Analysis Of Astaxanthin In Phaffia Rhodozyma Using Laser Tweezers Raman Spectroscopy

Laser tweezers Raman spectroscopy technique, which combines traditional Raman spectroscopy and laser tweezers technique, possesses the advantages of quickness, simplicity, accuracy and a wide detection range, and it can be used in captivity and manipulation of a single cell in the samples. Even it’s nondestructive for cells. So compared with the traditional ultraviolet visible spectrophotometric and high performance liquid chromatography, it’s more suitable for analysis of material composition and cell component.Astaxanthin, a carotenoid, has strong oxidation resistance and many uses. However, its structure is highly unstable. Light, high temperature, organic solvent can cause its degradation or isomerization. Therefore, the detection method of astaxanthin must be strict. The extraction process used in traditional detection methods affects the accuracy of the results. In contrast, laser tweezers Raman spectroscopy can be used without organic solvent extraction, without damage to cells. And it can give an accurate result in short time. So, it’s the best tool for analysis of astaxanthin.Astaxanthin has many sources. The cultivation conditions of Phaffia rhodozyma are relatively mild. As a kind of natural astaxanthin sources, Phaffia rhodozyma is a common strain in the study on astaxanthin producton condition optimization. The detection of astaxanthin in Phaffia rhodozyma using laser tweezers Raman spectroscopy can obtain the optimization results more conveniently. It’s suitable for optimization experimental.This paper,consisted of nine chapters. Chapter one is the introduction and chapter nine is the conclusion. The others can be summarized into two parts. One part is theory analysis, the other part is experiment.Chapter two tells about the history and principle of Raman spectroscopy, and discusses its advantages and disadvantages.Chapter three describes the history and mechanism of laser tweezers.Chapter four analyzes the specific composition and light path of laser tweezers Raman spectroscopy system.Chapter five introduces the other species of Raman spectroscopy and their purposes.Chapter six describes the related knowledge of astaxanthin. Chapter seven validates the accuracy of quantitative analysis of astaxanthin in Phaffia rhodozyma using laser tweezers Raman spectroscopy. First, the Raman spectra of astaxanthin standard solution in different concentrations were acquired and the standard curve for astaxanthin was plotted; And then the Phaffia yeast cells cultivated in different nitrogen source and carbon source medium were divided into two parts, one for the detection of Raman spectra, the other for the determination of ultraviolet visible spectrophotometry; Finally the relationship between the two methods was analyzed. The correlation coefficient of standard curve for astaxanthin was0.9983. Comparing the two methods in analyzing the content of astaxanthin in unit mass Phaffia rhodozyma and the yield of astaxanthin in unit volume fermentation broth of Phaffia rhodozyma, it was found that the data obtained had good linear relationship. And the correlation coefficients were0.9177and0.9054, respectively. Therefore, laser tweezers Raman spectroscopy is accurate in measurement. And it is efficient in the analysis of astaxanthin in Phaffia Rhodozyma cells.In chapter eight, we detected astaxanthin in Phaffia rhodozyma cultivated in different culture medium which had different carbon source or nitrogen source using laser tweezers Raman spectroscopy for optimizing production conditions. First, For the identification of the characteristic peak for astaxanthin we compared the Raman spectra of Phaffia Rhodozyma cells with that of astaxanthin standard solution; Then we contradistinguish growth curve of Phaffia Rhodozyma from content curve of astaxanthin in Phaffia Rhodozyma cells in different fermentation time for optimization fermentation time; Finally, we cultured Phaffia Rhodozyma cells in different carbon medium or nitrogen medium, and compared the Raman spectra of cells in different medium for the medium optimization. The results are as follows:The peak at1520cm-1is an appropriate characteristic raman peak for astaxanthin quantitation; The optimal fermentation time is72h; The optimal medium contain sucrose60g/L for carbon source and ammonium sulfate4g/L, potassium nitrate4g/L for nitrogen source. The above results suggest LTRS when used for optimizing fermentation condition of astaxanthin in Phaffia Rhodozyma possesses advantages of simple operation, short time, less sample consumption, no damage to the sample and so on. It makes experimental data more accurate and reliable. Therefore LTRS is an efficient approach for optimizing fermentation condition of astaxanthin in Phaffia Rhodozyma.