The Research Of Determination Of Astaxanthin In Haematococcus Pluvialis And Its Spatial Distribution Based On Spectroscopic Techniques

The astaxanthin accumulated in Haematococcus pluvialis is of high commercial value.Astaxanthin produced by the algae is widely added to products such as health care products,cosmetics and coloring agents,and it has an important application significance in pharmaceutical industry,food industry,agriculture Aquaculture and other fields of industry.In this study,the optimum conditions for astaxanthin accumulation in Haematococcus pluvialis were studied,and the quantitative analysis of astaxanthin in Haematococcus pluvialis and the spatial distribution of intracellular components were also studied based on spectroscopic techniques.The mainly conclusions as following:(1)The optimum accumulation of astaxanthin from Haematococcus pluvialis used in this study was obtained by orthogonal experiment,and the change of internal pigment in Haematococcus pluvialis under different stress conditions was studied.Nitrogen source,carbon source and salinity were used to analysis by orthogonal design.The optimum combination of astaxanthin accumulation of Haematococcus pluvialis used in this experiment was analyzed by the intuitive and orthogonal variance method.The combination of sodium nitrate:0.15g/L,sodium acetate:1.5g/L and sodium chloride:0.1%w/v,which is the reference of Haematococcus pluvialis in subsequent research.The change of algae pigment content during the stress process was also analyzed.The content of chlorophyll decreased,but astaxanthin and carotenoid accumulated when Haematococcus pluvialis was during the stress.(2)The spectral information of Haematococcus pluvialis was obtained by using the visible/near infrared spectroscopy technology and hyperspectral imaging technology.The quantitative analysis of astaxanthin content of Haematococcus pluvialis were carried out by using the quantitative models.The hyperspectral imaging technology was considered to be superior to the visible/near infrared spectroscopy technology.For the visible/near infrared spectroscopy system,the quantitative model established for astaxanthin content by using absorption spectra in the range of 425-750nm of 198 samples was obtained.It was found that the CARS-MLR model was optimal,Rcal was 0.976,Rval was 0.961,Rpre was 0.967,RMSEC was 0.473,RMSECV was 0.606,RMSEP was 0.510 and RPD was 3.796.The CARS-MLR model was also better than the PLS model using full-band variables,and the variables of the former was only 2.39%of the latter.Therefore,the absorption spectra in the range of 425?750nm can be used to detect astaxanthin in the algae suspension of Haematococcus pluvialis.The CARS-MLR model established by selecting the characteristic variables of CARS was the best,and the corresponding quantitative model formula was also obtained.For the hyperspectral imaging system,the quantitative model established for astaxanthin content by using the absorption spectra in the range of 425-1023nm of 198 samples was obtained.It was found that the CARS-PLS model was optimal,Rcal was 0.978,Rval was 0.967,Rpre was 0.983,RMSEC was 0.443,RMSECV was 0.544,RMSEP was 0.398 and RPD was 5.324.Therefore,the visible/near-infrared reflectance spectra of Hyperspectral imaging in the range of 425?1023nm can be used to detect astaxanthin in the algae suspension of Haematococcus pluvialis.The CARS-PLS model established by selecting the characteristic variables of CARS was the best,and the corresponding quantitative model formula was also obtained.By comparing the results of these two techniques,it was found that the hyperspectral imaging technique is superior to the visible/near-infrared spectroscopy technique.(3)The feasibility of visualization of carotenoid and astaxanthin components distribution in Haematococcus pluvialis was studied by the confocal microscopy Raman spectroscopy.The Raman spectra of Haematococcus pluvialis were compared with Raman spectra of astaxanthin standard,and the Raman intensity of C=C stretching bond was used as the main index of carotenoid.The information of astaxanthin mixed in the Raman spectrum of Haematococcus pluvialis was analyzed by MCR-ALS,and the spatial distribution of astaxanthin in Haematococcus pluvialis was visualized by the information.