The Study Of Haematococcus Pluvialis Culture Conditions And Astaxanthin Extraction And Purification

The thesis studied the culture of Haematococcus pluvialis and the accumulation of astaxanthin. The extraction and purification of astaxanthin from the Haematococcus pluvialis were studied as well. The process included the cell disruption of Haematococcus pluvialis, the extraction of crude astaxanthin, the hydrolysis of astaxanthin esters and the purification of free astaxanthin.As to the optimal culture conditions and the suitable medium for the inducement of astaxanthin were studied. The emphases were the effects of temperature, pH and illumination conditions on the growth of Haematococcus pluvialis, and the effects of the concentrations of NaNO3, FeSO4 and sodium acetate on the accumulation of astaxanthin. The results suggested that the conditions such as 24℃, illuminated continuously under the intensity of 1000～1500LX, and pH of about 8.0 were suitable for the increase of mobile cells. By orthogonal test, it suggested that medium without nitric salts were suitable for the accumulation of astaxanthin and high concentrations of FeSO4 and sodium acetate had no significant effects on the accumulation of astaxanthin. As to the extracting astaxanthin from Haematococcus pluvialis directly, the results suggested that the mixture of chloroform: ethanol (1:1) was proper to exact Astaxanthin from Haematococcus pluvialis . The optimal exacting temperature and time were 40℃ and 45min respectively. Due to the low extraction rate of direct extraction by organic solvent, the thesis investigated the various mechanical methods of extracting astaxanthin from Haematococcus pluvialis . The optimal conditions of high-pressure homogenization treatment, ultrasonic processing and repeated freezing and thawing method were gained respectively. The different methods on the cell disruption rate and astaxanthin extraction rate were studied. The results showed that high-pressure homogenization treatment was most suitable to disrupt cell wall and extract astaxanthin. The optimal pressure was 40MPa, and cycle index is 6. The disruption rate was 91.4%, astaxanthin extraction rate was 28.02μg/mg(dry cell), in comparision with the untreated, the extraction rate was increased by 56.3%.The astaxanthin of the Haematococcus pluvialis is made up of astaxanthin esters, the ratio of the astaxanthin esters is above 95% of the total carotenoid. Astaxanthin esters is more stable than free astaxanthin and some researches suggested that in view of the bio-availability,the distinguish of astaxanthin esters and free astaxanthin was minor. But on the other hand, the compositions of the esters are complex and the standard sample of astaxanthin esters are hard to gain. In order to facilitate the identification of the experiment results, we changed the astaxanthin esters into free astaxanthin by hydrolysis. The thesis investigated the effects of the alkali concentration, hydrolysis temperature and time on the product. The results suggested that by 4% KOH-methanol at the temperature of hydrolyzing for 10min, the content of free astaxanthin accounted for 81.9% of the product's total carotenoid, comparing to the content of unhydrolysis sample. In order to gain more pure product, the experiment purified the free astaxanthin by silica-gel chromatography. The final free astaxanthin product was examined by Infrared absorption spectroscopy. The results indicated that the product was identical with standard sample. The TLC results showed that free astaxanthin was the only spot. The results from HPLC showed that the degree of purification was above 98% and the yield rate was 16.5μg/mg(dry cell).