Aflatoxin M1 is caused by moldy food. Toxins produce during the metabolism of Aspergillus flavus after mildew of grains and peanuts. Regarding the aflatoxin in pure milk, it was caused by mildew in the feed. Toxins enrichment exists in organisms. As the food chain rises, toxins accumulate, so we human at the top of the food chain suffer.
The nationally stipulated maximum value of Aflatoxin M1 is 0.5μg/kg. Aflatoxin mainly damages the liver and is carcinogenic. The limit of Aflatoxin M1 detection in milk and dairy products is 0.5μg/kg. Aflatoxin B1 is 10 micrograms/kg. If it exceeds standard for 140%, it will cause terrible impact, especially on the human liver. If it is consumed once or twice, the human liver can detoxify it. If you eat it for over a week, you’d better go to the hospital for examination and follow the doctor's advice.
The methods for detecting Aflatoxin M1 are generally immune-affinity chromatography purification high performance liquid chromatography and enzyme-linked immunosorbent assay (kit). The former method mainly uses high-performance liquid phase, while the latter uses a microplate reader. Due to the high cost and complicated procedures of high performance liquid chromatography, the latter method is mainly used to detect Aflatoxin M1 on the market.
In addition, there is immune-affinity chromatography purification fluorescence spectrophotometry. Different from the HPLC method, after the sample purified by the immune-affinity column, the fluorescence intensity is directly measured by the fluorescence spectrophotometer, which is in direct proportion to the concentration of M1. This is also the national standard. Compared with the HPLC method, it is easy to operate and safer, but it still has high cost.
Aflatoxins are a group of compounds with similar chemical structures. At present, 12 species have been isolated and identified, including b1, b2, g1, g2, m1, m2, p1, q, h1, gm, b2a and toxic alcohol. Aflatoxin M1 is a metabolite derived from the hydroxylation of aflatoxin b1 in the body. Aflatoxin can not only reduce the milk production of dairy cows, but also make the milk contain transformed aflatoxins m1 and m2.
Therefore, the Aflatoxin M1 should be caused due to the aflatoxin being secreted into the milk after the aflatoxin is converted into m1 in the milk. The Aflatoxin M1 in raw milk (raw material of milk and dairy products) should be mainly related to the content of aflatoxin in the feed of dairy cows.
If the result reads that it does not contain aflatoxin, then there is no Aflatoxin M1. Aflatoxin is a highly toxic metabolism of Aspergillus and other fungi Aflatoxin M1 which is a metabolite of aflatoxin B1. Aflatoxin M1 is mainly caused by dairy cows being fed contaminated feed. After entering the body, the amount of aflatoxin in the liver is higher than other tissues and organs.
The liver may be most affected by aflatoxin. But for a long time, due to the backward detection technology, it has restricted the control of aflatoxin pollution in agricultural products, and also restricted the export of agricultural products. The three-square-circle Aflatoxin M1 rapid test card has the characteristics of convenience, rapidity, accuracy and sensitivity, suitable for on-site screening and testing of large quantities of samples and the law enforcement supervision.
Thin film chromatography and liquid chromatography are the methods currently used by most domestic testing institutions. Due to its long detection cycle, complex procedures and a large number of reagents required, it is far from being able to meet the modern detection requirements.
With the continuous development of modern science and technology, especially the continuous development of immunobiology, molecular biology, people have established fast, simple, specific, sensitive, low-consumption and applicable aflatoxin detection methods. Moreover, these methods represented by gold-labeled test strips have been widely used in advanced countries.
The introduction and digestion of these advanced methods is our top priority in the field of detection. The immune-affinity column method has many advantages, but it cannot be popularized due to the high cost. The one-step gold standard test paper method for aflatoxin detection seems to be more suitable for China and is worthy of promotion.
Aflatoxins CAS No. 1402-68-2, is a group of compounds with similar chemical structures, 12 species have been isolated and identified including B1, B2, G1, G2, M1, M2, P1, Q, H1, GM, B2a and toxic alcohol. The basic structure of aflatoxin is difuran ring and coumarin. B1 is a derivative of dihydrofuran oxaphthalone.
That is to say, it contains a difuran ring and an oxaphthalone (coumarin) The former is the basic toxic structure and the latter is related to carcinogenesis. M1 is a metabolite derived from the hydroxylation of aflatoxin B1 in the body. The main molecular types of aflatoxin include B1, B2, G1, G2, M1, M2, etc. Among them, M1 and M2 are mainly found in milk. B1 is the most toxic and carcinogenic substance.
CSY-E96H aflatoxin rapid detector (aflatoxin rapid detector/ aflatoxin detector| aflatoxin tester) adopts the principle of solid-phase enzyme-linked immunosorbent ELISA, that is, enzyme-linked immunoassay: it can quantitatively detect aflatoxins (B1, B2, G1, G2 M1 M2 Aflatoxin M1, AFP1, AFQ1, Aflatoxin B1-2, 3-epoxy) in food, feed, oil, dairy products, drugs, beverages, milk, wine and other products Chemicals.
And it can be connected to the food safety monitoring system. Aflatoxin rapid detectors and drug residue detectors are widely used in product quality supervision and inspection, hygiene and epidemic prevention, environmental protection, business management, aquatic product wholesale markets, flour production bases, farms, and grain Warehouses, supermarkets, shopping malls, major food safety monitoring systems and other departments.
The Aflatoxin M1 toxicity is mainly manifested as carcinogenicity and mutagenicity. It has a destructive effect on human and animal liver tissues. In severe cases, it can lead to liver cancer and even death.
Physiological studies on carcinogenic mechanisms have shown that the remote furan ring epoxy chemical structure of Aflatoxin M1 is covalently bound to the purine residues of DNA in the body, causing some damage to DNA, changes in DNA structure and function, and carcinogenesis. And its carcinogenicity is basically similar to aflatoxin B1, but its toxicity is lower than that of aflatoxin B1. However, compared with potassium cyanide and arsenic, it is still a particularly high toxic substance and a strong carcinogen.
The results of reactive epidemiological studies on the conversion of Aflatoxin B1 to Aflatoxin M1 show that the incidence of liver cancer in high-incidence areas is closely related to the intake of Aflatoxin B1 and the conversion rate of Aflatoxin M1 in urine. However, with the adjustment of dietary structure, the chance of Aflatoxin B1 entering human body is vague, and the existence of Aflatoxin M1 in animal milk is still critical to human harm.
Since Aflatoxin M1 is quite stable and cannot be killed by pasteurization, the detection of Aflatoxin M1 must not only be performed in feed ingredients, but also in the final product.
The aflatoxin in milk comes from the feed of dairy cows. Even a small amount of aflatoxin in milk can cause cancer as it accumulates in the human body. Aflatoxin was classified as a category 1 carcinogen by the Cancer Research Institute of the World Health Organization (WHO) in 1993, and it is a highly toxic substance.
On October 27, 2017, the list of carcinogens published by the International Agency for Research on Cancer of the World Health Organization was initially compiled for reference. Aflatoxin M1 was included in the list of 2B carcinogens.
Aflatoxin M1 belongs to aflatoxins, a class of compounds with similar structures, which are likely shown in food and feed in hot and humid regions.
Aflatoxin (AF) is mainly a metabolite of Aspergillus flavus and Aspergillus parasiticus. The reason why it is widely taken seriously is that it is an indangerous carcinogen. There are more than 20 kinds of aflatoxins and their derivatives isolated so far, and aflatoxin B1 is the most common in natural foods. In AF, the order of toxicity is Aflatoxin B1>Aflatoxin M1>AFG1>AFB2>AFG.
Aflatoxin M1 is a metabolite formed by the hydroxylation of aflatoxin B1 in the body. The toxicity is second only to Aflatoxin B1. The WHO lists it as a class II B carcinogen. Aflatoxin M1 is mainly found in animal milk, kidney, liver, eggs, meat and urine, among which milk is the most common.
If moldy grain is used as feed, the aflatoxin in it can enter the milk after 24 hours. After being eaten by animals, a part of aflatoxin will accumulate in the animal's body, and the other part will be converted into milk and urine. The conversion rate is generally 3.45%-11.39%, and the amount of Aflatoxin M1 excreted is 1-3% of the intake of Aflatoxin.
Once produced, Aflatoxin M1 is difficult to be eliminated because it is relatively stable to light, heat and acid, and its melting point (cracking temperature) is 299d℃. The three common disinfection methods of milk are helpless.
After entering human or animal body, Aflatoxin M1 not only inhibits the synthesis of DNA and RNA, but also inhibits the synthesis of liver protein, leading to human poisoning. Its hazards are mainly manifested in three aspects: damage to tissues and organs; carcinogenic, teratogenic, mutagenic, causing liver cancer, kidney cancer and gastric cancer in animals and humans; suppressing immune function. Its toxicity is 10 times that of potassium cyanide and 68 times that of arsenic. The main aflatoxin m1 side effects act on the liver.
The aflatoxin in milk comes from the feed of dairy cows. Even a small amount of aflatoxin in milk can cause cancer as it accumulates in the human body.
The limit of Aflatoxin M1 in milk depends on many factors in science, society and economy. These factors influence and restrict each other and comprehensively determine the limit of Aflatoxin M1 in milk. At present, countries all over the world have established strict limits on the content of Aflatoxin M1 in food.
The number of countries that adopt the standard of 0.05μg/kg in milk monopolize. Among them, most countries are EU member states and some countries in Africa, Asia, and Latin America that have trade with the EU. The EU stipulates that aflatoxin in infant formula, including Infant formula milk is limited to 0.025μg/kg.
A. Isotope dilution liquid chromatography-tandem mass spectrometry: Aflatoxin M1 and M2 in the sample are extracted with methanol-water solution, then the supernatant is diluted with water or phosphate buffer, and purified and enriched by an immune-affinity column.
After the purification solution is concentrated, constant volume and filtered, it is separated by liquid chromatography, detected by tandem mass spectrometry, and quantified by isotope internal standard method.
Advantages: simple pretreatment, fast detection speed and high sensitivity, which can meet the detection needs of small and batch samples;
Disadvantages: high requirements for instruments and high cost.
B. High performance liquid chromatography:
Aflatoxin M1 and M2 in the sample are extracted with methanol-water solution, the supernatant buffer is diluted, purified and enriched by the immune-affinity column, and the purification solution is concentrated.
Advantages: high sensitivity, strong selectivity, strong separation ability, and few interference peaks.
C. Enzyme-linked immunosorbent screening method:
The aflatoxin in the sample competes with the antigen-specific immobilized on the enzyme-labeled plate to form an antigen-antibody complex on the surface of the solid-phase carrier. Wash off the excess antibody components, add enzyme-labeled II antibody, reacting with the antibody to color through an enzyme-catalyzed color reagent, and determine the amunt of aflatoxin in the sample according to the depth of color development.
Advantages: safe to use, easy and fast to operate, suitable for screening large quantities of samples.
Disadvantages: Applicable enzymes and antibodies have certain requirements for storage conditions and time.
D. Colloidal gold method: With this method, the test sample solution is added to the sample hole on the rapid test card, and the Aflatoxin M1 in the test solution combines with the gold-labeled antibody on the gold-labeled card to form a complex.
If the concentration of Aflatoxin M1 in the test solution is lower than set value (negative sample), the unbound gold-labeled antibody will flow to the T zone, captured by the antigen immobilized on the membrane to form a visible T-line; if the Aflatoxin M1 concentration is higher than the set value, the target antibody will form a complex with Aflatoxin M1 and no longer binds to the antigen at the T line, which is invisible. Line C is the quality control line, if it appears, it means that the rapid test card is valid.
Advantages: convenient and fast;
Disadvantages: Mainly used for qualitative purposes.
The limit standard for aflatoxin in milk is strict. In fact, aflatoxin B1 may be in the food you eat, which is much stronger than that in milk, and the limit standard for aflatoxin B1 in food is higher than that in milk. So the Aflatoxin M1 in milk will not pose a great threat to the body.
If a person eats food with aflatoxin B1, it will be converted into Aflatoxin M1 in the body, which is generally stored in the liver. So it has a greater impact on the liver. But if dairy cows eat feed with aflatoxin B1, Aflatoxin M1 can be detected in the milk. I think there will be some Aflatoxin M1 in human metabolites.
The anti-mold and detoxification measures for food include:
A. Anti-mould:
Controlling the moisture content in the grain below 12~13% can prevent mould. Keep the rice grains and peanut shells intact, and use chemical fumigants to prevent mould infection.
B. Detoxification method:
C. Strengthen food hygiene monitoring.
Aflatoxins are metabolites of Aspergillus flavus and Aspergillus parasiticus. At present, there are 17 aflatoxins B1, B2, G1, G2, M1, M2, etc., among which B2 is the most toxic.
Aflatoxin is a highly toxic substance, which can damage the liver of humans and animals, cause hepatocellular necrosis and cirrhosis, induce liver cancer, gastric cancer, etc., and cause disorders of animal metabolism. It is one of the strongest carcinogens found so far. Acute poisoning can cause nausea, vomiting, death of livestock and poultry, and decrease in milk and egg production rates.
Arsenic is the common name of arsenic trioxide, which is a highly toxic inorganic substance. It mainly affects the permeability of the nervous system and capillaries, and has a stimulating effect on the skin and mucous membranes. Oral poisoning resulted in nausea, vomiting, abdominal pain, convulsions, respiratory paralysis and death. Chronic poisoning can cause liver and kidney damage, as well as lung cancer and skin cancer.
The median lethal dose of aflatoxin B1 is 0.36 mg/kg body weight, and the median lethal dose of arsenic is 14.6 mg/kg. Earlier reports also mentioned that the toxicity of aflatoxin is 10 times that of potassium cyanide and 68 times that of arsenic, but the source of the data is unclear.
As different foods contain different types and quantities of aflatoxins, swill oil from different sources and treatments is also a complex system. It is correct to resist inferior oils such as swill oil, but the toxicity of substances contained in it should not be irrationally magnified in order to misrepresent and cause panic.
Aflatoxins are mainly toxic metabolites produced by Aspergillus flavus and Aspergillus parasiticus. The growth and reproduction of molds require certain conditions of temperature (25℃～32℃) and humidity (80%-100%). When molds are in high temperature, high humidity, or competing with other molds, mycotoxins are produced.
Aflatoxin is a highly carcinogenic and extremely toxic substance. It is a metabolite of toxin-producing strains of Aspergillus flavus and Aspergillus parasitica. It is commonly found in moldy food and feed. Aflatoxin is resistant to high temperatures and can live after cooking and conventional processing. It is difficult to be destroyed, and it is extremely harmful to humans and animals.
The kit contains a small amount of Aflatoxin M1; the substrate contains tetramethylbenzidine; the stop solution contains dilute sulfuric acid to prevent skin and mucous membranes from contacting the stop solution; the above solvents accidentally drip onto the skin, please rinse with water.
The Aflatoxin M1 kit detects Aflatoxin M1 through Aflatoxin M1 antibody. Add the Aflatoxin M1 standard or sample to the microwell and react with the anti-Aflatoxin M1 high-affinity antibody coated on the bottom of the microwell.
If Aflatoxin M1 existed in the sample, it will bind to the coated antibody. This process is incubated for 30 minutes and then the plate should be washed. Then add HRP (horseradish peroxidase) labeled toxin, and the enzyme-labeled toxin is combined with the standard or the part where the antibody does not bind. Incubate for 60 minutes, pour out the liquid in the well, add the coloring substrate after washing for 20 minutes, and the well will appear blue under the action of the enzyme.
The intensity of the color is directly proportional to the amount of bound enzyme conjugate, and inversely proportional to the amount of Aflatoxin M1 in the standard or sample. Therefore, the higher the concentration of Aflatoxin M1 in the standard or sample, the lighter the blue color will be. Acid was added to terminate the reaction, and the color of the substrate changed from blue to brown.
Put the microplate into the microplate reader and read the absorbance value at OD450nm. The absorbance value of the sample will be compared with the standard OD value in the kit, and the corresponding result is obtained.
Aflatoxin M1 test kit use a sensitive immunological detection method for quantitative detection of Aflatoxin M1. It is suitable for the detection of Aflatoxin M1 in milk and some dairy products. If necessary, it can be confirmed by HPLC and GC/MS methods. Store at 4-8°C. The temperature of the kit should be restored to room temperature (20-25°C) before use. The reagents can be used within the validity period.
In the European Union, the content of Aflatoxin M1 in milk and milk powder is limited to 0.05mg/L or 50ppt. In China, the nationally prescribed maximum value of Aflatoxin M1 is 0.5μg/kg. National and regional milk specifications for Aflatoxin M1 content in the EU (Belgium, Switzerland, Sweden) is less than 0.05 mg/ton, in Asia, the Americas (China, Japan, Brazil, Mexico, the United States) less than 0.5 mg/ton.
Aflatoxin M1 is a metabolite formed by the hydroxylation of aflatoxin B1 in the body. Its toxicity is second only to Aflatoxin B1. The WHO lists it as a class IIB carcinogen. Aflatoxin M1 is mainly found in animal milk, kidney, liver, eggs, meat and urine, among which milk is the most common.
If moldy grain is used as feed, the aflatoxin in it can enter the milk after 24 hours. After being eaten by animals, a part of aflatoxin will accumulate in the animal's body, and the other part will enter into milk and urine. The conversion rate is generally 3.45%-11.39%, and the amount of Aflatoxin M1 excreted is 1-3% of the intake of Aflatoxin B1.
In milk:
aflatoxin B1 is 0.002μg/kg,
aflatoxin B2 is 0.001μg/kg,
aflatoxins G1 and G2 are 0.003μg/kg,
aflatoxins M1 and M2 are 0.005μg/kg;
In milk powder:
Aflatoxin B1 is 0.02μg/kg,
aflatoxin B2 is 0.01μg/kg,
aflatoxins G1 and G2 are 0.03μg/kg,
and aflatoxins M1 and M2 are 0.05μg/kg.
The immune-affinity column can selectively adsorb the Aflatoxin M1 in the sample solution, so as to specifically purify the Aflatoxin M1 sample. The sample solution after purification by the column can be directly used for high performance liquid chromatography for detection of Aflatoxin M1 content. The combination of affinity column and HPLC can achieve the rapid determination to improve the signal-to-noise ratio and improve the accuracy of the detection method.
Aflatoxin is a well-known carcinogen, a small amount of ingestion may cause cancer, but not necessary.
When a dairy cow eats a diet containing Aflatoxin B1, it can quickly convert the Aflatoxin B1 absorbed into the blood into Aflatoxin M1, which is then secreted into the milk through the mammary glands, causing Aflatoxin M1 contamination.
Some previous studies have reported the transformation law of Aflatoxin B1 into milk. After 24 hours of consumption of Aflatoxin B1, Aflatoxin M1 in milk can reach a stable level. Aflatoxin M1 in milk can be eliminated if the contaminated feed is suspended for 3-4 days.
The Aflatoxin M1 content in goat milk reached a peak in 4-8 hours after eating Aflatoxin B1 (1 mg), and then decreased quickly. The maximum average conversion rate of Aflatoxin B1 to Aflatoxin M1 at each time was 0.19%, which occurred in 4 h. The sum of the average conversion rate of Aflatoxin M1 within 24 h after feeding Aflatoxin B1 accounted for 81% of the total conversion rate of Aflatoxin M1.
It can be seen that the Aflatoxin M1 in the milk can reach a stable level after the dairy cows consume the contaminated diet continuously for 2-4 days, and the Aflatoxin M1 in the milk can be eliminated if the Aflatoxin B1 is stopped for 3-4 days.
Many researchers have carried out studies on the correlation between Aflatoxin B1 intake and Aflatoxin M1 in milk. For example, Battacone et al. (2003) used lactating ewes to study the relationship between daily intake of Aflatoxin B1 and Aflatoxin M1 concentration in sheep milk.
Most experiments show that the transfer rate of Aflatoxin M1 in milk is between 0.1% and 6%, with an average of 1.7%. If calculated according to the 1.7% conversion rate, when the dietary Aflatoxin B1 content exceeds 30μg/kg, the Aflatoxin M1 content in the milk will reach the safety limit of 0.5μg/kg in the United States and other countries. Similarly, when the diet contains more than 3μg/kg, the content of Aflatoxin M1 in the milk will reach the safety limit of 0.05μg/kg in the EU.
There are many factors affecting the conversion rate of AFs, such as nutritional or physiological factors such as health status, milk production, and individual differences in sensitivity to mycotoxins. In addition, there are also big differences between different dates and different stages of milk production. The toxin transfer rate of high-yielding dairy cows during lactation reached 6.2%.
The conversion rate of Aflatoxin M1 was 2.32% for high-yield and high somatic cell array cows (milk yield>30 kg/head/d, somatic cell count>350,000), and the conversion rate of high-yield and low cell array Aflatoxin M1 was 2.7 %, the conversion rate of Aflatoxin M1 in the low-yield and high somatic cell group was 1.48%, and the conversion rate of the low-yield and low somatic cell array Ml was 1.29%.
It is believed that milk production is the main factor affecting the conversion efficiency of Aflatoxin M1, and the number of somatic cells used as an index for evaluating mastitis is not related to the conversion rate of Aflatoxin M1.
Masoero et al. (2007) established a regression equation between milk production and conversion rate based on the experimental results; conversion rate (%) = -0.326+0.077* milk production (kg) (RSEM=0.692; R2=0.58).
In addition, the conversion rate of Aflatoxin M1 can also be reduced by adding a mycotoxin adsorbent. When the Aflatoxin B1 concentration in TMR was 112μg/kg, the conversion rate of Aflatoxin M1 was 2.7%; while the conversion rate was 1.48% when the inorganic mycotoxin hydrated calcium sodium alumino-silicate adsorbent was used.
The conversion rate is 2.5% when using organic mycotoxin yeast cell wall. It shows that adding mycotoxin adsorbent to dairy cow feed can reduce the conversion rate of Aflatoxin M1, but the adsorption effect of different types of mycotoxin adsorbent varies.