Study On Enzymatic Synthesis And Purification Of Medium/Long Chain Fatty Acids Triglycides

Cooking oil can provide heat energy and essential fatty acids for human. It caneffectively promote the absorption of fat-solibe vitamins. But with the increasingof the living standard, the intake of hidden fat (animal food, high fat foods) isincreasing in people’s diet cooking oil, in addition to normal cooking oil. Incidenceof related disease such as adiposity, hypertention and hyperlipaemia is higher andhigher. So study on the function structure directional catalytic reorganization ofcooking oil becomes one of the hot topics. Medium/Long Chain Fatty AcidsTriglycides (MLM-type structured lipids), synthesized by a replacement of originalfatty acids in rapeseed oil with medium chain fatty acid，can supply low energy incomparison to rapeseed oil. Therefore, they are widely popular with consumers.In this paper, the synthesis of MLM-type structured lipids was obtained bylipase-catalyzed reaction of rapeseed oil with caprylic acid by one-/two-stepenzymatic process. After the technique of refinement of MLM-type structuredlipids with molecular distillation, the optimum conditions of reaction, the gaschromatographic method of analysing the fatty acid composition and content ofrapeseed oil was established, with which the analysis and evaluation on assessmentcriteria of MLM-type structured lipids were conducted. The main research resultsare as follows:(1) A gas chromatographic method for efficient analysing the fatty acids inrapeseed oil was developed and methyl caprylate was selected as the externalstandard substance according to the order of fatty acid methyl esters’ peaks. Thearea normalization method was adopted to calculate the content of fattyacids. Recovery and precision tests were suggested that the repeatability and theaccuracy of this method were both well. This method is simple, fast and accuratefor synthesis and purification of MLM-type structured lipids. (2) With the reponse surface methodology, the optimized conditions forone-step lipase-catalyzed acidolysis of rapeseed oil with caprylic acid were molarratio of rapeseed oil to caprylic acid1:3.57,10.57%of the total weight ofsubstrates for immobilized lipase,5.2%of the total weight of immobilized lipase,reaction12h at48.6℃，the caprylic acid incorporation reached23.19%, which wasclose to the the model prediction25.77%. The results showed that the process ofone-step lipase-catalyzed acidolysis of rapeseed oil with caprylic acid to produceMLM-type structured lipids tended to be mild and simple. The product was easy toseparate. It was a kind of environment friendly technology, but the caprylic acidincorporation was lower.(3) The product obtained by one-step lipase-catalyzed acidolysis was purifiedby two-step molecular distillation. The optimum distillation process parameters bythe orthogonal experiment design were showed as follows: the feed materielquantity1mL/min, temperature155℃, Rotating speed150r/min, pressure0.4-0.6Pa. Under this condition, the purity of MLM-type structured lipids reachedto93.11%, and acid value was1.28mgKOH/g. Other quality indexes were peroxidevalue4.32mmol/kg, saponification value202.17mgKOH/g, iodine value95.75g/100g. MLM-type structured lipids was less lighter than rapeseed oil, butyellower than rapeseed oil.(4) During two-step enzymatic process of MLM-type structured lipids, theoptimum alcoholysis process conditions gained by the orthogonal experimentdesign based on single factor experiment was shown as the follows：the molar ratioof rapeseed oil to anhydrous ethanol1:5, the amount of lipase7%(w/w ofsubstrates)，the reaction was carried out at35℃for18h，total percent of MAGsreached to38.82%. The purity of MAGs in alcoholysis products was90.76%through third-step molecular distillation. According to the results of orthogonalexperiments based on single factor experiment, the optimum conditions foresterification were the amount of Lipozyme RM IM lipase9%(w/w of substrates),the molar ratio of MAGs to caprylic acid1:6, temperature35℃, reaction time14h.U n d e r t h i s c o n d i t i o n, c a p r y l i c a c i d i n c o r p o r a t i o n w a s u p t o60.48%.(5) After two-step molecular distillation, the purity of the product obtained bytwo-step lipase-catalyzed reaction reached to91.52%, and acid value was2.35 mgKOH/g. Other quality indexes were peroxide value4.47mmol/kg, saponificationvalue246.65mgKOH/g, iodine value86.15g/100g. MLM-type structured lipids wasclose to rapeseed oil, but yellower than rapeseed oil. So MLM-type structuredlipids comply with the Grade4standards of the state for rapeseed oil at least byquality indexes analysis.