雨生红球藻的培养及其虾青素提取
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摘要
本文研究了雨生红球藻的培养和虾青素诱导累积的条件，并研究了从累积了虾青素的雨生红球藻中提取虾青素的工艺。该工艺包括雨生红球藻细胞破壁，虾青素的提取，虾青素酯的水解和游离虾青素的纯化分离。
实验探讨了雨生红球藻（Haematococcus pluvialis）712株的适宜培养条件及藻体诱导累积虾青素的培养基条件。重点研究了温度、pH和光照条件对雨生红球藻营养生长的影响，以及NaNO3、Fe2＋盐和乙酸钠浓度对雨生红球藻诱导累积虾青素含量的影响。结果表明，24℃、1000～1500LX连续光照，pH控制在8.0左右的生长条件适合雨生红球藻游动细胞增殖，生物量的增加。通过正交试验表明缺氮培养基对于雨生红球藻细胞累积虾青素最为有利，而高的FeSO4和乙酸钠浓度对虾青素的累积无显著性的影响。经过缺氮的BBM培养基，在24℃，24h连续光照，光照强度为5000LX的条件下进行验证实验，结果雨生红球藻细胞内累积的虾青素含量达到6.72μg/ml。
实验研究了直接用溶剂提取虾青素的工艺条件，结果表明，氯仿∶乙醇（1∶1，v/v）的混合溶剂最有利于从雨生红球藻孢子态细胞中提取出虾青素，较适的提取温度为40℃，较适的提取时间为45min。由于直接对雨生红球藻的细胞进行溶剂提取的提取率低，实验又研究了几种常用的机械破壁方法对雨生红球藻厚壁孢子细胞的破壁效果。结果表明, 高压均质法最适合于雨生红球藻厚壁孢子的破碎和虾青素的提取。高压均质的优化条件为：40MPa, 循环3次, 室温, 破壁率达到91.4％, 虾青素提取率为28.0μg/mg(细胞干重), 比未经破壁处理的提取率提高了10.1μg/mg(细胞干重)，提高了56.3%。
由于雨生红球藻中虾青素95%以上都是以虾青素酯的形式存在，虽然虾青素酯比游离虾青素更为稳定，且部分报道认为虾青素酯生理活性与游离虾青素相当，但是由于虾青素酯的脂肪酸构成复杂，缺少标准样品对照，较难定性进行研究。因此，我们利用酯类的皂化反应将虾青素酯水解成游离虾青素，实验研究了碱浓度、皂化温度和时间对游离虾青素得率的影响。结果表明：4%KOH－甲醇皂化液
在15℃下皂化10min的工艺，得到的游离虾青素占总类胡萝卜素的含量从未皂化前的4.1%提高到81.9%。
为了得到更纯的游离虾青素，实验通过硅胶柱层析进一步纯化了游离虾青素。最终得到的游离虾青素样品经过红外光谱检测，结果表明其分子结构与标准样品完全相同。未经柱层析时游离虾青素得率为21.7μg/mg(细胞干重)，经200目硅胶G柱层析后，经TLC分析只有游离虾青素一个色素斑点，经HPLC结合折光指数检测器分析，游离虾青素的纯度达到98%以上，但经柱层析后的游离虾青素得率下降至16.5μg/mg(细胞干重)。
The thesis studied the culture of Haematococcus pluvialis and the accumulation of astaxanthin. The extraction and purification of astaxanthin from the Haematococcus pluvialis were studied as well. The process included the cell disruption of Haematococcus pluvialis, the extraction of crude astaxanthin, the hydrolysis of astaxanthin esters and the purification of free astaxanthin.
As to the optimal culture conditions and the suitable medium for the inducement of astaxanthin were studied. The emphases were the effects of temperature, pH and illumination conditions on the growth of Haematococcus pluvialis, and the effects of the concentrations of NaNO3, FeSO4 and sodium acetate on the accumulation of astaxanthin. The results suggested that the conditions such as 24℃, illuminated continuously under the intensity of 1000～1500LX, and pH of about 8.0 were suitable for the increase of mobile cells. By orthogonal test, it suggested that medium without nitric salts were suitable for the accumulation of astaxanthin and high concentrations of FeSO4 and sodium acetate had no significant effects on the accumulation of astaxanthin.
As to the extracting astaxanthin from Haematococcus pluvialis directly, the results suggested that the mixture of chloroform: ethanol (1:1) was proper to exact Astaxanthin from Haematococcus pluvialis . The optimal exacting temperature and time were 40℃ and 45min respectively. Due to the low extraction rate of direct extraction by organic solvent, the thesis investigated the various mechanical methods of extracting astaxanthin from Haematococcus pluvialis . The optimal conditions of high-pressure homogenization treatment, ultrasonic processing and repeated freezing and thawing method were gained
respectively. The different methods on the cell disruption rate and astaxanthin extraction rate were studied. The results showed that high-pressure homogenization treatment was most suitable to disrupt cell wall and extract astaxanthin. The optimal pressure was 40MPa, and cycle index is 6. The disruption rate was 91.4%, astaxanthin extraction rate was 28.02μg/mg(dry cell), in comparision with the untreated, the extraction rate was increased by 56.3%.
The astaxanthin of the Haematococcus pluvialis is made up of astaxanthin esters, the ratio of the astaxanthin esters is above 95% of the total carotenoid. Astaxanthin esters is more stable than free astaxanthin and some researches suggested that in view of the bio-availability，the distinguish of astaxanthin esters and free astaxanthin was minor. But on the other hand, the compositions of the esters are complex and the standard sample of astaxanthin esters are hard to gain. In order to facilitate the identification of the experiment results, we changed the astaxanthin esters into free astaxanthin by hydrolysis. The thesis investigated the effects of the alkali concentration, hydrolysis temperature and time on the product. The results suggested that by 4% KOH-methanol at the temperature of hydrolyzing for 10min, the content of free astaxanthin accounted for 81.9% of the product’s total carotenoid, comparing to the content of unhydrolysis sample.
In order to gain more pure product, the experiment purified the free astaxanthin by silica-gel chromatography. The final free astaxanthin product was examined by Infrared absorption spectroscopy. The results indicated that the product was identical with standard sample. The TLC results showed that free astaxanthin was the only spot. The results from HPLC showed that the degree of purification was above 98% and the yield rate was 16.5μg/mg(dry cell).
实验探讨了雨生红球藻（Haematococcus pluvialis）712株的适宜培养条件及藻体诱导累积虾青素的培养基条件。重点研究了温度、pH和光照条件对雨生红球藻营养生长的影响，以及NaNO3、Fe2＋盐和乙酸钠浓度对雨生红球藻诱导累积虾青素含量的影响。结果表明，24℃、1000～1500LX连续光照，pH控制在8.0左右的生长条件适合雨生红球藻游动细胞增殖，生物量的增加。通过正交试验表明缺氮培养基对于雨生红球藻细胞累积虾青素最为有利，而高的FeSO4和乙酸钠浓度对虾青素的累积无显著性的影响。经过缺氮的BBM培养基，在24℃，24h连续光照，光照强度为5000LX的条件下进行验证实验，结果雨生红球藻细胞内累积的虾青素含量达到6.72μg/ml。
实验研究了直接用溶剂提取虾青素的工艺条件，结果表明，氯仿∶乙醇（1∶1，v/v）的混合溶剂最有利于从雨生红球藻孢子态细胞中提取出虾青素，较适的提取温度为40℃，较适的提取时间为45min。由于直接对雨生红球藻的细胞进行溶剂提取的提取率低，实验又研究了几种常用的机械破壁方法对雨生红球藻厚壁孢子细胞的破壁效果。结果表明, 高压均质法最适合于雨生红球藻厚壁孢子的破碎和虾青素的提取。高压均质的优化条件为：40MPa, 循环3次, 室温, 破壁率达到91.4％, 虾青素提取率为28.0μg/mg(细胞干重), 比未经破壁处理的提取率提高了10.1μg/mg(细胞干重)，提高了56.3%。
由于雨生红球藻中虾青素95%以上都是以虾青素酯的形式存在，虽然虾青素酯比游离虾青素更为稳定，且部分报道认为虾青素酯生理活性与游离虾青素相当，但是由于虾青素酯的脂肪酸构成复杂，缺少标准样品对照，较难定性进行研究。因此，我们利用酯类的皂化反应将虾青素酯水解成游离虾青素，实验研究了碱浓度、皂化温度和时间对游离虾青素得率的影响。结果表明：4%KOH－甲醇皂化液
在15℃下皂化10min的工艺，得到的游离虾青素占总类胡萝卜素的含量从未皂化前的4.1%提高到81.9%。
为了得到更纯的游离虾青素，实验通过硅胶柱层析进一步纯化了游离虾青素。最终得到的游离虾青素样品经过红外光谱检测，结果表明其分子结构与标准样品完全相同。未经柱层析时游离虾青素得率为21.7μg/mg(细胞干重)，经200目硅胶G柱层析后，经TLC分析只有游离虾青素一个色素斑点，经HPLC结合折光指数检测器分析，游离虾青素的纯度达到98%以上，但经柱层析后的游离虾青素得率下降至16.5μg/mg(细胞干重)。
The thesis studied the culture of Haematococcus pluvialis and the accumulation of astaxanthin. The extraction and purification of astaxanthin from the Haematococcus pluvialis were studied as well. The process included the cell disruption of Haematococcus pluvialis, the extraction of crude astaxanthin, the hydrolysis of astaxanthin esters and the purification of free astaxanthin.
As to the optimal culture conditions and the suitable medium for the inducement of astaxanthin were studied. The emphases were the effects of temperature, pH and illumination conditions on the growth of Haematococcus pluvialis, and the effects of the concentrations of NaNO3, FeSO4 and sodium acetate on the accumulation of astaxanthin. The results suggested that the conditions such as 24℃, illuminated continuously under the intensity of 1000～1500LX, and pH of about 8.0 were suitable for the increase of mobile cells. By orthogonal test, it suggested that medium without nitric salts were suitable for the accumulation of astaxanthin and high concentrations of FeSO4 and sodium acetate had no significant effects on the accumulation of astaxanthin.
As to the extracting astaxanthin from Haematococcus pluvialis directly, the results suggested that the mixture of chloroform: ethanol (1:1) was proper to exact Astaxanthin from Haematococcus pluvialis . The optimal exacting temperature and time were 40℃ and 45min respectively. Due to the low extraction rate of direct extraction by organic solvent, the thesis investigated the various mechanical methods of extracting astaxanthin from Haematococcus pluvialis . The optimal conditions of high-pressure homogenization treatment, ultrasonic processing and repeated freezing and thawing method were gained
respectively. The different methods on the cell disruption rate and astaxanthin extraction rate were studied. The results showed that high-pressure homogenization treatment was most suitable to disrupt cell wall and extract astaxanthin. The optimal pressure was 40MPa, and cycle index is 6. The disruption rate was 91.4%, astaxanthin extraction rate was 28.02μg/mg(dry cell), in comparision with the untreated, the extraction rate was increased by 56.3%.
The astaxanthin of the Haematococcus pluvialis is made up of astaxanthin esters, the ratio of the astaxanthin esters is above 95% of the total carotenoid. Astaxanthin esters is more stable than free astaxanthin and some researches suggested that in view of the bio-availability，the distinguish of astaxanthin esters and free astaxanthin was minor. But on the other hand, the compositions of the esters are complex and the standard sample of astaxanthin esters are hard to gain. In order to facilitate the identification of the experiment results, we changed the astaxanthin esters into free astaxanthin by hydrolysis. The thesis investigated the effects of the alkali concentration, hydrolysis temperature and time on the product. The results suggested that by 4% KOH-methanol at the temperature of hydrolyzing for 10min, the content of free astaxanthin accounted for 81.9% of the product’s total carotenoid, comparing to the content of unhydrolysis sample.
In order to gain more pure product, the experiment purified the free astaxanthin by silica-gel chromatography. The final free astaxanthin product was examined by Infrared absorption spectroscopy. The results indicated that the product was identical with standard sample. The TLC results showed that free astaxanthin was the only spot. The results from HPLC showed that the degree of purification was above 98% and the yield rate was 16.5μg/mg(dry cell).
引文
[1] Martin Guerin, Mark E. Haematococcus astaxanthin: applications for human health and nutrition. Trends in Biotechnology. 2003, 21(5): 210-216.
[2] R. Todd Lorenz, Gerala R. Rysewski. Commercial potential for Haematococcus microalgae as a natural source omf astaxanthin. Trends in Biotechnology. 2000, 18(4): 160-167.
[3] 陈峰, 姜悦. 微藻生物技术. 北京: 中国轻工业出版社, 1999.56-58.
[4] 李浩明, 高蓝. 虾青素的结构、功能与应用. 精细化工. 2003, 20(1): 32-37.
[5] 吴彩娟．天然虾青素提取和纯化工艺研究：［硕士学位论文］．浙江：浙江大学材料与化学学院，2003．
[6] Andrewes, Phaff. Carotenoids of bacteria – structure and biosynthesis.Arch Microbiol, 1976, 17: 79-88.
[7] Shibata A, Kiba Y, Alati N, et al. Molecular characteristics of astaxanthin and beta-carotene in the phospholipids, monolayer and their distributions in the phospholipids bilayer. Chemistry Physics Lipids, 2001, 113(1): 11-22.
[8] Hua-Bin Li, Feng Chen, Preparative isolation and purification of astaxanthin from the microalga Chlorococcum sp. By high-speed counter-current chromatography. Journal of Chromatography A, 2001, 925: 133-137.
[9] Jian-Ping Yuan, Feng Chen. Purification of trans-astaxanthin from a high-yielding astaxanthin ester-producing stain of the microalga Haematococcus pluvialis . Food Chemistry, 2000, 68: 443-448.
[10] 庄惠如, 卢海声. 雨生红球藻(Haematococcus pluvialis Flotow)在胁迫条件下累积虾青素过程的超微结构研究. 电子显微学报. 2000, 19(2): 137-142.
[11] 刘建国, 殷明焱. 雨生红球藻的细胞周期初探. 海洋与湖沼, 2000, 31(2): 145-149.
[12] 邱保胜, 刘其芳. 红球藻研究进展. 水生生物学报, 2000, 24(5): 546-554.
[13] Kobayashi M, Kakizono T. Effects of light intensity, light quality, and illumination cycle on astaxanthin formation in a green alga, Haematococcus pluvialis . Journal of Fermentation and
Bioengineering, 1992, 74(1): 61-63.
[14] Kobayashi M, Kakizono T. Growth and astaxanthin formation of Haematococcus pluvialis in heterotrophic and mixotrophic conditions. Journal of Fermentation and Bioengineering, 1992, 74(1): 17-20.
[15] 邱保胜, 刘其芳. 雨生红球藻(Haematococcus pluvialis -4B)培养条件研究. 华中师范大学学报(自然科学版). 1999, 33(1): 112-118.
[16] Kobayashi M, Kurimura Y. Light-independent, astaxanthin production by the green microalga Haematococcus pluvialis under salt stress. Biotechnology Letters. 1997, 19: 507-509.
[17] Hagen C, Grunewald K, et al. Effect of cultivation parameters on growth and pigment biosynthesis in flagellated cells of Haematococcus pluvialis . J Appl Phycol. 2001, 13: 79-84.
[18] Kobayashi M. In vivo antioxidant role of astaxanthin under oxidative stress in the green alga Haematococcus pluvialis . Apply Microbiol Biotechnol, 2000, 54: 550-554.
[19] 魏东, 吴汪黔生. 雨生红球藻(Haematococcus pluvialis )在诱导条件下积累虾青素的调控机理研究新进展. 中国海洋药物. 2002, 2: 60-64.
[20] Boussiba S, Vonshak A. Astaxanthin accumulation in the green alga Haematococcus pluvialis . Plant Cell Physiol, 1991, 32: 1077-1082.
[21] Sammy Boussiba, Wang Bing. Changes in pigments profile in the green alga Haematococcus pluvialis exposed to environmental stresses. Biotechnology Letters. 1999, 21: 601-604.
[22] Orosa, D. Franqueira. Carotenoid accumulation in Haematococcus pluvialis in mixotrophic growth. Biotechnology Letters. 2001, 23: 373-378.
[23] Olaozola M. Commercial production of astaxanthin from Haematococcus pluvialis using 25,000-liter outdoor photobioreaction. Journal of Applied Phycology. 2000, 12:499-505.
[24] www.aquasearch.com
[25] www.cyanotech.com
[26] 魏东, 臧晓南. 大规模培养雨生红球藻生产天然虾青素的研究进展和产业化现状. 中国海洋药物. 2001, 5: 4-8.
[27] www.pharmnet.com.cn
[28] Grunewald K, Hagen C. Extrusion of secondary carotenoid containing vesicles from flagellates of Haematococcus pluvialis .
Journal of Apply Botany-Angewandte Botanik, 2000, 74: 141-144.
[29] 蔡明刚, 王杉霖. 利用雨生红球藻生产虾青素的研究进展. 台湾海峡. 2003, 22(4): 98-82.
[30] 章宗涉, 黄祥飞编. 淡水浮游生物研究方法. 北京: 科学出版社, 1991.90-98.
[31] 金传萌, 宋立荣. 红球藻水生748株(Haematocococcus sp. HB748)培养基的选择与对维生素B12的需求. 应用与环境生物学报. 1997, 3(2): 177-179.
[32] 沈萍, 范秀容. 微生物学实验(第三版). 北京: 高等教育出版社, 2000. 52-56.
[33] 叶勇, 应巧兰. 雨生红球藻3个株系的生长及对光盐反应初探. 应用与环境生物学报. 2001, 7(1): 33-36.
[34] 庄惠如, 卢海声. 雨生红球藻营养细胞的虾青素累积. 水生生物学报. 2001, 25(4): 376-379.
[35] 林维宣. 试验设计方法. 大连: 大连海事大学出版社. 1995, 192-211.
[36] 余叔文, 汤章城主编. 植物生理与分子生物学(第二版). 北京: 科学出版社, 2001.55-60.
[37] 徐明芳, 郭宝江. 高效培养螺旋藻封闭式光生物反应器系统的结构单元分析. 食品与发酵工业. 1998, 24(2): 72-78.
[38] Harker M, Hirschberg J. Biosynthesis of ketocarotenoids in transgenic cyanobacteria expressing the algal gene orβ-C4-oxygenase，crtO. FEBS Letters. 1997, 404: 129-134.
[39] 毛忠贵. 生物工业下游技术. 北京: 中国轻工业出版社，1999.10-45.
[40] 高昆玉. 色谱法在精细化工中的应用. 北京: 中国石化出版社, 1997.45-62.
[41] 刘珍. 化验员读本——仪器分析(下册, 第三版). 北京: 化学工业出版社, 2000.46-53.
[42] 张珏, 华聘聘. 羊毛脂皂化工艺的研究. 中国油脂. 2000, 25(2): 64-67.
[43] 张书圣, 李明. 用柱层析法分析辣椒色素中的黄色素. 青岛化工学院学报. 1998，19(4): 347-351.
[44] 邓从蕊. 柱层析法分离辣椒色素与辣椒素. 济南大学学报. 1997, 7(1): 81-83.
[45] 张汉辉，郑威, 等. 波谱学. 厦门：厦门大学出版社, 1998.23-25.
[46] P. H.radman. 红外吸收光谱. 中国化学会：人民教育社,1980.52-58.
[2] R. Todd Lorenz, Gerala R. Rysewski. Commercial potential for Haematococcus microalgae as a natural source omf astaxanthin. Trends in Biotechnology. 2000, 18(4): 160-167.
[3] 陈峰, 姜悦. 微藻生物技术. 北京: 中国轻工业出版社, 1999.56-58.
[4] 李浩明, 高蓝. 虾青素的结构、功能与应用. 精细化工. 2003, 20(1): 32-37.
[5] 吴彩娟．天然虾青素提取和纯化工艺研究：［硕士学位论文］．浙江：浙江大学材料与化学学院，2003．
[6] Andrewes, Phaff. Carotenoids of bacteria – structure and biosynthesis.Arch Microbiol, 1976, 17: 79-88.
[7] Shibata A, Kiba Y, Alati N, et al. Molecular characteristics of astaxanthin and beta-carotene in the phospholipids, monolayer and their distributions in the phospholipids bilayer. Chemistry Physics Lipids, 2001, 113(1): 11-22.
[8] Hua-Bin Li, Feng Chen, Preparative isolation and purification of astaxanthin from the microalga Chlorococcum sp. By high-speed counter-current chromatography. Journal of Chromatography A, 2001, 925: 133-137.
[9] Jian-Ping Yuan, Feng Chen. Purification of trans-astaxanthin from a high-yielding astaxanthin ester-producing stain of the microalga Haematococcus pluvialis . Food Chemistry, 2000, 68: 443-448.
[10] 庄惠如, 卢海声. 雨生红球藻(Haematococcus pluvialis Flotow)在胁迫条件下累积虾青素过程的超微结构研究. 电子显微学报. 2000, 19(2): 137-142.
[11] 刘建国, 殷明焱. 雨生红球藻的细胞周期初探. 海洋与湖沼, 2000, 31(2): 145-149.
[12] 邱保胜, 刘其芳. 红球藻研究进展. 水生生物学报, 2000, 24(5): 546-554.
[13] Kobayashi M, Kakizono T. Effects of light intensity, light quality, and illumination cycle on astaxanthin formation in a green alga, Haematococcus pluvialis . Journal of Fermentation and
Bioengineering, 1992, 74(1): 61-63.
[14] Kobayashi M, Kakizono T. Growth and astaxanthin formation of Haematococcus pluvialis in heterotrophic and mixotrophic conditions. Journal of Fermentation and Bioengineering, 1992, 74(1): 17-20.
[15] 邱保胜, 刘其芳. 雨生红球藻(Haematococcus pluvialis -4B)培养条件研究. 华中师范大学学报(自然科学版). 1999, 33(1): 112-118.
[16] Kobayashi M, Kurimura Y. Light-independent, astaxanthin production by the green microalga Haematococcus pluvialis under salt stress. Biotechnology Letters. 1997, 19: 507-509.
[17] Hagen C, Grunewald K, et al. Effect of cultivation parameters on growth and pigment biosynthesis in flagellated cells of Haematococcus pluvialis . J Appl Phycol. 2001, 13: 79-84.
[18] Kobayashi M. In vivo antioxidant role of astaxanthin under oxidative stress in the green alga Haematococcus pluvialis . Apply Microbiol Biotechnol, 2000, 54: 550-554.
[19] 魏东, 吴汪黔生. 雨生红球藻(Haematococcus pluvialis )在诱导条件下积累虾青素的调控机理研究新进展. 中国海洋药物. 2002, 2: 60-64.
[20] Boussiba S, Vonshak A. Astaxanthin accumulation in the green alga Haematococcus pluvialis . Plant Cell Physiol, 1991, 32: 1077-1082.
[21] Sammy Boussiba, Wang Bing. Changes in pigments profile in the green alga Haematococcus pluvialis exposed to environmental stresses. Biotechnology Letters. 1999, 21: 601-604.
[22] Orosa, D. Franqueira. Carotenoid accumulation in Haematococcus pluvialis in mixotrophic growth. Biotechnology Letters. 2001, 23: 373-378.
[23] Olaozola M. Commercial production of astaxanthin from Haematococcus pluvialis using 25,000-liter outdoor photobioreaction. Journal of Applied Phycology. 2000, 12:499-505.
[24] www.aquasearch.com
[25] www.cyanotech.com
[26] 魏东, 臧晓南. 大规模培养雨生红球藻生产天然虾青素的研究进展和产业化现状. 中国海洋药物. 2001, 5: 4-8.
[27] www.pharmnet.com.cn
[28] Grunewald K, Hagen C. Extrusion of secondary carotenoid containing vesicles from flagellates of Haematococcus pluvialis .
Journal of Apply Botany-Angewandte Botanik, 2000, 74: 141-144.
[29] 蔡明刚, 王杉霖. 利用雨生红球藻生产虾青素的研究进展. 台湾海峡. 2003, 22(4): 98-82.
[30] 章宗涉, 黄祥飞编. 淡水浮游生物研究方法. 北京: 科学出版社, 1991.90-98.
[31] 金传萌, 宋立荣. 红球藻水生748株(Haematocococcus sp. HB748)培养基的选择与对维生素B12的需求. 应用与环境生物学报. 1997, 3(2): 177-179.
[32] 沈萍, 范秀容. 微生物学实验(第三版). 北京: 高等教育出版社, 2000. 52-56.
[33] 叶勇, 应巧兰. 雨生红球藻3个株系的生长及对光盐反应初探. 应用与环境生物学报. 2001, 7(1): 33-36.
[34] 庄惠如, 卢海声. 雨生红球藻营养细胞的虾青素累积. 水生生物学报. 2001, 25(4): 376-379.
[35] 林维宣. 试验设计方法. 大连: 大连海事大学出版社. 1995, 192-211.
[36] 余叔文, 汤章城主编. 植物生理与分子生物学(第二版). 北京: 科学出版社, 2001.55-60.
[37] 徐明芳, 郭宝江. 高效培养螺旋藻封闭式光生物反应器系统的结构单元分析. 食品与发酵工业. 1998, 24(2): 72-78.
[38] Harker M, Hirschberg J. Biosynthesis of ketocarotenoids in transgenic cyanobacteria expressing the algal gene orβ-C4-oxygenase，crtO. FEBS Letters. 1997, 404: 129-134.
[39] 毛忠贵. 生物工业下游技术. 北京: 中国轻工业出版社，1999.10-45.
[40] 高昆玉. 色谱法在精细化工中的应用. 北京: 中国石化出版社, 1997.45-62.
[41] 刘珍. 化验员读本——仪器分析(下册, 第三版). 北京: 化学工业出版社, 2000.46-53.
[42] 张珏, 华聘聘. 羊毛脂皂化工艺的研究. 中国油脂. 2000, 25(2): 64-67.
[43] 张书圣, 李明. 用柱层析法分析辣椒色素中的黄色素. 青岛化工学院学报. 1998，19(4): 347-351.
[44] 邓从蕊. 柱层析法分离辣椒色素与辣椒素. 济南大学学报. 1997, 7(1): 81-83.
[45] 张汉辉，郑威, 等. 波谱学. 厦门：厦门大学出版社, 1998.23-25.
[46] P. H.radman. 红外吸收光谱. 中国化学会：人民教育社,1980.52-58.