| Summary: | 博士 === 國立中興大學 === 食品暨應用生物科技學系所 === 99 === In this study, the wild strains of X. dendrorhous CBS 6938 and P. rhodozyma BCRC 21346 were treated with mutagenic agent N-methyl-N''-nitro-N-nitroso-guanidine (NTG) and plated on yeast malt (YM) agar containing b-ionone as a selective medium. One of mutant isolate (X. dendrorhous NCHU-FS701) was found to contain more than threefold of astaxanthin content compared with the parental strain X. dendrorhous CBS 6938. The total carotenoids content of the mutant NCHU-FS701 was 1,508 ug g-1 yeast, and 87% of the total carotenoids was astaxanthin. The mutant NCHU-FS701 could produce higher astaxanthin concentration, but it grew slower than parental strain CBS 6938 in YM broth.
The cost-down medium including molasses, urea, and CaCl2 were selected and optimized by using Box-Behnken design under shaker flask cultivation of X. dendrorhous NCHU-FS701. Maximum astaxanthin production of 11 g ml-1 was obtained with the medium composition of 43.4 g L-1sugar as glucose equivalent in molasses, 2.49 g L-1 urea, and 0.73 g L-1 CaCl2. Based on the media composition, cell growth and astaxanthin production were enhanced when scale up of astaxanthin production was conducted in a 5-L stirred tank reactor with continuous air supply and pH control.
Cell disruption was needed because the astaxanthin and transfructosylating enzyme are intracellular. The optimal setting for 30 g-DCW L-1 yeast cell disruption was found to consist of 0.5-mm-diameter glass bead at 10°C by using a bead beater for 20 min, in which more than 97% yeast disruption could be observed under microscopy. The disrupted cell culture was then extracted with hexane/ethyl acetate, and 72% total carotenoids in the organic solvent phase and 446 U mg-1 protein of transfructosylating enzyme activity in the water phase was obtained, indicating a simple step for simultaneously obtaining astaxanthin and transfructosylating enzyme.
For the production of fructooligosaccharides (FOS), the transfructosylating enzyme was immobilized onto chitosan using Tris (hydroxymethyl) phosphine (THP) as coupling agents. The optimal pH was 5.0 and the optimal temperature was 60oC for both free and immobilized enzyme. The THP-immobilized enzyme had better thermal stability, with 70% residual activity even after 10 repeated runs at 60oC. Metal ions, such as K+ and Fe3+ had positive effect on the enzyme activity.
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