摘 要

目的建立禽蛋中抗生素药物残留的方便快速的检测方法，采用微生物检测法，利用一些敏感微生物在抗生素存在下生长受到抑制，及其代谢产物与微孔板中氧化还原试剂发生显色反应的原理。用嗜热脂肪芽孢杆菌作为指示菌，从营养菌体和芽孢两个方向，研究在微孔板中其最佳培养方法，确定菌的最佳包埋量，优化包埋培养基的配方，确定指示剂最佳包埋比例，为进一步开发禽蛋抗生素药物残留检测试剂盒提供依据和参考。我们发现营养菌体最佳生长培养时间为18小时，最佳包埋数为5×10⁷个/ml浓度，产芽孢率较高的培养基配方为0.4%的蛋白胨，0.2%的牛肉膏，0.1%的葡萄糖，0.2%的磷酸氢二钾，0.2%的MnCl₂，2%的琼脂，pH7.2±0.2。芽孢最适包埋浓度范围在2.5×10⁵个/ ml—5×10⁵个/ ml之间，包埋培养基成分0.2% 蛋白胨，0.1% 牛肉膏，0.3% 葡萄糖，2% 琼脂，pH7-7.5。选择溴甲酚紫作为指示剂，采用50:4的比例。对于包埋的菌液浓度需进一步实验精确。

关键词：嗜热脂肪芽孢杆菌；营养体；芽孢；抗生素残留

Development of screening kit for detection of antibiotic residues in eggs

ABSTRACT

The study is aim to establish a convenient and rapid method for the detection of antibiotic drug residues of tolerance levels in poultry eggs by using microorganism detection method. The mechanism is based on growth inhibition of some sensitive microorganisms in the presence of antibiotics. While the control without the antibiotics will change color due to the pH change caused by the metabolite of sensitive microorganism and redox reagents in solid medium. Using Bacillus stearothermophilus as indicator bacteria, the best culture method was studied one is nutrient body and the other spore. The optimum embedding amount was determined, the formula of embedding medium was optimized and the optimum embedding ratio of indicator was determined. It was concluded that the best growth time of the vegetative bacteria was 18 hours, the best embedding number was 5 × 10⁷ /ml concentration, the medium with high sprouts spore rate was 0.4% peptone, 0.2% beef paste, 0.1% glucose, 0.2% phosphoric acid two potassium, 0.2%MnCl₂, 2% agar, pH7.2 ± 0.2. The optimum concentration range of the spore was between 2.5 × 10⁵ /ml - 5 × 10⁵/ml. The embedding medium was 0.2% peptone, 0.1% beef extract, 0.3% glucose, 2% agar, and pH7-7.5. The ratio of pH indicator of bromine cresol violet in medium was 4:50. The best detection limit of antibiotics needs to be further investigated.

Key words：Bacillus stearothermophilus；Nutritional body；endospore；Antibiotics residue

目 录

1 前言 - 1 -

1.1 中国鸡蛋消费现状 - 1 -

1.1.1 中国鸡蛋消费现状及抗生素的使用 - 1 -

1.1.2 抗生素残留的危害 - 2 -

1.1.3 中国对鸡蛋检测的标准 - 2 -

1.2 中国对鸡蛋的检测方法 - 3 -

1.2.1 高效液相色谱串联质谱法 - 3 -

1.2.2 电极法 - 3 -

1.2.3 抑菌圈法 - 4 -

1.2.4 酶联免疫法 - 4 -

1.2.5 薄层层析法 - 4 -

1.2.6 高效毛细管电泳法 - 4 -

1.3 研究的目的、内容及改进方向 - 5 -

2 材料与方法 - 6 -

2.1 实验材料、试剂与仪器 - 6 -

2.1.1 实验材料 - 6 -

2.1.2 实验试剂的配制 - 7 -

2.1.3 实验仪器 - 7 -

2.2 实验方法 - 7 -

2.2.1 实验流程 - 7 -

2.2.2 嗜热脂肪芽孢杆菌营养菌体的生长状况检测 - 8 -

2.2.3 嗜热脂肪芽孢杆菌营养菌体最佳包埋数的实验 - 8 -

2.2.4 嗜热脂肪芽孢杆菌芽孢生成条件优化 - 9 -

2.2.5 芽孢悬液浓度优化 - 10 -

2.2.6 包埋培养基优化 - 11 -

3 结果与讨论 - 13 -

3.1 结果与分析 - 13 -

3.1.1 营养菌体的生长状况 - 13 -

3.1.2 营养体最佳包埋数 - 13 -

3.1.3 芽孢悬液浓度优化 - 16 -

3.1.4 包埋培养基指示剂添加量优化 - 21 -

3.2 讨论 - 22 -

结 论 - 23 -

致 谢 - 24 -

参考文献 - 25 -

1 前言