基于微孔板比色法的微生物胞内油脂快速检测方法的建立及应用

摘要

To achieve a rapid and accurate determination method for single-cell lipid,sulfo-phospho-vanillin (SPV)reaction conditions were optimized and the application of SPV reaction in determination of microbial intracellular lipid was discussed.The optimal SPV reaction conditions were that oil samples were added with 2 mL of 98％ H2SO4 keeping in 90 ℃ water bath for 20 min,after they were cooled to room temperature,3 mL of vanillin-phosphoric acid solution(prepared with 34％ phosphoric acid solution)was added for color development and absorbance measurement was made at 530 nm after 20 min of color reaction to eliminate the non-oil components interference.Under the optimal conditions,lipid contents of Schizochytrium sp.and Crypthecodinium cohnii were detected,and there was no significant difference in lipid determination comparing with macro-gravimetric method.Thus,SPV reaction was proved to be a simple,accurate,rapid method for detecting microbial intracellular lipid which could be used as a routine analytical method for the determination of single-cell lipid.%对磷酸香草醛显色法的反应条件进行优化研究,建立了磷酸香草醛显色法快速检测微生物胞内油脂的方法,并探讨该方法在微生物油脂测定上的应用.经过实验确定最佳比色条件为:将油脂样品蒸干溶剂后加入2 mL 98％硫酸,于90℃水浴20 min,冷却至室温后添加3 mL 0.25 g/L香草醛磷酸溶液(34％的磷酸溶液配制),摇匀室温显色20 min,扣除非油脂成分干扰,在530 nm波长下测定吸光度,经验证该反应体系稳定性良好.在裂殖壶茵和隐甲藻两种产DHA油脂微生物中,通过称重法与磷酸香草醛显色法比较,进一步证实建立的方法分析结果准确可靠,分析过程简便、快速,稳定性好,适合作为常规分析检测方法.