尼罗红染色法筛选产油酵母及定量检测胞内油脂含量的研究

Study on screening of oleaginous yeast and determination of intracellular lipid content by nile red dyeing

【目的】建立产油酵母筛选以及胞内油脂含量测定的简便方法。【方法】利用尼罗红与胞内油脂成分结合后在紫外光照射下发出荧光且荧光强弱与油脂含量相关的原理。通过在添加尼罗红的培养基中培养酵母,并观察菌落荧光的方法对385株深海酵母进行产油脂菌株筛选,利用26S rDNA D1/D2区序列分析方法对筛选获得的产油酵母菌株进行鉴定,并以其中的一株高产油脂酵母(2A00015)为试验菌株,建立了一套尼罗红染色快速测定油脂含量的方法。【结果】获得22株产油酵母,其中油脂含量最高可达62.9%,经分子鉴定后显示这22株酵母分别属于(Candida viswanathii)、近平滑假丝酵母(Candidaparapsilosis)、粘质红酵母(Rhodotorula mucilaginosa)、汉逊德巴利酵母(Debaryomyceshansenii)、季也蒙毕赤酵母(Pichia guilliermondii)以及Rhodosporidium paludigenum酵母。尼罗红染色快速测定油脂含量方法的最佳检测条件为:菌悬液OD600小于1.2,尼罗红浓度0.5 mg/L,染色时间5 min,激发波长488 nm,发射波长570 nm。该测定方法得到相对荧光强度与称重法得到油脂含量呈正相关性,R2=0.9637。

[Objective] To establish a simple method to screen oleaginous yeast and determine the intracellular lipid content. [Methods] The study is based on the theory that the combination of nile red and intracellular oil will emit fluorescence when induced by UV light and the fluoresence indensity is associated with the lipid content. We cultivate yeast in the culture medium added with nile red, and screen the oleaginous yeast strains from the 385 deep-sea yeasts by measns of obeserving the fluorescence of the yeast colony. We have identified the screened oleaginous yeast strains by the 26S rDNA D1/D2 series analysis method. Designating one of the oleaginous yeasts （2A00015） as the test strain, the lipid content rapid determination method by nile red dyeing was established. [Results] 22 oleaginous yeasts were obtained with the lipid content reaching up to 62.9%. Based on the molecular identification, it showed that the 22 yeasts are separately belong to Candida viswanathii, Candida parapailosis, Rhodotorla mucilaginosa, Debaryomyces hansenii, Pichia guilliermondii and Rhodosporidium paludi-genum. The optimum condition for lipid content determination by nile red dyeing is： bacterium suspension OD600 lower than 1.2, nile red concentration 0.5 mg/L, dyeing time 5 min, excit-ing wavelength 488 nm, emission wavelength 570 nm. The relative fluorescence intensity ob-tained by this method exhibits a positive association with the lipid content obtained by weigh-ing method, which can be explained as R2=0.9637.