基于单细胞拉曼光谱的酵母乙醇发酵动态过程研究

Kinetic monitoring ethanol fermentation of yeast using laser tweezers raman spectroscopy

应用光镊拉曼光谱技术以15%葡萄糖为乙醇发酵底物,在发酵过程中定时取样,收集发酵液及酵母单细胞的拉曼光谱,通过分析胞内主要生物大分子及葡萄糖、乙醇的特征性拉曼信号变化来监测酵母发酵乙醇的动态过程。结果显示,酵母细胞在进入发酵培养基后代谢很活跃,表征线粒体呼吸水平的1600cm-1信号在0h^3h很强,但在乙醇开始大量产生后迅速减弱;葡萄糖在第3h^18h大量消耗,乙醇在第3h开始产生,第4h^18h大量生成;RNA在3h后迅速增加,同时蛋白质和脂类物质的特征峰亦增强,在8h、9h后基本达到较高水平;酵母细胞蛋白质二级结构在发酵前6h以α螺旋为主,之后稳定以无规则卷曲为主。结果表明,利用光镊拉曼光谱技术可便捷获取胞内外信息,能有效地监测乙醇动态发酵过程,获取底物消耗、产物生成及胞内其他成分变化等。

The process of alcohol fermentation by Saccharomyces cerevisiae is complex. The Raman spectral changes of medium and trapped S. cerevisiae in the fermentation with initial 15% （w/v） of glucose were monitored using laser tweezers Raman spectroscopy （LTRS） in this paper. Biological macromolecules such as nucleic acids, protein, lipids and carbohydrates can generate specific Raman spectra, which could provide biochemical information on the molecular composition and structure. The results showed that the intensity of band 1600cm-1 which characterized the metabolism of mitochondria respiration increased rapidly in initial 3h, and decreased sharply with the production of ethanol. The spectral intensity of RNA began to increase after 3h of fermentation and reached highest value at 9 h. The intensity of protein and lipids increased after 3h and obtained maximum value at 8h-9h. The secondary structure of proteins in major Raman signals were α-helix before 6 h fermentation, after which random coil predominated gradually. These results demonstrated that LTRS could provide some valuable information and used to monitor the ethanol fermentation.