【作者】 孙兆敏；

【导师】 许加超； 薛长湖；

【作者基本信息】 中国海洋大学 ， 水产品加工及贮藏工程， 2010， 硕士

【摘要】 以DHA和EPA为代表的n-3多不饱和脂肪酸在人体的营养、发育和健康等方面起着重要作用。DHA和EPA食用的最好形式为甘油三酯,但是天然甘油三酯型鱼油中DHA和EPA含量较低,本文利用生物酶法催化酯交换反应制备含DHA和EPA含量在45%的甘油三酯,主要研究内容如下：1测定了包括脂肪酶J、U、P、T、B、L、M、N、Lipozyme TL IM和Lipozyme RMIM在内的10种脂肪酶的含水量和水解酶活,并建立了在无溶剂体系中测定脂肪酶酯交换酶活的方法,在此基础上比较了各脂肪酶的酯交换酶活。在10种脂肪酶中,来自褶皱假丝酵母(Candida Rugosa)的脂肪酶M水解酶活最高(为4702.7 U/g),其次为脂肪酶L, Lipozyme TL IM和Lipozyme RM IM的水解酶活分别为356.7 U/g和407.2 U/g。酯交换酶活的测定以正辛酸乙酯和三油酸甘油酯为底物,反应条件为：正辛酸乙酯和三油酸甘油酯的质量比为1：1,脂肪酶添加量为1%(占底物总质量),反应温度为60℃,反应时间为2h。利用该方法测得脂肪酶M的酯交换酶活最高,大约是常用于催化酯交换反应的Lipozyme RM IM的两倍。因此选用脂肪酶M作为酯交换反应的催化剂,制备含45%DHA和EPA的甘油三酯。通过对脂肪酶水解活力和酯交换活力的比较,发现脂肪酶的水解活力与酯交换活力之间的相关性不高。2建立了一种正相高效液相色谱-蒸发光散射检测器联用的方法,用于测定鱼油中甘油酯组成。色谱柱为Rx-SIL型正相硅胶色谱分析柱(250mm×4.6mm i.d.,5μm),柱温为35℃；流动相为正己烷-异丙醇-乙酸(90：10：0.2,V/V),流速为0.8mL/min;蒸发光散射检测器漂移管温度60℃,载气流速1.4 L/min,可在12min内完成一个样品的测定。标准品的色谱峰与进样质量成线性关系,峰面积变异系数小于1.5%,最低检出限为0.02-0.05μg,加标回收率为98.25%-103.08%。该方法可以较为快速准确地测定甘油酯混合物中甘油三酯、甘油二酯和单甘油酯的含量,而不受脂肪酸组成的影响。3利用筛选出的脂肪酶M催化酯交换反应,在无溶剂体系中制备DHA和EPA含量达45%的甘油三酯。首先比较了酯交换反应中酸解和酯-酯交换反应的优缺点,在相同的反应条件下,酸解反应的速度略快于酯-酯交换反应。综合考虑酸解反应和酯-酯交换反应工艺过程的优缺点,选择酯-酯交换法用于制备EPA和DHA总含量在45%的甘油三酯。利用鱼油乙酯和甘油三酯(EPA和DHA含量为28.87%)作为底物,考察了温度(40℃-70℃)、反应时间(6-60h)、加酶量(40-200U)、底物质量比(2：1-1：3)、加水量(0.5%-8%)以及乙酯鱼油中EPA和DHA的含量对酯交换反应的影响,通过正交试验发现,在选定的因素水平范围内,反应时间对酯交换结果的影响最大,其次是加酶量,对酯交换反应影响最小的是底物质量比,得到的较优的工艺条件为：反应温度60℃,反应时间24h,底物质量比为5：4,加酶量为80U,不向反应体系中加入水分。在该条件下酶反应得到的甘油三酯中EPA和DHA的含量分别为32.40%和13.10%,在该条件下脂肪酶重复利用7次仍能达到工艺目标。通过对底物与产物中EPA和DHA的含量的变化可推断出在无溶剂体系中,脂肪酶M对EPA的选择性大于DHA。更多还原

【Abstract】 n-3 polyunsaturated fatty acids, such as ecosapeatanolic acid (EPA) and docosahexaenoic acid (DHA), play an important role in human nutrition, health and development. As the natural form of EPA and DHA, the type of triglycerides is the best form to consume, which contains a small quantity of EPA and DHA. This paper aims at preparing triglycerides containing 45% EPA and DHA through transesterification catalyzed by lipases in solvent-free system, which mainly includes:1 The water content and hydrolysis activity of 10 kinds of lipases including lipase J, U, P, T, B, L, M, N, Lipozyme TL IM and Lipozyme RM IM are estimated, and the method of estimating the transesterification activity of lipases in solvent-free system is established, on whose basis the transesterification activity of 10 lipases are determined. The lipase M from Candida Rugosa has the highest hydrolysis activity of 10 lipases mentioned, which is 4702.7 U/g; lipase L from Candida Tropicalis has the second highest hydrolysis activity. Lipozyme TL IM and Lipozyme RM IM have hydrolysis activity of 356.7 U/g and 407.2 U/g, respectively. The reaction of measurement with ethyl caprylate and glycerol trioleate as the substrates is under the conditions as follows:the weight ratio of ethyl caprylate and glycerol trioleate is 1:1, enzyme dosage is 1%, the temperature is 60℃, and the time of reaction is 12h. The lipase M, whose hydrolysis activity is highest, also has the highest transesterification activity, which is about twice of Lipozyme RM IM (usually used as the catalyst of transesterification). So the lipase M is chosen as the catalyst of transesterification to prepare triglycerides containing 45% EPA and DHA. It is also found that the hydrolysis activity and the transesterification activity of lipases are absent of general correlation by comparison.2 A method for the determination of mono-, di-, and triglycerides by a high performance liquid chromatography (HPLC) with evaporative light scattering detector (ELSD) is established. Chromatographic analysis is carried out on a Rx-SIL column (250mm X 4.6mm id,5μm) with hexane- isopropyl alcohol-acetic acid (90:10:0.2, V/V) as the mobile phase. The flow rate is 0.8 mL/min. The drift tube temperature of ELSD and flow rate of carrier gas(nitrogen) are set at 60℃and 1.4 L/min respectively. It takes only 12 min to finish the analysis of a sample. The peak area and quality of sample injected have a linear relationship, and the C.V. is less than 1.5%. The detection limit is from 0.02μg to 0.05μg and the average recovery is 98.25%-103.08%. The method can be used for rapid and accurate determination of mono-, di-, and triglycerides, despite of the composition of fatty acids.3 The chosen lipase M is used as the catalyst of transesterification to prepare triglycerides which contains 45% DHA and EPA totally. The advantages and disadvantages of acidolysis and interesterfication are compared and interesterfication is chosen although acidolysis has a little faster reaction speed. Transesterification is carried out with fish oil ethyl esters and triglycerides (containing 28.87% EPA and DHA) as substrates. The effect on transesterification of temperature (40℃-70℃), reaction time (6-60h), the enzyme dosage(40-200U), substrate weight ratio (2:1-1:3), water dosage (0.5%-8%) and the content of EPA and DHA in ethyl esters are investigated, and it can be conclude through orthogonal experiment that reaction time has the greatest effect on transesterification, which is followed by enzyme dosage, the substrates weight ratio has the least effect. The favorable conditions obtained are: reaction temperature 60℃, reaction time 24h, the substrate weight ratio 5:4, enzyme dosage 80U, without adding water to the reaction system. Under these conditions, the triglycerides obtained contain 32.40% EPA and 13.10% DHA, and the immobilized enzyme can be used seven times. It is found that lipase M favors EPA by comparison between the content of EPA and DHA in products and substrates.更多还原