Research On Lipase-catalyzed Synthesis Of Deglyceride From Corn Oil

Diglyceride is situable for human consumption of functional oils and fats. Itsnutritional value is higher, which not only has the characteristic of security, nutrition,high compatiblity and processing eligibility; but also has the functional of regulationof blood lipids, prevention of atherosclerosis, treatment for fatty liver, relievingdiabetes and losing weight. Currently, the chemical synthesis methods of diglyceridehave the characteristic of high energy and material consumptions, seriousenvironmental pollution, poor sensory organ and flavor. Therefore, the enzymaticsynthesis of diglyceride has been a hot area of research in recent years. Enzymaticsynthesis of diglyceride was studied by using the immobilized lipase, corn oil andglycerol as substrates in organic solvent system. The main topics of this work are asfollows:1. Using corn oil as raw materials, preparing mixed fatty acid by saponificationreaction, and then preparing linoleic acid of high content through the uring bagreaction. The best process of uring bag reaction were confirming by single factorexperiment and orthogonal test. The results showed that:（1） The physical and chemical properties of corn oil were investigated, includingwater content, acid value, saponification value, peroxide value, iodine value andunsaponifiables value, laying the foundation date for the research of saponificationreaction and uring bag reaction.（2） Based on the results of single factor analysis, the best process of uring bagreaction are fatty acid/urea/ethonal（W/W/V）1:2.5:10, crystallization temperature5℃,crystallization time12h.（3） Orthogonal test showed that: the quality ratio of urea and fatty acid3:1, theratio of urea quality and ethonal volume1:4, crystallization temperature20℃,crystallization time12h, the enrichment effect of linoleic acid is better under theseconditions. Iodine value and the recovery rate of linoleic acid can reach155.16（gI2/100g） and44.81%.2. Enzymatic synthesis of diglyceride was studied by using the immobilizedlipase as the catalyst, linoleic acid and glycerol as substrates in organic solvent system.Optimizing the processing parameters of preparation of diglyceride by single factorexperiments and response surface experiments. The results showed that: （1） The enzyme activity and water content of immobilized lipase Lipozyme TLIM are138EU/g and6.2%.（2） The paper select normal hexane as the anxiliary solvent for esterificationreaction.（3） The immobilized lipase Lipozyme TL IM reacts3batches, continuously,while the relative enzyme activity is47.8%.（4） Based on the results of single factor analysis, the paper determined theoptimal synthesis conditions of diglyceride: In anhydrous condition, reactiontemperature60℃, the molar ratio of linoleic acid and glycerol2:1, reaction time12h,the amount of enzyme20%and shaker rotating speed150r/min.（5） The optimum conditions were as follows: the molar ratio of linoleic acid andglycerol2.10:1, reaction temperature61.16℃, reaction time12.17h, the amount ofenzyme20.09%, the conversion rate of the linoleic acid is75.69%under theseconditions.（6） Optimizing the process conditions of enzymatic synthesis of diglyceride,throughing center composite design（four factors and five levels）, combining with theDesign-7.0software expert, the research got the following regression equation:3. The thermodynamic equation and reaction mechanism of enzyme-catalyzed innormal hexane solvent system was investigated. The thermodynamic analysis usingthe Arrhenius model indicated that the activation energy（Ea） of the enzymatic reactionwas58.08kJ/mol. The reaction rate constants and temperature of esterificationreaction between40-70℃using the Arrhenius model could be represented as:The kinetics equation of enzyme-catalyzed in normal hexane solvent system wasinvestigated. When the concentration of linoleic acid and glycerol were0.08⁰.11mol/L and0.01⁰.04mol/L, the synthesis method accords with thePing-Pong Bi-Bi reaction mechanism inhibited by substrates. The kinetics analysisindicated that the esterification reaction could be inhibited by high concentration ofglycerol. The reaction kinetics values for Vmax, KA, KB, KiAand KiBwere2.7709×10-4mol/（L·min）,0.0465mol/L,0.4390mol/L,0.0572mol/L and0.1296mol/L,respectively. The kinetics equation was established: V=2.7709×10^-4C_AC_B/0.0465C_B[1-C_B/0.1296]+0.4390C_A[C_A/0.0572]+C_AC_B4. Silica gel column chromatography was investigated to purify diglyceride, weobtained the optimum process condition of purification. It was widely used for oilindustry of TBHQ, VE and citric acid to compound. It could get effective compositeantioxidants to delay the reaction of oxidation. The results showed that:（1） Petroleum ether/aether/acetic acid（80:20:2,v/v/v） was used as an eluantsystems to get favourable separation effect of product components; adding2%aceticacid of eluant systems can increase the migration rate of product components.（2） The optimum process conditions were as follows: At room temperature,petroleum ether/aether（4:1,v/v） was used as eluant systems, the silica gel was columnmaterial, the column specification was Φ20×300mm, the elution rate was2ml/min,the sample loading was3g, the yield of diglyceride was33.64%. After4cyclesrepeated usage, the silica gel column chromatography showed high reuse effect andperfect tomographic regeneration effect, the yield of diglyceride was more than31.13%.（3） Diglyceride purified by silica gel column chromatography was in line withnational standard.（4） The effect for the oxidation stability of diglyceride was TBHQ> VE>citricacid.（5） In the orthogonal experiment, the optimal amount of TBHQ, VE and citricacid were150mg/kg,250mg/kg and500mg/kg, respectively. The stability ofdiglyceride can increase5times.