Studies On Haematococcus Pluvialis Culture Methods And Extraction, Stability And Application Of Astaxanthin

Astaxanthin,a naturally occurring carotenoid pigment,is a powerful biological antioxidant with high economic value for feedstuff,food,nutraceutical and cosmetics industries.Green alga Haematococcus pluvialis,which is the richest source of natural astaxanthin,has become the focus in recent years.In this thesis,culture methods of Haematococcus pluvialis,extraction of astaxanthin from Haematococcus pluvialis, and stability of astaxanthin were studied.Finally,bioactivity of astaxanthin was studied in an ornamental fish model.Benefits of different carbon nutrition for Haernatococcus pluvialis were investigated under mixotrophic and heterotrophic cultures.Results showed microalga grew better on acetate than on malonate in both culture modes.The optimum concentrations of acetate for its growth and accumulation of astaxanthin under mixotrophic and heterotrophic were 1.0 g·L^-1 and 2.0 g·L^-1,respectively.The specific growth rates of mixotrophic and heterotrophic were 0.29 d^-1 and 0.136 d^-1.Cells dry weight and astaxanthin concentrations of mixotrophic or heterotrophic after 8-days' induction were 0.987±0.101g·L^-1,20.304±0.705 mg·L^-1 or 0.763±0.051 g·L^-1, 14.928±0.518 mg·L^-1,respectively.Compared with phototrophic,the specific growth rate,cells dry weight and astaxanthin concentration of mixotrophic increased by 35%, 25%,21%.However,these parameters were decreased in heterotrophic.So mixotrophic with 1.0 g·L^-1 acetate is a good choice for culturing Haematococcus pluvialis.Semicontinuous cultures were compared with the batch culture under the former optimized trophic mode.Results showed the first stage of semicontinuous culture with daily renewal rates of 20%and 30%obtained 2.2 and 3.7 fold higher biomass than batch culture;in the second stage,the survival of alga under any renewal rates was remarkably higher than that of the control(p＜0.05),while there were no significant differences between different renewal rates(p＞0.05).The highest yield of astaxanthin was 22.94 mg·L^-1,obtained at a renewal rate of 30%,which was not significant to that of 20%renewal culture(p＞0.05).Besides,Haematococcus pluvialis cultured in the photobioreactor grew faster, enhanced the biomass and reduced stress cycle,however,the yield of astaxanthin was not higher.A study of supercritical CO₂ extraction of astaxanthin from Haematococcus pluvialis was carried out.The effects of pressures,temperatures,CO₂ flow rates and time on extraction efficiency of astaxanthin were studied by the central composite experimental design principles and analyzed with Response Surface Method(RSM). The optimal conditions were determined:the highest extraction efficiency of astaxanthin was 1.028%under the following conditions,the pressure of 44.6 MPa, the temperature of 64.2℃,the CO₂ flow rate of 7.1 L·h^-1,extracting for 3.5 h.Effects of temperatures,light,alkali concentrations and reducing agents on the stability of astaxanthin from Haematococcus pluvialis were studied.The results were as follows:the absorption value of astaxanthin decreased when temperatures were higher than 40℃;illumination and alkali concentrations can lead to the degradation of free astaxanthin;reducing agents can protect astaxanthin from oxidation,so astaxanthin contents did not changed in a week with 0.2%BHT.Effects of astaxanthin from Haematococcus pluvialis on the growth, pigmentation and antioxidant capacity of ornamental fish Cichlasoma citrinellum x C. synspilum were studied.Compared with control,the fish fed with astaxanthin-supplemented diet for 50 days gained over 50%higher weight,174% and 184%higher astaxanthin contents in the scales and skin,207%and 256% higher total carotenoid contents in the scales and skin,and significantly higher total antioxidant capacity(TAC) in the muscle.