UPLC-MS/MS法测定广叶绣球菌不同生长阶段栽培基质中有机酸含量及其对菌丝生长的影响

Determination of organic acids in culture substrate of Sparassis latifolia at different growth stages by UPLC-MS/MS method and its effect on mycelia growth

为探究绣球菌生长发育过程中有机酸的代谢情况,采用超高效液相色谱串联质谱法(UPLC-MS/MS),以负离子多反应监测(MRM)模式测定广叶绣球菌栽培基质未接种阶段(0 d)、菌丝阶段(40 d)、原基阶段(60 d)及子实体生长阶段(80 d)26种有机酸的含量,并研究主要有机酸对菌丝生物量和菌丝生长速度的影响。结果表明:各有机酸在其线性范围内与峰面积呈良好的线性关系(r2≥0.99),检出限在2.0~100.0 ng/mL;有13种有机酸在各阶段均能检测到,其中苹果酸、L-焦谷氨酸、柠檬酸、D-葡萄糖醛酸和酒石酸变化趋势明显,浓度变化分别为113.222~13.826μg/g、41.349~1.418μg/g、16.821~46.399μg/g、15.653~96.850μg/g和4.125~66.550μg/g;酒石酸和L-焦谷氨酸在试验浓度0.02~0.20 g/L范围内均对菌丝生长有显著促进作用,柠檬酸和苹果酸则在添加浓度较高时对菌丝生长促进作用显著。研究有机酸在栽培过程中的变化趋势有利于探究绣球菌基质降解机理,同时也可将其作为安全的营养添加剂用于配方优化。

In order to investigate the metabolism of organic acids during the growth and development of Sparassis latifolia,the content of 26 organic acids in the cultivation substrate of Sparassis crispa during the uninoculated stage(0 d),mycelium stage(40 d),primordium stage(60 d),and fruiting body growth stage(80 d)were determined by by ultra high performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)and negative ion multiple reaction monitoring(MRM)mode.The effects of organic acids on mycelial biomass and mycelial growth were analyzed.The results showed that the chemical constituents fitted linear relationship well with the peak area in the linear range(r2≥0.99),and the detection limit was between 2.0~100.0 ng/mL.13 organic acids could be detected at all stages of Sparassis crispa culturation.Among them,five organic acids,namely malic acid,L-pyroglutamic acid,citric acid,D-glucuronic acid,and tartaric acid,exhibited notable changes in their concentrations,with their initial-to-final concentration ranges being 113.222~13.826μg/g,41.349~1.418μg/g,16.821~46.399μg/g,15.653~96.850μg/g and 4.125~66.550μg/g,respectively.Tartaric acid and L-pyroglutamic acid can significantly promote the growth of mycelia in the range of 0.02~0.20 g/L,while citric acid and malic acid can promote the growth of mycelia in higher concentration.Studying the variation trend of organic acids in cultivation process is conducive to exploring the substrate degradation mechanism of the matrix of Sparassis latifolia,and can also be used as a safe nutrient additive for formula optimization.