Microsoft Word _编辑二校出3066

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1 7 8 Vol. 7 No Journal of Food Safety and Quality Aug., 2016 庞世琦, 刘青 *, 刘朝霞, 李志勇, 张嘉俊 (,, ) 摘要 : 目的方法 5%, A (50:0.2:49.8, V:V:V) B (1:0.2:98.8, V:V:V),,,,, 结果 mg/l 93.5%~99.2%, 1.62~5.74%(n=6), 50 mg/l 结论,, 关键词 : ; ; Determination of lysozyme in wines by high performance liquid chromatography PANG Shi-Qi, LIU Qing *, LIU Zhao-Xia, LI Zhi-Yong, ZHANG Jia-Jun (Inspection and Quarantine Technology Center of Guangdong Entry-Exit Inspection and Quarantine Bureau; Guangdong Key Laboratory of Import and Export Technical Measures of Animal, Plant and Food, Guangzhou ,China) ABSTRACT: Objective To establish a method for the determination of lysozyme content in wines by high performance liquid chromatography (HPLC). Methods Wine samples were acidified by 5% hydrochloric acid solution. The gradient elution solvents were composed of the mobile phase A acetonitrile: trifluoroacetic acid: water (50:0.2:49.8, V:V:V) and the mobile phase B acetonitrile: trifluoroacetic acid: water(1:0.2:98.8, V:V:V). The samples were separated through a column of reverse phase with phenyl functional groups and determined by HPLC with fluorescence detector (FLD) or ultraviolet detector (UV). The results were qualitative by retention time and quantified by external standard method. Meanwhile, the ratio of mobile phase, gradient elution program and acidification conditions were optimized. Results The recoveries of lysozyme spiked at the levels of 50, 200, 500 and 750 mg/l were ranged from 93.5% to 99.2% with the relative standard deviations (s) of 1.62%~5.74% (n=6), and the limit of quantitation (LOQ) of lysozyme was 50 mg/l. Conclusion The method has high efficiency on separation and good reproducibility with the measured results confirming to the results of enzyme-linked immunosorbent assay (ELISA), which is also accurate, reliable and sensitive, and can meet the requirements for the detection of lysozyme in wines. 基金项目 : (2015GDK36) (2013B ) (2014A ) Fund: Supported by Guangdong Inspection and Quarantine Project (2015GDK36), Guangdong Provincial Department of Science and Technology International Cooperation Project (2013B ) and Guangdong Provincial Department of Science and Technology Public Welfare Project (2014A ) * 通讯作者 :,,, gdciqlq@163.com. *Corresponding author: LIU-Qing, Master, Guangdong Entry-Exit Inspection and Quarantine Bureau, No.66, Huacheng Road, Tianhe District, Guangzhou , China. gdciqlq@163.com

2 8, : 3067 KEY WORDS: lysozyme; high performance liquid chromatography; wine 1 引言, N-,, [1],, [2,3],, [4-7],,,, [8],,, [9] [10],, GB [11] , 500 mg/kg,,,, [12-17],, ph,,,,,, 2 材料与方法 2.1 样品 2.2 仪器与试剂 E2695 (, Waters ); Promax 2020 ( Heidolph ); KQ-500E ( ); VORTEX 4 digital ( IKA ); ( 0.1 mg, ) (CAS: , Sigma ) (, Fisher ); (, ); 0.22 μm ( ); Milli-Q 2.3 实验方法标准溶液的配制 :,,, 2 g/l, 0~4 :,, mg/l, 样品前处理 10.0 ml ( 100 ml,, ) 100 ml, 5% 5 min, 0.22 μm, 液相色谱条件 : Toso Bioscience (TSK-get Pheny1 5PW RP), 7.5 cm 4.6 mm; : A: (50:0.2:49.8, V:V:V), B: (1:0.2:98.8, V:V:V); : 0.8 ml/min; : 50 μl; : 30 ; : : 280 nm, : (λ ex ): 276 nm, (λ em ): 345 nm; : 0~3 min, 100%B; 3~10 min, 100%~70%B; 10~15 min, 70%~60%B; 15~20 min, 60%~50%B; 20~25 min, 50%~0%B; 25~34 min, 0%B; 34~36 min, 0%~100%B; 36~40 min, 100%B 液相色谱测定,,,,,, 3 结果与分析 3.1 前处理条件优化样品酸化条件优化,

3 ph, HCl ph, HCl, 5% 10% 15% 20% HCl, 1, 5%HCl, HCl 5% 标准溶液溶剂的选择 [18], (PBS: 0.27 g 1.42 g 8.0 g 0.2 g 1000 ml, 1:1 ph 7.4, 1000 ml ) 0.9%NaCl 1 g/l 250 mg/l,,,,, 3.2 色谱条件优化检测器波长的选择 :, (λ ex ): 276 nm, (λ em ): 345 nm, : 200~800 nm, 280 nm, 2 Fig. 1 1 HCl Comparison of the chromatograms of the same spiked sample under different concentrations of HCl Fig. 2 2 The ultraviolet scan image of the standard solution of lysozyme

4 8, : 流速的选择,,, ml/min, 0.8 ml/min 柱温的选择 [19], 30~ , 17.6 mg/l 250 mg/l,, 30, 洗脱程序的选择, 80%,, 10%~13%, 1000, 300,,, : 0~3 min, 100%B; 3~10 min, 100%~65%B; 10~15 min, 65%B; 15~27 min, 65%~40.5%B; 27~29 min, 40.5%~0%B; 29~34 min, 0%B; 34~36 min, 0%~100%B; 36~40 min, 100%B 30 min,,,, : 0~3 min, 100%B; 3~10 min, 100%~50%B; 10~20 min, 50%~0%B; 20~25 min, 0%B; 25~30 min, 0%~100%B, 15 min,,,,, : 0~3 min, 100%B; 3~10 min, 100%~70%B; 10~15 min, 70%~60%B; 15~20 min, 60%~ 50%B; 20~25 min, 50%~0%B; 25~34 min, 0%B; 34~36 min, 0%~100%B; 36~40 min, 100%B 24 min,,,, 光化学衍生对信号的影响,, Toso Bioscience (TSK-get Pheny1 5PW RP) 7.5 cm 4.6 mm,,, 3 4,, 3.3 方法线性范围及定量限 5.0~60.0 mg/l, 0.99,, 50.0 mg/l Table 1 表 1 方法的回归方程相关系数 The regression equation and correlation coefficients of the method Y=7.32e+004X-8.53e Y=7.94e+002X+7.48e Fig. 3 3 (UV ) The chromatogram of the standard solution (UV detector)

5 回收率及精密度实验 ( mg/l), 2 6, 93.5%~99.2%, 1.62%~5.74%,, 60%,, 2~ 4 Fig. 4 4 (FLD ) The chromatogram of the standard solution (FLD detector) 表 2 Table 2 红葡萄酒中的溶菌酶回收率和相对标准偏差 (n=6) Recoveries and s of lysozyme in red wine (n=6) 100 ND ND ND ND 表 3 Table 3 白葡萄酒中的溶菌酶回收率和相对标准偏差 (n=6) Recoveries and s of lysozyme in white wine (n=6) 50 ND ND ND ND

6 8, : 3071 表 4 Table 4 起泡葡萄酒中的溶菌酶回收率和相对标准偏差 (n=6) Recoveries and s of lysozyme in sparkling wine (n=6) 50 ND ND ND ND 实际样品测定 ,,, 4 结论, (International Organization of Vine and Wine, OIV),,,,, 参考文献 [1],,,. [J]., 2010, 28(1): Zhou GC, Liu QF, Lu Y, et al. Stability studies of lysozyme for preformulation [J]. J Pharm Pract, 2010, 28(1): [2],. [J]., 2008, 24(1): Zhao HY, Wang AP. Application of lysozyme in the food industry [J]. Occup Health, 2008, 24(1): [3],. [J]., 2003, 24(2): Ni Y, Zhong LR. Natural preservatives the study of lysozyme [J]. Food Res Dev, 2003, 24(2): [4],. [J]., 2003, (5): Ni Y, Zhong LR. Application of lysozyme in the wine production [J] Sino-Overseas Grapevine and Wine, 2003, (5): [5] Delfini C, Cersosimo M, Prete VD, et al. Resistance screening essay of wine lactic acid bacteria on lysozyme: efficacy of lysozyme in unclarified grape musts [J]. J Agric Food Chem, 2004, 52(7): [6] Marchal R, Chaboche D, Douillard R, et al. Influence lysozyme treatments on champagne base wine foaming properties [J]. J Agric Food Chem, 2002, (50): [7],,,. [J]., 2006, 22(1): Xu JY, Xu YP, Liu S, et al. Research advances in egg bioactive component-lysozyme and its applications in food industry [J]. Food Mach, 2006, 22(1): [8] The practical application of directive 2003/89/EC on ingredient and allergen labelling [S]. [9] [EB/OL]. jiuwenhua-jiuzhishi/63712.h tml Lysozyme of egg white in wine may cause irritation[eb/ol] tv/news/jiuwenhua-jiuzhishi/63712.h tml [10] [EB/OL] Introduction to wine add lysozyme induce allergies[eb/ol]. tv/putaojiu/ [11] GB [S]. GB National Food Safety Standards Standards for uses of food additives [S]. [12],,,. [J]., 2011, 39(7): Ji ZP, Wang J, Han J, et al. Detection of lysozyme by flow injection chemiluminescence [J]. Chin J Anal Chem, 2011, 39(7): [13] [EB/OL]. jiuwenhua-jiuzhishi/63712.html [14] [S]. :, Drug standard 6 copies [S]. Beijing: People's Medical Publishing House, [15],,. [J]., 2012, 10(1): Dong WX, Liu SX, Wang T. Research progress of determination method of lysozyme [J]. Central South Pharm, 2012, 10(1): [16],,. [J]., 2011, 8: Li BY, Zhang CM, Zhu XB. Simple measurement for determination of totally sozyme in bee products [J]. Beijing Agric, 2011, 8: [17],,,. [J]., 2010, 35(4):

7 Wang TX, Liu ZH, Xia LY, et al. Determination of total lysozyme in honey with ultraviolet spectrophotometry [J]. Food Sci Technol, 2010, 35(4): [18] International Organisation of Vine and Wine. Compendium of international methods of wine and must analysis [S]. [19],,,. [J]., 2008, 40(12): Zuo RY, Guo JL, Ren GZ, et al. Lateral secretion of spore bacillus and the change rule of lysozyme enzymology characteristics analysis [J]. Anim Husband Vet Med, 2008, 40(12): ( 责任编辑 : 姚菲 ) 作者简介庞世琦, 学士, 工程师, 主要研究方向为食品葡萄酒质量安全检测, 色谱色谱质谱等检测技术应用等 psqciq@163.com 刘青, 硕士, 高级工程师, 主要研究方向为食品葡萄酒质量安全检测, 色谱色谱质谱等检测技术应用等 gdciqlq@163.com

g ml 10% ph 色谱条件 kinetex C μm 100 A 4. 6 mm 150 mm 25 5 μl A B 10 mmol /L ml /min

g ml 10% ph 色谱条件 kinetex C μm 100 A 4. 6 mm 150 mm 25 5 μl A B 10 mmol /L ml /min 25 4 2013 8 监测技术 5 1 2 1 ( 1. 江苏省疾病预防控制中心, 江苏南京 210009; 2. 南京医科大学第二附属医院, 江苏南京 210029) 5 Phenomenex kinetex C 18 2. 6 μm 4. 6 mm 150 mm 10 mmol /L ph 2. 5 + 60 40 DAD 280 nm 5 20. 0 mg /L ~ 100 mg /L 0.

Microsoft Word _编辑 二校出3066

Microsoft Word _编辑二校出3066