: β- 1829,,,, 2 - A,,, β- β-, β-,,, [4 6],, β-,,, β-, 1 材料和方法 1.1 材料菌株 :,, [3] 培养基 : [3] 试剂 :β-,, Sigma 仪器 :Waters1525 ( Wate

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1 微生物学通报 NOV 20, 2008, 35(11): 1828~1832 Microbiology 生物实验室 2008 by Institute of Microbiology, CAS 用 β- 紫罗兰酮筛选虾青素高产菌株 1 伊文刚肖安风李利君倪辉 * 蔡慧农 ( ) ( ) 摘要 : 用 β- 紫罗兰酮作为筛选剂选择性分离海洋红酵母虾青素高产突变菌株实验结果表明, 在 β- 紫罗兰酮存在的情况下, 由于类胡萝卜素合成受到抑制, 海洋红酵母的生物量和虾青素合成量都减少 ; 当平板培养基中 β- 紫罗兰酮浓度达到 370 mg/l 时, 海洋红酵母的致死率为 92.3%; 在这种平板培养基上涂布经甲基磺酸乙酯诱变的海洋红酵母, 随机筛选 200 个菌落, 结果表明生物量虾青素体积产率和细胞产率均有所提高的正突变株占 18%, 生物量虾青素体积产率和细胞产率的单项指标有所提高的正突变株分别占 22.5% 45% 和 46% 该实验结果表明在分离培养基中添加 β- 紫罗兰酮可选择性地分离海洋红酵母虾青素高产菌株, 提高虾青素高产突变株的筛选效率关键词 : 海洋红酵母, 虾青素, β- 紫罗兰酮, 高产菌株, 筛选模型 Screening Astaxanthin-hyperproducing Strains with β-ionone YI Wen-Gang 1 XIAO An-Feng LI Li-Jun NI Hui CAI Hui-Nong * (1. School of Biotechnology Engineering, Jimei University, Xiamen ) (2. Research Center of Food Biological Engineering Technology of Xiamen City, Xiamen ) Abstract: The astaxanthin-hyperproducing mutants of oceanic red yeast were efficiently screened with β-ionone. As shown by the result, the biomass and astaxanthin content of oceanic red yeast was reduced due to the presence of β-ionone which inhibited the synthesis of carotenoids. The lethality rate of oceanic red yeast would attained 92.3% if 370 mg/l of the β-ionone concentration was contained in the plate medium. The oceanic red yeast mutated by ethyl methane sulfonate was cultured on plate medium containing 370 mg/l of the β-ionone. 200 mutants were randomly screened, positive mutants with increased the biomass, astaxanthin volumetric yield and astaxanthin specific yield accounted for 18% of the total strain, the mutants with higher biomass, astaxanthin volumetric yield and astaxanthin specific yield higher occupied 22.5%, 45% and 46%, respectively. The results indicated that the astaxanthin-hyperproducing strains of oceanic red yeast could be efficiently isolated on the selective medium containing β-ionone. Keywords: Oceanic red yeast, Astaxanthin, β-ionone, Hyperproducing strain, Screening model [], [3],, 基金项目 :(No. B ); (No. C19005) * 通讯作者 :Tel: ; : chn@jmu.edu.cn 收稿日期 : ; 接受日期 :

2 : β- 1829,,,, 2 - A,,, β- β-, β-,,, [4 6],, β-,,, β-, 1 材料和方法 1.1 材料菌株 :,, [3] 培养基 : [3] 试剂 :β-,, Sigma 仪器 :Waters1525 ( Waters ) UV-2006 ( ) FA2004 ( ) ZHWY-2102 ( ) TDL-40B () 5415D (Eppendorf Co. Ld.) HH-4 ( ) SW-CJ-2FD ( ) CL-40L (ALP.Co.Ltd) 1.2 方法培养方法 :, 22 C 3 d, 4 C; 100 μl,, 22 C 3 d; [7], 22 C, 160 r/min 48 h, 4%, 22 C, 160 r/min 4 d 诱变方法 5 ml 3500 r/min 5 min, (ph 7.2) 3 5 ml(ph 7.2) 150 μl(ems) [8], 30 min 3500 r/min 5 min (ph 7.2) 3 5 ml (ph 7.2), 分离方法 :, β- [9,10] 370 mg/l,, 筛选方法 :,,, ;,, 4, 分析测定方法 :(1) [11] (2) [12-14] 7 ml 3500 r/min 5 min,, 3 75 C 2 ml,, 5 ml, 3500 r/min 5 min, 10 ml, (3) [14], 474 nm,, (4) [14], Waters 1525, WatersNovapad C 18 (3.9 mm 150 mm, 4 μm), 1

3 1830 微生物学通报 2008, Vol.35, No.11 Table 1 Time (min) 表 1 液相色谱操作条件 Operation parameters of high performance liquid chromatography Velecity (ml/min) Methanol (%) Urtrapure water(%) Note: λ=474 nm; Pump mode: gradient; High pressure limit: 结果和讨论 2.1 β- 紫罗兰酮对海洋红酵母的生长及虾青素的合成具有抑制作用 1 β-, 1, β- β-,,, β-,,,,, β-,,, 图 1 β- 紫罗兰酮对摇瓶发酵的影响 Fig. 1 The effect of β-ionone on flask fermentation Note Aastaxanthin volume yield = Astaxanthin content per 1 liter fermentation broth(mg/l); Aataxanthin specific yield = Astaxanthin content per 1 gram dry cell(mg/g); Carotenoid volume yield = Carotenoid content per 1 liter fermentation broth(mg/l); Carotenoid specific yield = Carotenoid content per 1 gram dry cell(mg/g) 2.2 选择适于平板分离虾青素高产菌株的 β- 紫罗兰酮浓度 2 β-, 2, β-,, β mg/l, 92.3%, 463 mg/l, 100% 92%, β- 370 mg/l,, β-

4 : β 图 2 β- 紫罗兰酮浓度与致死率的关系 Fig. 2 The relationship between the concentration of β-ionone and lethality rate 2.3 用 β- 紫罗兰酮可选择性分离虾青素高产菌株 (EMS), β-, 200, 36, 18% , 22.5% 45% 46%, 36 9, 2 2, 9 ; B079, 8 B079, 1.01 mg/g 3.71 mg/, 62.90% 35.90%,,,, β-, Number value (g/l) 表 2 以 β- 紫罗兰酮为筛选剂获得的正突变菌株 Table 2 Positive mutant strains with β-ionone as a screening agent Biomass rate(%) Astaxanthin volume yield value (mg/l) rate (%) Astaxanthin specific yield value (mg/g) rate (%) B c c b B d a a B c b b B a b b B b b b B c c b B b c b b B b c c b B b b b Original strain 4.42 c 2.73 d 0.62 c 4, 5% Note: Each digital value is expressed as a mean value of four replicates. Means with different superscript letters within the same column are significantly different (P<0.05) 3 结论 β- β-,, β-, β-,

5 1832 微生物学通报 2008, Vol.35, No.11 β-, - 200, 36, 18%, 参考文献 [1] Miki W. Biological functions and activities of animal carotenoids. Pure Appl Chem, l99l, 63: l4l l46. [2] Woodall AA, Britton G, Jackson MJ. Carotenoids and protections of phospholipids in solution or in liposomes against oxidation by peroxyl radicals. Biochim Biophys Acta, 1997, 1336(6): 578. [3],,,.., 2007, 27(1): [4] Lewis MJ, Ragot N, Berlant MC, et al. Selection of astaxanthin-overproducing mutants of phaffia rhodozyma with β-ionone. Applied and Environmental Microbiology, 1990, 56(9): [5] Fang TJ, Chiou TY. Batch cultivation and astaxanthin production by a mutant of the red yeast phaffia rhodozyma NCHU -FS501. Journal of Industrial Microbiology, 1996, biology, 1996, 16(3): [6],,., 2003, 22(1): [7],, 2002, 29(8): [8] Calo P, Velázquez JB, Sieiro C, et al. Analysis of astaxanthin and other carotenoids from several phaffia rhodozyma mutants. Agric Food Chem, 1995, 43(5): [9],,.., 2002, 29(6): [10],,.., 2006, 4(1): [11],,,.., 2002, 29(1): [12] Johnson EA, Lewis MJ. Astaxanthin formation by the yeast phaffia rhodozyma. Journal of General Microbiology, 1979, 115: [13],, 玥.., 2005, 20(1): [14],,,.., 2005, 10(4): 征订启事 2009 年腐植酸杂志征订启事 1979,,, 16, 60, ISSN ; CN /TQ,, 1979, (), 2009, (), 6, (), 1 : : : chaia@126.com :

: 307, [], [2],,,, [3] (Response Surface Methodology, RSA),,, [4,5] Design-Expert 6.0,,,, [6] VPJ33 ph 3,, ph, OD, Design-Expert 6.0 Box-Behnken, VPJ3

: 307, [], [2],,,, [3] (Response Surface Methodology, RSA),,, [4,5] Design-Expert 6.0,,,, [6] VPJ33 ph 3,, ph, OD, Design-Expert 6.0 Box-Behnken, VPJ3 微生物学通报 FEB 20, 2008, 35(2) 306~30 Microbiology 2008 by Institute of Microbiology, CAS tongbao@im.ac.cn 生物实验室响应面分析法优化副溶血性弧菌生长条件刘代新宁喜斌 * 张继伦 2 (. 200090) (2. 20202) 摘要

: β- 1829,,,, 2 - A,,, β- β-, β-,,, [4 6],, β-,,, β-, 1 材料和方法 1.1 材料 菌株 :,, [3] 培养基 : [3] 试剂 :β-,, Sigma 仪器 :Waters1525 ( Wate

: β- 1829,,,, 2 - A,,, β- β-, β-,,, [4 6],, β-,,, β-, 1 材料和方法 1.1 材料菌株 :,, [3] 培养基 : [3] 试剂 :β-,, Sigma 仪器 :Waters1525 ( Wate