高效液相色谱仪法检测乳酸链球菌素含量的研究
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摘要
乳酸链球菌素效价检测法有琼脂扩散法、显色法和酶联免疫吸附法等,前两种方法检测周期长,操作过程复杂,结果误差大;酶联免疫吸附法灵敏度高,检测准确快速,但前期需制备免疫原、多克隆抗体,周期长,成本高。为提高检测效率,节约成本,本实验建立了高效液相色谱仪法快速检测乳酸链球菌素含量(效价)的方法。本法在乳酸链球菌素质量浓度1～20mg/mL范围内线性关系良好,其回归方程为Y=340.118973x+35.202113,相关系数为r~2=0.99973,检出限为0.5mg/mL,样品回收率达90%以上。本实验建立的分析方法简便快捷、具有较好的灵敏度和可靠性,可用于乳酸链球菌素的定量分析。
The titer detectionmethod of nisin included agar diffusion method, coloration method, enzyme-linked immunosorbent assay and others. The detecting period of former two methods was long, and operation process was complex, andresult error wasbig;The sensitivity of enzyme-linked immunosorbent assay(ELISA) was high, and detection was accurate and rapid, but immunogen and polyclonal antibody needed to be prepared at prophase, and period was long, and cost was high. In order to improve detection efficiency and save cost, the method of rapid detection of content of nisin(titer) by high performance liquid chromatography was established. The linear relationship of this method waswell in the nisin mass concentration range of 1-20 mg/mL, and regression equation was Y = 340.118973 x +35.202113, and correlation coefficient was r~2 = 0.99973, anddetection limit was 0.5 mg/mL, andrecovery rate of samplewasmore than 90%.The analysis method established was simple and rapid, and it hadbetter sensitivity and reliability, and it could be applied in quantitative analysis of nisin.
The titer detectionmethod of nisin included agar diffusion method, coloration method, enzyme-linked immunosorbent assay and others. The detecting period of former two methods was long, and operation process was complex, andresult error wasbig;The sensitivity of enzyme-linked immunosorbent assay(ELISA) was high, and detection was accurate and rapid, but immunogen and polyclonal antibody needed to be prepared at prophase, and period was long, and cost was high. In order to improve detection efficiency and save cost, the method of rapid detection of content of nisin(titer) by high performance liquid chromatography was established. The linear relationship of this method waswell in the nisin mass concentration range of 1-20 mg/mL, and regression equation was Y = 340.118973 x +35.202113, and correlation coefficient was r~2 = 0.99973, anddetection limit was 0.5 mg/mL, andrecovery rate of samplewasmore than 90%.The analysis method established was simple and rapid, and it hadbetter sensitivity and reliability, and it could be applied in quantitative analysis of nisin.
引文
1 黄现青,张建威,高晓平等.乳酸链球菌素检测及其在肉品中的应用[J].肉类工业,2011,359(3):38-40
2 罗晶.Nisin抑菌稳定性研究与应用[D].天津:天津科技大学硕士学位论文,2015.
3 MARIA C,PILAR R,CARMEN M M.Combined effects of nisin,lactic acid and modified atmosphere packaging on the survival of Listeria monocytogenes in raw ground pork[J].International Journal of Food Science and Technology,2007,(42):562-566
4 RESA C P O,GERSCHENSON L N,JAGUS R J.Natamycin and nisin supported on starch edible films for controlling mixed culture growth on model systems and Port Salut cheese[J].Food Control,2014,(44):146-151
5 GHARSALLAOUI A,OULAHAL N,JOLY C,et al.Nisin as a food preservative:part 1:physicochemical properties,antimicrobial activity and main uses[J].Critical Reviews in Food Science and Nutrition,2016,(56):1262-1274
6 刁静静.肉类保藏技术(十二)生物防腐剂在肉品保鲜中的应用(一)——乳酸链球菌素和纳他霉素[J].肉类研究,2009,12(3):64-69
7 BALCIUNAS E M,MARTINEZ F A C,TODOROV S D,et al.Novel biotechnological applications of bacteriocins:a review[J].Food Control,2013,(32):134-142
8 高彩红,宋杰,崔倩等.高效液相色谱法检测食品中防腐剂乳酸链球菌素[J].农业科技与装备,2011,203(5):26-28
9 徐俊进,金陈斌,陈小龙.新型绿色防腐剂乳酸链球菌素[J].浙江农业科学,2017,58(6):1005-1007
10 姜旭德,张春华.乳酸菌素及其乳酸链球菌素在食品中的应用[J].中国乳业,2017,12(2):60-62
11 周莉,刘明春.乳酸链球菌素-天然抗菌素[J].肉类研究,2008,12(9):75-77
12 GB2760-2010食品添加剂使用卫生标准[S].
13 张倩.提高乳酸链球菌素(Nisin)热稳定性的研究[D].天津:天津科技大学硕士学位论文,2016.
14 J.Tramer,GG.Fowler.Estimation of Nisin in foods[J].Science and Food Agriculture,1964,15(8):522-528
15 洪超群.浅述国内外乳酸链球菌素(Nisin)的效价检测方法异同[J].食品安全导刊,2015,12(6):91-93
16 刘珈伶,廖强,蒋定之等.鲜湿米粉中两种乳酸链球菌素的HPLC-MS/MS检测方法[J].分析测试学报,2018,37(7):820-824
17 黄明发,刘君.乳酸链球菌素在肉制品中的应用[J].肉类研究,,2006,12(9):39-42
18 李克柱.天然食品防腐剂乳酸链球菌素(Nisin)在低温肉类食品中的应用[J].肉类研究,2005,12(9):31-34
19 孟茹,赵贵明,贾瑜等.酶联免疫法检测食品中乳酸链球菌素[J].食品与生物技术学报,2013,32(5):509-515
20 王茜.高效液相色谱的应用与展望[J].食品安全导刊,2018,36(27):186
21 丁峰.高效液相色谱(HPLC)法在食品检验中的应用[J].科技与创新,2017,24(24):148-149
22 郑伟,孙东方.高效液相色谱柱的选择、使用与维护[J].口腔护理用品工业,2011,21(6):37-39
23 崔莹.HLPC色谱柱的使用和维护[J].科技风,2011,36(16):88
24 宋学英,杨华.色谱柱的选择及应用[J].分析仪器,2016,06(6):84-88
25 全红娜,金松子,雷勇胜等.反相高效液相色谱中流动相选择与优化的研究进展[J].现代药物与临床,2014,29(10):1190-1194
26 STEVENS K A,SHELDON B W,KLAPES N A,et al.Nisin treatment for inactivation of Salmonella species and other gram-negative bacteria[J].Appl Environ Mrcrobiol,1991,57(12):3613-3615
27 MASSCHALCK B,DECKERS D,MICHIELS C W.Sensitization of outer-membrane mutants of Salmonella typhimurium and Pseudomonas aeruginosa to antimicrobial peptides under highpressure[J].J Food Prot,2003,66(8):1360-1367
28 云雪霞,胡静,陈清.乳酸链球菌素对产肠毒素大肠杆菌的抑菌作用观察[J].中国消毒学杂志,2006,23(6):529-531
29 赵源慧,王艳萍.液相色谱柱的修护[J].科技与企业,2015,24(10):182
30 孟凡超,刘兵兵,胡朋举等.高效液相色谱柱的使用与维护[J].轻工科技,2018,12(12):93-94
31 张子杰.新型乳酸链球菌素抗菌体系的构建与性能研究[D].北京:北京化工大学硕士学位论文,2014.
32 杜琨.食品成分和环境对乳酸链球菌素抑菌活性的影响[J].粮食与饲料工业,2018,12(3):28-30
33 熊玲,张立彦,刘启莲.不同阴离子对乳酸链球菌素抑菌稳定性的影响研究[J].食品工业科技,2012,33(23):306-308,313
34 GB1886.231-2016食品安全国家标准食品添加剂乳酸链球菌素[S].
35 李健武,余瑞元,袁明秀等.生物化学实验原理和方法[M].北京:北京大学出版社,2000.
36 Rongguang Yang,MontyC Johnson,et al.Novel method extract amounts of bacteriocins from Lacticacid bacteria[J].Appl EnvironMicrobiol,1999,58(10):3355-3359
37 李红,赵春燕.乳酸链球菌素的研究进展[J].食品科技,2006,12(1):75-78
2 罗晶.Nisin抑菌稳定性研究与应用[D].天津:天津科技大学硕士学位论文,2015.
3 MARIA C,PILAR R,CARMEN M M.Combined effects of nisin,lactic acid and modified atmosphere packaging on the survival of Listeria monocytogenes in raw ground pork[J].International Journal of Food Science and Technology,2007,(42):562-566
4 RESA C P O,GERSCHENSON L N,JAGUS R J.Natamycin and nisin supported on starch edible films for controlling mixed culture growth on model systems and Port Salut cheese[J].Food Control,2014,(44):146-151
5 GHARSALLAOUI A,OULAHAL N,JOLY C,et al.Nisin as a food preservative:part 1:physicochemical properties,antimicrobial activity and main uses[J].Critical Reviews in Food Science and Nutrition,2016,(56):1262-1274
6 刁静静.肉类保藏技术(十二)生物防腐剂在肉品保鲜中的应用(一)——乳酸链球菌素和纳他霉素[J].肉类研究,2009,12(3):64-69
7 BALCIUNAS E M,MARTINEZ F A C,TODOROV S D,et al.Novel biotechnological applications of bacteriocins:a review[J].Food Control,2013,(32):134-142
8 高彩红,宋杰,崔倩等.高效液相色谱法检测食品中防腐剂乳酸链球菌素[J].农业科技与装备,2011,203(5):26-28
9 徐俊进,金陈斌,陈小龙.新型绿色防腐剂乳酸链球菌素[J].浙江农业科学,2017,58(6):1005-1007
10 姜旭德,张春华.乳酸菌素及其乳酸链球菌素在食品中的应用[J].中国乳业,2017,12(2):60-62
11 周莉,刘明春.乳酸链球菌素-天然抗菌素[J].肉类研究,2008,12(9):75-77
12 GB2760-2010食品添加剂使用卫生标准[S].
13 张倩.提高乳酸链球菌素(Nisin)热稳定性的研究[D].天津:天津科技大学硕士学位论文,2016.
14 J.Tramer,GG.Fowler.Estimation of Nisin in foods[J].Science and Food Agriculture,1964,15(8):522-528
15 洪超群.浅述国内外乳酸链球菌素(Nisin)的效价检测方法异同[J].食品安全导刊,2015,12(6):91-93
16 刘珈伶,廖强,蒋定之等.鲜湿米粉中两种乳酸链球菌素的HPLC-MS/MS检测方法[J].分析测试学报,2018,37(7):820-824
17 黄明发,刘君.乳酸链球菌素在肉制品中的应用[J].肉类研究,,2006,12(9):39-42
18 李克柱.天然食品防腐剂乳酸链球菌素(Nisin)在低温肉类食品中的应用[J].肉类研究,2005,12(9):31-34
19 孟茹,赵贵明,贾瑜等.酶联免疫法检测食品中乳酸链球菌素[J].食品与生物技术学报,2013,32(5):509-515
20 王茜.高效液相色谱的应用与展望[J].食品安全导刊,2018,36(27):186
21 丁峰.高效液相色谱(HPLC)法在食品检验中的应用[J].科技与创新,2017,24(24):148-149
22 郑伟,孙东方.高效液相色谱柱的选择、使用与维护[J].口腔护理用品工业,2011,21(6):37-39
23 崔莹.HLPC色谱柱的使用和维护[J].科技风,2011,36(16):88
24 宋学英,杨华.色谱柱的选择及应用[J].分析仪器,2016,06(6):84-88
25 全红娜,金松子,雷勇胜等.反相高效液相色谱中流动相选择与优化的研究进展[J].现代药物与临床,2014,29(10):1190-1194
26 STEVENS K A,SHELDON B W,KLAPES N A,et al.Nisin treatment for inactivation of Salmonella species and other gram-negative bacteria[J].Appl Environ Mrcrobiol,1991,57(12):3613-3615
27 MASSCHALCK B,DECKERS D,MICHIELS C W.Sensitization of outer-membrane mutants of Salmonella typhimurium and Pseudomonas aeruginosa to antimicrobial peptides under highpressure[J].J Food Prot,2003,66(8):1360-1367
28 云雪霞,胡静,陈清.乳酸链球菌素对产肠毒素大肠杆菌的抑菌作用观察[J].中国消毒学杂志,2006,23(6):529-531
29 赵源慧,王艳萍.液相色谱柱的修护[J].科技与企业,2015,24(10):182
30 孟凡超,刘兵兵,胡朋举等.高效液相色谱柱的使用与维护[J].轻工科技,2018,12(12):93-94
31 张子杰.新型乳酸链球菌素抗菌体系的构建与性能研究[D].北京:北京化工大学硕士学位论文,2014.
32 杜琨.食品成分和环境对乳酸链球菌素抑菌活性的影响[J].粮食与饲料工业,2018,12(3):28-30
33 熊玲,张立彦,刘启莲.不同阴离子对乳酸链球菌素抑菌稳定性的影响研究[J].食品工业科技,2012,33(23):306-308,313
34 GB1886.231-2016食品安全国家标准食品添加剂乳酸链球菌素[S].
35 李健武,余瑞元,袁明秀等.生物化学实验原理和方法[M].北京:北京大学出版社,2000.
36 Rongguang Yang,MontyC Johnson,et al.Novel method extract amounts of bacteriocins from Lacticacid bacteria[J].Appl EnvironMicrobiol,1999,58(10):3355-3359
37 李红,赵春燕.乳酸链球菌素的研究进展[J].食品科技,2006,12(1):75-78