【作者】 孟祥河；

【导师】 毛忠贵；

【作者基本信息】 江南大学 ， 发酵工程， 2004， 博士

【摘要】 甘油二酯(DAG)是常见的表面活性剂,其优良的可生物降解特性以及无臭、无味、无毒、无腐蚀及非离子特性是其广泛应用的主要原因。同时DAG也是天然植物油脂的微量成分及体内脂肪代谢的内源中间产物。它是公认安全(GRAS)的食品成分,可用于面团增强剂、润滑剂、脱模剂、表面装饰剂、增稠剂、稳定剂及组织形成剂等(FDA,2000)。此外近年来发现膳食DAG具有降低内脏脂肪、抑制体重增加、减少血脂的作用,因而受到广泛的关注。然而,长期以来DAG的大规模生产还主要依赖于有机化学、化工过程。化学方法通常是利用碱性催化剂在高温下进行反应,其缺点是耗能高,产品易褪色,常会出现苦味及反应缺乏专一性,而且由于化学催化剂不易降解、有毒使得产品在食品、化妆品及制药方面的应用受到限制。酶法由于具有专一性强、反应条件温和及环境友好等优点日益受到人们的重视,因此为DAG的合成提供了有效的替代方法。研究开发了一个新颖、有效的酶法合成DAG的生产过程。生产中直接以非吸附的甘油、脂肪酸为起始原料,操作方便,时间短,产率高,而且产物组成简单,利于下游纯化工艺。合成反应中,生物催化剂的选择是至关重要的。酶筛选结果表明Lipozyme RMIM、Novo435不仅酯化活力高,而且可反复利用,用于生产DAG是较合适的。结合考虑位置专一性及成本经济性,实验选择Lipozyme RMIM作为优化的对象。酯化特性研究表明Lipozyme RMIM在40℃优先作用SCFA、MCFA,而60℃对LCFA选择性更强。醇底物选择性研究发现Lipozyme RMIM对伯羟基要优于仲羟基,而对叔羟基或芳环上的羟基活力很低或不起作用。无溶剂体系中,最适水分活度为0.11,当Aw≥0.54时,酯化活力明显降低。非水相中酶活力研究表明,异辛烷是较佳的反应介质,然后依次为正庚烷、正己烷、丁酮、丙酮。为监测酯化反应中间过程及分析终产品,分别采用TLC、HPLC/ELSD、HPLC/RID对甘油酯产物进行分离、定性定量分析。结果表明1,3-DAG与1,2-DAG异构体分离良好。实验详细研究了间歇反应条件下,无溶剂体系中Lipozyme RMIM催化亚油酸、甘油酯化反应,以及各因素对酯化产物组成的影响。并采用二次旋转组合设计对最适条件进行了优化,对因素的重要性作了适当的评价,并给出了拟合良好、回归显著、可靠性具佳的经验性模型方程。研究表明真空脱水可有效增加亚油酸的转化率,使反应平衡向合成方向移动。为减少能耗、降低脱水成本,成功地采用离子交换树脂吸附替代真空减压脱水,效果良好。<WP=11>不同的反应温度、底物摩尔比、酶添加量等热力学因素条件下的酯化进程曲线对比研究表明,温度、底物摩尔比、酶添加量不仅影响酯化反应的初速度、转化率,还与产物组成密切相关。响应面优化结果表明63℃、亚油酸甘油摩尔比为2.1,固定化酶添加量为18EU/g底物,0.01MPa真空脱水,反应8hr亚油酸酯化率最高可接近100%。三因子对亚油酸转化率、1,3-DAG/1,2-DAG比例的影响大小依次分别为:酶添加量>温度>底物比例;温度>底物比例>酶添加量。动力学因素研究表明,多相酯化反应中传质至关重要。添加适当的乳化剂,可明显增加底物接触面积,改善传质,提高反应初速度,减少反应达到平衡所需时间,但不会改变反应平衡。本实验中单甘酯是最佳的乳化剂,添加量为0.3%。适当的搅拌会改善传质,增加脂肪酸、甘油两相的接触,加快反应速度。试验发现雷诺准数小于10000时,外部传质是主要的限速步骤;当雷诺准数大于10000后,内扩散及酶催化反应速度是主要限速步骤。而且搅拌速度过高,可能会对固化酶造成机械损伤,影响酶活。在雷诺准数为10000左右时,内外传质及催化反应速度达到统一,因而反应速度最快。通过对实验条件下酶操作稳定性的研究,给出了Lipozyme RMIM的失活动力学方程及时间、产量关系模型。采用Matlab对实验数据进行拟合,求得Lipozyme RMIM的半衰期为594小时,酶失活常数Kd为0.001062hr-1,生产能力为2000-2500g产物/g酶。两次采用不同的方法拟合误差很小,仅为2.26%,而且根据模型求得预测值与真实值拟合良好,模型是极为有效的。实验进一步研究了填充柱反应器中酶催化亚油酸、甘油连续酯化反应的影响因素。发现填充柱内径,亚油酸、甘油的比例,反应温度,进料速度不仅影响酯化反应的转化率、反应速度,还影响酯化产物中各组分的比例。通过对数据的分析确定固定化酶填充柱长径比7.8(id16mm, L125mm),亚油酸、甘油摩尔比1:2,进料速度1.5ml/min(保留时间12min),65℃条件下酯化反应可实现最终的产量、产品纯度及生产效率三者的统一。填充床反应器中固定化酶连续催化酯化反应的一个主要问题即体系水分清除困难。实验研究了采用过量甘油吸附脱水的可行性,模拟试验表明甘油的干燥脱水作用良好。稳定性研究确定65℃条件下,亚油酸、甘油摩尔比为1:2,进料速度1.2ml/min,可明显提高酯化反应的转化率,改善固定化酶的稳定性,增加Lipozyme RMIM的使用寿命。连续运行10天,残余酶活仍保持在80%以上,而对照组则仅为52%。对酯化的产品,尝试了采用液液萃取方式清除单甘酯的研究。实验发现乙醇水溶液可方便地萃取产品中的单甘酯,萃取的选择性随温度增加、载荷量的减少、水比例的加大而增加。载荷量增大,萃取效率增加但选择性下降。多段萃取可实现萃取效率更多还原

【Abstract】 Diacylglycerols (DAGs) are well known as bio-surfactants. Their excellent biodegradability as well as the behaviors of tasteless, odorless, nontoxic, non-irritant and non-ionic has been paid more and more attraction in numerous industrial fields. It is found that, DAGs are a minor natural component in vegetable oils and fats, as well as an endogenous product of triglyceride metabolism. DAGs are generally recognized as safe as direct human food ingredients and are approved for use in food as dough strengtheners, formulation acid, lubricant and release agents, vehicles, surface finishing agents, stabilizers and thickeners, texturizers (FDA, 2000). In addition, the long-term ingestion of dietary DAG, in contrast to TAG, prevented the accumulation of visceral fat and body weight gain in obesity-prone mice, and decreased postprandial blood lipid levels of rats administration with lipid emulsion. On the other hand, for a long time, large scale’s production of DAG remained mainly in the realms of organic chemistry and chemical processing. Chemical methods are mainly performed at high temperatures in the presence of alkaline catalysts. High energy’s consumption, coloring of products and low selectivity are major disadvantages of these methods. Moreover, some chemicals are toxic and not readily biodegradable, thus causing their limited application in cosmetics, food industry and pharmaceutics. In this case, the enzymatic synthesis offers an alternative way.In the present work, a novel and effective enzymatic method which could eliminate most of the above-mentioned problems, was developed for the production of DAGs. Non-adsorptive glycerol and free fatty acids were directly used as starting materials in order to increase production yield. The selection of biocatalyst is very crucial. It turned out that Rhizopusmucor miehei lipase (Lipozyme RMIM) and Candida antarictica lipase (Novo 435) are the only suitable biocatalysts for the production of 1,3-DAG, not only for their higher esterification activities, but also for the easy reuse. In view of regiospecificity (1,3-specific) and economics, Rhizopusmucor miehei lipase is more suitable than Candida antarictica lipase. Studies on esterification characteristics of Lipozyme RMIM suggested that the selectivity for LCFA is higher than for MCFA and SCFA at 60 degree, but the situation is reversed at 40 degree. The optimum alcoholic substrates for Lipozyme RMIM are primary alcohols, followed by the secondary alcohols, and then tertiary alcohols. However, Rhizopusmucor <WP=14>miehei lipase hardly catalyst phenol hydroxyl group. The optimum water activity is 0.11. When water activity is more than 0.54, the enzymatic activity decrease significantly. Studies on non-aqueous phase suggested that isooctane is the optimum medium, followed by heptane and hexane, and then EMK and acetone, as their polarities are strong enough to peel off essential water for enzyme activity.For monitoring of intermediate process and analysis of products, thin-layer chromatography, normal-, reverse-phase high-performance liquid chromatography were adopted to isolate neutral lipid. Mono-, di-, and tri-acylglycerol were determined with density colorimetric, evaporative light-scatting detector (ELSD) and refractive index detector (RID) individually. It was found that the 1,3-DAGs were evidently resolved from the 1,2-DAG position isomers. Intermediate and final product can be assayed according to methods mentioned above. Esterification reaction of linoleic acid and glycerol catalyzed by Lipozyme RMIM in solvent-free batch system were studied in detail. The conversion rate of fatty acid was obviously affected by reaction conditions such temperature, molar ratio of substrate, enzyme load, emulsifier, agitation and water-removal methods, and so was the composition of esterification products. In order to get high conversion yield and concentration of 1,3-DAG, some important parameters such as substrate ratio (R, acyl donor to glycerol), reaction temperature (T), enzyme load ([E], based on s更多还原