牛奶中β-内酰胺类抗生素残留检测试剂盒的研制

Development of a Test Kit for β-lactam Antibiotic Residues in Milk

摘要:

以牛奶中残留的β-内酰胺类抗生素为检测目标，开发一种能快速检测其残留的试剂盒。利用嗜热脂肪芽孢杆菌BS 10208对酸和β-内酰胺类抗生素敏感的特点，优化其最优的生长条件，并制成在2.5 h内能发生显色反应的检测试剂盒。单因素结果表明，当菌液接种量5%、生长初始pH 8.0、培养温度55 ℃、蛋白胨添加量0.50%，其菌落数对数值分别为2.9、7.5、7.3、5.8。正交结果表明，在外加入0.5%蛋白胨营养肉汤中，接种5%种子液，在65 ℃恒温振荡器中培养24 h，其菌落数对数值为7.6。优化后检测试剂盒在2.5 h内能检测牛奶中?-内酰胺类抗生素残留，其各项参数为：反应体积为0.5 mL，1.5%琼脂，0.01 g/L溴甲酚紫浓度，BS 10208细胞总数为1.30×108 cfu，最适反应温度65 ℃，青霉素钠的限值为2?4 ?g/L。该试剂盒还能检测牛奶中残留的四环素类、磺胺类、大环内酯类、氨基糖苷类等抗生素。

Abstract:

We used β-lactam antibiotic residues in milk as the target to develop a rapid detection kit. Based on sensitivity of Bacillus stearothermophilus 10208 to acid and β-lactam antibiotics, growth conditions were optimized and a test kit that could show a color reaction within 2.5 h was developed. The results of the single-factor experiment showed that when the inoculum was 5%, initial growth pH was 8.0, culture temperature was 55 ℃, and the quantity of peptone was 0.50%, the logarithm values of growth for the bacterium were 2.9, 7.5, 7.3, and 5.8, respectively. The results of an orthogonal test showed that when a nutrient broth containing 0.50% peptone was added and the mixture was inoculated with 5% seed broth and incubated for 24 h at 65 ℃, the logarithm value of growth for the bacterium was 7.6. This optimized test kit could be used to detect residual β-lactam antibiotics in milk within 2.5 h. The parameters were as follows: reaction volume, 0.5 mL; agar, 1.5%; bromocresol purple, 0.01 g/L; total BS 10208 cell number, 1.30 × 108 cfu, and optimum reaction temperature, 65 ℃. The detection limit on penicillin sodium was 2~4 ?g/L. The test kit could also detect residual tetracyclines, sulfonamides, macrolides, aminoglycosides, and other antibiotics in milk.