ω-3多不饱和脂肪酸甘油酯富集的研究
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摘要
鱼油中ω-3多不饱和脂肪酸(ω-3PUFA)在人体中具有重要的生物学意义,其特征脂肪酸EPA和DHA的生理活性己经明确,EPA具有预防冠心病、降血压、消除疲劳、预防动脉粥样硬化和脑血栓、抗癌等生理活性,DHA能显著地促进婴儿的智力发育,改善大脑机能,提高记忆力,是生产预防心血管疾病和婴儿益智食品的良好基料。前期研究证明ω-3PUFA甘油酯产品在消化吸收和利用方面优于其乙酯和游离型产品,但天然鱼油中ω-3PUFA甘油酯的含量偏低,影响了产品的保健功效,为了提高产品中ω-3PUFA甘油酯的纯度和浓度,提升产品的功能保健价值,本文以甘油和鱼油经水解后分离纯化出来的ω-3PUFA为原料,通过脂肪酶在有机相中催化合成ω-3PUFA甘油酯产品,达到富集ω-3PUFA甘油酯的目的。
本论文首先对鱼油水解样中的ω-3PUFA(EPA、DHA)的分析检测和定量方法进行研究,并与国标气相色谱测定方法进行比较,确定了EPA、DHA的HPLC定量分析条件:色谱柱Sunfire-C18(5μm,250mm×4.6mm i.d);PDA检测波长205nm;流动相为甲醇-0.2%甲酸水溶液体系;洗脱方式为二元梯度洗脱;柱温35℃,进样量5μL。
然后对鱼油的水解工艺以及水解后游离ω-3PUFA(EPA、DHA)的分离纯化进行研究,比较了化学水解和酶催化水解两种水解方式,发现化学水解在水解率和EPA、DHA得率上都优于酶水解,化学水解优化的工艺条件为:皂化时间60min,皂化温度50℃,乙醇/水比例(v/v)6:1;振荡频率150r/min,此条件下水解率为95.5%,EPA得率为91.2%,DHA得率为88.9%。鱼油水解后,利用C18柱对水解液进行层析分离纯化,适宜的分离条件为:流动相为甲醇/水,70%、80%、90%、100%甲醇梯度洗脱,流速2mL/min,上样量为2g鱼油水解液,每管10mL进行分部收集,并采用质谱检测对层析样品进行快速分析,利用HPLC检验EPA、DHA收集液的纯度,经分离纯化后EPA、DHA纯度分别达到92.9%和93.2%。
随后利用甘油和分离纯化得到的EPA/DHA作为合成反应原料,对有机相脂肪酶催化合成ω-3PUFA甘油酯的间歇生产工艺进行了优化研究。通过单因素实验,确定了有机相脂肪酶催化合成EPA/DHA甘油酯的间歇反应最佳工艺条件:反应溶剂为正己烷,EPA/DHA的浓度为0.2mol/L,EPA/DHA与甘油摩尔比为3:1,分子筛添加在反应进行了24h时,脂肪酶添加量为17g/L,40℃振荡水浴反应48h,经反应合成后,EPA甘油酯的酯化率达到95%,其中甘油三酯含量达93.3%,DHA甘油酯的酯化率为94.5%,其中甘油三酯含量为93.2%,取得了较好的富集效果。通过质谱及1H-NMR、13C-NMR的检测,验证了合成产物EPA甘油三酯和DHA甘油三酯的结构。
最后,为了提高化学合成效率,自行设计并组装了一个小型的连续化合成反应装置,初步研究了EPA/DHA甘油酯的连续化生产工艺,探讨了在连续化合成反应过程中脱水装置加入与否、EPA/DHA与甘油的摩尔比、酶催化反应的温度、反应液在酶柱中的滞留时间等因素对产物生成的影响,结果表明,选用1,4-二氧六环溶剂,ω-3PUFA(EPA、DHA)甘油酯的连续化合成在反应温度45℃,EPA/DHA与甘油摩尔比为3:1,反应底物在酶柱中滞留时间为2h的工艺条件下,EPA和DHA甘油酯的酯化率分别为31.4%和29.7%,甘油三酯含量分别为9.3%和8.8%。ω-3PUFA甘油酯连续化合成反应的初步研究结果显示,连续化工艺尽管在反应效率上大幅度提高,对于工业化生产应用有一定参考价值,但是在如何提高甘油三酯产率问题还有待于进一步研究。
It is by now well established that the polyunsaturated fatty acids (ω-3 PUFA), especially eicosapentaenoic (EPA), docosahexaenoic(DHA) acids , are beneficial to health. EPA and DHA have been recognized have different physiological function. EPA was recently shown to have such highly beneficial effects as preventing coronary heart diseases, hypertriglyceridem, blood platelet aggregation and lowering blood cholesterol, thus reducing the risk of arteriosclerosis, inflammation and various carcinomas. DHA is important in the development of the central nervous system of infants. In this work, glyceride of EPA DHA was synthesized by glycerol and EPA/DHA which was separated from hydrolytic fish oil.
First, a high performance liquid chromatography (HPLC) method was developed for determination of EPA and DHA in hydrolytic fish oil.Chromatogram with high resolution was obtained using the column of Sunfire-C18 (5μm,250×4.6mm)with PDA detector at 205nm and the mobile phase of methanol and water with 0.2% formic acid at a flow rate of 1mL/min at 35°C.Then, this analysis method was compared with GC which was prescribed in GB.
Chemical hydrolysis is better than enzymatic hydrolysis in degree of hydrolysis and the yield of EPA/DHA, thus optimizing the conditions of chemical hydrolysis. The optimum conditions: saponification time 60min, 50℃, ethanol/water 6:1(v/v), 150r/min, the degree of hydrolysis was 95.46% and the recovery of EPA and DHA were 91.24% and 88.87%. C18 was used to isolated EPA/DHA in hydrolysis fish oil, it was identified by MS. The purity of EPA was identified by HPLC.
The conditions for lipase-catalyzed synthesis of EPA/DHA and glycerol in a batch reactor were optimized. Hexane was confirmed as the lipase-catalyzed reaction solvent, and 4A molecular sieve as desiccant. The molar ratio of EPA/DHA to glycerol was 3:1, the concentration of EPA/DHA was 0.2mol/L, the amount of lipase was 17g/L, the molecular sieve was added after 24h. The condensation of EPA/DHA and glycerol was conducted with vigorously shaking in a water bath at 40℃for 48 hours. The degree of esterification on EPA was 95% and the content of tri-EPA was 93.3%,and for DHA,the degree of esterification was 94.5% and the content of tri-DHA was 93.2%. And then, the structures of tri-EPA and tri-DHA were validated by MS and 1H-NMR, 13C-NMR.
For large-scale production in industry, a continuous reaction would be preferred. In this study, a packed-bed reaction system was designed to continuously synthesize EPA/DHA and glycerol. The influences of the addition of molecular sieves, the molar ratio of substrates, and the superficial residence time of substrate solution were emphatically studied in detail. The degree of esterification on EPA and DHA were 31.4% and 29.7% when the molar ratio of EPA/DHA to glycerol was 3:1,the superficial residence time was 2hours at 45℃in 1,4-dioxane.9.3% and 8.8% tri-EPA and tri-DHA were detected. Although the results can provide practicality for industry with its increased reaction efficiency, more work have to do to improve the yield of synthesization.
本论文首先对鱼油水解样中的ω-3PUFA(EPA、DHA)的分析检测和定量方法进行研究,并与国标气相色谱测定方法进行比较,确定了EPA、DHA的HPLC定量分析条件:色谱柱Sunfire-C18(5μm,250mm×4.6mm i.d);PDA检测波长205nm;流动相为甲醇-0.2%甲酸水溶液体系;洗脱方式为二元梯度洗脱;柱温35℃,进样量5μL。
然后对鱼油的水解工艺以及水解后游离ω-3PUFA(EPA、DHA)的分离纯化进行研究,比较了化学水解和酶催化水解两种水解方式,发现化学水解在水解率和EPA、DHA得率上都优于酶水解,化学水解优化的工艺条件为:皂化时间60min,皂化温度50℃,乙醇/水比例(v/v)6:1;振荡频率150r/min,此条件下水解率为95.5%,EPA得率为91.2%,DHA得率为88.9%。鱼油水解后,利用C18柱对水解液进行层析分离纯化,适宜的分离条件为:流动相为甲醇/水,70%、80%、90%、100%甲醇梯度洗脱,流速2mL/min,上样量为2g鱼油水解液,每管10mL进行分部收集,并采用质谱检测对层析样品进行快速分析,利用HPLC检验EPA、DHA收集液的纯度,经分离纯化后EPA、DHA纯度分别达到92.9%和93.2%。
随后利用甘油和分离纯化得到的EPA/DHA作为合成反应原料,对有机相脂肪酶催化合成ω-3PUFA甘油酯的间歇生产工艺进行了优化研究。通过单因素实验,确定了有机相脂肪酶催化合成EPA/DHA甘油酯的间歇反应最佳工艺条件:反应溶剂为正己烷,EPA/DHA的浓度为0.2mol/L,EPA/DHA与甘油摩尔比为3:1,分子筛添加在反应进行了24h时,脂肪酶添加量为17g/L,40℃振荡水浴反应48h,经反应合成后,EPA甘油酯的酯化率达到95%,其中甘油三酯含量达93.3%,DHA甘油酯的酯化率为94.5%,其中甘油三酯含量为93.2%,取得了较好的富集效果。通过质谱及1H-NMR、13C-NMR的检测,验证了合成产物EPA甘油三酯和DHA甘油三酯的结构。
最后,为了提高化学合成效率,自行设计并组装了一个小型的连续化合成反应装置,初步研究了EPA/DHA甘油酯的连续化生产工艺,探讨了在连续化合成反应过程中脱水装置加入与否、EPA/DHA与甘油的摩尔比、酶催化反应的温度、反应液在酶柱中的滞留时间等因素对产物生成的影响,结果表明,选用1,4-二氧六环溶剂,ω-3PUFA(EPA、DHA)甘油酯的连续化合成在反应温度45℃,EPA/DHA与甘油摩尔比为3:1,反应底物在酶柱中滞留时间为2h的工艺条件下,EPA和DHA甘油酯的酯化率分别为31.4%和29.7%,甘油三酯含量分别为9.3%和8.8%。ω-3PUFA甘油酯连续化合成反应的初步研究结果显示,连续化工艺尽管在反应效率上大幅度提高,对于工业化生产应用有一定参考价值,但是在如何提高甘油三酯产率问题还有待于进一步研究。
It is by now well established that the polyunsaturated fatty acids (ω-3 PUFA), especially eicosapentaenoic (EPA), docosahexaenoic(DHA) acids , are beneficial to health. EPA and DHA have been recognized have different physiological function. EPA was recently shown to have such highly beneficial effects as preventing coronary heart diseases, hypertriglyceridem, blood platelet aggregation and lowering blood cholesterol, thus reducing the risk of arteriosclerosis, inflammation and various carcinomas. DHA is important in the development of the central nervous system of infants. In this work, glyceride of EPA DHA was synthesized by glycerol and EPA/DHA which was separated from hydrolytic fish oil.
First, a high performance liquid chromatography (HPLC) method was developed for determination of EPA and DHA in hydrolytic fish oil.Chromatogram with high resolution was obtained using the column of Sunfire-C18 (5μm,250×4.6mm)with PDA detector at 205nm and the mobile phase of methanol and water with 0.2% formic acid at a flow rate of 1mL/min at 35°C.Then, this analysis method was compared with GC which was prescribed in GB.
Chemical hydrolysis is better than enzymatic hydrolysis in degree of hydrolysis and the yield of EPA/DHA, thus optimizing the conditions of chemical hydrolysis. The optimum conditions: saponification time 60min, 50℃, ethanol/water 6:1(v/v), 150r/min, the degree of hydrolysis was 95.46% and the recovery of EPA and DHA were 91.24% and 88.87%. C18 was used to isolated EPA/DHA in hydrolysis fish oil, it was identified by MS. The purity of EPA was identified by HPLC.
The conditions for lipase-catalyzed synthesis of EPA/DHA and glycerol in a batch reactor were optimized. Hexane was confirmed as the lipase-catalyzed reaction solvent, and 4A molecular sieve as desiccant. The molar ratio of EPA/DHA to glycerol was 3:1, the concentration of EPA/DHA was 0.2mol/L, the amount of lipase was 17g/L, the molecular sieve was added after 24h. The condensation of EPA/DHA and glycerol was conducted with vigorously shaking in a water bath at 40℃for 48 hours. The degree of esterification on EPA was 95% and the content of tri-EPA was 93.3%,and for DHA,the degree of esterification was 94.5% and the content of tri-DHA was 93.2%. And then, the structures of tri-EPA and tri-DHA were validated by MS and 1H-NMR, 13C-NMR.
For large-scale production in industry, a continuous reaction would be preferred. In this study, a packed-bed reaction system was designed to continuously synthesize EPA/DHA and glycerol. The influences of the addition of molecular sieves, the molar ratio of substrates, and the superficial residence time of substrate solution were emphatically studied in detail. The degree of esterification on EPA and DHA were 31.4% and 29.7% when the molar ratio of EPA/DHA to glycerol was 3:1,the superficial residence time was 2hours at 45℃in 1,4-dioxane.9.3% and 8.8% tri-EPA and tri-DHA were detected. Although the results can provide practicality for industry with its increased reaction efficiency, more work have to do to improve the yield of synthesization.
引文
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