【作者】 刘园；

【导师】 苏文金； 倪辉；

【作者基本信息】 集美大学 ， 微生物学， 2011， 硕士

【摘要】 维生素A是一种脂溶性维生素,是人体必需的营养素之一。但维生素A不稳定,且对皮肤有刺激性。在动物z来源的食品中,维生素A的主要存在形式是维生素A酯,最常见的是维生素A棕榈酸酯,它们在小肠内被转化成维生素A。目前,市售维生素A棕榈酸酯都是化工合成,能耗高、污染多,这都将限制化工合成在现代经济中的应用。因而,建立一套条件温和且污染少的酶法制备维生素A棕榈酸酯的工艺有重要意义。本论文主要目的是初步建立一套酶法制备维生素A棕榈酸酯的工艺。利用Novozym 435脂肪酶催化维生素A醋酸酯和棕榈酸合成维生素A棕榈酸酯,系统研究了合成条件对分批式反应影响,并利用中心组合设计优化了影响反应的重要因素温度和时间,初步建立了一套填充柱式酶合成维生素A棕榈酸酯工艺和从酶反应体系中分离纯化维生素A棕榈酸酯的方法。在分批式反应工艺中,有机溶剂的极性对反应有影响,疏水性强的溶剂更有利于维生素A棕榈酸酯的合成。在维生素A醋酸酯浓度一定的前提下,棕榈酸存在影响反应的上限浓度。整个反应进程中,合成会逐步趋于稳定。固定化脂肪酶有饱和加入量。随着底物浓度的升高,转化率呈负幂指数形式降低,用黑箱模型拟合的乘幂函数模型能更准确地描述底物浓度对反应初速度的影响。初步确定了固定化脂肪酶Novozym 435转酯化合成维生素A棕榈酸酯的工艺条件:石油醚为反应溶剂,棕榈酸与维生素A醋酸酯摩尔比为3:1,底物浓度0.0042 g/mL,酶用量为维生素A醋酸酯质量的20%,反应时间6 h,转化率可达91%。中心组合实验结果说明温度和时间存在明显的交互作用,拟合的模型能很好地描述转化率与温度和时间的关系。初步建立了一套填充柱式反应工艺:维生素A醋酸酯浓度0.015 g/mL,棕榈酸与维生素A醋酸酯摩尔比2:1,温度30℃,反应液流速0.13 mL/min/ mL,转化率达到90.9%,且工艺稳定性良好,连续反应7批次,转化率仍在87%以上。放大后工艺能达到小试水平,且稳定性很好。就底物浓度对转化率的影响进行数学模型拟合,结果表明,随着底物浓度升高,转化率呈直线下降。初步确立了一套从酶反应体系中分离纯化维生素A棕榈酸酯的方法。反应液先在4℃下放置20 h,然后在-20℃放置8 h,除去过量的棕榈酸。利用液-液萃取分离技术,以84%乙醇/水:石油醚=3:1为萃取溶剂,萃取温度为4℃,连续萃取4次,维生素A棕榈酸酯纯度达到98.3%,这已经满足食品级维生素A的纯度要求,此时回收率为65.2%。更多还原

【Abstract】 Vitamin A, which is one of the nutrients necessary to human, is a kind of fat-soluble vitamin and is unstable and irritating to skins. In foods of animal origin, the major form of vitamin A is an ester, primarily retinyl palmitate, which is converted to the retinol (chemically an alcohol) in the small intestine. At present, commercial retinol palmitate is synthesized by a chemical process, which would be restricted in application due to its high energy consumption and more pollution in modern economy. Thus it is important to construct a process of lipase-catalyzed synthesis of retinol palmitate with mild conditions and less pollution.The main purpose of the thesis was to establish a new enzymatic process by using Novozym 435 lipase that catalyzes the synthesis of retinol palmitate through ester exchange reaction between retinyl acetate and palmitic acid. At first, we studied the effects of synthetic conditions on batch reaction and optimized the important influence factors (temperature and reaction time) using a central composite design. Then we primitively established the process of ester synthesis of retinol palmitate in a packed column reactor. Furthermore, an effective method was developed to separate and purify retinol palmitate from the reaction system.In batch reaction process, the polarity of organic solvents had effect on synthesis reaction, and the strong hydrophobic solvent was beneficial to synthesis. In the premise of fixed concentration of Vitamin A acetate, there was a threshold level of palmitic acid concentration to affect reaction. Throughout the reaction process, the synthesis tended to be stable gradually. It had a saturated enzyme dosage. With the concentration of retinol acetate increased, the conversion rate decreased in the negative exponent form. The power function fitted by black-box model could depict relation between the initial reaction rate and substrate concentration well. The optimized reaction conditions of Novozym 435 lipase-catalyzed synthesis of retinol palmitate: petroleum ether used as reaction media, the molar ratio of palmitic acid to retinol acetate was 3:1 with 0.0042 g/mL retinol acetate, the enzyme dosage accouted for 20% of Vitamin A acetate dosage, the conversion rate could reach to 91% in 6 hours. As illustrated by the result of central composite design experiment, there had visible interaction between temperature and reaction time, the fitted model could describe relation between conversion rate and temperature, reaction time well.The established chromatography column process was that the molar ratio of palmitic acid to retinol acetate was 2:1 with 0.015 g/mL retinol acetate. The column temperature was kept at 30 ℃with a flow rate at 0.13 mL/min/mL. Under these conditions, the highest conversion rate reached to 90.9% with satisfied stability. In all the 7 batches of continuous reaction, the conversion rate was not lower than 87%. The further amplified process showed that it could reach to the small-scale level with good stability. The mathematical model relation between conversion rate and substrate concentration was fitted, the result showed that with substrate concentration increased, conversion rate decreased in the linear form.A new method of separation and purification of retinol palmitate from enzyme catalyzed reaction system was built. In order to eliminate the excessive palmitic acid, the reaction solution was firstly placed at 4℃for 20 h and then changed the temperature to -20℃for another 8 h, In liquid-liquid extraction, 84% ethanol/distilled water: petroleum ether (3:1) was used as extraction solvent. After 4 consecutive extractions at 4℃, the purity of retinol palmitate reached to 98.3% with recovery rate of 65.2%. This purity is complete accord with needs of food-grade Vitamin A.更多还原