【作者】 王艳；

【导师】 辛嘉英；

【作者基本信息】 哈尔滨商业大学 ， 食品科学， 2013， 博士

【摘要】 中长链脂肪酸淀粉酯是一类新型的化学改性淀粉,因其具有较好的粘度、透明度、疏水性、乳化性、冻融稳定性、抗凝沉性和可生物降解性,它的合成现已成为变性淀粉工业中的一个新兴研究热点。但是在目前的研究中,中长链脂肪酸淀粉酯的合成主要采用化学法。由于化学法所需的极端pH值、高温高压高机械条件以及有机溶剂的加入,不仅造成了环境污染和能源浪费,同时危害到人体健康,因此大大限制了其发展与应用。所以,开展生物法合成中长链脂肪酸淀粉酯的研究,生产纯天然、绿色更适合应用于食品、医药以及精细化学品等行业的中长链脂肪酸改性淀粉具有十分重要的现实意义。目前主要在有机溶剂介质中进行酶催化法制备中长链脂肪酸淀粉酯,有机溶剂的使用不仅限制了产品的应用,同时也对酶的催化活性产生一定的抑制作用。发展至今,限制酶促中长链脂肪酸淀粉酯合成的关键问题主要包括：淀粉颗粒结构紧密、活性羟基深藏在分子内部、淀粉和中长链脂肪酸的极性相差较大不易混溶,这就使酶促酯化反应很难进行。为解决以上问题,本论文主要从淀粉的预处理活化、酶的筛选、酰化试剂的选择和反应体系的建立等方面进行深入研究。以天然玉米淀粉为原料,选用各种碳链长度的中长链脂肪酸为酰化剂,脂肪酶为催化剂,在无溶剂体系中进行中长链脂肪酸淀粉酯的绿色合成,并对处理淀粉和各中长链脂肪酸淀粉酯的理化性质进行分析。通过对酶促反应条件的优化、反应机理的探索和反应动力学模型的建立,以棕榈酸为例,揭示酶促玉米淀粉与中长链脂肪酸发生酯化反应所需的必要条件和一般规律。本研究所获得的实验数据不仅将补充和完善现有非水相体系酶催化反应理论,同时也为中长链脂肪酸淀粉酯的实际生产和应用提供一定的理论依据。主要研究内容和结论如下：1、玉米淀粉的预处理活化及对酶促酯化反应的影响。为解决原玉米淀粉颗粒结构紧密不易与中长链脂肪酸发生酯化反应的难题。用NaOH/尿素水溶液在低温条件下对玉米淀粉进行预处理。选择碳链长度适中、熔点在酶最适反应温度范围内的棕榈酸作为中长链脂肪酸的代表性底物,以酶催化预处理淀粉与棕榈酸发生酯化反应的酶促反应初速度和产物取代度为考察指标,获得玉米淀粉处理过程的重要参数值为：氢氧化钠/尿素水溶液的浓度为9%、氢氧化钠与尿素的质量比为2、氢氧化钠/尿素水溶液的预冷温度为-9℃、乙醇的添加量为50%。在此条件下淀粉的冷水溶解度高达96.42%,平均颗粒直径小于0.10μm,结晶度由原来的24.82%降低至10.32%,此时酶的催化反应速度最大(0.34mmol·h-1·mg-1)比催化原玉米淀粉发生酯化反应的速度高出4个数量级,并且棕榈酸预处理淀粉酯的取代度(0.82)明显高于原玉米淀粉酯(0.23×10-2)。原玉米淀粉经预处理后,使酶促酯化反应活性提高的机理在于：预处理后淀粉颗粒粒径越小酶活性中心对其的识别能力越强；结晶度越小活性羟基暴露的越完全,越易进入到酶周围的水化层与分布在水化层表面的棕榈酸发生酯化反应；从而使酶促酯合成的反应速度加快,产物取代度变大。2、甲醇醇解-气相色谱分析测定中长链脂肪酸淀粉酯取代度方法的建立。甲醇醇解-气相色谱分析测定取代度的方法分为两部分：其一,中长链脂肪酸淀粉酯的甲酯化,甲酯化的程度在取代度的测定中起到至关重要的作用。以棕榈酸淀粉酯为例对甲酯化的条件进行了优化,并以棕榈酸淀粉酯的测定值与真实值的拟合度为指标,获得最佳甲酯化条件为：甲醇用量2mL/30mg、甲醇钠用量8mg/30mg、甲酯化温度为70℃、甲酯化时间为40min；其二,脂肪酸甲酯在气相色谱仪中的定量测定,以棕榈酸淀粉酯为例,首先将棕榈酸甲酯的量转化成与淀粉发生酯化反应的棕榈酸的量,再通过公式计算出棕榈酸淀粉酯的取代度。该方法与广泛应用的皂化-滴定法相比,误差小、重复性好更适合用于中长链脂肪酸淀粉酯取代度的测定。3、以棕榈酸淀粉酯的酶催化合成为例,构建酶促中长链脂肪酸淀粉酯的合成体系。在所研究的脂肪酶中,Novozym435在非水相体系中表现出较高的催化预处理淀粉与棕榈酸发生酯化反应的催化活性。以棕榈酸为酯化剂,固定化脂肪酶Novozym435为催化剂,分别在无溶剂体系和微溶剂体系下进行预处理玉米淀粉与棕榈酸的酶促酯化反应。并以取代度和酶酯化比活力为指标考察不同反应体系中酶的催化反应活性。在微溶剂体系中,由于高极性有机溶剂夺取了脂肪酶周围的必须水,酶的刚性增强从而使酶酯化比活力下降,因此在无溶剂体系下可以获得取代度高达1.04的棕榈酸淀粉酯,而在微溶剂体系下只能获得取代度为0.72×10-2的棕榈酸淀粉酯。最终选定无溶剂体系作为Novozym435催化预处理玉米淀粉与中长链脂肪酸发生酯化反应的反应介质。4、无溶剂体系酶促中长链脂肪酸淀粉酯的合成。以平均粒径<0.10μm的预处理玉米淀粉为原料,不同碳链链长度的中长链脂肪酸(C8-C20)为酯化剂,固定化脂肪酶Novozym435为催化剂,在无溶剂体系下进行各中长链脂肪酸淀粉酯的合成。研究发现无溶剂体系中,固定化脂肪酶Novozym435对碳链长度在C8-C16之间的脂肪酸均具有很好的催化能力,酶的酯化比活力基本保持在1.30mmol·h-1·mg-1左右。5、以棕榈酸淀粉酯的酶催化反应合成为例,探讨无溶剂体系酶促中长链脂肪酸淀粉酯的合成机理。在无溶剂体系中,底物分子周围没有有机溶剂,因此不会对酶活力产生影响；同时在反应过程中酶与底物直接接触,无需去溶剂效应这一过程而使反应速度加快；通过控制反应体系的水活度不仅可以为脂肪酶提供发挥其催化活性所必需的水,同时也可以改变酶促反应的平衡,避免水解反应的发生。反应体系适度的水活度使固定化脂肪酶表面形成一层均匀连续的、厚度适宜的水化层。由于脂肪酶催化玉米淀粉与脂肪酸的酯化反应属于界面反应,水化层的连续性和厚度直接影响着底物在油水界面的分布和向脂肪酶活性中心的扩散；预处理淀粉分子上的羟基(亲水性)和棕榈酸上的羧基(疏水性)具有相反的极性,因而适宜比例的两底物可以有序地分布排列在固定化脂肪酶分子的表面；从而在酶分子表面形成水分子层和底物分子层的油水界面,这种水分子层和底物分子层的形成不仅有利于底物分子流动,同时油水界面的形成也为脂肪酶发挥其催化活性提供了必要条件；在对无溶剂体系脂肪酶催化玉米淀粉与棕榈酸酯化反应的反应进程研究中发现,反应速度出现两次突然加快的现象,第一次加速主要是由于底物最大程度的克服了体系中的外扩散和内扩散限制从而使酶促反应速度达到最大；而第二次加速是由于棕榈酸淀粉酯的生成和积累使其发挥了良好的表面活性剂效应,有利于脂肪酶周围油水界面的形成和底物的分布与排列。同时,棕榈酸淀粉酯具有疏水性有助于其生成后迅速脱离酶的催化活性中心,从脂肪酶表面的水化层逃离出来,从而促进酶促酯化反应的进行,避免水解反应的发生,提高酶催化酯化反应活性使反应速度突然加快。产物棕榈酸淀粉酯的HLB值和取代度对脂肪酶Novozym435的催化活性具有一定的影响,产物的HLB(亲水亲油平衡值)越小,取代度越高酶促反应的初速度越大,其对酶酯化活性的促进作用越强。6、以棕榈酸淀粉酯的酶催化反应合成为例,对无溶剂体系酶促预处理玉米淀粉与中长链脂肪酸发生酯化反应的动力学进行研究。对无溶剂体系中影响酶催化活性的条件进行优化,最佳条件为：底物摩尔比1：5；温度65℃；时间24h；酶用量5%；转速180r/min;初始水活度为0.57。在该反应条件下无底物扩散限制,因此可以采用底物摩尔数与反应初速度的关系对反应动力学模型进行推导。研究表明,无溶剂体系Novozym435催化预处理玉米淀粉与棕榈酸的酯化反应符合乒乓反应机制。反应动力学模型为V=(1.7350×Cfatty-acid×Cstarch)/(Cfatty-acid×Cstarch+0.0156×Cstarch+2.3947×Cfatty-acid)。酶促反应顺序为：酰基供体棕榈酸(A)首先与酶结合形成棕榈酸-酶复合体(EA),EA再转化成棕榈酸酰基-酶复合体(EI),此时释放H20(Q)。然后EI再与酰基受体预处理玉米淀粉形成另一个二元复合体(EIB),由于EIB不稳定,最终释放出棕榈酸淀粉酯(P)以及酶。7、预处理淀粉及各中长链脂肪酸淀粉酯理化性质分析。原玉米淀粉经NaOH/尿素水溶液处理后其冷水溶解度和淀粉乳的透明度、凝沉稳定性、冻融稳定性提高,但粘度下降。预处理淀粉经酯化改性后,使其具有良好的乳化性和乳化稳定性,且乳化效果明显好于明胶和蔗糖酯,但与单甘脂相似。同时进一步提高了淀粉乳的粘度、凝沉稳定性和冻融稳定性。与辛烯基琥珀酸淀粉酯和醋酸淀粉酯相比,低取代度中长链脂肪酸淀粉酯的乳化性和冻融稳定性要优于辛烯基琥珀酸淀粉酯和醋酸淀粉酯。且随取代度的升高,乳化性先增强而后降低到40%左右,而冻融稳定性则随取代度的升高而升高。在对中长链脂肪酸淀粉酯乳化性的评价中得出,随中长链脂肪酸淀粉酯质量浓度的增加其乳化性和乳状液稳定性增强；随乳化油量的增多,乳状液稳定性下降；贮存温度越高,乳状液越不稳定；中长链脂肪酸淀粉酯乳状液具有降低油-水界面张力的能力,并随其质量浓度的升高而升高。但在不同油水界面产生的界面压力不同,经实验发现在橄榄油-水界面产生的界面压比在大豆油-水中产生的界面压大。更多还原

【Abstract】 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.更多还原