固定化乳酸菌制备生物传感器测定抗生素
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摘要
用海藻酸钠-淀粉凝胶作固定剂,将乳酸菌固定到两片核微孔膜中间制成"三明治"式传感膜,然后将其固定到玻碳电极上制成生物传感电极。通过电化学工作站检测3种抗生素分别在不同质量浓度条件下的响应电流,结果表明:该传感器固定瑞士乳杆菌的最适量为0.05 g,此时对青霉素、链霉素、四环素的最低检测限分别为1×10-10、1×10-9、1×10-9 g/m L,检出时间为4 min,明显优于国内外对抗生素残留量的要求。而且经测定表明该电极在37℃的MRS培养基中至少可以稳定保存7 d,低温保藏应该可以保存更长时间,说明电极性能比较稳定。总之,固定化乳酸菌制备的生物传感器提供了一种新的定量测定抗生素的方法,其检测灵敏度高、成本低、简单、快速,不仅适用于乳制品中抗生素残留的定量化快速检测,而且可以实现对多种类型的抗生素进行检测。
Using sodium alginate-starch gel as a fixing agent, lactobacilli were immobilized between two nuclear microporous membranes to make a sandwich-type sensing membrane, which was then fixed to a glassy carbon electrode to make a biosensor electrode. Based on the current responses of three antibiotics at different concentrations tested by electrochemical workstation, the calculation results showed that the most appropriate quantity of lactobacilli fixed by the sensor was 0.05 g, and now the limits of detection concentration(LDC) of the biosensor for penicillin, streptomycin and tetracycline were 1 × 10-10, 1 × 10-9 and 1 × 10-9 g/m L, respectively. The detection time was 4 min, which was obviously better than the requirement for the analysis of residual antibiotics at home and abroad. The electrode in 37 ℃ MRS medium could remain stable for 7 days at least, and the low temperature preservation time was extended, suggesting that the electrode performance was stable. In total, the microbial sensor by immobilized lactobacilli provides a new method for quantitative determination of antibiotics with high sensitivity, low cost, simple operation and fast detection. Therefore, it is not only applicable to the quantification and rapid detection of residual antibiotics in milk products, but also can detect a variety of types of antibiotics.
Using sodium alginate-starch gel as a fixing agent, lactobacilli were immobilized between two nuclear microporous membranes to make a sandwich-type sensing membrane, which was then fixed to a glassy carbon electrode to make a biosensor electrode. Based on the current responses of three antibiotics at different concentrations tested by electrochemical workstation, the calculation results showed that the most appropriate quantity of lactobacilli fixed by the sensor was 0.05 g, and now the limits of detection concentration(LDC) of the biosensor for penicillin, streptomycin and tetracycline were 1 × 10-10, 1 × 10-9 and 1 × 10-9 g/m L, respectively. The detection time was 4 min, which was obviously better than the requirement for the analysis of residual antibiotics at home and abroad. The electrode in 37 ℃ MRS medium could remain stable for 7 days at least, and the low temperature preservation time was extended, suggesting that the electrode performance was stable. In total, the microbial sensor by immobilized lactobacilli provides a new method for quantitative determination of antibiotics with high sensitivity, low cost, simple operation and fast detection. Therefore, it is not only applicable to the quantification and rapid detection of residual antibiotics in milk products, but also can detect a variety of types of antibiotics.
引文
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[11]曾卫军,李杰.孢子型乳酸菌对常用抗生素耐药性及最低抑菌浓度测定[J].湖北畜牧兽医,2012(3):18-20.
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[14]高寅生,庞广昌,李家鹏.基于乙醇氧化酶型酶膜的生物传感器的研制和应用[J].食品与发酵工业,2009,35(5):105-109.
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[21]王庆忠,蒲彪.乳品中抗生素的检测方法[J].食品工业科技,2003,24(11):79-82.
[22]张增福.基于微型表面等离子共振系统的牛奶中抗生素残留检测研究[D].天津:天津大学,2009.
[23]BERTRAM G K,ANGELIKA S,DIETRICH R,et al.Automated microarray system for the simultaneous detection of antibiotics in milk[J].Analytical Chemistry,2004,76(3):646-654.
[24]邓斌,邓春来,张曦.高效液相色谱法检测牛奶中青霉素残留的研究进展[J].中国兽药杂志,2003,37(6):36-40.
[25]韩洪莉,高长玲.牛奶中抗生素残留来源及几种常用检测方法[J].安全监测,2009(8):66-68.
[26]SCHNAPPINGER P,SCHNEIDER E,MARTLBAUE E,et al.Rapid detection of streptomycin and dihydrostreptomycin in milk by enzymelinked immunofiltration assay[J].Food and Agricultural Immunology,1996,8(4):269-272.
[27]秦燕,陈毓芳,林峰,等.固相萃取高效液相色谱法同时测定牛奶中的多种四环素残留[J].食品科学,2005,26(3):202-204.
[28]张素霞,李俊锁,钱传范.牛奶中四环素多残留分析方法研究[J].畜牧兽医学报,2002,33(1):51-54.
[29]李利东,宓晓黎,袁建兴,等.酶联免疫检测试剂盒应用于牛奶中四环素残留的测定[J].乳业科学与技术,2004,27(2):52-54.
[2]赵先晟,赵伯川,魏学良.牛奶中抗生素残留的危害及控制对策[J].中国动物保健,2009(5):63-65.
[3]王宏博,高雅琴,杜天庆,等.牛奶中抗生素残留的危害及检测方法的研究进展[J].畜牧与饲料科学,2010,31(4):158-160.
[4]丁洁,陈晓蔚,曾理,等.牛奶中抗生素残留两种检测方法的论证和应用[J].中国卫生检验杂志,2004,14(5):639-640.
[5]沈永聪,李守军,杨林.牛奶中抗生素残留检测技术进展[J].食品科学,2006,27(5):87-88.
[6]朱奎,丁双阳.牛奶中抗生素残留检测技术[J].乳品加工,2008(6):56-59.
[7]章银良,伍季,鲍彤华.牛奶中抗生素残留检测方法的研究现状[J].郑州轻工业学院学报,2006,21(4):21-25.
[8]刘军安,高美虹,黄运丽.牛奶中抗生素残留检测方法的研究进展及展望[J].湖北农业科学,2010,49(6):1481-1483.
[9]余奇飞,陈翠莲,孙莉娜.牛奶中抗生素残留检测方法的研究进展[J].农产品加工:学刊,2005(11):72-75.
[10]张珍,李波清.乳酸菌主要代谢产物及其作用研究进展[J].滨州医学院学报,2012,35(4):274-276.
[11]曾卫军,李杰.孢子型乳酸菌对常用抗生素耐药性及最低抑菌浓度测定[J].湖北畜牧兽医,2012(3):18-20.
[12]杨吉霞,陈芝兰,贺稚非,等.牦牛奶酪中乳酸菌对抗生素的药敏性[J].食品科学,2014,35(17):176-180.doi:10.7506/spkx1002-6630-201417034.
[13]左瑞雨,常娟,郑秋红,等.不同抗生素对乳酸菌抑制作用的研究[J].中国畜牧兽医,2011,38(8):39-42.
[14]高寅生,庞广昌,李家鹏.基于乙醇氧化酶型酶膜的生物传感器的研制和应用[J].食品与发酵工业,2009,35(5):105-109.
[15]GOMBOTZ W R,WEE S F.Protein release from alginate matrices[J].Advanced Drug Delivery Rcviews,1998,31:267-285.
[16]ZACTITI E M,KIECKBUSCH T G.Release of potassium sorbate from active films of sodium alginate crosslinked with calcium chloride[J].Packaging Technology and Science,2009,22(6):349-358.
[17]REISS M,HEIBGES A,METZGER J,et al.Determination of BODvalues of starch-containing waste water by a BOD-biosensor[J].Biosensors and Bioelectronics,1998,13:1083-1090.
[18]BIERHALZ A C K,da SILVA M A,BRAGA M E M,et al.Effect of calcium and/or barium crosslinking on the physical and antimicrobial properties of natamycin-loaded alginate films[J].LWT-Food Science and Technology,2014,57:494-501.
[19]SHI K,SHIU K K.Scanning tunneling microscopic and voltammetric s t u d i e s o f t h e s u r f a c e s t r u c t u r e s o f a n e l e c t r o c h e m i c a l l y activated glassy carbon electrode[J].Analytical Chemistry,2002,74(4):879-885.
[20]NAGAOKA T,YOSHINO T.Surface properties of electrochemically pretreated glassy carbon[J].Analytical Chemistry,1986,58(6):1037-1042.
[21]王庆忠,蒲彪.乳品中抗生素的检测方法[J].食品工业科技,2003,24(11):79-82.
[22]张增福.基于微型表面等离子共振系统的牛奶中抗生素残留检测研究[D].天津:天津大学,2009.
[23]BERTRAM G K,ANGELIKA S,DIETRICH R,et al.Automated microarray system for the simultaneous detection of antibiotics in milk[J].Analytical Chemistry,2004,76(3):646-654.
[24]邓斌,邓春来,张曦.高效液相色谱法检测牛奶中青霉素残留的研究进展[J].中国兽药杂志,2003,37(6):36-40.
[25]韩洪莉,高长玲.牛奶中抗生素残留来源及几种常用检测方法[J].安全监测,2009(8):66-68.
[26]SCHNAPPINGER P,SCHNEIDER E,MARTLBAUE E,et al.Rapid detection of streptomycin and dihydrostreptomycin in milk by enzymelinked immunofiltration assay[J].Food and Agricultural Immunology,1996,8(4):269-272.
[27]秦燕,陈毓芳,林峰,等.固相萃取高效液相色谱法同时测定牛奶中的多种四环素残留[J].食品科学,2005,26(3):202-204.
[28]张素霞,李俊锁,钱传范.牛奶中四环素多残留分析方法研究[J].畜牧兽医学报,2002,33(1):51-54.
[29]李利东,宓晓黎,袁建兴,等.酶联免疫检测试剂盒应用于牛奶中四环素残留的测定[J].乳业科学与技术,2004,27(2):52-54.