Enzymatic Synthesis And Properties Analysis Of Medium-long Chain Fatty Acid Starch Ester

Chemical modification starch is often required to better suit its properties to specific applications. Many reports exist in literature pertaining to the preparation of starch esters or its components with the ultimate aim of significantly modifying the physical-chemical properties of starches and imparting suitable mechanical characteristics so as to render them more useful as engineering materials than the native starch. Although the introduction of an ester group into starch is an important chemical modification task, it is very difficult to synthesize high substituted starch derivatives, mainly because of the almost impossible proposition of dissolving granular starch in a suitable medium. Sophisticated experimental techniques and systems of solvents are used to achieve a homogeneous reaction medium for modification of the chosen starch. Unlike chemical esterification modification, an enzymatic one is an environmentally friendly method which occurs under milder conditions. The use of lipase as catalyst for ester production has a great potential. In fact, using a biocatalyst eliminates the disadvantages of the chemical process by producing very high purity compounds with less fewer or no downstream operations. However, the intact starch granules inhibit medium-long chain fatty acids from making contact with the molecules in the crystalline region, thus the chemical reactivity and reaction efficiency of native starch is usually low. And the starch and medium-long chain fatty acids are very difficult to immiscible, because of their polarity are opposite. So the enzymatic esterification of starch with medium-long chain fatty acid is extreme difficult to conduct. The aim of this work was to modify the structure in the crystalline region, or decrease the size of crystalline regions to increase reaction activity of starch and biosynthesize medium-long chain fatty acid esters of starch (MLFES) using lipase Novozym435as catalyst in non-aqueous system. The main contents and conclusions were as followed:In order to improve the esterification activity of native corn starch (NS), NS was pretreated by using NaOH/urea/HbO solution. The optimum pretreatment conditions were the ratio of NaOH to urea is2, the concentration of the NaOH/urea/H2O solution is9%, the amount of ethanol is50%, the pre-cooling temperature is-9℃, and the concentration of starch is5%. It has been found that the average particle size of pretreatment corn starch (PS) decreased to less than0.1Oμm, smaller than those of NS (4-15μm). XRD revealed that crystalline pattern of PS was VH-type, which was different from that of NS (A-type). The effects of pretreatment on esterification activity of corn starches were investigated by analyzing the initial rate of enzymatic esterification and the degrees of substitutions (DS) of the esterification products. The initial rate of enzymatic esterification of PS with palmitic acid was0.34mmol·h-1·mg-1, it was high to four orders of magnitude than enzymatic esterification of NS with palmitic acid. The maximum DS of pretreatment starch palmitate was0.82, while the DS of native starch palmitate was very low and even could not be detected.The methanolysis-GC method was established to determine the DS of MLFES. Transesterification of acetyl groups from MLFES to methanol has been employed, where the resulting methyl acetate was distilled, then analysed with GC. Once the MLFES was quantified, the average mol of acyl groups per anhydroglucose unit was calculated to give the DS of MLFES. The optimum methanolysis reaction conditions were30mg of starch palmitate dissolved in1mL DMSO and mixed with1mL of sodium methoxide (0.07M) in methanol solution, refluxed for40min at70℃. The accuracy and reproducibility of the method was tested by replicate analysis of MLFES that DS was0.20, showing a standard deviation of less than3%.Enzymatic synthesis of starch palmitate was used for as an instance to establishment of the reaction system of enzymatic synthesis MLFES. The activities of a number of commercially available lipases such as Novozym435, PPL and Lipozyme TL IM were evaluated, amongst which Novozym435was found to be the most active. The esterification specific activity of enzyme and the DS of starch palmitate were employed to investigate the enzymatic activity in different reaction system. In micro-solvent system, the DS of starch palmitate was only0.72x10-2, it was distinct lower than the DS of starch palmitate that synthesis in solvent-free system. This mainly because of the essential water of lipase was carried off by high polar solvent that made the rigidity of enzyme increasing. The increase of rigidity of enzyme must result in the esterification specific activity of enzyme weakening. So, the solvent-free system was selected as the reaction medium for enzymatic esterification of PS with medium-long chain fatty acids.Study on the solvent-free synthesis mechanism of MLFES catalyzing by Novozym435. In solvent-free system, the enzyme activity could not be inhibited by solvent and could contact with substrate directly without the process of removing solvent from the surface of enzyme and substrate, so the reaction rate was accelerated. Novozym435could catalyze esterification of PS with medium-long chain fatty acid (Cg-C)6) in solvent-free system, and the esterification specific activity of enzyme was about1.3mmol·h-1·mg-1. The positive factors for enzymatic synthesis of MLFES include suitable aw. right proportion of substrates, the generation and accumulation of MLFES in solvent-free system. The suitable aw (0.57) made the formation of a continuous, uniform and moderate thickness water layer on the surface of immobilized lipase. Two substrates with suitable proportion (1:5) could been arranged on the surface of immobilized lipase orderly, because of the opposite polarity of the hydroxyl (hydrophilic) of PS and the carboxyl (hydrophobic) of palmitic acid. Thus, there was a substrate molecules layer on the water molecules layer of lipase that not only conduced to the flow of substrate molecules, but also contributed to the formation of oil-water interface. The formation of oil-water interface is the necessary condition for lipase to play its catalytic activity. The suddenly acceleration phenomenon happened two times in the enzymatic synthesis process. The first acceleration was mainly due to the external and internal diffusion limitation of reaction system had been overcome completely, so the reaction rate was speed up suddenly. And the second acceleration was mainly because of the generation and accumulation of starch palmitate. Starch palmitate could play a good surfactant role that not only contributed to the formation of oil-water interface, but also affected the distribution and arrangement of substrates on oil-water interface. The HLB (Hydrophile-Lipophile Balance Number) value and DS of MLFES affected the catalytic activity of Novozym435. The smaller HLB value and the higher DS of MLFES could speed up the enzymatic initial rate. The starch palmitate could separate itself from the enzyme catalytic activity site and escaped from the hydration layer of lipase as quickly as it had been generated, because of its hydrophobic. So that also accelerated the enzymatic esterification reaction and avoided the occurrence of hydrolysis reaction.Kinetic studies of esterification reactions catalyzed by Novozym435leading to the synthesis of starch palmitate from PS and palmitic acid in solvent-free system were investigated in detail. Initial reaction rates were determined from kinetic runs involving the molar ratio of substrate (PS:palmitic acid=1:5), the reaction temperature (65℃), amount of lipase (5%), rotate speed (180r/min), initial aw(0.57). Graphical double reciprocal plots showed that the kinetics of the enzyme catalyzed reactions exhibited Ping-Pong Bi-Bi mechanism. An attempt to obtain the best fit of this kinetic model through computer simulation yielded in good approximation, the kinetic equation was v=(1.7350×Cfatty-acid×Cstarch)/(Cfatty-acid×Cstarch+0.0156×Cstarch+2.3947×Cfatt)-acid).The enzymatic reaction order was expounded as follow. First, acyl donor-palmitic acid (A) combined with enzyme (E) into a compound of palmitic acid-enzyme (EA). Then, EA transformed into another compound of palmitoyl-enzyme (EI) and released H2O (Q). Second, El combined with acyl acceptor-pretreatment starch (B) into a dualistic compound (EIB). At last, EIB break up into palmitic acid ester of starch (P) and E, because of the instability of EIB.The physical-chemical properties of PS and MLFES were studied. The crystallinity decrease and hydrophilic group exposure of PS allowed water enter into the interior of starch, that made the improvement of its cold-water solubility and transparency, but the viscosity of PS decreased. In addition, MLFES exhibited higher freeze-thaw stability and retrogradation stability. The introduction of medium-long chain fatty acid endowed starch with better emulsifiability and emulsifiability stability. And the emulsifying effectiveness of MLFES was better than gelatin and sucrose ester but similar to monostearin. Compare with octenyl succinic starch ester and acetic acid starch ester, the emulsifiability and freeze-thaw stability of low DS of MLFES were superior to octenyl succinic anhydride starch ester and acetate starch ester. The results of emulsifiability evaluation experiment showed that the emulsifiability and the emulsification stability of MLFES increased with the increase of its concentration, storage temperature, but decreased with the increase of amount of emulsified oil. The MLFES could reduce the oil-water interfacial tension, and the reduce capacity increased with the increase of its concentration. But, the reduce capacity was different, when applying to emulsify different oil.