法夫酵母虾青素发酵条件的优化及提取与分析研究
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摘要
虾青素(3,3’-二羟基-β,β’-胡萝卜素-4,4’二酮)是生物界广泛存在的一种类胡萝卜素类物质,在生物体内虾青素具有抗氧化、抗肿瘤、抗辐射、抗衰老、提高免疫力、抗光敏作用及显色等多种生物学功能,在食品、医药、化妆品、饲料等行业中有广泛地需求。法夫酵母是一种重要的天然虾青素资源,为了利用法夫酵母生产天然虾青素,本课题对法夫酵母虾青素的分析及提取方法、法夫酵母突变育种、虾青素的累积模型、培养基、培养条件进行了研究,并对高产虾青素的法夫酵母突变株的发酵经济学进行了初步的评价。
在丙酮二甲亚砜混合溶液中,虾青素的最大吸收波长范围为470-490 nm,β-胡萝卜素的最大吸收波长范围为450—465nm。以虾青素为标准样,487nm为测定波长,利用分光法测定法夫酵母类胡萝卜素总量工作曲线的回归模型为y=0.0913x+0.0037,回归模型的R~2值超过了0.999,样品的回收率范围为98.85%～102.60%,RSD值为0.30%～7.00%。该法能快速、准确地测定法夫酵母的总类胡萝卜素含量。
在很大的波长范围内,虾青素和β-胡萝卜素的分光光谱严重重叠,用传统的分光法不能分别定量法夫酵母中的虾青素素和β-胡萝卜素。比导数光谱法是近年来发展起来的一种新型分光分析方法,它可以消除混合物中结构类似物的干扰。利用比导数光谱法测定虾青素的波长为466nm,求导间隔为2nm,除数因子为2mg/l;测定β-胡萝卜素的波长为461nm,求导间隔为2nm,除数因子为2mg/l。在此条件下,虾青素和β-胡萝卜素标准曲线的回归方程分别为y=0.0146x-0.0006和y=-0.0082x-0.0002,模型的R~2值都超过了0.999,虾青素和β-胡萝卜素的回收率都在99%-101%之间,标准偏差都小于5%。该法适用于快速对大量法夫酵母样品的虾青素和β-胡萝卜素进行定量分析。
法夫酵母含有多种类胡萝卜素成分,利用色谱方法可以同时对多种类胡萝卜素组分进行分析测定。薄层色谱是一种快速、微量的分析方法,适用于硅胶薄层分析法夫酵母类胡萝卜素的溶剂极性为1.45左右,乙醚、三氯甲烷、二氯甲烷三种极性混合溶剂的比例为0.148:0.501:0.350,由于受到仪器及类胡萝卜素特殊性质的限制,建立的薄层色
Astaxanthin (3,3-dihidroxy-β-carotene-4,4-dione) is an unique carotenoid widely distributed in nature. Recently, there has been growing interest in nature astaxanthin produceing in consideration of its health benefits to human beings in light of its roles in cancer prevention, enhancement of immune response, and serving as a free radical quencher. As a result, astaxanthin possesses a high market value both to the pharmaceutical and food industries. Phaffia rhodozyma is an important source of natural astaxanthin, in this thesis, methods for analysis carotenoids and procedures for extracting astaxanthin from Phaffia rhodozyma were optimized, method of mutant genesis and model of astaxanthin accumulation were investigated, media components and conditions for Phaffia rhodozyma cultivation were studied and fermentation economics was evaluated.In mixed solution of acetone and dimethylsulphoxide in ratio of 2 to 1, The absorptive peaks in the spectra of astaxanthin was in range of 470 - 487 nm, and the absorptive peaks of β-carotene was in range of 450-465 nm. experiments carried out at 487nm with astaxanthin as standard showed that R~2 value of regressive model of calibration curve was in excess of 0.999, recoveries were in range of 98.85%~ 102.60%, RSD values ranged from 0.30% to 7.00%, which all indicated the proposed method was accurate and decisive for quantitative analyzing total carotenoids of Phaffia rhodozyma.Astaxanthin and β -carotene in extracts of Phaffia rhodozyma could be simultaneously analyzed by ratio spectrometric method, wavelength selected for determination astaxanthin and β -carotene by ratio spectrometric method were 466nm and 461nm, wavelength interval used for calculate ratio spectrometric values was 2nm, divisor was 2mg/l. Calibration graphs were established for p-carotene within 0 - 6.0 ug/ml and for astaxanthin within 0 - 5.0 μg/ml with their corresponding regressive equations in: y = -0.0082x-0.0002 and y = 0.0146x -0.0006, respectively. R square values in excess of 0.999 indicated the good linearity of the calibration graphs. Sample recovery rates were found satisfactory (99% - 101%) with the
relative standard deviations (RSD) less than 5%. This method can be conveniently used to determine p-carotene and astaxanthin simultaneously in large sample analysis.There were several carotenoids contents in Phaffia rhodozyma; it is reasonable to analyze these different carotenoids composition simultaneously by chromatography method. Thin layer chromatography was a rapid microanalysis method, polarity suitable for analysis carotenoids of Phaffia rhodozyma on the silicon gel plate was so far 1.45, optimal solution for developing carotenoids of Phaffia rhodozyma on silicon gel plate was mixed with 7.7% ether, 17.7% chloroform, 16.4% dichloromethane, and 58.2% n-Hexane. The newly developed thin layer method was not so accurate and of precision to quantitatively determinate carotenoids of Phaffia rhodozyma, it was convenient to qualitatively analyze carotenoids of Phaffia rhodozyma.Optimal organic solvent components for isolating carotenoids of Phaffia rhodozyma on Novapak Cig reverse chromatography column were'composite with 33.33% methanol, 33.33%tetrahydrofuran and 33.33%acetonitrile. controlling developing phase flow at 1.2ml/min, column temperature at 40°C, column pressure at 0—3000psi,optimized gradient condition was that increasing ratio of organic phase ascended from 0 to 70% in the first 6 minutes, keeping 70% for 12 minutes, increasing the ratio of organic phase to 90% in 4 minutes, keeping the ratio for 12 minutes, then increased the ratio to 100% in 2minutes and followed by decreasing to 0% in 2 minutes. Under the optimal organic solvent and gradient procedure, 19 carotenoids were isolated, regressive equations were r=93768x-32214 for astaxanthin and r=39975x+2793.6 for P-carotene, respectively. R square values both in excess of 0.999 indicated the good linearity of the calibration graphs. Sample recovery rates were found satisfactory in range of 98.5%~ 105.6% for astaxanthin and 90.5%
在丙酮二甲亚砜混合溶液中,虾青素的最大吸收波长范围为470-490 nm,β-胡萝卜素的最大吸收波长范围为450—465nm。以虾青素为标准样,487nm为测定波长,利用分光法测定法夫酵母类胡萝卜素总量工作曲线的回归模型为y=0.0913x+0.0037,回归模型的R~2值超过了0.999,样品的回收率范围为98.85%～102.60%,RSD值为0.30%～7.00%。该法能快速、准确地测定法夫酵母的总类胡萝卜素含量。
在很大的波长范围内,虾青素和β-胡萝卜素的分光光谱严重重叠,用传统的分光法不能分别定量法夫酵母中的虾青素素和β-胡萝卜素。比导数光谱法是近年来发展起来的一种新型分光分析方法,它可以消除混合物中结构类似物的干扰。利用比导数光谱法测定虾青素的波长为466nm,求导间隔为2nm,除数因子为2mg/l;测定β-胡萝卜素的波长为461nm,求导间隔为2nm,除数因子为2mg/l。在此条件下,虾青素和β-胡萝卜素标准曲线的回归方程分别为y=0.0146x-0.0006和y=-0.0082x-0.0002,模型的R~2值都超过了0.999,虾青素和β-胡萝卜素的回收率都在99%-101%之间,标准偏差都小于5%。该法适用于快速对大量法夫酵母样品的虾青素和β-胡萝卜素进行定量分析。
法夫酵母含有多种类胡萝卜素成分,利用色谱方法可以同时对多种类胡萝卜素组分进行分析测定。薄层色谱是一种快速、微量的分析方法,适用于硅胶薄层分析法夫酵母类胡萝卜素的溶剂极性为1.45左右,乙醚、三氯甲烷、二氯甲烷三种极性混合溶剂的比例为0.148:0.501:0.350,由于受到仪器及类胡萝卜素特殊性质的限制,建立的薄层色
Astaxanthin (3,3-dihidroxy-β-carotene-4,4-dione) is an unique carotenoid widely distributed in nature. Recently, there has been growing interest in nature astaxanthin produceing in consideration of its health benefits to human beings in light of its roles in cancer prevention, enhancement of immune response, and serving as a free radical quencher. As a result, astaxanthin possesses a high market value both to the pharmaceutical and food industries. Phaffia rhodozyma is an important source of natural astaxanthin, in this thesis, methods for analysis carotenoids and procedures for extracting astaxanthin from Phaffia rhodozyma were optimized, method of mutant genesis and model of astaxanthin accumulation were investigated, media components and conditions for Phaffia rhodozyma cultivation were studied and fermentation economics was evaluated.In mixed solution of acetone and dimethylsulphoxide in ratio of 2 to 1, The absorptive peaks in the spectra of astaxanthin was in range of 470 - 487 nm, and the absorptive peaks of β-carotene was in range of 450-465 nm. experiments carried out at 487nm with astaxanthin as standard showed that R~2 value of regressive model of calibration curve was in excess of 0.999, recoveries were in range of 98.85%~ 102.60%, RSD values ranged from 0.30% to 7.00%, which all indicated the proposed method was accurate and decisive for quantitative analyzing total carotenoids of Phaffia rhodozyma.Astaxanthin and β -carotene in extracts of Phaffia rhodozyma could be simultaneously analyzed by ratio spectrometric method, wavelength selected for determination astaxanthin and β -carotene by ratio spectrometric method were 466nm and 461nm, wavelength interval used for calculate ratio spectrometric values was 2nm, divisor was 2mg/l. Calibration graphs were established for p-carotene within 0 - 6.0 ug/ml and for astaxanthin within 0 - 5.0 μg/ml with their corresponding regressive equations in: y = -0.0082x-0.0002 and y = 0.0146x -0.0006, respectively. R square values in excess of 0.999 indicated the good linearity of the calibration graphs. Sample recovery rates were found satisfactory (99% - 101%) with the
relative standard deviations (RSD) less than 5%. This method can be conveniently used to determine p-carotene and astaxanthin simultaneously in large sample analysis.There were several carotenoids contents in Phaffia rhodozyma; it is reasonable to analyze these different carotenoids composition simultaneously by chromatography method. Thin layer chromatography was a rapid microanalysis method, polarity suitable for analysis carotenoids of Phaffia rhodozyma on the silicon gel plate was so far 1.45, optimal solution for developing carotenoids of Phaffia rhodozyma on silicon gel plate was mixed with 7.7% ether, 17.7% chloroform, 16.4% dichloromethane, and 58.2% n-Hexane. The newly developed thin layer method was not so accurate and of precision to quantitatively determinate carotenoids of Phaffia rhodozyma, it was convenient to qualitatively analyze carotenoids of Phaffia rhodozyma.Optimal organic solvent components for isolating carotenoids of Phaffia rhodozyma on Novapak Cig reverse chromatography column were'composite with 33.33% methanol, 33.33%tetrahydrofuran and 33.33%acetonitrile. controlling developing phase flow at 1.2ml/min, column temperature at 40°C, column pressure at 0—3000psi,optimized gradient condition was that increasing ratio of organic phase ascended from 0 to 70% in the first 6 minutes, keeping 70% for 12 minutes, increasing the ratio of organic phase to 90% in 4 minutes, keeping the ratio for 12 minutes, then increased the ratio to 100% in 2minutes and followed by decreasing to 0% in 2 minutes. Under the optimal organic solvent and gradient procedure, 19 carotenoids were isolated, regressive equations were r=93768x-32214 for astaxanthin and r=39975x+2793.6 for P-carotene, respectively. R square values both in excess of 0.999 indicated the good linearity of the calibration graphs. Sample recovery rates were found satisfactory in range of 98.5%~ 105.6% for astaxanthin and 90.5%
引文
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Wery J., Verdoes J. C. and Van O. A. J. J., 1998. Efficient transformation of the astaxanthin-producing yeast Phaffia rhodozyma. Biotechnol. Tech.., 12: 399-405.
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姜启兴,夏文水,2003.甲壳类加工下脚料中虾青素的提取研究.食品科技,(12):85-88.
吕玉华,金征宇,2000.虾青素高产突变株的选育.食品与发酵工业,26(5):22-27.
施安辉,贾朋辉,张治国等,2003.酵母菌发酵虾青素研究的进展.山东饲料,(11):13-15.
田小群,朱明军,梁世中,2003.高产虾青素红发夫酵母的诱变和筛选方法.武汉工业学院学报,22(1):8-11.
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郑裕国,沈寅初,2002.微生物发酵生产虾青素.生物工程进展,22(2):19-23.
许培雅,章银金,2003.虾壳提取虾青素工艺的研究.粮食与饲料工业,(2):27-29.
张亮,朱湘民.一株产虾青素的黄杆菌C_(F-60)的研究.微生物学通报.1999,26(5):332-335.