尼罗红荧光光谱法快速测定解脂耶氏酵母油脂含量的研究

为了建立快速简便检测解脂耶氏酵母油脂含量的方法,探讨了尼罗红-光谱法测定解脂耶氏酵母油脂含量的检测条件。通过研究解脂耶氏酵母最佳发射光波长、细胞密度、尼罗红染液用量、染色时间、不同助溶剂效能及最佳体积分数对荧光强度的影响,确定了最佳检测条件,得到细胞油脂含量与荧光强度的线性关系。解脂耶氏酵母在激发光560 nm、发射光650 nm处有最高荧光值,每毫升菌液加入质量分数为0.1 mg/m L的尼罗红染液20μL,加入体积分数为15%的异丙醇,黑暗染色5 min,在细胞OD600=0¹.3的范围内,菌液的油脂含量（X）与荧光值（Y）呈较好的线性关系,线性关系式为Y=6.3651X+10.097,R2=0.9902,灵敏度达0.0001 g。该方法能够准确地反映出解脂耶氏酵母胞内油脂含量。尼罗红-荧光法可成为一种快速检测解脂耶氏酵母胞内油脂含量的新方法。 

Study on a Nile Red fluorescence spectrometry for rapid lipid determination in Yarrowia lipolytica

In order to establish a rapid lipid determination method in Yarrowia lipolytica, the determination conditions were studied with the Nile Red fluorescence spectrometry. The optimal determination conditions were confirmed by researching the optimal emission wavelength in Yarrowia lipolytica, optimal volume fraction, dosage of Nile Red dye solution, dyeing time and efficiency of different penetrating agents, the linearity between lipid content in cell and fluorescence intensity were affirmed.Yarrowia lipolytica had maximum fluorescence value in excitation wavelength 560 nm, emission wavelength 650 nm, 20 μL0.1 mg/m L mass fraction of Nile Red dye solution and 15% volume fraction of isopropanol were added in per milliliter of Yarrowia lipolytica suspension respectively, dyeing time 5 min in darkness, in the range of cell with OD600 of 0¹.3, there was a good linear relationship between lipid content ( X) of solution and fluorescence intensity ( Y) , and the linear formula was Y =6.3651 X + 10.097 ( R2= 0.9902) , the sensitivity of this method was 0.0001 g. The Nile Red fluorescence spectrometry could reflect the lipid content accurately in Yarrowia lipolytica and be used as a new method for rapid lipid determination in Yarrowia lipolytica.