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With the development of dairy farming industry, antibiotics have been widely used in the prevention and treatment of dairy cow diseases. The main sources of antibiotics in fresh milk are: first, the antibiotics used in the treatment of lactating cows will migrate from the cows to the breast residues and enter the milk. The data shows that the milk of the cows after treatment is within 5 days. Antibiotic residues; second, in order to prevent cow diseases and increase production, the addition of antibiotics in dairy cow feed will also cause antibiotic residues in milk; third, due to poor farm management, there is no strict sanitation system and supporting milking and storage milk Facilities, artificially add or cause pollution of milk antibiotics.
Milk contains antibiotics, which not only cause great harm to human health, but also bring economic losses to dairy processing companies (because they cannot produce yogurt and cheese). Therefore, antibiotic residues in milk must be strictly controlled. In addition to scientific feeding and careful management; correct milking and disease prevention, it is also necessary to regulate the use of antibiotics. According to the relevant provisions of the national standard, the milk produced by cows after 5 days of medication It can be used as raw milk, and its residue should be tested. The World Food and Agriculture Organization (FAO), the World Health Organization (WHO), the European Union (EC) and the US Food and Drug Administration (FDA) have clear regulations on the maximum amount of antibiotic residues in food. China also has antibiotic residues in fresh milk Quantity inspection standard (GB4689.27-94).
TTC method
The TTC method is the detection method of the antibiotic residue test standard (GB4689.27-94) in fresh milk in China, and belongs to the biological detection method. Its measuring principle is based on the inhibitory effect of antibiotics on microorganisms. If the milk contains antibiotics, add the strain (Streptococcus thermophilus) and after 2.5 to 3 hours of incubation, the addition of the TTC indicator (triphenyltetrazolium) does not cause a reduction reaction, so the sample is colorless; if the milk Without antibiotics, the sample is red. In this way, the color of the sample is positive after the experiment, and the sample dyed red is negative.
The specific operation steps of TTC method:
1. Preparation of bacterial solution: use single-fat (Streptococcus thermophilus) with skim milk as the culture medium, incubate in a 36 ± 1 ℃ incubator for 15 hours, and then use skim milk so that it is diluted 1: 1 for use;
2. Take 9mL of the sample solution to be tested, heat it in an 80 ° C water bath for 5 minutes, then cool to below 37 ° C, add 1mL of viable bacterial solution, and add a 4% TTC indicator 0.3mL, 36mL at 36 ° C ± 1 ° C Incubate in water bath at ℃ ± 1 ℃ for 30 minutes;
3. If the color of the sample solution does not change to positive, red is negative; if the positive sample solution is placed in a water bath for 30 minutes, the colorless is positive, and the red is negative.
The sensitivity of various antibiotics measured by TTC method is: penicillin: 4ppb, streptomycin: 500ppb, gentamicin: 400ppb, kanamycin: 5000ppb. It has the advantages of low cost and easy development; the disadvantage is that it takes a long time and requires The operator needs to have certain professional knowledge, and the preparation of bacterial liquid during the experiment and the control of the water bath process must strictly abide by the operating rules, otherwise false positives will easily occur, resulting in the instability of the test results.
Delvo Test
The law was first introduced to Guangdong in Hong Kong, and its use was based on the requirement in the early 1980s that Hong Kong required raw milk exported from Guangdong to be “non-resistance†and required to be tested using the Delvotest method. This method is also a bioassay, and its reagents are produced by the Dutch DSM company and certified by AOAC. The principle is that the microorganism-Bacillus thermophilus will produce acid after culturing at 64 ℃ for 2.5 to 3 hours, and the acid causes the indicator BCP (bromocresol purple) to turn yellow; if the milk sample does not contain antibiotics, the sample after cultivation Yellow, if the sample contains antibiotics, the growth of Bacillus thermophilus is inhibited and cannot produce acid, the indicator will not change color.
Operation steps of Delvo Test:
1. Put a piece of nutritional tablet into a small test tube by aseptic operation;
2. Use a micropipette to inject 0.1mL of milk sample into a small test tube;
3. Put the small test tube into the water bath or thermostat that has been preheated to 64 ℃ for cultivation;
4). Take out for 3 hours to observe the color change. If the bottom 2/3 solid medium is yellow, it is negative, if the bottom 2/3 solid medium is purple, it is positive.
The Delvo test is broad-spectrum and can detect more antibiotics including lactam antibiotics, such as sulfonamides, tetracyclines, macrolides, aminoglycosides, chloramphenicol, etc., of which penicillin and sulfonamide The antibiotics are particularly sensitive. The sensitivity is: penicillin: 3ppb, streptomycin: 300ppb, gentamicin: 400ppb, kanamycin: 2500ppb. The Delvo test method is easy to operate, strict and practical, easy to judge, reliable, and moderate in cost; but it is also prone to false positives. Experiments have shown that when milk samples are added with microbial preservatives (such as nisin-Nisn) Or if there is enough detergent residue in the sample, it can affect the growth of Bacillus thermophilus and make the test positive.
law
This method is an enzyme-linked immunoassay method. The detection analyzer and its kit produced by the American IDEXX company are all certified by AOAC. It uses competitive enzyme-linked immunoassay technology. The basic principle is to activate the solid-phase carrier with a specific antibody, add a solution containing the antigen to be tested and a certain amount of enzyme-labeled antigen to keep them together at 45 ℃ ± 5 ℃, so that the antibiotics in the sample compete with the built-in antibiotic markers. The immobilized antibody is bound, followed by washing and color development. The complex formed by the combination of the built-in antibiotic marker and the immobilized antibody is decomposed by the action of an enzyme to form a colored substance. By measuring the color and comparing it with a reference, the result can be determined Is positive or negative.
Operation steps of enzyme-linked immunoassay:
1. Add the milk sample to the sample tube, shake well, and heat the sample and the detection plate for 5 minutes;
2. Add the milk sample to the sample well, when the activation ring starts to recede, press the Snap key;
3. After 4 minutes of reaction, the results are read and printed by the enzyme-linked immunoassay reader. When the test reading is less than 1.05, it is negative, and when it is greater than 1.05, it is positive.
Enzyme-linked immunoassay is a method that combines the sensitivity of enzyme chemical reactions with the specificity of antigen-antibody immune reactions. It has good sensitivity and specificity, and the sensitivity of detection is based on the commonly used? -Lactams: penicillin : 5ppb, Amoxicillin: 10ppb, Ampicillin: 10ppb, Cefacillin: 8ppb. For the detection of other antibiotics such as tetracycline, etc., you need to purchase the corresponding reagents. The results of enzyme-linked immunoassay are fast and accurate, within 9 minutes Detects the residual content of antibiotics such as lactams, tetracyclines, sulfonamides, etc. in the milk, and has a semi-quantitative reading, which can monitor the use of drugs in the pasture; the detection instrument has good stability, high reproducibility of results, and the entire detection process It is simple and convenient; but special equipment and reagents need to be purchased, and the cost is higher.
HPLC detection method
It is a physical and chemical detection method, which uses the special reaction of the groups in the antibiotic molecule to determine its content. The detection process uses gas chromatography theory, high pressure liquid phase and high sensitivity detector, the separation speed is fast, High efficiency and automated operation. Generally, it needs to go through the steps of sample extraction, deproteinization, centrifugation, chromatographic column purification, derivatization, etc. It can detect the specific content of antibiotics, which has high sensitivity, but the detection procedure is complicated and the cost is high. , Not suitable for small inspection rooms.
There are many traditional antibiotic detection methods, some operations are cumbersome, some require high experimental conditions, and some inspection time is too long; these will not only cause economic and time losses to dairy production enterprises, but the test results will often be Factors such as raw and auxiliary materials and man-made operations. In view of the fact that antibiotic residues in milk are a public health problem involving human health, dairy companies and farms should pay attention to and strengthen the detection work, and apply some simple, fast, and highly accurate methods to monitor products The quality guarantees the health of consumers. For example, the quick test strips produced by our company are convenient and quick to use, and the actual measurement effect is good. The antibiotic content in milk can be detected in 3-5 minutes.
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