Study On The Characterization Of Emulsion System And The Mechanism Of Demulsification During Simultaneous Separation Of Peanut Oil And Protein By Aqueous Enzymatic Method

Compared with the traditional press and solvent leaching methods,the extraction of vegetable oils by the aqueous enzymatic method has a mild condition,environmental and safety,and the in-depth systematic research on the extraction of peanut oil and protein by the aqueous enzymatic method meets the demands of resource and environment-friendly society development.In view of the high cost of enzyme preparation and the formation of a large number of stable emulsions in the process of aqueous enzymatic oil extraction,the industrial application of aqueous enzymatic method is severely limited.In this study,a new type process by cell wall degrading enzyme was used to separate peanut emulsions and aqueous phase,and a large amount of peanut protein in the aqueous phase could be obtained without hydrolysis.Then the composition of the two peanut emulsion produced by the new process was compared and characterized,and their physicochemical properties were explored.Finally,peanut emulsion was demulsified with fatty acid of peanut oil,and the demulsification process was optimized and the demulsification mechanism was explored.The purpose is to provide a theoretical basis for the industrial application of aqueous enzymatic method.First,the three-phase separation process of peanut emulsion,aqueous phase and precipitate phase during aqueous enzymatic method was studied.By comparing different crushing methods,the peanut oil obtained from wet crushing is more stable and easy to store and has the least amino acid in aqueous phase loss,and it is finally determined to use wet crushing.By comparing the effects of compound polysaccharide enzyme,cellulase,pectinase and compound plant hydrolase on aqueous and slag residue rate,we found that the compound polysaccharide enzyme ase had the best effect on oil extraction.Through the influence of the different ratio of cellulase and pectinase on the emulsion oil extraction rate and aqueous protein extraction rate,the 1∶3 compound ratio is the best on the extraction effect of oil and protein.The first step extracted the emulsion,and the second step separated the emulsion and the aqueous phase.The conditions were optimized as follows:Step 1:temperature 40℃,solid-liquid ratio 1∶6,crushing time 100 s,and reaction time 10 min;Step 2:temperature44℃,enzyme concentration 0.9%,enzymatic hydrolysis time 67 min.After Step 1,the extraction rate of peanut oil was 76.41%.Following Step 2,the highest yields obtained for oil and protein were 95.68%and 81.07%,respectively.Secondly,the system characteristics of the emulsion generated by the optimal process are further explored.The compositions and structures of the emulsions produced in the first and second steps（E₁and E₂,respectively）were explored using SDS-PAGE,³¹P-NMR,and CLSM.Peanut emulsion proteins were mainly composed of oleosin,steroleosin,caleosin,lipoxygenase,and arachin.The composition of phospholipids in the emulsions were essentially the same as that in peanut raw materials,with the most abundant phospholipid being phosphatidylcholine.A surface protein wrapped the oil droplets.The film formation and emulsification of phospholipids also stabilized the emulsion.Theζ-potential,particle size,and surface protein concentration properties of E₂were consistent with those produced using the conventional aqueous enzymatic method,whereas E₁was more prone to unstable aggregation between oil droplets.The mechanical properties of shear deformation resistance（viscosity,storage modulus,and loss modulus）were better in E₁than in E₂.Next,by comparing the demulsification effect and cost value of fatty acid,oleic acid was selected as the de-emulsifier,and citric acid was selected to assist in demulsification.We investigated the effect of citric acid-assisted oleic acid on the demulsification of peanut emulsion and the corresponding phase transformation mechanism.After single-factor optimization,the following optimal conditions were obtained:the temperature was 70℃,the sample emulsion-citric acid aqueous solution ratio was 1:4.5,the oleic acid addition amount was 1.5%,and the reaction time was 15 min.The free oil yield was 95.87%under optimal conditions.Sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed that the protein composition of the emulsion treated with citric acid and oleic acid was similar to that of the sample emulsion.The addition of citric acid made the particle size of emulsion from the original 2.34μM increased to 9.03μm andζ-potential increased from-11.4 mV to-0.09 mV.When the content of oleic acid reached 1.5%,the conductivity of emulsion increased from114.5μS/cm reduced to 0.301μS/cm and Fourier-transform infrared spectroscopy demonstrated that the intensity of the hydroxyl absorption peak decreased significantly.Gas chromatography analysis revealed that the content of free oil oleic acid increased by only0.4–0.5%.The phase transformation process during demulsification was investigated by optical and confocal laser scanning microscopy.Oleic acid combined with phospholipids changed the surface hydrophilic–lipophilic balance,and the emulsion changed from oil-in-water to water-in-oil.Small-Angle X-ray scattering analysis deduced that the droplets in the demulsified emulsion were approximately spherical reverse micelles.Finally,the quality characteristics of peanut oil and protein simultaneous separation by a aqueous enzymatic method were investigated.Gas chromatography indicated that the fatty acid composition of peanut oil emulsified with combination of oleic and citric acids was roughly similar compared to freeze thawing demulsification,organic solvent extraction,protease demulsification.Free fatty acid content,acid value,peroxidation values of the oil produced by different methods of demulsification:combination of oleic and citric acids>protease demulsification>organic solvent extraction>freeze thawing demulsification;The opposite was true for oxidative stability.HPLC indicated that tocopherol content:organic solvent extraction>freeze thawing demulsification>protease demulsification>combination of oleic and citric acids.The amino acid composition was generally similar between all three groups:peanut protein powder by aqueous enzymatic method,peanut protein isolate powder,and commercially available peanut protein powder.Scanning electron microscopy showed that the peanut protein fraction by aqueous enzymatic method exhibited a globular structure,and the fraction was lumpy;Peanut protein isolate was almost blocky;Commercially available peanut protein exhibited a lamellar structure.Derived from Fourier infrared spectroscopy compared to commercially available peanut protein and peanut protein isolate,the aqueous enzymatic method of peanut proteinβ-turns and random coil structure.The solubility,water holding,oil holding,emulsifying activity index,emulsification stability,bubble and foam stability of peanut protein were aqueous enzymatic method peanut protein>peanut protein isolate>commercially available peanut protein.