RP-HPLC测定保加利亚乳杆菌腺苷酸方法的建立与应用
详细信息Determination of adenosine phosphates in L.bulgaricus by RP-HPLC method and its application
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摘要: 建立适用于测定保加利亚乳杆菌腺苷酸的反相高效液相色谱方法,为评价代谢组学研究中淬灭液的淬灭效果提供方法支持。采用SinoChrom ODS-BPC18色谱柱,甲醇-30mmol/L磷酸钾（V/V=5∶95）缓冲液（pH7.0）作为流动相,流速0.7mL/min,柱温25℃,紫外检测波长254nm。结果表明该方法具有较好的线性和较高的精密度,回收率在94.12%～109.6%之间。利用该方法测定了使用最广泛的60%（v/v）冷甲醇/水溶液淬灭保加利亚乳杆菌的效果,结果发现ATP的泄露率高达70.43%±2.3%,能荷值是0.42±0.015,从而推断该淬灭方案不但会造成胞内代谢物的大量泄漏,而且菌体的代谢反应也没有完全停止,故该淬灭方案不适合保加利亚乳杆菌。Abstract: To evaluate the quenching effect of the quencheing solutions in the metabolomics research, a RP- HPLC method was developed for the determination of adenosine phosphates in L.bulgaricus. The chromategrap-hic separation was achieved on an SinoChrom ODS- BP C 18 column and a mobile phase of methanol30mmol /L phosphate ( V/V = 5 ∶ 95 ) buffer solution ( pH7 ) with a flow rate of 0.7mL /min. The column temperature and UV detection wavelength were set as 25℃ and 254nm, respectively. The method had the good linearity and higher accuracy.The recovery rates of adenosine phosphates were in the range of 94.12% ～ 109.6% .The intracellular metabolite leakages of L.bulgaricus was evaluated by the RP- HPLC method, when 60% ( V/V) methanol /water at -40℃ that was the most popular method to quench the metabolism.The results showed that the leak rate of ATP was as high as 70.43% ± 2.3% , EC value was 0.42 ± 0.015. It was inferred that the quenching fluid could not only cause severe leakage of intracellular metabolites but also not completetly quench the metabolism .The quenching procotol was not suitable for quenches L.bulgaricus.
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