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  

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

作　　者：赵爱飞 黄旭镇 叶晓锋[1] 朱军莉[1] 励建荣[2] 

机构地区：[1]浙江工商大学食品与生物工程学院,浙江杭州310018 [2]辽宁省食品安全重点实验室渤海大学,辽宁锦州121013

出　　处：《微生物学通报》2016年第2期343-350,共8页Microbiology China

基　　金：国家自然科学基金项目(No.31271954);浙江省自然科学基金项目(No.LY15C200001);浙江工商大学研究生创新项目(No.3100XJ1514133)~~

摘　　要：【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 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.

关 键 词：群体感应 二酮哌嗪信号分子 气相色谱-质谱联用 腐败菌 

分 类 号：TS254.7[轻工技术与工程—水产品加工及贮藏工程] O657.63[轻工技术与工程—食品科学与工程]