Research On The Identification,Optimization Of Fermentation Conditions And Preservation Stability Of Marine Yeast YS-185 Producing Astaxanthin

Marine yeast strain YS-185 preserved by Marine Products and Enzyme Engineerine Laboratory was isolated to greatly produce astaxanthin. It was studied that identification of the yeast strain by testing 26S rDNA, optimization of fermentation conditions using single-factor and orthogonal test and optimization of protectants formula of marine yeast by the method of factorial experiment and Box-Behnken test according the content of astaxanthin.26S rDNA sequences analyses showed that the yeast strain preserved by our laboratory belongs to Rhodotorula glutinis. During the experiment, the astaxanthin was extracted with ultrasonication of yeast. After ultrasonication, at 4 degree centigrade it was centrifugated at 1000 r/min for 25 minutes to get rude astaxanthin solution. The solution was purified using reduced pressure distillation and organic solvent extraction. In this paper, pigment contents from yeast strain was analysed by HPLC. Chromatographic column: symmetry C18 column (150mm×2.1 mm,5μm); mobile phase: methanol: acetonitrile: tetrahydrofuran (volume ratio 80:5:15); detection wavelength:478nm. The results showed that the pigments contained astaxanthin. The linear range of astaxanthin was 1-6μg/ml. The regression equation was y=120308x-35246 and R2=0.9904. And we obtained that astaxanthin was 69.3% of the total pigments, the content of total pigments was 24.3μg per g dry powder and recovery rate was 98.1% by calculating peak area.The contents of astaxanthin extracted with ultrasonication of yeast, which was used to judge the fernmentation conditions, were analysed by HPLC. The strain was the original strain,with which an experimental study was conducted to determine the optimized culture condition in order to improve the contents of astaxanthin. The fermentation optimization of marin yeast YS-185 was studied.The effect of the culture medium components and fermentation conditions on the marine yeast growth and pigment yield were investigated by shake flask.The rusult indicated that the optimum culture medium for growth consisted of glucose 8g/L,peptone 8g/L and the optimum fermentation conditions for marine yeast YS-185 growth were intitial pH value 5.5,shaking speed 200r/min,inoculum’s level 8%(v/v)and tem-perature 20℃. The optimum culture medium for astaxanthin producing consisted of glucose 8g/L,peptone 8g/L and the optimum fermentation conditions for marine yeast YS-185 astaxanthin producing were intitial pH value 5.5, shaking speed 220 r/min,inoculum’s level 8%(v/v) and tem-perature 25℃.And we found that the temperature had significant influence on growth and astaxanthin production with the analysis of the data on orthogonal experiment .There was an overall increase of 69.7% astaxanthin producing yield(2.670μg/ml) compared with that ofthe original conditions (1.572μg/ml).Tested with spectrophotometry and with astaxanthin content for index, the study aimed to research the conditions of optimizing protectants formula of red yeast astaxanthin by using by-factor test and Box-Behnken experiment .The obtained process optimization model was y=-0.08175+ 3.613x1 +1.715x2+0.2844x3 +0.4000x1x2- 0.5250x1x3+ 0.8000x2x3 -8.250x12- 10.35x22- 0.3781x32 analyzed by Design-Expert software. Respectively for partial derivative again to independent variable to get the optimized protectants formula: :x1= 0.241, x2=0.405, x3 = 0.699,through by converting the actual protectants formula ,and the best protectants formulaes were : 0.22% citric acid, 0.11% vitamin C, 0.41% TP. The result of repeated experiment was 0.401±0.003(n=3)in the most optimal protectants formula , consisting with the software estimation value OD4800.404. It shows that the conjunction of by-factor and Box-Behnken can be well used in the red yeast astaxanthin preservation process, response of pigment 100 days after optimization of preservation effect was improved 44.24%.In this paper, antioxidant properties of red yeast pigment was evaluated by verifying the ability of reduction and the ability of scavenging Hydroxyl radical with recognized antioxidantα-tocopherol as a control.Use the lyophilization pigment powder solution in the experiment, the solution was configured to 0.1-0.4 mg/mL sample fluid.The results showed that the ability of reduction and the ability of scavenging Hydroxyl radical of red yeast pigment was greater thanα-tocopherol’s, the ability of scavenging hydroxyl radical was greater thanα-tocopherol in the experimental concentration rang and the scavenging capacity increases with the sample concentration strengthened.