利用突变与较适化培养条件提升Xanthophyl Lomyces Dendrorhous虾红素的生产

虾红素是类胡萝素的一种,不但作为增色剂常添加于水产养殖的饲料中(Torrissenetal,1989年)1],同时具有较强的抗氧化功能(Naguib,2000年)2],亦有抗癌作用(Chewetal,1999年)。但天然的虾红素产量不多,如红酵母Xanthophyllomycesdendrorhous(旧名Phaffiarhodozyma)的天然菌产的虾红素只有200￣600mg/kg-drycell,为了提升原始菌株的虾红素生产能力,使用突变法并筛选出高产量的菌株,当利用致突变剂NTG处理X.dendrorhous原始菌种后,再经由含有β-ionone的YMagar上培养筛选,成功筛出数株具有生产高虾红素的突变株,有来自CBS6938的NCHU-FS301(1516mg/kg-drycel)l,NCHU-FS501(1289mg/kg-drycel)l与来自BCRC21346的NCHU-FS701(1372mg/kg-drycel)l,比原始菌种CBS6938(396mg/kg-drycell)与BCRC21346(392mg/kg-drycel)l都高出3倍￣4倍。X.dendrorhous所产的虾红素为胞内色素,为使色素萃取方便,利用能分泌细胞壁分解酵素的细菌BacilluscirculansBCRC11590与突变种X.dendrorhousNCHU-FS501混合培养,并以小型发酵槽进行两阶段批式之混合培养方式,开发有效及简便之虾红素萃取法。B.circulansBCRC11590在最适的氮源YNB和X.dendrorhous混菌培养,在温度为30￣34℃,萃取率可高达97%以上。为降低扩大培养的成本,利用廉价之糖蜜及尿素为培养时的碳源与氮源,X.dendrorhousNCHU-FS701以2.88%的糖蜜,0.82gL-1总尿素氮源,0.68gL-1CaCl2在5L发酵槽中通气搅拌培养120h后,所生产之虾红素可达8.52mg/L;1380mg/kg-drycell,当增加培养之碳源浓度时,虾红素产量更可达20.8mg/L;1763mg/kg-drycell。

Improvement of Astaxanthin Production By Xanthophyllomyces Dendrorhous Through Mutation and Optimization of Culture Conditions

Astaxanthin is not only an important constituent of aquacultural feeds(Torrissen et al,1989),but also a strong antioxidant(Naguib,2000). Red yeast Xanthophyllomyces dendrorhous(formerly Phaffia rhodozyma)is the most promising microbial source of natural astaxanthin for animals. However,X. dendrorhous strains with increased astaxanthin contents are needed for commercial development. In order to this purpose,X. dendrorhous strains were treated with the mutagenic agent NTG several times and plated onto YM agar containing β-ionone as a selective medium. The type strains of X. dendrorhous NCHU-FS301(1 516 mg/kg-dry cell),NCHU-FS501(1 289 mg/kg-dry cell)produced considerably more astaxanthin than the parent CBS(6 938 286 mg/kg-dry cell),and NCHU-FS701(1 372 mg/kg-dry cell)produced considerably more astaxanthin than the parent BCRC21346(282 mg/kg-dry cell). In order to extract astaxanthin from X. dendrorhous more easily, we evaluated the productivity and extractability of astaxanthin in a mixed culture of X. dendrorhous NCHU FS-501 and B. circulans BCRC 1159 using a two-stage batch fermentation technique. The first stage was for X. dendrorhous NCHU FS-501 cultivation. The second stage was the mixed fermentation of the red yeast and B. circulans BCRC 11590. The highest lytic enzyme activity of B. circulans BCRC 11590 was found at 24 h when yeast nitrogen base(YNB)was used as the nitrogen source and incubated at 30 ℃ in a pure culture. Glucose concentration of 45 g/L in YNB supported high X. dendrorhous NCHU FS-501 growth and astaxanthin production,with astaxanthin production of 9 010 mg/L was observed during the first stage. The optimal temperature for pigment extraction in the mixed culture was 30~34 ℃. Under this condition,about 97% of the total pigment could be extracted after a 48 h incubation time. X. dendrorhous NCHU-FS701 was cultivated in low-cost medium for producing astaxanthin. Molasses,urea,and CaCl2 were found to be suitable for pigment production. During cultivation in shaker flasks a maximum astaxanthin production of 6.78 mg/l or 1 206 mg/kg-dry cell was reached at the following medium composition:2.88% sugar as glucose equivalent in molasses,0.82 g/L total urea nitrogen,and 0.68 g/L CaCl2. Astaxanthin production increased further(8.52 mg/L;1 380 mg/kg-dry cell)when X. dendrorhous NCHU-FS701 was cultivated in a stirred tank reactor with pH control and a continuous air supply at dissolved oxygen >40%. When the optimized medium was supplemented with a two-fold increase in molasses concentration,higer astaxanthin was obtained in the fermentor(20.8 mg/L; 1 763 mg/kg-dry cell).