高效液相色谱法检测发酵液中四氢嘧啶及羟基四氢嘧啶

doi:10.3969/j.issn.1001-1803.2022.11.013

摘要/Abstract

摘要：

为实现发酵液样品中四氢嘧啶和羟基四氢嘧啶的分离检测，使用高效液相色谱（HPLC）技术建立一种测定中度嗜盐菌发酵液中四氢嘧啶及羟基四氢嘧啶含量的方法。使用乙醇抽提法提取样品，采用Vertex^TM NH₂（4.6×250 mm，5 μm）液相色谱柱对中度嗜盐菌发酵液中四氢嘧啶及四氢嘧啶进行了分析，选用紫外（UV）检测器，对比不同检测条件。通过色谱条件优化，选定了70%乙腈作为流动相，柱温30 ℃，检测波长210 nm，流速0.8 mL/min。发酵液样品中四氢嘧啶和羟基四氢嘧啶在各自范围内线性关系良好，两种样品的平均回收率在99.2%~102%，精密度偏差在1%~2%范围内，检出限为1.5 μg/mL，定量限为5 μg/mL，分离度为1.70。该方法检测范围大，具有较好的重复性、分离度及稳定性，适用于发酵液中四氢嘧啶及羟基四氢嘧啶的分离检测。

关键词: 中度嗜盐菌, 高效液相色谱, 四氢嘧啶, 羟基四氢嘧啶

Abstract:

A method was developed for the determination of ectoine and hydroxyectoine in fermentation broth samples of moderately saline bacteria using high performance liquid chromatography （HPLC）. Samples were extracted using ethanol extraction, and ectoine and hydroxyectoine standards were dissolved and diluted in ultrapure water. Vertex^TM NH₂ （4.6×250 mm, 5 μm） liquid chromatographic column was used to analyse ectoine and hydroxyectoine in the fermentation broth of moderately halophilic bacteria. Waters 1515 high performance liquid chromatograph with ultraviolet （UV） detector was selected to compare the different detection conditions. The chromatographic conditions were optimized by using acetonitrile （70%） as the mobile phase with a column temperature of 30 ℃, a detection wavelength of 210 nm and a flow rate of 0.8 mL/min. The linearity of ectoine and hydroxyectoine in the fermentation broth samples is good in their respective ranges. The average recoveries of the two samples are in the range of 99.2% to 102%, the precision deviations are in the range of 1% to 2%, the limit of detection is 1.5 μg/mL, the limit of quantification is 5 μg/mL and the separation degree is 1.70. The method is suitable for the separation and determination of ectoine and hydroxyectoine in fermentation broth and for the quantitative analysis of ectoine and hydroxyectoine under laboratory conditions.

Key words: moderately halophile, HPLC, ectoine, hydroxyectoine

中图分类号:

TQ658

刘紫寒,郭秋爽,周超,孙杨,李华,刘宇鹏. 高效液相色谱法检测发酵液中四氢嘧啶及羟基四氢嘧啶[J]. 日用化学工业（中英文）, 2022, 52(11): 1236-1240.

Liu Zihan,Guo Qiushuang,Zhou Chao,Sun Yang,Li Hua,Liu Yupeng. Determination of ectoine and hydroxyectoine in fermentation broth by high performance liquid chromatography[J]. China Surfactant Detergent & Cosmetics, 2022, 52(11): 1236-1240.

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参考文献 14

[1]	Chen Weichuan, Hsu Ching Cha, Lan John Chi Wei, et al. Production and characterization of ectoine using a moderately halophilic strain Halomonas salina BCRC17875[J]. Journal of Bioscience and Bioengineering, 2017, 125 (5) : 578-584. doi: 10.1016/j.jbiosc.2017.12.011
[2]	Liu Mengshuang, Liu Hui, Shi Meng, et al. Microbial production of ectoine and hydroxyectoine as high-value chemicals[J]. Microbial Cell Factories, 2021, 20 (1) : 76. doi: 10.1186/s12934-021-01567-6 pmid: 33771157
[3]	Harishchandra R K, Sachan A K, Kerth A, et al. Compatible solutes: ectoine and hydroxyectoine improve functional nanostructures in artificial lung surfactants[J]. Biochimica et Biophysica Acta (BBA) -Biomembranes, 2011, 1808 (12) : 2830-2840. doi: 10.1016/j.bbamem.2011.08.022
[4]	Zhang Xin, Liu Jing, Zhu Derui. Advances in bioengineering and medical application of natural products ectoine and hydroxyectoine[J]. Natural Products Research and Development, 2017, 29 (5) : 882-887.
[5]	Joachim B, Joachim D, Hansjürgen D. The protective function of compatible solute ectoine on the sking, skin cells and its biomolecules with respect to UV-radiation, immunosuppression and membrane damage[J]. IFSSC Magazine, 2001, 4: 359-365.
[6]	Ruediger G, Soheila A, Joachim B, et al. The multifunctional role of ectoine as a natural cell protectant[J]. Clinics in Dermatology, 2008, 26 (4) : 326-333. doi: 10.1016/j.clindermatol.2008.01.002
[7]	José M P, Manuel S, Montserrat A, et al. Ectoines in cell stress protection: Uses and biotechnological production[J]. Biotechnology Advances, 2010, 28 (6) : 782-801. doi: 10.1016/j.biotechadv.2010.06.005 pmid: 20600783
[8]	Buenger J, Driller H. Ectoine: an effective natural substance to prevent UVA-induced premature photoaging[J]. Skin Pharmacology Andphysiology, 2004, 17 (5) : 232-237.
[9]	Pfluecker F, Buenger J, Hitzel S, et al. Complete photo protection: Going beyond visible endpoints[J]. SOFW Journal, 2005, 131: 20-30.
[10]	Li Haiyu, Peng Tong, Bai Linhan. Optimization of UPLC-MS method for determination of ectoine and hydroxyectoine in moderately halophilic bacteria[J]. Journal of Sichuan University (Natural Science Edition), 2016, 53 (4) : 919-924.
[11]	Li Yaodong, Long Qifu, Li Wenjun, et al. Determination of ectoine in halophilic bacteria from Qinghai Lake by high performance liquid chromatography[J]. Environmental Chemistry, 2013, 32 (9) : 1687-1692.
[12]	Alexander S, Reshetnikov, Valentina N, et al. Genes and enzymes of ectoine biosynthesis in Halotolerant methanotrophs[J]. Methods in Enzymology, 2011, 495: 15-30. doi: 10.1016/B978-0-12-386905-0.00002-4 pmid: 21419912
[13]	Zhu Derui, Han Rui, Shen Gouping, et al. Recombinant co-expression of the ectoine biosynthesis gene cluster ectABC in Halomonas from Qinghai Lake[J]. Acta Hydrobiologica Sinica, 2015, 39 (2) : 358-367.
[14]	Anne U K, Erhard B. Osmotically regulated synthesis of the compatible solute ectoine in Bacillus pasteurii and related Bacillus spp.[J]. Appl. Environ. Microbiol., 2002, 68 (2) : 772-783. doi: 10.1128/AEM.68.2.772-783.2002

流速/（mL/min）	分离度R
流速/（mL/min）	60%	65%	70%	75%	80%
0.6	0.63	0.71	1.81	0.55	0.89
0.8	0.44	0.53	1.70	0.43	0.75
1.0	0.24	0.32	1.55	0.29	0.63

组分	检出限/（μg/mL）	线性范围/（g/L）	回归方程	相关系数（R²）
四氢嘧啶	1.50	0.005~1.0	Y=43 653 700X+332 830.157 81	0.999
羟基四氢嘧啶	1.50	0.005~1.0	Y=2.321 000X-135 776.073 1	0.998

样品组分	样品质量浓度/（g/L）	加标量/（g/L）	回收率/%	精密度/%
四氢嘧啶	0.25	0.25	101.60	1.01
四氢嘧啶	0.25	0.75	99.50	1.40
羟基四氢嘧啶	0.10	0.40	99.20	1.61
羟基四氢嘧啶	0.10	0.90	102.0	1.17

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