The Study Of Astaxanthin High-producing Measure

Astaxanthin is a nonprovitamin A oxygenated carotenoid, which has multi-pHysiologicalfunctions including antioxidant, antitumor and immuin enhancement effects, and has beenused for the cultivation, food, cosmetics, medicine industry. PHaffia rhodozyma is the mostpotential microorganism source to produce natural astaxanthin for commercial production, butit was restricted by relativity low productivity of astaxanthin. The application of new theoriesand new technologies is the foundation of enhancing synthetic level of astaxanthin by Phaffiarhodozyma. In order to improve the production of astaxanthin, the rational selection ofastaxanthin-overproducing strain、optimization of medium composition and fermentationaccelerator and the process of fermentation were system investigated. The main resultsachieved from this research are as follows:1. The high production strain of astaxanthin was rational screened using thedipHenylamine and2-D-deoxy-glucose by Ultrasonic mutagenesis, ultrasonic-LiCl complexmutagenesis and NTG mutagenesis. A mutant strain N-22, with a higher astaxanthinproduction and better genetic stability, was achieved finally. The production of astaxanthinwas949.19μg/g that was4.37times higher than the initial strain.2. According to single-factor test, the Plackett-Burman design and Box-Benhnken designwas used to optimize the medium composition. The optimized medium was composed ofglucose40g/L, sucrose55.55g/L,（NH₄）₂SO₄0.8g/L, yeast extract2.22g/L, KH₂PO₄1.95g/L, MgSO₄·7H₂O2.07g/L, CaCl2·H₂O0.1g/L. The production of astaxanthin was improvedby28.66%higher than that before optimization.3. The results shown, appropriate2-D-deoxy-glucose and lycopene supplyment is benefitfor the accumulation of astaxanthin. On the basis of the results from single-factor test, thefermentation accelerant was investigated using the design of orthogonal experiment. Aoptimal fermentation accelerant component was obtained, which was composed of Na⁺0.4g/L、Fe³⁺0.01g/L、Cu²⁺0.1g/L、Co²⁺0.03g/L、Mn²⁺0.1g/L、sodium citrate6g/L、sodiumacetate1g/L. The astaxanthin production reached10.32mg/L and yeast biomass reached17.02g/L.The astaxanthin production were64.78%higher than that before optimization.4. The optimum fermentation conditions were gained by orthogonal experiment designthat were temperature20℃、 rotation speed190r/min、inoculation amount of7%、liquidmedium volume30mL/250mL and the initial pH was6.0. Based the above conditions, the production of astaxanthin was12.59mg/L, which was22%higher than that beforeoptimization.5. The process of fermentation was studied for N-22mutation in5L fermentor. Theoptimal fermentor process conditions were the fermentation medium volume3L/5L,temperature20oC, inoculation10%, pH（1－84h，pH5.0、84－132h，pH6.0）, dissolvedoxygen（1－36h,50%、36－84h,40%、84－132h,30%）. In the process, the production ofastaxanthin reached16.67mg/L that were increased by32.41%higher than that in theflask-shaking fermentation.