With the development of dairy industry, antibiotics are widely used in the prevention and treatment of dairy diseases. The main sources of antibiotics in fresh milk are: firstly, antibiotics used to treat sick cows during lactation will migrate from cows to breast residues and enter milk. The data showed that the treated cows had antibiotic residues in their milk within 5 days. Secondly, in order to prevent diseases in dairy cows and increase production, adding antibiotics to dairy cows' feed will also cause antibiotic residues in milk; Thirdly, due to poor pasture management, there is no strict health system and supporting facilities for milking and milk storage, which artificially adds or causes antibiotic pollution in milk.
Antibiotics in milk not only do great harm to people's health, but also bring economic losses to dairy processing enterprises (because yogurt and cheese cannot be produced). Therefore, in addition to scientific feeding and careful management, it is necessary to strictly control the antibiotic residues in milk; In addition to correct milking and disease prevention, it is also necessary to standardize the use of antibiotics. According to the relevant regulations in national standards, the milk produced by cows after 5 days of medication can only 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) all have clear regulations on the maximum residue of antibiotics in food, and China also has the Inspection Standard for Antibiotic Residues in Fresh Milk (GB 4689.27-94).
At present, the detection methods of antibiotic residues in fresh milk can be roughly divided into three categories: bioassay (microbial assay, radiation receptor assay), immunoassay (radioimmunoassay, fluorescence immunoassay, enzyme-linked immunoassay), and physical and chemical analysis (spectroscopy, chromatography and combined technology). Here are several commonly used detection methods of antibiotic residues in milk.
TTC method
TTC method is a biological detection method, which is the detection standard of antibiotic residues in fresh milk in China (GB 4689.27-94). Its determination principle is based on the inhibitory effect of antibiotics on microorganisms. If milk contains antibiotics and bacteria (Streptococcus thermophilus) are added for 2.5~3 hours, TTC indicator (triphenyltetrazole) will not be reduced, so the sample is colorless. If the milk contains no antibiotics, the sample is red. In this way, the color of the sample remains positive after the experiment, and the sample dyed red is negative.
Specific operation steps of TTC method:
1. Preparation of bacterial liquid: culture a single strain (Streptococcus thermophilus) in an incubator at 36 1℃ for 15 hours, and then dilute it with skim milk to 1: 1 for later use;
2. Take 9mL of sample solution to be tested, heat it in water bath at 80℃ for 5 minutes, then cool it to below 37℃, add 1mL live bacteria solution, take water bath at 36℃ 1℃ for 2 hours, add 0.3ml 4% TTC indicator, and take water bath at 36℃ 1℃ for 30 minutes;
3. If the color of the sample solution does not become positive, it will be negative if it is red; The sample solution is positive, and it is positive if it is cultured in water bath for 30 minutes, and negative if it is not colored.
The sensitivity of TTC method to determine various antibiotics is: penicillin: 4ppb, streptomycin: 500ppb, gentamicin: 400ppb and 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 to have certain professional knowledge. During the experiment, the preparation of bacterial liquid and the control of water bath process must strictly abide by the operating rules, otherwise false positives will easily occur, leading to unstable test results.
Delvotest？ Sp method (Delvaux test method)
This method was first introduced to Guangdong in Hong Kong, and its application was based on the requirement that raw milk exported from Guangdong must be "non-resistant" in the early 1980s, and it should be tested by the Delvotest method. This method is also a bioassay, and its reagent is produced by DSM company in the Netherlands and certified by AOAC. The principle is that the microorganism Bacillus thermophilus will produce acid after being cultured at 64℃ for 2.5 ~ 3 hours, and the acid will make the indicator BCP (bromocresol purple) yellow; If the milk sample does not contain antibiotics, the sample will be yellow after culture. If the sample contains antibiotics, the growth of Bacillus thermophilus will be inhibited, acid will not be produced, and the indicator will not change color.
Delvotest？ Operating steps of sp method:
1. Sterile operation: Put the nutrient tablets into a small test tube;
2. Inject 0. 1 ml milk sample into a small test tube with a micropipette;
3. Put the small test tube into a water bath box or a constant temperature box preheated to 64℃ for culture;
4) Take it out every 3 hours and observe the color change. If the bottom 2/3 solid medium is yellow, it is negative, and if the bottom 2/3 solid medium is purple, it is positive.
Delvotest？ Sp method has broad spectrum and detectability. -More antibiotics include lactams, such as sulfonamides, tetracyclines, macrolides, aminoglycosides and chloramphenicol. Penicillin and sulfonamides are particularly sensitive. Sensitivity: penicillin: 3ppb, streptomycin: 300ppb, gentamicin: 400ppb, kanamycin: 2500ppb. Delvotest？ Sp method is simple in operation, rigorous and practical, easy to judge, reliable in results and moderate in cost. However, it is also prone to false positives. The experiment shows that the growth of Bacillus thermophilus can be affected when microbial preservatives (such as —Nisn) are added to the milk sample or there is enough detergent residue in the sample, and the experiment will be positive.
Hey? law
This method is an enzyme-linked immunosorbent assay (ELISA). The detection analyzer and its kit produced by IDEXX company in the United States have obtained AOAC certification and adopted competitive ELISA technology. The basic principle is to activate the solid carrier with specific antibody, add a solution containing the antigen to be detected and a certain amount of enzyme-labeled antigen, keep the temperature at 45℃+/-5℃ * *, make the antibiotics in the sample compete with the built-in antibiotic markers to bind the immobilized antibody, and then wash and develop color. The complex formed by the combination of built-in antibiotic markers and immobilized antibodies can be decomposed by enzymes to form colored substances. By measuring the chromaticity and comparing it with a reference object, it can be determined whether the result is positive or negative.
Hey? Method operation steps:
1. Add the milk sample into the sample tube, shake it evenly, and heat the sample and the detection plate for 5 minutes;
2. Add the milk sample into the sample hole, and when the activated ring begins to retreat, press the Snap key;
3. react for 4 minutes and press Snap? The reader reads and prints the results. When the reading is less than 1.05, it is judged to be negative, and when it is greater than 1.05, it is judged to be positive.
Hey? This method combines the sensitivity of enzymatic chemical reaction with the specificity of antigen-antibody immune reaction. It has good sensitivity and specificity, and the sensitivity of detection is widely used. -lactams: penicillin: 5ppb, amoxicillin: 10ppb, ampicillin: 10ppb, cefotaxime: 8ppb. For the detection of other antibiotics such as tetracycline, it is necessary to buy corresponding reagents for detection. Hey? This method is rapid and accurate, and can detect milk within 9 minutes. -Residues of antibiotics such as lactams, tetracyclines and sulfonamides, with semi-quantitative readings, can monitor the drug use in pasture; The detector has good stability, high reproducibility of results and simple whole detection process. However, it is necessary to purchase special instruments and reagents, and the cost is high.
High performance liquid chromatography detection method
It is a physical and chemical detection method, which uses the special reaction of groups in antibiotic molecules to determine its content. The detection process adopts the principle of gas chromatography, and through high-pressure liquid phase and high-sensitivity detector, the separation speed is fast, the efficiency is high and the operation is automatic. Generally, the specific content of antibiotics can be detected by sample extraction, deproteinization, centrifugation, column purification, derivatization and other steps. The sensitivity is high, but the detection procedure is complicated and the cost is high, so it is not suitable for small-scale inspection rooms.
There are many traditional methods to detect antibiotics, some of which are cumbersome to operate, some of which require high experimental conditions, and some of which take too long. These will not only cause economic and time losses to dairy production enterprises, but also the test results are often affected by raw and auxiliary materials and human operation. In view of the fact that antibiotic residues in milk are a public health problem involving human health, dairy enterprises and pastures should attach importance to and strengthen the detection work, and apply some simple, rapid and accurate methods to monitor product quality to protect consumers' health.
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