Blakeslea trispora产番茄红素菌株的选育及优化
摘要
番茄红素属于不具有环状结构的一种类胡萝卜素，具有很强的抗氧化活性和多种生理活性，是一种新型功能性天然色素，在食品、药品及医疗保健等方面有良好的应用前景。采用微生物发酵法生产番茄红素，其产品质量和生理活性与天然提取产品完全相同，且采用发酵法能降低成本，污染相对较小。因此，保证菌株高产、优化发酵条件和得到较高的提取率，是实现工业化生产较理想的途径。
本论文主要从以下四个方面进行研究：
1．从保存的192支沙土管和6支冻干管三孢布拉氏霉菌(Blakeslea trispora，B．t．，下同)菌种中，经划线分离、纯化筛选，通过形态观察，显微结构观察，获得了6~+、80~+、83~+、75~-、94~-、G_3~+和F_2~-共7株菌。经发酵培养，干菌体含油率为45.1％～62.5％，发酵液分别含453.4～821.2 mg／Lβ-胡萝卜素(β-carotene)和29.2～65.4 mg／L番茄红素(Lycopene)。根据菌株生长情况以及发酵实验结果，从中选出83~+、94~-、G_3~+和F_2~-共4株菌作为诱变育种的出发株。
2．对分离筛选到的B．t．菌株进行两次~(60)Co-γ辐射处理。经过菌体形态、平皿生长、发酵培养和色素实验等研究，选育出能稳定遗传的MG_3~+和MF_2~-两株产番茄红素的突变株作为发酵生产实验用菌株，其番茄红素的产量达661.8mg／L。
3．设定发酵培养基中的玉米淀粉、黄豆饼粉、植物油和葡萄糖作为实验的4个因素，分别取3个水平，以发酵菌体菌丝量和番茄红素产量为指标，进行正交实验。根据正交表L_9(3~4)设计正交实验，以筛选出最优培养基条件。实验结果用SPSS软件分析，根据直观及方差分析，最后得出最优发酵培养基中的配比为A_3B_1C_2D_2。番茄红素产量(与最佳方案最接近的处理组)比优化前提高了30％以上，达到968.2 mg／L。
4．在完成了B．t．诱变选育及其摇瓶发酵实验的基础上，进行了B．t．突变株30 L罐的发酵实验，以便为该菌株的大规模发酵探明工艺流程和技术条件。经过连续5批发酵，番茄红素产量最高达到1295.6 mg／L。
Lycopene is a unique caroteniod with non-ring structure. It demonstrates strong antioxdative reactivity and multi-physiological effects. It has great potential in food industry, drug production and medical care. Producing lycopene with fermentation method of microbion, the quality and physiological activityof products are identical with the natural products. It would be to reduce the cost and pollution by applying fermentation method. Accordingly the rather ideal way of implementing industrialized production is to ensure highly producing strain, optimize fermentation condition and get higher extraction ratio.
In this paper, the production of lycopene by Blakeslea trispora was investigated in four parts.
1. Using 192 sand soil test tubes and 6 freeze dried test tubes, Blakeslea trispora strains 6~+, 80~+, 83~+, 75~-, 94~-, G_3~+and F_2~- were obtained by separating, purifying and screening. After fermentation, the content of lipid in dry mycelia was 45.1～62.5%,β-carotene and lycopene were 453.4～821.2 mg/L and 29.2～65.4 mg/L respectively. According to growth state and experimental result of fermentation, 83~+、94~-、G_3~+and F_2~-strains were chosen as original strains for mutagenesis breeding.
2. Blakeslea trispora that had been isolated was treated twice with ~(60)Co-γirradiation. According to the research of thalli figure、living in culture utensil、fermentation culture and pigment test, we bred two strains with MG_3~+ and MF_2~- for fermentability production. Its genetic characteristic was stable and its yield of lycopene reached 661.8mg/L.
3. In order to raise the mutant strain production, we designed an orthogonal experiment according to L_9(3~4) table for screening the optimization medium. The experiment took the following four factors for each at three different levels. And the four factors were corn flour, bean cake flour, vegetable oil and glucose of main medium. The targets of experiment was mycel weight and lycopene yield. Result was analyzed by SPSS. According to the analysis of direct-viewing and variance, we concluded that in the optimization medium the content of four factors was A3B1C2D2. The lycopene yield raised more than 30%, was 968.2 mg/L.
4. At the base of mutagenesis selection and shake flask fermentation experiment of Blakeslea trispora, in order to try to find out technology flow-sheet and specification, we completed the experiment by 30 L fermentor. The highest lycopene yield is 1295.6 mg/L through continuously fermenting 5 times.
本论文主要从以下四个方面进行研究：
1．从保存的192支沙土管和6支冻干管三孢布拉氏霉菌(Blakeslea trispora，B．t．，下同)菌种中，经划线分离、纯化筛选，通过形态观察，显微结构观察，获得了6~+、80~+、83~+、75~-、94~-、G_3~+和F_2~-共7株菌。经发酵培养，干菌体含油率为45.1％～62.5％，发酵液分别含453.4～821.2 mg／Lβ-胡萝卜素(β-carotene)和29.2～65.4 mg／L番茄红素(Lycopene)。根据菌株生长情况以及发酵实验结果，从中选出83~+、94~-、G_3~+和F_2~-共4株菌作为诱变育种的出发株。
2．对分离筛选到的B．t．菌株进行两次~(60)Co-γ辐射处理。经过菌体形态、平皿生长、发酵培养和色素实验等研究，选育出能稳定遗传的MG_3~+和MF_2~-两株产番茄红素的突变株作为发酵生产实验用菌株，其番茄红素的产量达661.8mg／L。
3．设定发酵培养基中的玉米淀粉、黄豆饼粉、植物油和葡萄糖作为实验的4个因素，分别取3个水平，以发酵菌体菌丝量和番茄红素产量为指标，进行正交实验。根据正交表L_9(3~4)设计正交实验，以筛选出最优培养基条件。实验结果用SPSS软件分析，根据直观及方差分析，最后得出最优发酵培养基中的配比为A_3B_1C_2D_2。番茄红素产量(与最佳方案最接近的处理组)比优化前提高了30％以上，达到968.2 mg／L。
4．在完成了B．t．诱变选育及其摇瓶发酵实验的基础上，进行了B．t．突变株30 L罐的发酵实验，以便为该菌株的大规模发酵探明工艺流程和技术条件。经过连续5批发酵，番茄红素产量最高达到1295.6 mg／L。
Lycopene is a unique caroteniod with non-ring structure. It demonstrates strong antioxdative reactivity and multi-physiological effects. It has great potential in food industry, drug production and medical care. Producing lycopene with fermentation method of microbion, the quality and physiological activityof products are identical with the natural products. It would be to reduce the cost and pollution by applying fermentation method. Accordingly the rather ideal way of implementing industrialized production is to ensure highly producing strain, optimize fermentation condition and get higher extraction ratio.
In this paper, the production of lycopene by Blakeslea trispora was investigated in four parts.
1. Using 192 sand soil test tubes and 6 freeze dried test tubes, Blakeslea trispora strains 6~+, 80~+, 83~+, 75~-, 94~-, G_3~+and F_2~- were obtained by separating, purifying and screening. After fermentation, the content of lipid in dry mycelia was 45.1～62.5%,β-carotene and lycopene were 453.4～821.2 mg/L and 29.2～65.4 mg/L respectively. According to growth state and experimental result of fermentation, 83~+、94~-、G_3~+and F_2~-strains were chosen as original strains for mutagenesis breeding.
2. Blakeslea trispora that had been isolated was treated twice with ~(60)Co-γirradiation. According to the research of thalli figure、living in culture utensil、fermentation culture and pigment test, we bred two strains with MG_3~+ and MF_2~- for fermentability production. Its genetic characteristic was stable and its yield of lycopene reached 661.8mg/L.
3. In order to raise the mutant strain production, we designed an orthogonal experiment according to L_9(3~4) table for screening the optimization medium. The experiment took the following four factors for each at three different levels. And the four factors were corn flour, bean cake flour, vegetable oil and glucose of main medium. The targets of experiment was mycel weight and lycopene yield. Result was analyzed by SPSS. According to the analysis of direct-viewing and variance, we concluded that in the optimization medium the content of four factors was A3B1C2D2. The lycopene yield raised more than 30%, was 968.2 mg/L.
4. At the base of mutagenesis selection and shake flask fermentation experiment of Blakeslea trispora, in order to try to find out technology flow-sheet and specification, we completed the experiment by 30 L fermentor. The highest lycopene yield is 1295.6 mg/L through continuously fermenting 5 times.
引文
1．陈锦屏，孙灵霞，段玉峰．番茄红素的性质及提取方法研究进展．粮食与油脂，2004，8：50-53
2．陈涛，陈宗胜，马国华，朱湘民，童骁，宋冬林，姜文侯，单志萍，孟妤，孙冬梅．三孢布拉氏霉发酵生产β-胡萝卜素的研究．微生物学通报，1998，25(2)：79-82
3．陈涛，童骁，宋冬林，朱湘民，陈宗胜，马国华．三孢布拉氏霉菌中胡萝卜素的提取方法．中国专利，ZL97109357．1．2003-05-21
4．陈晓，张亚楼，张月明，牙生买买提，阿孜古丽，温浩．番茄红素干预食管癌细胞系(Eca 9706)及表皮生长因子表达的研究．安徽医药，2005，9：19-20
5．成坚，曾庆孝，何宇峰．番茄红素的稳定性研究．仲恺农业技术学院学报，2001，14(2)：15
6．成坚，曾庆孝．番茄红素的性质及生理功能研究进展．食品与发酵工业，1999，26(2)：75-79
7．董殿军，郭秀云．番茄红素的生理学作用与机制的研究进展．中国慢性病预防与控制，2006，6(3)：220-222
8．范永仙，许兴．微生物生产类胡萝卜素的研究进展．食品与发酵工业，2003，29(7)：69-74
9．范永仙．番茄红素的生产工艺研究进展．食品科技，2002，3：53-551
10．冯晓梅，韩玉谦，隋晓，管华诗．番茄红素稳定性的研究．青岛海洋大学学报，2003，33(6)：875-880
11．傅若农．色谱分析概论．北京：化学工业出版社，1999．9
12．国家卫生部，国家标准化管理委员会．中华人民共和国国家标准GB／T 5009．7-2003．食品中还原糖的测定．中国标准出版社，2003
13．侯纯明，何美，谢颖．番茄红素提取效果的进一步研究．沈阳化工学院学报，2005，19(1)：75-78
14．侯纯明，何美，郭志学．番茄红素提取工艺的研究．沈阳化工学院学报，2004，18(2)：108-111
15．惠军，韩彬，单艳丽，惠寿年．番茄红素提取工艺及其稳定性初步研究．新疆农业科学，2004，41(4)：204-207
16．冀智勇，吴荣书，刘智梅，番茄红素的保健作用及生产工艺的研究进展．中国调味品，2005，10：4-8
17．姜雨，赵广华，胡小松．皂化法番茄红素粗提物的稳定性研究．中国公共卫生，2004，20(9)：1085-1086
18．李琳，吴永娴．番茄红素的研究进展．食品科学，2000，21(6)：8-11
19．李伟，丁霄霖．差示扫描量热法测定番茄红素的纯度．食品科学，2002，23(9)：87-89
20．李向荣，方晓，陈青俊，黄百芬．高效液相色谱法同时测定果蔬与血浆中的番茄红素和β-胡萝卜素．浙江大学学报(农业与生命科学版)，2000，26(4)：444-446
21．李玉环，王锋等．皂化法提取番茄红素的研究．食品科学，2002，23(8)：146
22．李增光，吴骥陶．番茄酱中番茄红素的测定．食品与发酵工业，1991，16(2)：82-84
23．刘慧，闫树刚，仝其根．功能性番茄红素的提取生产技术．农产品加工，2003，6：16-17
24．刘欣，冯杰，番茄红素的生理功能及其在饲料工业中的应用．资源开发利用，2005：28-29
25．齐国鹏，赵锁奇．超临界流体色谱分析番茄红素．分析化学研究简报，2002，30(12)：1477-1480
26．任云霞，刘海洋，丰淑红，郑洋，肖宇星，张坤生．番茄红素提取工艺的优化研究．食品工业科技，2002，23(5)：33-35
27．尚德军，王军，番茄红素研究现状与展望．检验检疫科学，2004，14(2)：59-61
28．盛龙生，何丽一等．药物分析，北京：化学工业出版社，2002：12
29．孙国新，王德才，孙长颢．反相高效液相色谱法测定番茄红素．中国公共卫生，2003，19(12)：15-17
30．孙庆杰，丁宵霖．番茄红素的研究进展．中国食品添加剂，1998，1：1-6
31．孙庆杰，丁霄霖．．超临界CO_2萃取番茄红素的初步研究．食品与发酵工业，2004，1：3-6
32．孙庆杰，丁霄霖．番茄红素稳定性的初步研究．食品与发酵工业，1997，24(2)：47-49
33．孙庆杰，番茄红素稳定性的研究．食品与发酵工业，1998，2：47-51
34．孙庆杰，张运涛，肖刚．番茄油树脂中番茄红素的HPLC测定．无锡轻工大学学报，2001，20(5)：506-509
35．孙庆杰．超临界CO2萃取番茄红素的初步研究．食品与发酵工业，2000，24(1)：3-61
36．孙庆杰．番茄红素的研究．无锡轻工业大学博士学位论文，1999
37．孙新虎，李伟．超临界流体萃取技术在番茄红素提取中的应用．中国食品添加剂，2003，1：69-67
38．谭新平，王银娜，刘晰．番茄红素与癌．天然产物研究与开发，2001，13(4)：71-75
39．王宝东，李垚，王艳波，付晶．番茄红素的研究进展．饲料博览，2005，12：13-15
40．王海曼，西瓜汁加工及其天然色素提取工艺．中国农业大学，1998，3
41．王俊卿，张肇铭．Rhodobacter sphaeroides的番茄红素含量的研究．山西大学学报(自然科学版)，2004，27(4)：406-409
42．王强，韩雅珊，戴蕴青．反相高相液相色谱法同时测定番茄中5种类胡萝卜素．色谱，1997，15(6)：534-538
43．王强，韩雅珊，广田才之，戴蕴青．食品中多种类胡萝卜素的测定(HPLC法)．营养学报，1997，19(2)：212-215
44．王燕燕，邸进申，郑宇，郑辉杰．皂化对番茄红素提取的影响研究．食品与发酵工业，2003，29(3)：71-73
45．王业勤，李勤生．天然类胡萝卜素—研究进展．北京：中国医药科技出版社，1997：1-2
46．王业勤．天然类胡萝卜素—研究进展、生产、应用．中国医药科技出版社，1997
47．维塔特内有限公司．一种生产番茄红素的方法．CN：1370241A，2002-09-18
48．夏传涛，袁秉祥．无空列正交试验的设计及SPSS软件的数据处理．数理医药学杂志，2006，19(1)：91-92
49．徐冠仁，植物诱变育种学，中国农业出版社，1996
50．徐娜，郑珩，许激扬．微生物生产番茄红素及其发酵促进剂的研究进展．中国生化药物杂志，2006，27(2)：114-116
51．薛颖，武兴德，陈杭．高相液相色谱法测定番茄及其制品中的番茄红素．实验技术与方法，2001，14(5)：17-19
52．闫春兰，刘子贻．番茄红素保健功能的研究现状．浙江大学学报，2002，31(2)：139-142
53．杨铃，宁正祥．番茄红素提取工艺研究进展．粮油食品科技，2004，12(5)：44-45．
54．于文利；舒伯；赵亚平；胡卫乐；汤健俭．番茄红素生理功能的动物试验评价．无锡轻工大学学报，2005，24(1)：99-101
55．张连富，丁霄霖．番茄红素简便测定方法的建立．食品与发酵工业．2001，27(3)：51-55
56．章名春，工业微生物诱变育种，科学出版社，1984
57．张卫强，邓宇．微波辐射萃取番茄红素的研究．食品工业科技，2002，1(23)：36-37
58．张文成，王磊．纯天然番茄红素的超临界CO_2萃取研究．安徽化工，1999(5)：28-29
59．赵文恩，乔宪生，俞宏．番茄红素的生物学性质．生物学杂志，2000，17(4)：4-6
60．赵文恩，张劲强．高效液相色谱法测定苹果果皮的类胡萝卜素．果树学报，2001，18(2)：95-97
61．周静，提取番茄皮色素的初步研究．新疆农垦科技，1992，4：28-29
62. Agarwal SR, Akkinappally V. Tomato lycopene and its role in human health and chronic diseases. Assoc Med Can, 2000,163(6): 739-744
63. Anocha K, et al. Analysis of Carotendid. Food Chemistry, 1998, 63(4): 577-584
64. Britton G, Over view of carotenoid biosynt hesis. In Carotenoids: Biosynthesis and Metabolism, vol3 Edited by Britton G, Pfander H, Liaaen-Jensen S. Basel: Birkha user, 1998.143-147
65. Clinton S K, Emenhiser C, Schwartz SJ, et al. Cis-trans lycopene isomers carotienoids, and retirol in the huranprostate. Cancer Epidemiol Biomarkers Prev, 1996, 5(10): 823-833
66. Curt E, Nada S, et al. Separations of Geometrical Carotenoid Isomers in Biological Extracts Using a Polvmeric C30 Column in Reversed-phase Liquid Chromatography. Agric Food Chem, 1996,44:3887-3893
67. Darby S, Whitley E, Doll R, et al. Diet, smoking and lung cancer: a case-control study of 1000 cases and 1500 controls in south-west England. Br J Cancer, 2001, 84: 728-735
68. De Stefani E, Boffetta P, Oreggia F, Plant foods and risk of laryngeal cancer: a case-control study in Uruguay. Int J Cancer, 2000, 87: 129-132
69. Fuhramn B, Elis A, Aviram M. Hypocholesterolemic effect of lycopene and beta-carotene is related to suppression of cholesterol synthesis and augmentation of LDL receptor activity in macrophage. Biochem Biophys Res Commun, 1997, 233(3): 658-662
70. Goodwin T W. Chemistry and Biochemistry of Plant Pigments. London: Academic Press, 1965
71. Govind N S, Amin A R, Modi V V. Stimulation of carotenogenesis in Blakeslea trispora by cupric ions. Phytochemistry, 1982, 21(5): 1043-1044
72. Hirschberg J. Carotenoid biosynthesis in flowering plants. Curt Opin Plant Biol, 2001,4:210-218
73. Joseph Sehierle. Content and Isometric ratio lycopene in food and human blood plasma. Food Technology, 1997, 59(3): 459-465
74. K Jinno, Y lin. Separation of Carorenoids by High-Perfomance Chromatography with Polymeric and Monomeric Octadecylsitica Station. Chwma, 1995, 41(6): 311-317
75. Karas M, Amir H, Fishman D et al. Lycopene interferes with cell cycle progression and insulin-like growth factor I signaling in mammary cancer cells. Nutr Cancer, 2000, 36: 101-111
76. Klipstein-Grobusch K, Launer LJ, Geleijnse JM, et al. Serum carotinoids and atherosc lerosis, the Rotterdam study. Atherosclerosis, 2000,148(1): 49-56
77. Kobayashi T, Mitamula T, et al. Effects of lycopene, a carotenoid, on intrathymic T cell differentiation and peripheral CD4/ CD8 ratio in a high mammary tumor strain of SHN retired mice. Anticancer Drugs, 1996, 7(2): 195-198
78. Lan Y, Kumwenda N, Taha T E, et al. Carotenoid status of pregnant women with and without HIV infection in Malawi. East Afr Med J, 1999, 76: 133-137
79. Lauridsen V S T, Daneshvar B, Jakobsen J. Dose-response effects of lycopene on selected drug-metabolizing and antioxidant enzymes in the rat. Cancer Lett, 2000, 154: 201- 210
80. Lopez-Nieto M J, Costa J, Peiro E, et al. Biotechnological lycopene production by mated fermentation of Blakeslea trispora. Appl Microbiol Biotechnol, 2004, 66(2): 153-159
81. Mehta BJ, Obraztsova IN, Cerd aOlmedo E. Mutants and intersexual heterokaryons of Blakeslea trispora for production of beta-carotene and lycopene. Appl Environ Microbiol, 2003, 69(11): 4043-4048
82. Minhthy L Nguyen, Stven J. Lycopene: Chemical biologicalproperties. Food Tech, 1999, 53: 38-43
83. Mortensen A, et al. Comparative mechanisms and rates of free radical scavenging by carotenoid antioxidants. FEBS Lett, 1997, 418(12): 91-97
84. Nguyen M L, Schwartz S J. Lycopene stability during food processing. Proc Soc Exp Biol Med, 1998, 218(2): 101-105
85. Pan H, Shi G, Chen W, et al. Effect of lycopeneon the function of antioxidative enzyme system in rats. J Hyg Res, 2003,32 (5): 441-442
86. Ri baya, Mercado J D, Garmyn M, et al. Skin lycopene is destroyed prefer entially over β-carotene during gultra violet irradiationin human. Natr, 1995, 125(7): 1854-1859
87. Riso P, Pinder A. Dose tomato consumption effectively increase the resistance of lymphocyte DNA to oxidative damage. Am J Clin Nutr, 1999, 69(4): 712-718
88. Sandmann G. Genetic manipulation of carotenoid biosynthesis: strate-gies, problems and achievements. Trends Plant Sci, 2001, 6(1): 14-17
89. Sharma S K, Kinetic of lycopene degradation in tomato solids. IEF Annual Meeting, 1995, 77
90. Sies H, Stahl W. Vitamins E and C, p-carotene and other carotenoids as antioxidants. Am J Clin Nutr, 1995,62:1315-1321
91. Lacker T, Epler S, et al. Evaluation of Reversed-phase Liquid Chromatographic Columns for Recovery and Selectivity of Selected Carotenoids. Chroma, 1992, 595: 89-101
92. Teodor H, Carmen S, et al. Carotenoid Composition of Rosa Canina Fruits Determined by Thin-Layer Chromatography. Journal of Pharmaceutical and Biomedical Analysis, 1997, 16: 521-528
93. Tzonou A, Signorello LB, Lagiou P, et al. Diet and cancer of the prostate: a case-control study in Greece. Int J Cancer, 1999, 80: 704-708
94. Vandenl angenberg G M, Brady WE, Nebeling L C. Influence of using different sources of carotenoid data in epidem iologic study. Journal of the American Dietetic Association, 1996, 12: 1271-1275
95. Velmurugan B, Bhuvaneswari V, et al. Prevention of N-methyl-N'-nitro-N-nitrosouanidine and saturated sodium chloride-induced gastric carcinogenesis in Wistar rats by lycopene. Eur J Cancer Prev,2002, 11: 19-26
96. Watzl B, Bub A. Modulation of human T-lymphocyte functions by the consumption of carotenoid-rich vegetables. Br J Nutr, 1999, 82(5): 383-389
97. Yamano S Isshii T, Nakagawa M, et al. Metabolic engineering for production of β-carotene and lycopene in Saccharomyces cerevisae. Biosci Biotechnol Biochem, 1994,58(6): 1112-1114
98. Yutaka M, Keiji K, Hiroshi S, et al. Production of lycopene by the food yeast, Candida utilis that does not naturally synthesize carotenoid. Biotechnol Bioeng, 1998, 58(283): 306-308
2．陈涛，陈宗胜，马国华，朱湘民，童骁，宋冬林，姜文侯，单志萍，孟妤，孙冬梅．三孢布拉氏霉发酵生产β-胡萝卜素的研究．微生物学通报，1998，25(2)：79-82
3．陈涛，童骁，宋冬林，朱湘民，陈宗胜，马国华．三孢布拉氏霉菌中胡萝卜素的提取方法．中国专利，ZL97109357．1．2003-05-21
4．陈晓，张亚楼，张月明，牙生买买提，阿孜古丽，温浩．番茄红素干预食管癌细胞系(Eca 9706)及表皮生长因子表达的研究．安徽医药，2005，9：19-20
5．成坚，曾庆孝，何宇峰．番茄红素的稳定性研究．仲恺农业技术学院学报，2001，14(2)：15
6．成坚，曾庆孝．番茄红素的性质及生理功能研究进展．食品与发酵工业，1999，26(2)：75-79
7．董殿军，郭秀云．番茄红素的生理学作用与机制的研究进展．中国慢性病预防与控制，2006，6(3)：220-222
8．范永仙，许兴．微生物生产类胡萝卜素的研究进展．食品与发酵工业，2003，29(7)：69-74
9．范永仙．番茄红素的生产工艺研究进展．食品科技，2002，3：53-551
10．冯晓梅，韩玉谦，隋晓，管华诗．番茄红素稳定性的研究．青岛海洋大学学报，2003，33(6)：875-880
11．傅若农．色谱分析概论．北京：化学工业出版社，1999．9
12．国家卫生部，国家标准化管理委员会．中华人民共和国国家标准GB／T 5009．7-2003．食品中还原糖的测定．中国标准出版社，2003
13．侯纯明，何美，谢颖．番茄红素提取效果的进一步研究．沈阳化工学院学报，2005，19(1)：75-78
14．侯纯明，何美，郭志学．番茄红素提取工艺的研究．沈阳化工学院学报，2004，18(2)：108-111
15．惠军，韩彬，单艳丽，惠寿年．番茄红素提取工艺及其稳定性初步研究．新疆农业科学，2004，41(4)：204-207
16．冀智勇，吴荣书，刘智梅，番茄红素的保健作用及生产工艺的研究进展．中国调味品，2005，10：4-8
17．姜雨，赵广华，胡小松．皂化法番茄红素粗提物的稳定性研究．中国公共卫生，2004，20(9)：1085-1086
18．李琳，吴永娴．番茄红素的研究进展．食品科学，2000，21(6)：8-11
19．李伟，丁霄霖．差示扫描量热法测定番茄红素的纯度．食品科学，2002，23(9)：87-89
20．李向荣，方晓，陈青俊，黄百芬．高效液相色谱法同时测定果蔬与血浆中的番茄红素和β-胡萝卜素．浙江大学学报(农业与生命科学版)，2000，26(4)：444-446
21．李玉环，王锋等．皂化法提取番茄红素的研究．食品科学，2002，23(8)：146
22．李增光，吴骥陶．番茄酱中番茄红素的测定．食品与发酵工业，1991，16(2)：82-84
23．刘慧，闫树刚，仝其根．功能性番茄红素的提取生产技术．农产品加工，2003，6：16-17
24．刘欣，冯杰，番茄红素的生理功能及其在饲料工业中的应用．资源开发利用，2005：28-29
25．齐国鹏，赵锁奇．超临界流体色谱分析番茄红素．分析化学研究简报，2002，30(12)：1477-1480
26．任云霞，刘海洋，丰淑红，郑洋，肖宇星，张坤生．番茄红素提取工艺的优化研究．食品工业科技，2002，23(5)：33-35
27．尚德军，王军，番茄红素研究现状与展望．检验检疫科学，2004，14(2)：59-61
28．盛龙生，何丽一等．药物分析，北京：化学工业出版社，2002：12
29．孙国新，王德才，孙长颢．反相高效液相色谱法测定番茄红素．中国公共卫生，2003，19(12)：15-17
30．孙庆杰，丁宵霖．番茄红素的研究进展．中国食品添加剂，1998，1：1-6
31．孙庆杰，丁霄霖．．超临界CO_2萃取番茄红素的初步研究．食品与发酵工业，2004，1：3-6
32．孙庆杰，丁霄霖．番茄红素稳定性的初步研究．食品与发酵工业，1997，24(2)：47-49
33．孙庆杰，番茄红素稳定性的研究．食品与发酵工业，1998，2：47-51
34．孙庆杰，张运涛，肖刚．番茄油树脂中番茄红素的HPLC测定．无锡轻工大学学报，2001，20(5)：506-509
35．孙庆杰．超临界CO2萃取番茄红素的初步研究．食品与发酵工业，2000，24(1)：3-61
36．孙庆杰．番茄红素的研究．无锡轻工业大学博士学位论文，1999
37．孙新虎，李伟．超临界流体萃取技术在番茄红素提取中的应用．中国食品添加剂，2003，1：69-67
38．谭新平，王银娜，刘晰．番茄红素与癌．天然产物研究与开发，2001，13(4)：71-75
39．王宝东，李垚，王艳波，付晶．番茄红素的研究进展．饲料博览，2005，12：13-15
40．王海曼，西瓜汁加工及其天然色素提取工艺．中国农业大学，1998，3
41．王俊卿，张肇铭．Rhodobacter sphaeroides的番茄红素含量的研究．山西大学学报(自然科学版)，2004，27(4)：406-409
42．王强，韩雅珊，戴蕴青．反相高相液相色谱法同时测定番茄中5种类胡萝卜素．色谱，1997，15(6)：534-538
43．王强，韩雅珊，广田才之，戴蕴青．食品中多种类胡萝卜素的测定(HPLC法)．营养学报，1997，19(2)：212-215
44．王燕燕，邸进申，郑宇，郑辉杰．皂化对番茄红素提取的影响研究．食品与发酵工业，2003，29(3)：71-73
45．王业勤，李勤生．天然类胡萝卜素—研究进展．北京：中国医药科技出版社，1997：1-2
46．王业勤．天然类胡萝卜素—研究进展、生产、应用．中国医药科技出版社，1997
47．维塔特内有限公司．一种生产番茄红素的方法．CN：1370241A，2002-09-18
48．夏传涛，袁秉祥．无空列正交试验的设计及SPSS软件的数据处理．数理医药学杂志，2006，19(1)：91-92
49．徐冠仁，植物诱变育种学，中国农业出版社，1996
50．徐娜，郑珩，许激扬．微生物生产番茄红素及其发酵促进剂的研究进展．中国生化药物杂志，2006，27(2)：114-116
51．薛颖，武兴德，陈杭．高相液相色谱法测定番茄及其制品中的番茄红素．实验技术与方法，2001，14(5)：17-19
52．闫春兰，刘子贻．番茄红素保健功能的研究现状．浙江大学学报，2002，31(2)：139-142
53．杨铃，宁正祥．番茄红素提取工艺研究进展．粮油食品科技，2004，12(5)：44-45．
54．于文利；舒伯；赵亚平；胡卫乐；汤健俭．番茄红素生理功能的动物试验评价．无锡轻工大学学报，2005，24(1)：99-101
55．张连富，丁霄霖．番茄红素简便测定方法的建立．食品与发酵工业．2001，27(3)：51-55
56．章名春，工业微生物诱变育种，科学出版社，1984
57．张卫强，邓宇．微波辐射萃取番茄红素的研究．食品工业科技，2002，1(23)：36-37
58．张文成，王磊．纯天然番茄红素的超临界CO_2萃取研究．安徽化工，1999(5)：28-29
59．赵文恩，乔宪生，俞宏．番茄红素的生物学性质．生物学杂志，2000，17(4)：4-6
60．赵文恩，张劲强．高效液相色谱法测定苹果果皮的类胡萝卜素．果树学报，2001，18(2)：95-97
61．周静，提取番茄皮色素的初步研究．新疆农垦科技，1992，4：28-29
62. Agarwal SR, Akkinappally V. Tomato lycopene and its role in human health and chronic diseases. Assoc Med Can, 2000,163(6): 739-744
63. Anocha K, et al. Analysis of Carotendid. Food Chemistry, 1998, 63(4): 577-584
64. Britton G, Over view of carotenoid biosynt hesis. In Carotenoids: Biosynthesis and Metabolism, vol3 Edited by Britton G, Pfander H, Liaaen-Jensen S. Basel: Birkha user, 1998.143-147
65. Clinton S K, Emenhiser C, Schwartz SJ, et al. Cis-trans lycopene isomers carotienoids, and retirol in the huranprostate. Cancer Epidemiol Biomarkers Prev, 1996, 5(10): 823-833
66. Curt E, Nada S, et al. Separations of Geometrical Carotenoid Isomers in Biological Extracts Using a Polvmeric C30 Column in Reversed-phase Liquid Chromatography. Agric Food Chem, 1996,44:3887-3893
67. Darby S, Whitley E, Doll R, et al. Diet, smoking and lung cancer: a case-control study of 1000 cases and 1500 controls in south-west England. Br J Cancer, 2001, 84: 728-735
68. De Stefani E, Boffetta P, Oreggia F, Plant foods and risk of laryngeal cancer: a case-control study in Uruguay. Int J Cancer, 2000, 87: 129-132
69. Fuhramn B, Elis A, Aviram M. Hypocholesterolemic effect of lycopene and beta-carotene is related to suppression of cholesterol synthesis and augmentation of LDL receptor activity in macrophage. Biochem Biophys Res Commun, 1997, 233(3): 658-662
70. Goodwin T W. Chemistry and Biochemistry of Plant Pigments. London: Academic Press, 1965
71. Govind N S, Amin A R, Modi V V. Stimulation of carotenogenesis in Blakeslea trispora by cupric ions. Phytochemistry, 1982, 21(5): 1043-1044
72. Hirschberg J. Carotenoid biosynthesis in flowering plants. Curt Opin Plant Biol, 2001,4:210-218
73. Joseph Sehierle. Content and Isometric ratio lycopene in food and human blood plasma. Food Technology, 1997, 59(3): 459-465
74. K Jinno, Y lin. Separation of Carorenoids by High-Perfomance Chromatography with Polymeric and Monomeric Octadecylsitica Station. Chwma, 1995, 41(6): 311-317
75. Karas M, Amir H, Fishman D et al. Lycopene interferes with cell cycle progression and insulin-like growth factor I signaling in mammary cancer cells. Nutr Cancer, 2000, 36: 101-111
76. Klipstein-Grobusch K, Launer LJ, Geleijnse JM, et al. Serum carotinoids and atherosc lerosis, the Rotterdam study. Atherosclerosis, 2000,148(1): 49-56
77. Kobayashi T, Mitamula T, et al. Effects of lycopene, a carotenoid, on intrathymic T cell differentiation and peripheral CD4/ CD8 ratio in a high mammary tumor strain of SHN retired mice. Anticancer Drugs, 1996, 7(2): 195-198
78. Lan Y, Kumwenda N, Taha T E, et al. Carotenoid status of pregnant women with and without HIV infection in Malawi. East Afr Med J, 1999, 76: 133-137
79. Lauridsen V S T, Daneshvar B, Jakobsen J. Dose-response effects of lycopene on selected drug-metabolizing and antioxidant enzymes in the rat. Cancer Lett, 2000, 154: 201- 210
80. Lopez-Nieto M J, Costa J, Peiro E, et al. Biotechnological lycopene production by mated fermentation of Blakeslea trispora. Appl Microbiol Biotechnol, 2004, 66(2): 153-159
81. Mehta BJ, Obraztsova IN, Cerd aOlmedo E. Mutants and intersexual heterokaryons of Blakeslea trispora for production of beta-carotene and lycopene. Appl Environ Microbiol, 2003, 69(11): 4043-4048
82. Minhthy L Nguyen, Stven J. Lycopene: Chemical biologicalproperties. Food Tech, 1999, 53: 38-43
83. Mortensen A, et al. Comparative mechanisms and rates of free radical scavenging by carotenoid antioxidants. FEBS Lett, 1997, 418(12): 91-97
84. Nguyen M L, Schwartz S J. Lycopene stability during food processing. Proc Soc Exp Biol Med, 1998, 218(2): 101-105
85. Pan H, Shi G, Chen W, et al. Effect of lycopeneon the function of antioxidative enzyme system in rats. J Hyg Res, 2003,32 (5): 441-442
86. Ri baya, Mercado J D, Garmyn M, et al. Skin lycopene is destroyed prefer entially over β-carotene during gultra violet irradiationin human. Natr, 1995, 125(7): 1854-1859
87. Riso P, Pinder A. Dose tomato consumption effectively increase the resistance of lymphocyte DNA to oxidative damage. Am J Clin Nutr, 1999, 69(4): 712-718
88. Sandmann G. Genetic manipulation of carotenoid biosynthesis: strate-gies, problems and achievements. Trends Plant Sci, 2001, 6(1): 14-17
89. Sharma S K, Kinetic of lycopene degradation in tomato solids. IEF Annual Meeting, 1995, 77
90. Sies H, Stahl W. Vitamins E and C, p-carotene and other carotenoids as antioxidants. Am J Clin Nutr, 1995,62:1315-1321
91. Lacker T, Epler S, et al. Evaluation of Reversed-phase Liquid Chromatographic Columns for Recovery and Selectivity of Selected Carotenoids. Chroma, 1992, 595: 89-101
92. Teodor H, Carmen S, et al. Carotenoid Composition of Rosa Canina Fruits Determined by Thin-Layer Chromatography. Journal of Pharmaceutical and Biomedical Analysis, 1997, 16: 521-528
93. Tzonou A, Signorello LB, Lagiou P, et al. Diet and cancer of the prostate: a case-control study in Greece. Int J Cancer, 1999, 80: 704-708
94. Vandenl angenberg G M, Brady WE, Nebeling L C. Influence of using different sources of carotenoid data in epidem iologic study. Journal of the American Dietetic Association, 1996, 12: 1271-1275
95. Velmurugan B, Bhuvaneswari V, et al. Prevention of N-methyl-N'-nitro-N-nitrosouanidine and saturated sodium chloride-induced gastric carcinogenesis in Wistar rats by lycopene. Eur J Cancer Prev,2002, 11: 19-26
96. Watzl B, Bub A. Modulation of human T-lymphocyte functions by the consumption of carotenoid-rich vegetables. Br J Nutr, 1999, 82(5): 383-389
97. Yamano S Isshii T, Nakagawa M, et al. Metabolic engineering for production of β-carotene and lycopene in Saccharomyces cerevisae. Biosci Biotechnol Biochem, 1994,58(6): 1112-1114
98. Yutaka M, Keiji K, Hiroshi S, et al. Production of lycopene by the food yeast, Candida utilis that does not naturally synthesize carotenoid. Biotechnol Bioeng, 1998, 58(283): 306-308