| Currently,microalgae are considered as promising feedstock for edible oil production due to rapid growth rate and high lipid accumulation through photosynthesis.More importantly,microalgal oil rich in polyunsaturated fatty acids(PUFAs)possessing multiple biological activities.Aqueous enzymatic extraction(AEE),as a new type of oil production technology,has the advantages of mild conditions,high efficiency and safety.However,the emulsification system containing a large amount of oil was simultaneously formed in AEE,which results a lower oil precipitation and hinders the large-scale application of AEE.This study aims to investigate the factors that affect the precipitation of oil during the extraction process,and explore the molecular mechanism of emulsification system formation,thus providing theoretical and scientific basis for breaking through breaking through the bottleneck of AEE caused by emulsification and developing new demulsification technologies.Schizochytrium sp.was taken as raw material,and the extraction process was optimized based on free,emulsified and total oil recoveries.The optimal extraction conditions for microalgae oil were determined as follows: 675 W ultrasound for 15 min,solid-liquid ratio of 1:5,10 000 U/g(microalgae powder dry weight)papain,enzymatic hydrolysis for 1.5 h.Fatty acid compositions and physiochemical properties of crude/refined oils were determined.The results showed that both crude and refined oils contained PUFAs more than 50%,where DHA was the most abundant,indicating the nutritional value of microalgal oil obtained by AEE.The analysis of physiochemical properties suggested that crude and refined oil basically meet the standards of edible oil,suggesting that AEE has potential in the production of microalgae oil.Several microscopy techniques were utilized to observe the changes in the microstructure of emulsion droplets as the reaction progresses,and the data of particle size distribution and Zeta potential were used for further characterization of Emulsions at different hydrolysis time.The results showed that the largest oil droplets were produced by hydrolysis for 1.5 h.At this time,the interface membrane was ruptured the most,and oil overflowed.In order to explore the emulsification more deeply,the microstructure of interface film was observed by AFM and TEM,and the physical properties of interface film were characterized by contact angle,surface tension and interface rheological properties.The values of contact angle and surface tension showed that the physical stability of emulsion droplets was significantly reduced when hydrolyzed for 1.5 h.At this time,the interface film was rough and loose,where height and depth were significantly increased as compared to those before hydrolysis,as well as part of lipid surface was exposed.In addition,excessive hydrolysis led to a formation of a new interface film.Although its compactness was lower than that in ultrasonication group,its physical stability was increased as compared to emulsion droplets obtained at 1.5 h,reflecting as reduction of height,depth and roughness of interface film,and increase in viscosity.Protein adsorbed in interface of emulsions were separated,followed by comparing the effect of different hydrolysis time on protein conformation,thus analyzing the molecular mechanism of emulsification in hydrolysis process.The results showed that the molecular weight of peptides was less than 35 k D when hydrolyzed for 1.5 h,meanwhile,the exposure of amino acid residues was the most.Furthermore,the hydrophobicity of protein(1.5 h)was significantly increased,and the number of disulfide bonds was the least,as well as the interaction between peptides and lipids was strong,leading to a disorder of fatty acyl groups.In the case of excessive hydrolysis(2.0-3.0 h),due to the hydrophobic interaction and the non-covalent interaction of disulfide bonds,protein aggregates were produced,thus forming a protein membrane at the interface more stable than small peptides. |
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