Lipase-catalyzed Synthesis Of MLM-type Structured Lipids And Feruloylated Lysophospholipids

MLM-type structured lipids (MLM-SLs) are triacylglycerols with medium chain fatty aicds (MCFAs) on sn-1,3position of glycerol backbone and long chain fatty acids (LCFAs) on sn-2position of glycerol backbone. Comparing with conventional long chain triacylglycerols (LCTs) and medium chain triacylglycerols (MCTs), MLM-SLs can be metabolized easily in the body and provide the essential fatty acid to the body. MLM-SLs have been widely used as medical, functional lipids and nutraceuticals. Chemical transesterification results in desired randomized TAG molecular species. Enzymatic reactions are more specific, require less severe reaction conditions, and produce less waste. Lipases can be used as biocatalyst for producing MLM-SLs. Although enzymes have been used for several years to produce SLs, they have only recently become available for large-scale use in industry, mainly because of the high cost of enzymes and low stability of immobilized enzymes. To overcome these limits, the following works were done.Ferulic acid (4-hydroxy-3-methoxy cinnamic acid, FA) is a phenolic acid that is ubiquitous in grains, vegetables and fruits. FA has shown antioxidant activity, antimicrobial, anticarcinogenic and anti-inflammatory properties. FA was widely used in food, pharmaceutical and cosmetic industries. However, practical application of FA and its derivative was actually limited due to their poor solubility in both lipophilic and hydrophilic systems and low bioavailability. Phospholipids are good emulsifiers, stabilizers and antioxidants, and widely used in food, pharmaceutical and cosmetic industries. In addition, phospholipids are major constituents of cell membranes and play essential roles in biochemistry and physiology of cell functions. Due to the heat sensitivity and oxidation susceptibility of FA, chemical synthesis of the derivatives of FA is limits. Enzymatic synthesis strategy has been of great interest because of the mild operating conditions, the high substrate specificity and positional selectivity. To overcome these limits, lipase-catalyzed synthesis of novel feruloylated lysophospholipids were developed in this study to increasing solubility and stability of FA in hydrophilic media and the biological efficiency in the body. Meanwhile, the reaction media and lipases had been selected, the structure of1-FLP had been characterized and the reaction conditions had been optimized.Acidolysis, the transfer of an acyl group between caprylic acid/capric acid and soybean oil, was used to synthesize MLM-type SLs in solvent-free system. Lipozyme RM IM was chosen as the biocatalyst in this reaction, In this chapter, FE73403would be used as objective formula to synthesis the MLM-type SLs by enzymatic process. The optimal conditions generated were fatty acid molar ratio of4:1(caprylic acid to capric acid), a substrate molar ratio of6:1(fatty acids to soybean oil), a reaction temperature of55℃, an enzyme loading of7wt%(based on total substrates) and a reaction time of4h. Under these conditions, the incorporation of27%of caprylic acid and10%of capric acid could be reached. After molecular distillation, the triacylglycerols concents of the products were more than90%. The average hydroxyl value was9.71. The activation energy (Ea) value of this reaction was21.1kJ/mol. The reaction kinetic values for Km and Vm were2.28mol/L and18.52mol/(Lxmin), respectively.In order to increase the activity of lipases and reduce the reaction cost, the new reaction system had been built. The fatty acid ethyl esters, which was chemically synthesized by myself, were used as acyl donor for producing MLM-SLs. The cheap Lipozyme TL IM was chosen as the biocatalyst in the transesterifiaction between ethyl caprylate/ethyl caprate and soybean oil for synthesis of MLM-SLs in solvent-free system. In this chapter, FE73403would be used as objective formula to synthesis the MLM-SLs by enzymatic process. The optimal conditions generated were fatty acid molar ratio of3:1(ethyl caprylate to ethyl caprate), a substrate molar ratio of6:1(fatty acids to soybean oil), a reaction temperature of60℃, an enzyme loading of13wt%(based on total substrates) and a reaction time of2h. Under these conditions, the incorporation of27%of caprylic acid and10%of capric acid could be reached. The activation energy (Ea) value of this reaction was30.1kJ/mol. The reaction kinetic values for Km and Vm were4.02mol/L and4.94mol/(Lxmin), respectively.The fluidized bed reactor was designed and installed for synthesis MLM-SLs. The cheap Lipozyme TL IM showed similar interesterification degree (ID) and sn-1,3specificity to expensive Lipozyme RM EM in batch reactions. In packed-bed interesterification of soybean oil with MCT catalyzed by Lipozyme TL IM, the residence time had positive correlation with ID, whereas temperature had no significantly effect on ID during45-70℃. The sn-1,3specificity of Lipozyme TL IM was not satisfactory when reaction temperature was higher than60℃. The optimal residence time and temperature were30-40min and55℃, respectively. Solvents including acetone, isopropanol, tert-butanol, isobutanol were used to recover the activity of the used Lipozyme TL IM, and acetone was the most suitable with highest ID (44.38%).The methods of qualitative and quantitative analysis in the reaction of phosphatidylcholine (PC) and ethyl ferulate (EF) were developed. The novel two-step route established in this study was applied into the synthesis of FLPs, which were consisted of two consecutive steps, namely, hydrolysis of PC and transesterification of products of PC’s hydrolysis and EF. The product of1-FLP was gotten by preparative high performance liquid chromatography with high purity (over95%). The purified product was identified as1-FLP by TLC, HPLC, LC-MS and NMR. The optimal biocatalyst and reaction solvent were Novozym435and toluene, respectively.All kinds of single factors were optimized for increasing the FLPs conversion. The results showed that toluene/chloroform could obviously increase the conversion and the optimal combination of toluene and chloroform was90:10(v/v). The optimal conditions generated for FLPs production were a substrate molar ratio of5:1(PC/EF), a PC’s hydrolytic time of1.5h, an enzyme loading of60mg/mL, a solvent dosage of5mL and a molecular sieves concentration (4A) of100mg/mL. Under these conditions,40.51±1.25%of EF can be converted to FLPs during4days. To optimize the reaction conversion, dual response surface method was applied to determine the effects of three-level-five-factors and their reciprocal interactions on FLPs product. The optimal conditions were a substrate molar ratio of5:1(PC/EF), a PC’s hydrolytic time of1.5h, an enzyme loading of63mg/mL, a molecular sieves concentration (4A) of100mg/mL and the temperature of61℃.