酵母类胡萝卜素产生菌的筛选及其发酵条件的研究
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摘要
从本室保藏的5株红酵母菌株中筛选到1株高产类胡萝卜素的粘红酵母WR_(2’)通过碳源、氮源、pH、接种量、摇瓶装量、摇床转速等对WR_(2’)菌株生物量和类胡萝卜素产量的影响试验，以及蔗糖、蛋白胨、酵母膏三因子的正交试验，确定了WR_(2’)菌株在蔗糖7％，蛋白胨1.5％，酵母膏0.5％，pH6.0，接种量8％，装量40ml／250ml三角烧瓶，28℃，260rpm，振荡培养96h的条件下，其生物量、类胡萝卜素产量分分别高达30.37mg／ml、13.97μg／ml，迄今还未见国内有如此高产类胡萝卜素的酵母菌株的报道。同时，还研究了不同添加剂对WR_(2’)菌株的类胡萝卜素合成和积累的影响，试验结果表明，发酵培养基中添加一定浓度的番茄汁、核黄素、维生素C和MgSO_4对类胡萝卜素产量的提高都有—定的作用。此外，还研究了WR_(2’)菌株的动态生理学变化，研究表明，WR_(2’)菌株在12h左右进入对数生长期，48h左右进入稳定期，稳定期时间较长是该菌之特点，生物量在培养96h达到最高；类胡萝卜素产量的积累则主要在培养36h后的对数生长期的后期和稳定期，96h类胡萝卜素产量已达较高水平；蔗糖的消耗主要在培养48h前生物量大量生成之时，至96h已耗尽；经高效液相色谱仪(LC-4A)检测，WR_(2’)菌株所产色素是以β-胡萝卜素为主要成分的类胡萝卜素，其β-胡萝卜素含量占总色素的81.00％。可见，粘红酵母WR_(2’)是一株高产类胡萝卜素的优良菌株，因此，具有较高的应用价值和开发前景。
The Rhodotonda strain WR2vA\h. high carotenoids yield was obtained from 5 yeast strains of stock cultures in this laboratory yeast by common screening test Effect of carbon source, nitrogen source, initial pH value, inoculum size and additives on carotene production of strains Rhodotonda WR2 were studied The cell biomass, and yield of strain WR2 could reach 30.89 mg/ml, 13.97 //g/ml respectively under selected optimum conditions by an orthogonal design. The optimum fermentation conditions for carotenoid production were as follows: sucrose 7%, peptone 1.5%, yeast extract 0.5%, initial pH6.0, inoculum size 8%, medium 40ml(in 250ml flask) and culture time 96h at 28℃, 260rpm .The varied physiological conditions for carotenoid fermentation were studied by uniform design test method. The major component of WR2 is -carotene, & -carotene amounts to 81.00% Of the total carotene by HPLC (Shimadzu LCMA) detection.
The Rhodotonda strain WR2vA\h. high carotenoids yield was obtained from 5 yeast strains of stock cultures in this laboratory yeast by common screening test Effect of carbon source, nitrogen source, initial pH value, inoculum size and additives on carotene production of strains Rhodotonda WR2 were studied The cell biomass, and yield of strain WR2 could reach 30.89 mg/ml, 13.97 //g/ml respectively under selected optimum conditions by an orthogonal design. The optimum fermentation conditions for carotenoid production were as follows: sucrose 7%, peptone 1.5%, yeast extract 0.5%, initial pH6.0, inoculum size 8%, medium 40ml(in 250ml flask) and culture time 96h at 28℃, 260rpm .The varied physiological conditions for carotenoid fermentation were studied by uniform design test method. The major component of WR2 is -carotene, & -carotene amounts to 81.00% Of the total carotene by HPLC (Shimadzu LCMA) detection.
引文
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27．吕玉华，金征宇．虾青素高产突变株的选育．生物技术，1999，9(5)：22～26
28. KLH, Moo suk; Kim, Sang Moon; Chun SoonSai; Construction of astaxanthin overproduction strain of Phaffia rhodozyma by protoplast fusion J. Microbiol.. Biotechnology 1992, 2(1):46～49
29．张素琴，朱湘民．一株胡萝卜素高产菌C_r-1的研究[J]．食品与发酵工业，1989，5：1-5
30．张建法，黄为一．丝状真菌SC-2产β-胡萝卜素的研究[J]．微生物学通报，1996，23(1)：12-14
31．刘广发，楼士林等．一种(株)杜氏藻β-胡萝卜素的提取及含量比较[J]．厦门大学学报(自然科学版)1995，34(1)：94-98．
32．钦传光，丹．V~2．β-胡萝卜素高产从霉菌株的筛选及发酵[J]．生物技术，2000，10(1)：13-16
33．王岁楼，章银良．生物技术红酵母RY-98产类胡萝卜素培养基的优选及其发酵生理学研究[J]．生物技术，2000，10(4)：24-27
2． Nells H J, DeLeenheer A P.J.Appl. Becteriology,1991,70:181~191
3． Johnson E A,Schroeder W A.Advance in Biochemical Engineering Biotechnology ,1995,53:118-178
4． Mortensen A.J.Agric. Food Chem.,1997,45(8) :2970~2977
5． ninet 1,Renaut J,Carotenoids. In Microbiol. Technology, 2nd,Vol,New york:Academic Press, 1979
6． Tsushima M.Biol. Pharm. Bull.,1995,15(2)
7． Ausich R L.Pure and Appl. Chem.,1997,69(10) :2169~2173
8． Medwid R.U.S.Patent,5437997,1995
9． 1mura T.Japanese Patent,94152078,1994
10． Tsubokura A. U.S. Patent , 5496 709,1996
11. Kukharchuk V.V. Bull. Exp. Biol. Med.,1997,123(3):248～250
12. Kobayashi M.Appl. Envrion. Microbiol., 1993, 59:867
13. Borowitzka M A.J.Appl. Phycol., 1991, 3:295
14．姜文侯，单志萍．微生物产生的类胡萝卜素及其工业化．食品与发酵工业，1999，25(3)：46～51
15. A.G. Andrewes, H.J.Phah at.. Phytochemistry, 1996,10:1003～1007
16. W.A.Schtoeder and E.A. Johnson: Journal of biological Chemistry, 1995, 270(31): 18374～18379
17．施安辉，萧海杰．目前国外虾青素研究的进展，生物工程进展．1999，(1)：29～31
18. Johnson, E. A., An, Gi;-hwan, Astaxanthin from microbial sources. CRC, Critical Review in Biotechnology, 1991(4):297～326
19. FANG, T.J., CHENG, Y.S., Improvement of astaxanthin production by Phaffia rhodozyma through mutation and optimization of culture conditions. Journal of Fermentation and Bioengineering, 1993, 75(6): 466～469
20. Fontana, J.D., Guimaraes, M.F., et al.. Culture of the astaxanthinogenic yeast Phaffia rhodozyma in low cost media. Applied biochemistry and biotechnology. 1996, Spring, 57/58, 413～422
21．朱明军，宗敏华．虾青素研究进展．食品工业科技，2000，21(2)：79～81
22. Mancock I C, Willams KM.J>Gen. Microbiol., 1994, 40:780
23. Johnson, et al. US., Patent 5356809, 1994
24. Meyer, P.S., DuPreez, J.C.and Kilian S.G.Selection and evaluation of astaxanthin ji-Overproducing Mutants of Phaffia rhodozyma, World Journal of Microbiology and Biotechnology, 1993, 9:514～520
25．李永波，贺筱蓉，微波诱变和激光诱变相结合选育金霉素链霉菌的研究，生物工程学报，1998，14(4)：445～448
26．裘娟萍，沈寅初．红发夫酵母高产虾青素的选育方法。工业微生物．2000，30(4)：55～57
27．吕玉华，金征宇．虾青素高产突变株的选育．生物技术，1999，9(5)：22～26
28. KLH, Moo suk; Kim, Sang Moon; Chun SoonSai; Construction of astaxanthin overproduction strain of Phaffia rhodozyma by protoplast fusion J. Microbiol.. Biotechnology 1992, 2(1):46～49
29．张素琴，朱湘民．一株胡萝卜素高产菌C_r-1的研究[J]．食品与发酵工业，1989，5：1-5
30．张建法，黄为一．丝状真菌SC-2产β-胡萝卜素的研究[J]．微生物学通报，1996，23(1)：12-14
31．刘广发，楼士林等．一种(株)杜氏藻β-胡萝卜素的提取及含量比较[J]．厦门大学学报(自然科学版)1995，34(1)：94-98．
32．钦传光，丹．V~2．β-胡萝卜素高产从霉菌株的筛选及发酵[J]．生物技术，2000，10(1)：13-16
33．王岁楼，章银良．生物技术红酵母RY-98产类胡萝卜素培养基的优选及其发酵生理学研究[J]．生物技术，2000，10(4)：24-27
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