Technical Research On Lipid Extraction From Schizochytrium Sp.Using The Novel CO₂-Expanded Ethanol

CO₂-expanded ethanol（CXE）is a mixed fluid,formed by adding compressed CO₂ to ethanol,resulting in the rapid expansion of solvent,fast diffusivity and tunable solubility.Since the bioactivity of oxidizable or thermolabile components can be protected in the low temperature and moderated pressure,CXE is a novel benign medium,widely applied in chemical reaction and natural product separation.In this study,the CO₂-ethanol binary system was split into two-phase region,subcritical region as well as supercritical region based on the phase equilibrium principle.In each region,the extraction capacity and efficiency of lipids from Schizochytrium sp.using CXE was evaluated,simultaneously compared with that of conventional organic solvent extraction and supercritical CO₂extraction.Finally,the quality of lipids obtained by different methods was comprehensively evaluated on the aspects of physical and chemical properties,fatty acids composition and antioxidant activity.The major research works were mentioned as follows:（1）The process of lipid extraction from Schizochytrium sp.using organic solvent was optimized.Firstly,the lipid was extracted from microalgae matrix using three regular methods including Soxhlet extraction,acid-heating extraction and ultrasound assisted extraction.The lipid yields were determined,respectively.On basis of central composite design,a response surface methodology was employed to investigate the influence of three independent variables including temperature,ultrasonic power and the ratio of liquid to solid on the lipid-extracting process.The maximum lipid yield（24.6%）was obtained at the optimal operating condition including the temperature of 36?C,ultrasonic power of 225 w,the ratio of liquid to solid of 17:1.（2）A novel process for extracting lipids from Schizochytrium sp.using supercritical CO₂ with ethanol as entrainer was studied.The estimation of the molar fraction range of ethanol depended on the thermodynamic data of the vapor（CO₂）-liquid（ethanol）equilibrium reported in the literatures.The total amount of CO₂ added was determined based on the kinetic analysis of the extraction process.The model equation was constructed successfully according to the response surface trials on the basis of central composite design（CCD）,presenting the optimal operation conditions including temperature of 52?C,pressure of 38 MPa and ethanol molar fraction of 0.04.The maximum recovery of microalgae lipid using new method reached around 88.2%significantly higher than that of pure supercritical CO₂ extraction（71.9%）.Furthermore,Peng-Robinson equation was employed for calculating the densities of mixed fluid and the variation of fluid density with respect to the relevant parameters possibly influenced the solubility of microalgae lipid,partially explaining the impacts of operating conditions on lipid recovery.（3）Considering the variation of CO₂ state,the CXE system was finely split into two-phase region,subcritical region and supercritical region based on the isotherms of the CO₂-ethanol mixture,and in each region,the extraction capability of lipid from microalgae was comprehensively evaluated.The results indicated that in subcritical region,large amount of compressed liquid CO₂ was able to be dissolved into ethanol,forming a homogeneous fluid with given polarity,with the maximum recovery reaching 92.4%lipid content of dry biomass,comparable to the complete extraction with hexane.Furthermore,the expansion of ethanol might cause the variation of physicochemical properties such as viscosity as well as diffusivity,and eventually,could highly improve mass transfer during the extraction.Thus,continuous extraction of the fixed-bed matrix for 20 g of microalgae was performed,providing 15.6 mg of lipids per milliliter of fluid compared to 3⁶ mg/mL using either CXE in the other two regions or organic solvents.This study revealed the necessity of the consideration of key factors such as phase equilibrium during employing CXE for extracting specific products.（4）The quality of lipids obtained using different methods were evaluated by measuring physicochemical properties,fatty acid composition and antioxidant activity.The results indicated that both the quality of CXE extract and supercritical extract were superior to that of organic solvent extract over the aspects of unsaponifiable matter content,acid value and the level of lipid peroxidation products.Gas chromatography analysis showed that the DHA content in CXE extract reached 41.92%,higher than that in either supercritical CO₂ extract（37.09%）or organic solvent extract（38.96%）.The DPPH radical scavenging test result showed that the median inhibitory concentration（IC50）of CXE extract was 2.49 mg/mL,presenting higher antioxidant activity than the extracts obtained by the other two methods.In conclusion,this study revealed that subcritical CXE can be used as a promising alternative for the extraction of high-quality products with oxidizable or thermolabile properties.