Enzyme catalysed production of phospholipids with modified fatty acid profile

Phospholipider har stor anvendelse i levnedsmiddel-, kosmetik-, og farmaceutiske produkter for blandt andet deres emulgerende egenskaber samt evne til at danne liposomer. Interessen for at ændre på phospholipidernes struktur er stigende. Strukturændringer resulterer i ændret funktionalitet. Ved udskiftning af fedtsyrerne med andre ikke naturligt forekommende er det muligt at forbedre fysiske og kemiske, ernæringsmæssige, farmaceutiske og medicinske egenskaber. Indtil nu har der været relativt få forsøg på at optimere de funktionelle egenskaber ved at ændre fedtsyresammensætningen. Målet med projektet var at udvikle processer baseret på enzymatisk interesterificering til produktion af phospholipider med specifik fedtsyre profil (strukturerede phospholipider), og opsætning af membrane separationssystemer til oprensning af strukturerede phospholipider efter reaktion. Produktionen af strukturerede phospholipider blev udført i både ”packed bed”- og batchreaktorer med eller uden tilstedeværelse af solvent (organisk opløsningsmiddel). Under den praktiske udførelse af reaktionerne kan dannelsen af biprodukter, grundet sideløbende hydrolyse og acyl migrering, være et alvorligt problem. En grundig belysning af disse problemer og optimering af diverse reaktionssystemer blev udført i dette projekt. Til produktion i større skala er det praktisk at udføre reaktionerne i ”packed bed” reaktorer, da det tillader en kontinuerlig proces. Kontinuerlig proces i ”packed bed” reaktor blev dog vist at være vanskelig med et solvent frit system. En lang reaktionstid kombineret med hurtig deaktivering af enzymet gør processen ufavorabel. Et solvent system virker tilsyneladende til at være et godt valg for reaktionen, da høj inkorporering og udbytte opnås. Til adskillelse af strukturerede phospholipider fra fedtsyrer blev der under dette arbejde udviklet en ”downstream” proces, som involver ultrafiltrering. I apolære solventer har phospholipider tendens til a danne ”reverse micelles”, som kan adskilles fra fedtsyrer og solvent ved anvendelse af passende membraner. Ydermere blev fysiske egenskaber af specifikke strukturerede phospholipider undersøgt i emulsioner og liposom formuleringer. Projektet blev udført af Anders F. Vikbjerg ved BioCentrum-DTU under ledelse af Lektor Xuebing Xu. Andre deltagere af projektet var Huiling Mu (BioCentrum-DTU), Gunnar E. Jonsson (Institut for Kemiteknik, DTU) og LiPlasome Pharma A/S. This project is mainly a study on the enzyme catalyzed production of phospholipids with modified fatty acid profile (structured phospholipids). Besides production of structured phospholipids, membrane purification of structured phospholipids, and properties of selected structured phospholipids in emulsions and liposome formulations were also studied. Replacements of existing fatty acids in natural soybean phospholipids with others not natural occurring, were done by acidolysis using different commercial microbial lipases and porcine pancreatic phospholipase A2 (PLA2). Lipases were used for modification of sn-1 positioned fatty acids of the phospholipids, whereas PLA2 was used for modification of the sn-2 positioned fatty acids. Reactions were performed in both packed-bed and batch reactors with or without the presence of organic solvents. Effects of different reaction parameters, on primary- and side reactions, were examined for various reaction systems. TLC-FID method was developed during this work to assist the evaluation of product and byproduct formations. The incorporation of desired fatty acids into phospholipid and recovery in batch reactors was affected by enzyme load, reaction time, reaction temperature, water content, substrate molar ratio and solvent amount. Influence of temperature and substrate ratio seemed to depend on the particular reaction system. In solvent systems using immobilized Thermomyces lanuginosa lipase incorporation of desired fatty acid increased with increased temperature (35-55°) and substrate molar ratio (3-15 mol/mol), whereas in solvent free system using immobilized Rhizomucor miehei lipase, incorporation decreased with increase of these parameters in similar range. During PLA2 catalyzed acidolysis reaction, substrate ratio showed no effect on incorporation or yield, and maximum incorporation was observed at 45 °C. Individually, water content showed no effect on the incorporation in solvent-free system during lipase-catalyzed reactions; however it had significant effect during reactions involving PLA2. With both types of enzyme, the recovery of diacylphospholipids decreased with increase of watercontent due to parallel hydrolysis. Presence of solvent improves mixing in the system, and makes subsequent removal of enzyme more convenient; however increasing amounts of solvent was shown to reduce recovery of phospholipid more strongly than it increased fatty acid incorporation during batch operation. During lipase-catalyzed acidolysis reaction between phosphatidylcholine (PC) and acyl donor, the formation of glycerophosphorylcholine (GPC), the presence of acyl donor in the intermediate lysophosphatidylcholine (LPC) and migration into the sn-2 position of PC were observed, which are consequences of acyl migration. GPC formation increased especially with increasing water content in the reaction system; whereas incorporation into LPC and migration into sn-2 position increased with reaction time. Acyl migration should be minimized in the reaction system in order to achieve a high product yield and purity. Production of structured phospholipids in packed bed reactors was affected by the same reaction parameters tested during batch operation. Continuous operation in packed bed reactor was very difficult with a solvent free system. A long reaction time combined with rapid deactivation of the enzyme makes the process unfavorable. Solvent system seems to provide good choice for acidolysis reaction, as high incorporation and yields are achieved. Recovery of diacylphospholipids is considerably higher when reactions are performed in packed bed reactors as compared to batch operation. For the separation of structured phospholipids from free fatty acids, a downstream process involving ultrafiltration was developed during this work. In non-polar solvent phospholipids tend to form reverse micelles, which can be separated from free fatty acids and solvent by using appropriate membranes. Different commercial membranes with different cut-off values were screened in dead end operation. Polysulphone membrane with polyester support showed some good qualities in terms of flux and selectivity. Multiple steps with dilution of retentate to minimize the viscosity and fouling were done to improve the separation. Membrane performance was shown to be very dependent on the initial feed concentration, concentration factor in each step and applied pressure. Two individual studies were made to examine the physical and chemical properties of specific structured phospholipids. In the first study, the ability of enzymatically synthesized structured phosphatidylcholine containing caprylic acid to form and stabilize oil-in-water emulsions, prepared with different triacylglycerols, was examined and compared with natural soybean PC and deoiled lecithin. In the other study, oxidative properties of structured phospholipid containing highly unsaturated docosahexaenoic acid were examined in liposome formulations. The two studies demonstrate the potential usages of structured phospholipids, which have properties differing from natural soybean phospholipids. Overall, the study provides detailed information for practical application of enzyme catalyzed acidolysis of structured phospholipids including down stream processing, and property evaluation of specific structured phospholipids.