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%。
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.
引文
[1]王叶,刑杰.维生素A缺乏对儿童健康的影响[J].中国妇幼保健, 2008, 23(19): 2760-2761.
[2]窦清惠.维生素A与骨质疏松症[J].中国骨质疏松杂志, 2008, 14(2): 134-137.
[3]李雪梅,易宗容,曾饶琼.维生素A在动物中的生理学效应[J].四川畜牧兽医, 2008, (11): 25-29.
[4]刘胜勇.维生素A在神经母细胞瘤发生和治疗中的应用研究进展[J].实用医学杂志, 2008, 24(13): 2358-2359.
[5]薛延军,邵世和,李先茜,等.维生素A对Hela细胞凋亡的影响[J].山东医药, 2010, 50(1): 34-35.
[6] H Konig, K Lammerhirt, J Paust, et al. Chemistry and galenics of tretinoin[J]. Arzneimittelforschung, 1974, 24(8): 1184-1187.
[7] MJ Aurell, M Parra, A Tortajada, et al. Polyenolates of unsaturated carboxylic acids in synthesis. A straightforward synthesis of retinoic acids[J]. Tetrahedron Letters, 1984, 31(40): 5791-5794.
[8]吕国锋,高建荣,黄国东,等.合成维生素A醋酸酯新工艺的研究[J].浙江工业大学学报, 2006, 34(6): 614-616.
[9] T Maugard, MD Legoy. Enzymatic synthesis of derivatives of Vitamin A in organic media[J]. Journal of molecular catalysis B: Enzymatic, 2000, 8: 275-280.
[10] T Maugard, B Rejasse, MD Legoy. Synthesis of water-soluble retinol derivatives by enzymatic method[J]. Biotechnology Progress, 2002, 18(3): 424-428.
[11] B Rejasse, T Maugard, MD Legoy. Enzymatic procedures for the synthesis of water-soluble retinol derivatives in organic media[J]. Enzyme and Microbial Technology, 2003, 32(2): 312-320.
[12]刘涛,尹春华,谭天伟.脂肪酶催化合成维生素A酯[J].现代化工, 2005, 25(2): 37-40.
[13]高静,姜艳军,马丽,等.混合溶剂中酶促合成维生素A乳酸酯[J].分子催化, 2006, 20(4): 346-350.
[14]李宏亮,胡晶,谭天伟.固定化脂肪酶合成维生素A棕榈酸酯[J].生物工程学报, 2008, 24(5): 817-820.
[15]张文娟,朱凯,韩萍芳.超声酶促合成维生素A棕榈酸酯[J].食品工业科技, 2010, 31(1): 316-319.
[16]唐凤仙,李春,徐小琳,等. Aspergillus Sp. Li-38产脂肪酶发酵条件及催化反应的初步研究[J].石河子大学学报, 2007, 25(5): 615-618.
[17]阎金勇,杨江科,闫云君. Galactamyces geotrichum Y25产脂肪酶条件的优化[J].生物加工过程, 2007, 5(2): 46-51.
[18]罗珊珊,戴大章,夏黎明. Penicilium expansum PED-03固态发酵产脂肪酶及酶学性质研究[J].食品与发酵工业, 2006, 32(3): 14-17.
[19]戚薇,邵静,王晨,等.产低温碱性脂肪酶菌株Acinetobactor johnsonii Lp28的鉴定及发酵条件优化[J].天津科技大学学报, 2009, 24(6): 9-12.
[20]余琼,梁运祥.产低温脂肪酶假单胞菌的选育及产酶条件的优化[J].湖北农业科学, 2006, 45(5): 662-665.
[21]付建红,石玉瑚,欧阳平凯,等.产低温脂肪酶均值Geotrichum candida ch-3的选育、发酵条件及酶学性质研究[J].中国生物工程杂志, 2007, 27(10): 22-27.
[22]袁红玲,汤鲁宏,许正宏,等.产有机相催化酯合成活性的脂肪酶菌株的筛选[J].微生物学通报, 2007, 34(1): 19-23.
[23]周军.产脂肪酶菌株的筛选及脂肪酶发酵条件的研究[J].河北农业科学, 2008, 12(2): 1-2.
[24]季祥,蔡禄,王勇,等.产脂肪酶细菌的筛选及产酶条件优化[J].内蒙古科技大学学报, 2007, 26(4):359-361.
[25]张振乾,官春云.产脂肪酶细菌的筛选及培养条件优化研究[J].现代生物医学进展, 2008, 8(12): 2469-2472.
[26]朱绮霞,张博,陆雁,等.产脂肪酶粘质沙雷氏菌发酵产酶条件的优化[J].生物技术, 2009, 19(5): 61-65.
[27]陈林林,辛嘉英,张颖鑫,等.粗状假丝酵母产脂肪酶发酵条件的优化[J].食品工业科技, 2010, 31(1): 183-185.
[28]陈贵元,季秀玲,林连兵,等.低温脂肪酶产生菌的筛选与鉴定、产酶条件及酶学性质研究[J].云南大学学报, 2010, 32(1): 108-113.
[29]张立莹,魏东芝,童望宇.假丝酵母Candida rugosa产脂肪酶条件的优化[J].生命科学研究, 2003, 7(4): 320-323.
[30]沈芳,刘雄民,刘力恒,等.假丝酵母菌GXU08产脂肪酶发酵条件的优化[J].广西植物, 2009, 29(5): 689-693.
[31]刘瑞志,江晓路,牟海津.碱性脂肪酶高产菌株Fusarium Sp. N4-2的筛选及产酶条件研究[J].食品与发酵工业, 2005, 31(7): 31-34.
[32]王松,李长春,徐榕敏,等.碱性脂肪酶高产菌株的筛选及产酶条件的优化[J].西南大学学报, 2010, 32(6): 99-103.
[33]刘海洲,刘均洪,张媛媛,等.南极假丝酵母诱变育种及产脂肪酶条件的优化[J].青岛科技大学学报, 2009, 30(3): 226-229.
[34]孙宏丹,胡军,姚民昌.无根根霉脂肪酶的菌株筛选及产酶条件设置[J].大连医科大学学报, 2001, 23(2): 150-152.
[35]李迅,王友东,刘慧琴,等.洋葱伯克霍尔德菌0107产脂肪酶发酵条件优化研究[J].安徽农业科学, 2010, 38(12): 6122-6124.
[36]汪小锋,杨江科,闫云君.洋葱假单胞菌(Pseudomonas cepecia)PCL-3产脂肪酶发酵条件的研究[J].中国生物工程杂志, 2008, 28(10): 72-78.
[37]张博,杨江科,苏华武,等.脂肪酶产生菌的筛选、鉴定及其产酶条件的优化[J].生物技术, 2007, 17(1): 23-26.
[38]王咏华,杨博,黄海浪,等.酶解油脂生产天然奶味香精基料的方法.中国: 200610036800[P],2007-02-07.
[39]夏咏梅,章克昌,石贵阳,等.微水体系中荧光假单胞菌脂肪酶催化合成单甘酯[J].生物工程学报, 2002, 18(1): 84-88.
[40] M Arroyo, JM Sanchez-Monlero, JV Sinisterra. Thermal stabilization of immobilized Lipase B from C. antarctica on different supports: effect of water activity on enzymatic activity in organic media[J]. Enzyme and Microbial Technology, 1999, (24): 3-12.
[41]丰宗清.壳聚糖复合物固定化脂肪酶用于催化合成手性化合物[J].农业科学研究, 2008, 29(3): 40-43.
[42]邬显章,李江华.洗涤剂用酶的研究与开发-新兴碱性脂肪酶[J].无锡轻工业学院学报, 1994, 13(3): 201-207.
[43]坂口博修.脂肪酶在洗涤剂中应用[J].日用化学工业译丛, 1990, (2): 11-14.
[44] UN Das. Gamma-linolenic acid, Arachidonic acid, and Eicosapentaenoic acid as potential anticancer drugs[J]. Nutrition, 1990, 6(6): 429-434.
[45] K Hata, M Matsukura, H Taneda, et al. Mill-scale application of enzymatic pitch control during paper production[J]. ACS Symposium Series, 1996, (655): 280-296.
[46]秦梦华,陈嘉翔,余家鸾.马尾松磨木浆树脂的脂肪酶生物控制[J].中国造纸学报, 1997, (12): 29- 34.
[47]李俊奎,王芳,谭天伟,等.固定化脂肪酶催化小桐子毛油合成生物柴油[J].北京化工大学学报, 2010, 37(2): 100-103.
[48]赵希岳,陈巧丽,蔡志强,等.有机溶剂体系固定化脂肪酶催化合成生物柴油[J].中国粮油学报, 2010, 25(2): 74-77.
[49]李相,刘涛,杨江科.响应面优化洋葱伯克霍尔德菌固定化脂肪酶催化合成生物柴油工艺[J].北京化工大学学报, 2009, 36(5): 78-83.
[50]毛跟年,李丽维,齐凤.固定化脂肪酶应用研究进展[J].中国酿造, 2009, (8): 17-19.
[51]尹志娜.固定化酶及其在食品和生物领域的研究进展[J].生命科学仪器, 2009, 7(10): 11-15.
[52]曾淑华,杨江科,闫云君.固定化脂肪酶性质及其应用研究进展[J].生物加工过程, 2007, 5(1): 45-49.
[53]阎金勇,闫云君.脂肪酶非水相催化作用[J].生命的化学, 2008, 28(3): 268-271.
[54]曹淑桂.有机溶剂中酶催化研究的新进展[J].化学通报, 1995, (5): 5-12.
[55] MA Jackson, JW King. Lipase-catalyzed glycerolysis of soybean oil in supercritical carbon dioxide[J].Journal of the American Oil Chemists Society, 1997, 74(2): 103-106.
[56] H Nakaya, O Miyawaki, K Nakamura. Transesterification between triolein and stearic acid catalyzed by lipase in CO2 at various pressures[J]. Biotechnology Techniques, 1998, 12(12): 881-884.
[57]宋欣,曲音波,胡晓燕.非水介质中脂肪酶催化亚麻油醇酯合成的研究[J].高等学校化学学报, 1999, 20(10): 1578-1580.
[58]汤鲁宏,张浩.非水相脂肪酶催化合成L-抗坏血酸棕榈酸酯的研究Ⅰ[J].生物工程学报, 2000, 16(3): 363-367.
[59]杨本宏,吴克,刘斌,等.非水溶剂Rhizopus arrhizus脂肪酶催化合成三种酯的最佳条件[J].中国生物化学与分子生物学报, 2003, 19(5): 572-575.
[60]陈必强,叶华,谭天伟.固定化假丝酵母脂肪酶合成棕榈酸异辛酯[J].化工学报, 2004, 55(3): 422-425.
[61]赵天涛,高静,张丽杰,等.有机相中脂肪酶合成乳酸乙酯[J].催化学报, 2006, 27(6): 537-540.
[62]辛嘉英,柳眉,张蕾,等.有机相脂肪酶催化合成阿魏酸乙酯[J].食品科学, 2007, 28(9): 137-140.
[63] A Wolfson, A Atyya, C Dlugy, et al. Glycerol triacetate as solvent and acyl donor in the production of isoamyl acetate with Candida antarctica Lipase B[J]. Bioprocess and Biosystems Engineering, 2010, 33(3): 363-366.
[64] AV de Paula, GFM Nunes, JL Silva, et al. Screening of food grade lipases to be used in esterification and interesterification reactions of industrial interest[J]. Applid Biochemistry and Biotechnology, 2010, 160(4): 1146-1156.
[65]孟祥河,孙培龙,杨开,等.填充床反应器中酶法连续合成甘油二酯的研究[J].生物工程学报, 2005, 21(3): 425-429.
[66] G Carta, JL Gainer, ME Gibson. Synthesis of esters using a nylon- immobilized lipase in batch and continuous reactors[J]. Enzyme and Microbial Technology, 1992, 14(11): 904-910.
[67] C Lanne, S Boeren, K Vos, et al. Rules for optimization of biocatalysis in organic solvents[J].Biotechnology Bioengeering, 1987, 30(1): 81-87.
[68] DS Clark. Characteristics of nearly dry enzymes in organic solvents: implications for biocatalysis in the absence of water[J]. Phil Trans R Soc Lond B, 2004, (359): 1299-1307.
[69]王镜岩,朱圣庚,徐长法.生物化学[M].北京:高等教育出版社, 2002: 355-355.
[70] M Martinelle, K Hult. Kinetics of acyl transfer reactions in organic media catalyzed by Candida antarctica lipase B[J]. Biochimica et Biophysica Acta, 1995, 1251(2): 191-197.
[71] GD Yadav, AH Trivedi. Kinetic modeling of immobilized-lipase catalyzed transesterification of n-octanol with vinyl acetate in non-aqueous media[J]. Enzyme and Microbial Technology, 2003, 32(7): 783- 789.
[72]徐岩,赵成明,章克昌.固定化脂肪酶有机相中催化己酸乙酯反应动力学的研究[J].生物工程学报, 1999, 15(4): 533-536.
[73]孙志浩,许建和(译).生物催化-基础与应用[M].北京:化学工业出版社, 2006: 88-88.
[74] Bonrath W, Karge R, Netscher T. Lipase-catalyzed transesterifications as key-steps in the large-scale prep- aration of vitamins[J]. Journal of molecular catalysis, 2002, 19(2):67-72.
[75] K. R. Kiran, B. Manohar, S. Divakar. A central composite rotatable design analysis of lipase catalyzed synthesis of lauroyl lactic acid at bench-scale level[J]. Enzyme and Microbial Technology, 2001, (29):122-128.
[76] X Zhang, RJ Wang, XX Yang, et al. Central composite experimental design applied to the catalytic aromatization of isophorone to 3, 5-xylenol[J]. Chemometrics and Intelligent Laboratory systems, 2001, 89(1):45-50.
[77] S.H Krishna, A.P.Sattur, N.G.Karanth. Lipase-catalyzed synthesis of isoamyl isobutyrate–optimization using a central composite rotatable design[J]. Process Biochemistry, 2001, 37(1): 9-16.
[78] G.A.Macedo, G.M.Pastore, M.I.Rodrigues. Optimising the synthesis of isoamyl butyrate using Rhizopus sp.lipase with a central composite rotatable design[J]. Process Biochemistry, 2004, 39(6): 687-692.
[79]姜艳军.脂肪酶催化合成维生素A乳酸酯的研究[D].河北:河北工业大学, 2006.
[80] DK. Eggers, HW. Blanch, JM. Prausnitz. Extractive catalysis: solvent effects on equilibria of enzymatic reactions in two-phase systems[J]. Enzyme and Microbial Technology, 1989, 11(2): 84-89.
[81]王娜,王海娇,汪寅夫,等.液-液萃取-气相色谱法测定水中9种有机氯农药[J].岩矿测试, 2010, 29(5): 497-502.
[82]于振花,荆淼,王小如,等.液液萃取-高效液相色谱-电感耦合等离子体质谱同时测定海水中多种有机锡[J].光谱学与光谱分析, 2009, 29(10): 2855-2859.
[83]袁仲,杨继远.液液萃取-GC/MS法分析国产食醋的香味成分[J].中国调味品, 2009, 34(7): 102-105.
[84]张莉华,许新德,孙晓霞,等.硅胶柱层析法纯化天然维生素E[J].中国食品添加剂, 2008, (3): 105-109.
[85]袁华,吴莉,吴元欣,等.硅胶柱层析法提纯茶多酚的研究[J].华中师范大学学报, 2007, 41(4): 553-556.
[86]张颖颖,杨亦文,任其龙,等.硅胶柱层析法提纯大豆磷脂酰胆碱的研究[J].高校化学工程学报, 2006, 20(5): 685-690.
[87] DS Kinnory, Y Takeda, DM Greenberg. Chromatography of carboxylic acids on a silica gel column with a benzene-ether solvent system[J]. The Journal of Biological Chemistry, 1955, (212): 379-383.
[88] ST Ingalls, MS Kriaris, Y Xu, et al. Method for isolation of non-esterified fatty acids and several otherclasses of plasma lipids by column chromatography on silica gel[J]. Journal of Chromatography: Biomedical Applications, 1993, 619(1): 9-19.
[89] JG Alvarez, JC Touchstone. Separation of acidic and neutral lipids by aminopropyl-bonded silica gel column chromatography [J]. Journal of Chromatography B: Biomedical Sciences and Applications, 1992, 577 (1): 142-145.