气相色谱-质谱定量检测水产品腐败菌群体感应DKPs信号分子

【目的】为了分析水产品腐败菌群体感应的新型信号分子二酮哌嗪(DKPs)化合物,建立一种简便、灵敏的气相色谱-质谱(GC-MS)定量检测方法。【方法】通过优化气相色谱和质谱条件、培养基和提取溶剂建立定量检测方法,确定Cyclo-(L-Pro-L-Gly)、Cyclo-(L-Pro-L-Leu)、Cyclo-(L-Leu-L-Leu)和Cyclo-(L-Pro-L-Phe)4种DKPs标准品的特征离子,并检测水产品腐败菌荧光假单胞菌(Pseudomonas fluorescens)和波罗的海希瓦氏菌(Shewanella baltica)中的DKPs。【结果】4种DKPs化合物在1-200 mg/L范围内线性良好,检测限为0.06、0.10、0.06和0.04 mg/L,定量限为0.16、0.18、0.14和0.12 mg/L,回收率为51.8%-88.5%,标准偏差为1.4%-8.3%。以LB培养基为细菌培养基,氯仿作为萃取溶剂,检测的DKPs含量较高。两种水产品腐败菌都检测到DKPs活性,主要种类为Cyclo-(L-Pro-L-Leu)和Cyclo-(L-Pro-L-Phe)。随着两种细菌的生长,培养上清中DKPs含量显著增加,在12 h达到最高。【结论】建立了检测细菌DKPs的GC-MS定量方法,具有较高精密度、准确度,能够准确定量分析4种DKPs的含量。为探究水产品特定腐败菌DKPs的调控机制奠定基础。

Quantitative analysis of diketopiperazines of quorum sensing in spoilage bacteria of aquatic products by gas chromatography/mass spectrometry

Objective] To detect a novel signaling molecule of quorum sensing, diketopiperazines (DKPs), in spoilage bacteria of aquatic products, the simple and sensitive technique of GC-MS method was developed for the quantitative analysis. Methods] By optimizing the conditions of gas chromatography and mass spectrometry, media and extracting solvent, the quantitative measure of diketopiperazines (DKPs) was established. The characteristic ions of four kinds of DKPs standards, cyclo-(L-Pro-L-Gly), cyclo-(L-Pro-L-Leu), cyclo-(L-Leu-L-Leu) and cyclo-(L-Pro-L-Phe), was ensured. The DKPs activity in two spoilage bacterial of aquatic product, Pseudomonas fluorescens and Shewanella baltica, were detected. Results] Four DKPs had good linear at range of 1?200 mg/L. The limits of detection were 0.06, 0.10, 0.06 and 0.04 mg/L, respectively. The limits of quantification were 0.16, 0.18, 0.14 and 0.12 mg/L, respectively. The recovery of four DKPs in the medium was among 51.8%?88.5% with the RSDs of 1.4%?8.3%. The higher level of DKPs was detected with chloroform served as extracting solvent and LB used as medium. Two kinds of DKPs, including cyclo-(L-Pro-L-Leu) and cyclo-(L-Pro-L-Phe), were found in P. fluorescens and S. baltica. The level of DKPs in two spoilage bacteria increased with the bacterial growth, and arrived to the highest activity for 12 h of culture. Conclusion] The technique of GC-MS method developed could analyze quantitatively the levels of four kinds DKPs in spoilage bacteria, with high precision and accuracy.