosamine , and it is currently recognized as one of the most carcinogenic substances. According to the World Health Organization reported that aflatoxin content in 30-- is 50ug / kg and low toxicity, 50-- is poisoning 100ug / kg, 100 - when 1000ug / kg is highly toxic, 1000ug / kg or more is highly toxic. In view of the great harm of aflatoxin to humans, China has strictly regulated its content in food. Among them, the maximum allowable amount of aflatoxin in dairy products is 5ug/kg (ie 5ppb ).
Third, the type of aflatoxin
There are four main types of aflatoxins : B1 , B2 , C1 , and G2 . Among them, B1 is considered to be the main toxic substance, and there are two metabolites M1 and M2 of these toxins . Among them, aflatoxin B1 is mainly found in agricultural products, animal feed, traditional Chinese medicine and other products; aflatoxin M1 is a product of hydroxylation metabolism in the body after ingestion of aflatoxin B1 in animals , part of which is excreted from urine and milk, and part of which is present in The edible parts of animals, such as milk, liver, eggs, kidneys, blood and muscle, of which milk is most common. The toxicity and carcinogenicity of aflatoxin M1 is substantially similar to that of aflatoxin B1 . Since cow's milk and its products are the main foods of humans, especially babies, they are more harmful.
Fourth, the latest policy and national standard (including some foreign policies)
1, since 2003 August 1, where in the territory of China engaged in rice, flour, oil, soy sauce, vinegar production and processing enterprises, their products must be approved inspection before listing. The Notice of the General Administration of Quality Supervision, Inspection and Quarantine on Printing and Distributing the Implementation Regulations for Five Kinds of Food Production Licenses Such as Wheat Flour clearly states that aflatoxin B1 must be tested.
2. In the eighth issue of the "Gazette of the General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China", the latest national standard for the detection method of aflatoxin is published:
( 1 ), GB/T 18979-2003 "Determination of aflatoxin in food - immunoaffinity layer purification high performance liquid chromatography and fluorescence spectrophotometry"
( 2 ), GB/T 18980-2003 " Determination of aflatoxin M1 in milk and milk powder - Immunoaffinity layer purification by high performance liquid chromatography and fluorescence spectrophotometry"
More than two GB were implemented in 2003, August 1
3, February 4, 2002 EU resolution on special conditions for aflatoxin implement mandatory testing of imports from China or commissioned by imports from China peanuts and peanut products
V. Standards for testing aflatoxins in China and internationally
1. Main countries:
Product name | Chinese standard | American standard | EU |
Corn, peanuts, peanut oil, nuts and dried fruits (walnuts, almond) | ≤ 20 μg/kg | ≤ 20 μg/kg | ≤ 2 , 4 , 5 , 8, 10, 15μg/kg |
Corn and peanut kernel products (converted according to raw materials | ≤ 20 μg/kg | ≤ 20 μg/kg | ≤ 2 , 4, 5 , 8, 10, 15μg/kg |
Rice, other edible oils (sesame oil, rapeseed oil, soybean oil, sunflower oil, flax oil, tea oil, sesame oil, corn germ oil, rice bran oil, cottonseed oil) | ≤ 10 μg/kg | ≤ 10 μg/kg | ≤ 2 , 4 μg/kg |
Other foods (milk, flour, dried potato), fermented foods (soy sauce, vinegar, soybean meal, fermented products), starchy products (pastries, biscuits, bread, cakes) | ≤ 5 μg/kg (ppb) | ≤ 10 μg/kg (ppb) | Not checked out |
Milk and its products (sterilized milk, fresh raw milk, whole milk powder, evaporated milk, sweetened condensed milk, butter), butter, fresh pig tissue (liver, kidney, blood, lean meat) | ≤ 0.5 μg/kg (ppb) | ≤ 0.5 μg/kg (ppb) | ≤ 0.05 μg/kg (ppb) |
2 , other
(1) , WHO / FAO standards. World Health Organization (WHO) / Codex Alimentarius Commission (CAC) FAO belongs to recommend food, feed aflatoxin standards for the maximum allowable amount of the total amount (B1 + B2 + G1 + G2 ) of less than 15 μ g / kg; milk wherein M 1 is the maximum allowable amount of 0. 5 μ g / kg
(2) South African standards. The maximum allowable standard for aflatoxins was enacted in 1990 : the total amount of aflatoxin in food is less than 10 μg / kg , and aflatoxin B 1 is less than 5 μg / kg .
(3) , other standards. The Indian standard is that aflatoxin B 1 is less than 30 μg / kg in peanuts ; the standard for aflatoxin B 1 in Vietnam and Argentina is less than 20 μg / kg .
Six, aflatoxin detection method
The method for determining aflatoxin M1 is thin layer chromatography, high performance liquid chromatography, enzyme-linked immunosorbent assay, mass spectrometry, and radioimmunoassay. These methods have more or less the following deficiencies:
( 1 ) In the course of operation, the highly toxic aflatoxin M 1 is required as a calibration standard, which poses a huge risk of contamination to the operator, and the purchase of the aflatoxin M 1 standard substance is very difficult.
( 2 ) The operation process is cumbersome, complicated, long-term, and labor intensive.
( 3 ) Instruments and equipment are expensive, cumbersome, and complicated to operate, making it difficult to achieve rapid on-site analysis.
( 4 ), the sensitivity is poor, and it is difficult to obtain satisfactory results with repeatability.
The latest national standard method "immunoaffinity column method" mentioned in the fourth item above solves the above deficiencies better. The method can be detected by using a matching fluorometer or combined with high performance liquid chromatography. Solve problems such as standard contamination and complicated operation. At the same time, this method has good authority and versatility, and is recognized by many foreign organizations and is listed as a standard method, such as:
American Association of Public Analytical Chemists ( AOAC )
US Department of Agriculture Federal Grain Testing Center ( FGIS )
International Association of Pure and Applied Chemistry ( IUPAC )
US Food and Drug Administration ( US-FDA )
US Department of Agriculture ( USDA )
At the same time certified institutions in the country:
China Entry-Exit Inspection and Quarantine Bureau ( CIQ )
National Standard of the People's Republic of China ( GB )
Seven, aflatoxin detection - "immune affinity column method" method introduction
In order to develop a simple, rapid, sensitive and accurate method of analysis, the United States VICAM (Viacom) company and Harvard University, Massachusetts Institute of Technology, John - Johns Hopkins University, AOAC, FDA, FGIS, USDA and other famous The university and government agencies have worked together to develop a series of analytical methods using a monoclonal immunoaffinity column as a separation tool and a fluorometer and UV lamp as detection tools.
This technology has the following characteristics:
1. The certification body is extensive and versatile (see item 6)
2 , the analysis speed is fast, a sample takes only 10 - 15 minutes, other traditional methods to do quantitative analysis takes hours to days.
3, high sensitivity, a wide range of measurement (0 - 300ppb), can be determined 0.1ppb aflatoxin M1 fluorometer, it can be detected by HPLC 10ppt aflatoxin M 1.
4 , using monoclonal immunization technology, can specifically separate aflatoxins and other mycotoxins, high separation efficiency and recovery, correctness and reliability.
5 , The instrument is light and easy to carry, high degree of automation, simple operation, direct reading of test results, low requirements for experimental operators, can be used in small experimental sites.
6 , do not need highly toxic aflatoxin and other mycotoxins standards to calibrate, so it is absolutely safe and reliable throughout the experiment. At present, more than 90% of US peanut farmers, processing plants, and official testing organizations use Weikang technology to analyze aflatoxins. Weikang technology has been widely used in 110 countries and regions around the world .
Typical users in China are: inspection and quarantine systems (Shandong, Weihai, Rizhao, Linyi, Shidao, Weifang, Tianjin, Tanggu, Henan, Liaoning, Dayaowan, Jinzhou, Tangshan, Beijing, Shantou, Anhui, Shanghai, Xiamen, Pingliang, Lushan, Hubei, Guangzhou, Zhuhai), CDC (Beijing, Shanghai), quality inspection system (Shanghai, Shanxi, Weifang, Zibo, Xi'an, Harbin), National Feed Testing Center (Beijing), National Grain and Oil Testing Center (Beijing) ), Chengdu Institute of Food Science, Tianjin Academy of Agricultural Sciences, Agricultural Products Testing Center of the Ministry of Agriculture (Xinjiang), Shanghai Veterinary Station, Li Jinji (Guangdong), Liaoning Grain and Oil Foreign Trade, Shandong Huasheng, Donghai Grain and Oil, Anhui Hongan, Changsheng Vegetable Oil, Hongda Oil, Wendeng Cereals and Oils, Wendeng Kunyu, Rizhao Dongfang, Rizhao Dongsheng, Rizhao Zhonggu Cereals and Oils, Rizhao Cereals and Oils, Rizhao Dongyue, Rizhao Yatai, Wulian Cereals and Oils, Xing'an Grain and Oil, Jixian Shanfu, Pixian County Grain Bureau, Rizhao Haina, Rizhao Shengkang, Qixian Spring Garden, Yantai Pingfu, Yantai Fenglin, Yantai Jinling, Waterway Grain Management Office, Yantai Dacheng, Yantai Dajiang, Tobacco Xinrui, Yantai Tianfu, Yantai Aohua, Zhaoyuan Grain and Oil, Laiyang Luhua, Rongcheng Food, Zhongfu Grain and Oil, Weihai Zhengchang, Hongkun Food, Weihai Hualong, Weihai Huanghai, Runde Grain and Oil, Qingdao Jiaan, Qingdao Jiade, Qingdao Jiajing, Qingdao Oriental, Qingdao Xinghua, Qingdao Jinhua, Qingdao Ruisheng, Qingdao Shengde, Laixi Peanut, Laixi Songwang, Jiaozhou Hengsheng,
Jiaozhou Supply and Marketing, Linyi Yulong, Weinan Peanut Association, Weinan Yousheng, Weinan Zhengda, Weinan Sihai, Lishui Baosheng, Linyi Tianwang, Linyi Greenland, Linyi Zhongsheng, Zhucheng Cereals and Oils, Zhucheng Supply and Marketing, Anqiu Foreign Trade, Tangshan Nanguang, Tangshan Tianshen, Langfang Purina, Zhenjiang Cargill, Jiaxing Cargill, Shanghai Weihaomei, Tongrentang, etc. (In Yantai, Jinan, Guangdong, Ningbo, Jilin, Shanxi, Heilongjiang, Hebei, Jiangsu, Huai'an, Fujian and Gansu inspection and quarantine Bureau, SFDA , Shanghai Drug Testing, Shenzhen Nanshan CDC , Guangdong CDC , Shanghai SGS , Medical Institute, Tianjin Nestle, Commodity Inspection Institute, Zhejiang University Feed Institute, Dalian Quality Inspection, Liaoning Quality Inspection, Linyi Quality Inspection, Sunshine Quality Inspection, Qingdao Tongbiao, Qingdao Kerry, Henan Academy of Agricultural Sciences, and Hubei Green Product Testing Center use the affinity column + HPLC method).
Samples that can be tested include: rice, flour, soy sauce, vegetable oil, vinegar, milk, dairy products, peanuts, peanut butter, peanut oil, walnuts, walnuts, almonds, coffee, corn, honey, cereals, sorghum, soy, sauce, vegetables, pepper , pepper, Chinese herbal medicine, tobacco, beer, wheat, cottonseed, oats, feed, etc.
Eight other mycotoxins that can be tested
This method is widely applicable, and the same instrument and similar analytical methods can also detect ochratoxin, fumonisin, zearalenone, vomiting toxin, T-2 toxin and bilobamycin.
Quick and accurate detection of mycotoxins in food
The aflatoxin immunoaffinity column analysis method (IAC-AFLATEST) is a patented technology of the American company Vicam. It was approved by the AOAC ( American Association of Professional Analytical Chemists ) in 1994 as the latest official method ( No. 991.31) At the same time, it is also the Federal Grain Testing Center of the US Department of Agriculture ( Certificate No.: FGIS91-103-13), IUPAC ( International Association of Pure and Applied Chemistry ) US-FDA ( US Food and Drug Administration ) and USDA ( US Department of Agriculture ) official certification methods. Commodity Inspection Bureau in Shandong by the efforts of scientists, the aflatoxin analysis immunoaffinity column (IAC-AFLATEST) has been developed for the China Entry-Exit Inspection and Quarantine (CIQ) the new industry standard.
Previously, aflatoxin analysis was generally performed by thin layer chromatography (TLC) , high performance liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (ELISA) . Although TLC is simple, the sensitivity is poor. Although HPLC has high sensitivity, the sample processing is cumbersome, the operation is complicated, and the instrument is expensive. The ELISA has poor reproducibility and requires a low temperature storage. In addition, these methods have common shortcomings:
( 1 ) In the course of operation, highly toxic mycotoxins are required as calibration standards, posing a significant risk of contamination of the operator.
( 2 ) In the process of pretreatment of samples, it is necessary to use a variety of toxic and odorous organic solvents, which not only poisons the operators but also pollutes the environment.
( 3 ) The operation process is cumbersome, long-term, and labor intensive.
( 4 ) The equipment is complex and cumbersome, making it difficult to achieve rapid on-site analysis.
( 5 ) The sensitivity is poor and cannot meet the standards of the European Union and other countries.
In order to develop a simple, rapid, sensitive and accurate method of analysis, the United States Wellcome (Vicam) company with Harvard University, MIT, about Han - Johns Hopkins University, AOACFDA, FGIS, USDA and other famous universities and Government agencies have worked together to develop and invent a series of analytical methods using monoclonal immunoaffinity columns as separation methods , using fluorometers and UV lamps as detection tools . Weikang technology has the following characteristics :
1) is AOAC ( American Association of Professional Analysts ) , IUPAC ( International Association of Pure and Applied Chemistry ) , USDA-FGIS ( United States Department of Agriculture Federal Valley Testing Center ) , USDA-GIPSA ( US Department of Agriculture Grain Inspection, Packaging, Storage Administration UNDP) official certification method, the detection result can be recognized around the world.
2) The analysis speed is fast, a sample takes only 10-15 minutes, while other traditional methods take several hours to several days.
3) High sensitivity, wide measurement range (0.1-300 ppb) , 10 pct of aflatoxin M1 can be determined by HPLC .
4) Using monoclonal antibody immunoassay , aflatoxin or other mycotoxins can be isolated specifically , with high separation efficiency and recovery rate , correctness and reliability.
5) The instrument is light and easy to carry , with high degree of automation , simple operation , direct reading of test results , high school literacy level can be competent , can be used in small laboratory or on-site
6) does not require a highly toxic and other mycotoxin aflatoxin standard for calibration, the process throughout the test operation, absolutely safe and reliable.
7) Using the same instrument and similar analytical methods, it can also detect ochratoxin, fumonisin, zearalenone, deoxynivalenol ( DON or vomiting toxin), T-2 toxin and orange Penicillin.
No. Description Order No. Description Description Order No.
1 4 series fluorometer G8000 13 methanol dispenser 20501
2 fluted filter paper 31240 14 printing paper G8100
3 disposable plastic funnel 36020 15 disposable plastic pipette 20652
4 disposable plastic beaker 36010 16 500ml wash bottle 20700
5 stainless steel homogenizer 20200 17 disposable test tube 34000
6 glass mixer 20300 18 Kim-wipe paper 31967
7 pump flow operating frame 21020 19 color liquid distributor 20600
8 500ml measuring cylinder 20501 20 experimental operation guide videotape
9 microfiber filter paper 31955 21 English experimental operation manual
10 50 ml measuring cylinder 20600 22 test tube rack 21010
11 color liquid 3,201,023 standard 33020
12 electronic scale 20100 24 glass syringe 34010
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