The paper method, also known as the PD (Paper Disc) method, is a widely used technique for detecting β-lactam antibiotics in milk. Two commonly employed variations of this method are the *Bacillus subtilis* paper test and the *Astragalus thermophila* (or *Geobacillus thermophilus*) paper test. Both methods operate on similar principles but differ in the bacterial strains used, which affects their sensitivity and specificity.
The *Bacillus subtilis* method is effective for detecting β-lactam antibiotics, but it tends to produce false positives more frequently. To confirm whether the positive result is due to penicillin or another β-lactam antibiotic, the milk sample is treated with penicillinase, an enzyme that inactivates penicillin. After this treatment, the test is repeated, allowing detection at levels as low as 0.01 IU/mL. On the other hand, the *Astragalus thermophila* method not only detects β-lactam antibiotics but also helps identify the presence of other bacteriostatic substances. It has a lower detection limit, reaching below 0.008 IU/mL, and provides results within approximately 4 hours. As a result, the *Astragalus thermophila* method is more widely used in practice due to its higher accuracy and reliability.
In 1991, Shen Baosheng conducted further research and improved the paper method, enhancing both its ability to identify specific antibiotics and its overall detection accuracy. The improved method can detect various antibiotics at very low concentrations, such as chloramphenicol at 0.01 mg/kg, oxytetracycline at 0.05 mg/kg, streptomycin at 1 mg/kg, erythromycin at 0.05 mg/kg, and penicillin at 0.0025 mg/kg.
Another common microbiological detection method is the TTC (triphenyltetrazolium chloride) method, which is specified in China's food hygiene standards (GB5409-85). This method is simple, fast, and does not require special equipment, making it ideal for field use in dairy farms and food inspection departments. It typically takes 3–4 hours to complete, and its sensitivity varies depending on the antibiotic being tested. For example, the minimum detectable amounts are 0.004 U/mL for penicillin, 0.5 U/mL for streptomycin, 0.4 U/mL for gentamicin, and 5 U/mL for kanamycin.
While traditional microbiological methods like the paper and TTC tests are cost-effective and accessible, they have some limitations, including long testing times, subjective color interpretation, and complex procedures. To address these issues, researchers are continuously developing more sensitive, accurate, and rapid microbial detection techniques. In the U.S. and Canada, methods such as the swab method and the bovine antibiotic and sulfonamide test (CAST) are gaining popularity. Additionally, the Rapid Antibiotic Screening Test (FAST) has been introduced in recent years, offering faster results, although it may occasionally produce false positives, which can be confirmed through additional testing.
Physical and chemical detection methods, such as high-performance liquid chromatography (HPLC), gas chromatography, and mass spectrometry, provide more precise and quantitative analysis of antibiotic residues. These methods are highly sensitive and capable of identifying and quantifying multiple compounds simultaneously. However, they often involve more complex procedures and higher costs.
HPLC is one of the most commonly used physical and chemical methods for analyzing antibiotic residues in milk. It offers high efficiency, speed, and automation, making it suitable for a wide range of compounds, including polar and ionic analytes. Reversed-phase HPLC, using C18 or C8 stationary phases, is particularly popular due to its stability, compatibility with water-based mobile phases, and ability to handle complex matrices. UV detectors are the most commonly used, though pre-column derivatization is often necessary to improve sensitivity, especially when dealing with low-concentration residues.
For instance, Peng Li et al. used HPLC with a UV detector at 278 nm to detect chloramphenicol in milk after extraction with ethyl acetate. The method achieved an average recovery rate of 94.8% with a coefficient of variation less than 5%, demonstrating its effectiveness in residue analysis.
Overall, the choice of detection method depends on factors such as required sensitivity, available resources, and the type of antibiotic being tested. As technology advances, more efficient and accurate methods continue to emerge, improving the safety and quality of dairy products.
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