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【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。
【Objective】 To establish a simple and sensitive GC-MS method for the quantitative analysis of diketopiperazine (DKPs), a novel signaling molecule induced by the spoilage population of aquatic products. 【Method】 Quantitative determination methods of Cyclo- (L-Pro-L-Gly), Cyclo- (L-Pro-L-Leu) and Cyclo- (L) were established by optimizing the conditions of gas chromatography and mass spectrometry, (Leu-L-Leu) and Cyclo- (L-Pro-L-Phe), and the detection of the spoilage bacteria Pseudomonas fluorescens and Shewanella Shewanella baltica). 【Result】 The results showed that the four DKPs showed good linearities in the range of 1-200 mg / L with detection limits of 0.06, 0.10, 0.06 and 0.04 mg / L, with limits of quantitation of 0.16, 0.18, 0.14 and 0.12 mg / L, respectively. The recoveries were 51.8% -88.5%, standard deviation of 1.4% -8.3%. LB medium as a bacterial culture medium, chloroform as an extraction solvent, the detection of higher DKPs content. DKPs activity was detected in both spoilage aquatic products, with the major species being Cyclo- (L-Pro-L-Leu) and Cyclo- (L-Pro-L-Phe). With the growth of both bacteria, the DKPs content in the culture supernatant increased significantly and reached the highest at 12 h. 【Conclusion】 A GC-MS method for the determination of bacterial DKPs was established. It has high precision and accuracy, and can accurately and quantitatively analyze the content of four kinds of DKPs. Which laid the foundation for exploring the regulatory mechanism of DKPs in particular spoilage organisms of aquatic products.