Research On Enzymatic Synthesis Of Partial Glyceride And Its Physical And Chemical Properties

This thesis researched on the preparation, separation, purification and physicochemicalproperties of partial glycerides rich in α-linolenic acid, thus providing a theoretical basis ofthe production and application of polyunsaturated diglycerides and monoglycerides. Thecontent of triglycerides, diglycerides and monoglycerides in mixed esters was detected byHPLC-RID method and then calculated the percentage of them in the sample separately. Theoptimum conditions of preparing diglycerides and monoglycerides with glycerol and linseedoil by lipozyme435using single factor experiment and Box-Behnken experiment design waspresented. After purifying the reaction mixture by molecular distillation, high puritydiglycerides and monoglycerides products rich in α-linolenic acid were obtained.1. The determination method of the percentage composition in mixed ester samples waspresented in this paper, and the accuracy and precision of the method were investigated. Theresults showed that the standard curve linear regression equation of triglycerides,1,3-Dilinolenin and Monolinolenin were y1=1.3423x1, y2=1.5722x2and y3=1.4055x3respectively, the corresponding correlation coefficients were0.999,0.998and0.999respectively, showed that the method of detecting triglycerides, diglycerides andmonoglycerides by HPLC-RID, then calculating their percentage in mixed ester sample viaconcentration is very well if the tested substances is in proper mass concentration. Comparedwith the method of calculating weight percentage via peak area ratio directly, the percentageof triglycerides and monoglycerides were0.81%and1.73%higher, the diglycerides contentswas2.54%lower with the method of calculating via concentration, nevertheless，the results ofthe two method are very close to each other.2. The technology of preparing diglycerides by lipozyme435with glycerol and linseedoil under solvent-free systems was researched. Factors research and response surfaceexperiment design were used to assess the effect of substrate molar ratio，reaction time,enzyme load and reaction temperature to diglycerides yield and optimize the preparingcondition. The best condition were as follows: the substrate ratio of linseed oil to glycerol is5:3, add880PLU per gram linseed oil lipase and react at58.3℃for9.1h. Under this condition,the diglycerides content in reaction mixture reached50.21%， its theoretical purity couldreach60.12%. Fatty acid analysis showed that the diglycerides contained high amounts ofunsaturated fatty acids (87.37%) in which the major fatty acid was α-linolenic acid (46.41%).3. The technology of preparing monoglycerides by lipozyme435with glycerol and linseed oil under tert-Butyl alcohol system was researched. Factors research and responsesurface experiment design were used to assess the effect of substrate molar ratio，reaction time,enzyme load，solvent adding and reaction temperature to monoglycerides yield and optimizethe preparing condition. The best condition were as follows: substrate molar ratio of linseedoil to glycerol is1:3, the oil to t-butyl alcohol ratio is39.7%(m/v), add601PLU per gramlinseed oil lipase, and react at56.11℃for10.5h. Under this condition, the monoglyceridescontent in reaction mixture reached71.1%. Fatty acid analysis showed that themonoglycerides contained high amounts of unsaturated fatty acids (89.38%) in which themajor fatty acid was α-linolenic acid (51.36%).4. The suitable molecular distillation temperature for purifying the reaction mixture wasstudied, the results showed that the first time and second time molecular distillationtemperature should be close to120℃and220℃separately, the purity of diglycerides andmonoglycerides in products could reach56.5%and82.3%respectively. The diglyceridesproduct is transparent and no suspension in25℃，has0.13%water and volatile matter,0.17%free glycerol; the acid value, peroxide value, saponification value is2.10mg KOH/g,2.76mg KOH/g and182.60mg KOH/g respectively; the red value and yellow value are4.0and8.3separately detected by lovbind colorimetry (25.4mm). The monoglycerides product ispaste like in25℃，has0.10%water and volatile matter,2.08%free glycerol; the acid value,peroxide value, saponification value is3.20mg KOH/g,0.50mg KOH/g and168.21mgKOH/g respectively; the red value and yellow value are0.1and10.0separately detected bylovbind colorimetry (25.4mm). Both of the diglycerides and monoglycerides product meetthe relevant standards. The oxidation stability research results indicated that the storagestability of diglycerides product is slightly better than linseed oil, whereas worse than soybeanoil, the storage stability of monoglycerides product is worse than soybean oil, diglyceridesand linseed oil. The storage stability of the diglycerides and monoglycerides productcontaining much polyunsaturated fatty acid can be significantly enhanced by addingantioxidants.