High-efficient Culture Of Haematococcus Pluvialis And Extraction And Purification Of Astaxanthin

Astaxanthin’s powerful antioxidant properties endow it many importantphysiologic functions, such as, improving animal immunity, being a foe of freeradicals and reactive oxygen, inhibiting tumor and so on. Therefore, the naturalastaxanthin has a widespread application and prospect. Furthermore, Haematococcuspluvialis is regarded as the best biological source of astaxanthin. In view of this, theresearch about increasing the cell concentration and the accumulation of astaxanthin,efficiently extracting astaxanthin from Haematococcus pluvialis and thepurifyication of astaxanthin is of important significance. The contents andconclusions of this dissertation mainly include the following aspects:To explore the effects of spectrum on cell growth and astaxanthin production ofHaematococcus pluvialis, absorption spectrum of cells on different growth phases(cell-growing and astaxanthin-producing stages), plus with the spectrum of differentilluminants were measured. And the effects of different light intensity of diverseilluminants were studied on cell growth and accumulation of astaxanthin. The resultswere as follows: Haematococcus pluvialis has good absorption on its absorptionpeaks’ wavelength light. That is, red light could accelerate the growth of green cellsand blue light was more effectively to promote astaxanthin accumulation in red cells.And combined illuminants could be more conducive to cell growth and astaxanthinaccumulation: in condition that the light intensity was2500lx and ratio of red light and white light was2:1, the cell concentration reached0.98g/L which increased by36.11%and15.91%compared with illumination under only fluorescent lamps and redLEDs, respectively; While in the phase of astaxanthin production, under illuminationwith combined illuminants at7000lx, and proportion of blue light to white light3:1,the astaxanthin concentration reached39.79mg/L, which was apparently higher thanthat illuminated only with fluorescent lamps or blue LEDs.The fed-batch culture of Haematococcus pluvialis in photobioreactor was studied.The results showed that fed-batch culture could accelerate the growth ofHaematococcus pluvialis. Furthermore, the concentration of glucose10times that ofthe original medium, while the other ingredients5times the original was the optimumfed-batch addition. And50mL addition was supplied three times at4d,5d,6d,respectively, with a result that, under such processing conditions, the dry cell weightscould reach0.92g/L, increasing by26.03%compared with the control group. But atthe later stages, the growth of Haematococcus pluvialis was inhibited because of thephotobioreactor’s limited conditions. In view of this, other large-scale equipmentswith high-efficiency lighting system were requied in future work.The optimal solvent of extracting astaxanthin from Haematococcus pluvialis wasstudied with the result that the solvet of dimethyl sulfoxide/acetone (4/1, v/v) was theoptimized solvent, by which the astaxanthin yield was34.73mg/L, increasing by15.05%compared with the solvent of ehyl acetate/ethanol(1/2, v/v). And then, basedon the single factor experiment, the optimal conditions to extract astaxanthin fromHaematococcus pluvialis were got through response surface methodology. Theresults showed that extracting conditions were optimum when extracting temperaturewas51.72℃; extracting time was43.32min; ratio of material to solution was1:6.33.Under the condition, the astaxanthin yield was35.49mg/L.Extracting from the Haematococcus pluvialis, the total carotenoid’s ingredients were studied by thin-layer chromatography and high-performance liquid chromatogr-aphy. The results showed that astaxanthin monoester was its main ingredient,followed by astaxanthin diesters, small amount of free astaxanthin and other rarecompounds. And then, the influence of different ratio of n-hexane to acetone onastaxanthin’s TLC was investigated. The results showed that the optimal matchingbetween them was7/3(v/v). Under this developer, the Rf value of free astaxanthin is0.21and all the components can be well separated.The technology of astaxanthin ester’ saponification condition was studied.Taking free astaxanthin yield as the evaluation index, the alkali concentration,temperature and time for saponification were researched. Furthermore, based on thesingle factor experiment, the optimum conditions of astaxanthin esters’ saponificationwere got through response surface methodology. The results showed that conditionswere optimical when the alkali concentration was40.27g/L; temperature was25.91℃;time was25.56min. Under this condition, the free astaxanthin yield is5.12mg/L.And then, the sample after saponification was analysed by TLC and HPLC with theresult that, the free astaxanthin accounts for a large part of the total sample, followedby a small part of astaxanthin esters and other components.In order to get a further pure sample of free astaxanthin, the sample, which wasafter saponification, was pured by200-screen mesh’s silica gel. The free astaxanthinafter column chromatography was only one stain by TLC analysis. And the purity isabout90%by HPLC.