碱法异构和生物法制备共轭亚油酸的研究
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摘要
共轭亚油酸(conjugated linoleic acids，简称：CLA)是一系列含有共轭双键、具有位置和构象异构的十八碳二烯酸的总称，是必需脂肪酸亚油酸的异构体。共轭亚油酸是具有许多生理活性的天然脂肪酸，具有抗癌、抗粥样动脉硬化、减肥、抗氧化、促进生长发育、增强免疫能力等生理活性。
采用KOH为催化剂，丙二醇为溶剂，以核桃油为原料，碱法异构转化共轭亚油酸；用分光光度法对CLA及其产率进行分析计算。考察了反应温度、反应时间、溶剂用量、催化剂用量等因素对CLA产率的影响。并利用二次正交组合实验设计优化方法，建立了相应的数学模型，得到了较适宜的异构条件：反应温度174℃，反应时间2.87h，m(核桃油)：m(KOH)=1.6，该条件下，CLA产率可达85.0％以上。
对碱法异构反应机理进行探讨，由反应机理推导速率方程，并以实验验证。建立了表观动力学模型，确定了速率方程：dc_r／dt=0.7167c_A。计算出碱法共轭异构反应的表观活化能E_a=67.22kJ／mol；反应速度常数与温度的关系式为：k=1.47456×10~4s~(-1)e~(67220／RT)。
从畜禽鸡鸭和兔的肠道、酸奶及泡菜中分离纯化得到四株转化CLA的菌株，初步鉴定均为乳杆菌。在MRS培养液中加入0.1％亚油酸，分别接入分离得到的五株乳杆菌和干酪乳杆菌(Lactobacillus casei)，37℃微好氧进行发酵24h，发酵液用正己烷萃取纯化，测定其紫外吸收，并计算得到发酵液中CLA的含量及CLA产率。结果表明：从酸奶分离的菌种CLA产率最高，产率为0.853％。
Conjugated linoleic acids (CLA) comprise a mixture of positional and geometric isomers of octadecadienoic acid with conjugated double bonds. CLA are isomers of essential fatty acid linoleic acid(cis-9, cis-12, octadecadienoic acid). CLA are the natural fatty acids ,which have many important physiological activities, such as anticancer, antiatherosclerosis, reducing body fat, antioxidant, strengthening immune function, antidiabetic and accelerating growth.
Conjugated linoleic acids(CLA) were prepared by alkali isomerization using KOH as catalyst, 1 ,2-propanediol as solvent and walnut oil as raw materials. The concentration and yield of CLA were determined at 233nm by spectrophotometric analysis. Effects of the factors on the yield of CLA have been studied and the mathematical models have been established by using the 23 factorial experiment. A fitted second-order response model in three variables were obtained and the optimum conditions were found: reaction temperature at 174 , reaction time in 2.87 hours, and m(walnut oil): w(KOH) =1.6,when the condition that m(l,2-propanediol): w(KOH) = 3 : 1 , stirring rate at 500r/min and nitrogen protection was given. At the optimum conditions yield of CLA can exceed
85.0%.
The reaction mechanism of alkali isomerization was discussed. The equation of velocity was speculated by reaction mechanism, and the conclusion was validated by experiment. The apparent kinetic model was also established and equation of velocity
was deduced as dcB/dt = 0.71 67cA. The apparent energy of activation (Ea) is quantified as Ea = 67.22kJ/mol. The constant of reaction rate (k) vs the temperature formula is that
67220
k = 1. 47456 104s-1e.
The strains of bacteria identified as Lactobacillus were isolated from yoghourt, pickle and intestine of domestic animal such as chook, duck and rabbit. 0.1 percent of linoleic acid was added to MRS medium, strains of Lactobacillus isolated and strain of Lactobacillus casei were cultured under microaerobic conditions at 37 for 24h in MRS medium. CLA were extracted from the culture by hexane. The levels of CLA formation were determined by spectrophotometric analysis. The result showed that four strains can produce CLA from linoleic acid, and yield of CLA produced from strain isolated from yoghourt is maximal, which is 0.853%.
采用KOH为催化剂，丙二醇为溶剂，以核桃油为原料，碱法异构转化共轭亚油酸；用分光光度法对CLA及其产率进行分析计算。考察了反应温度、反应时间、溶剂用量、催化剂用量等因素对CLA产率的影响。并利用二次正交组合实验设计优化方法，建立了相应的数学模型，得到了较适宜的异构条件：反应温度174℃，反应时间2.87h，m(核桃油)：m(KOH)=1.6，该条件下，CLA产率可达85.0％以上。
对碱法异构反应机理进行探讨，由反应机理推导速率方程，并以实验验证。建立了表观动力学模型，确定了速率方程：dc_r／dt=0.7167c_A。计算出碱法共轭异构反应的表观活化能E_a=67.22kJ／mol；反应速度常数与温度的关系式为：k=1.47456×10~4s~(-1)e~(67220／RT)。
从畜禽鸡鸭和兔的肠道、酸奶及泡菜中分离纯化得到四株转化CLA的菌株，初步鉴定均为乳杆菌。在MRS培养液中加入0.1％亚油酸，分别接入分离得到的五株乳杆菌和干酪乳杆菌(Lactobacillus casei)，37℃微好氧进行发酵24h，发酵液用正己烷萃取纯化，测定其紫外吸收，并计算得到发酵液中CLA的含量及CLA产率。结果表明：从酸奶分离的菌种CLA产率最高，产率为0.853％。
Conjugated linoleic acids (CLA) comprise a mixture of positional and geometric isomers of octadecadienoic acid with conjugated double bonds. CLA are isomers of essential fatty acid linoleic acid(cis-9, cis-12, octadecadienoic acid). CLA are the natural fatty acids ,which have many important physiological activities, such as anticancer, antiatherosclerosis, reducing body fat, antioxidant, strengthening immune function, antidiabetic and accelerating growth.
Conjugated linoleic acids(CLA) were prepared by alkali isomerization using KOH as catalyst, 1 ,2-propanediol as solvent and walnut oil as raw materials. The concentration and yield of CLA were determined at 233nm by spectrophotometric analysis. Effects of the factors on the yield of CLA have been studied and the mathematical models have been established by using the 23 factorial experiment. A fitted second-order response model in three variables were obtained and the optimum conditions were found: reaction temperature at 174 , reaction time in 2.87 hours, and m(walnut oil): w(KOH) =1.6,when the condition that m(l,2-propanediol): w(KOH) = 3 : 1 , stirring rate at 500r/min and nitrogen protection was given. At the optimum conditions yield of CLA can exceed
85.0%.
The reaction mechanism of alkali isomerization was discussed. The equation of velocity was speculated by reaction mechanism, and the conclusion was validated by experiment. The apparent kinetic model was also established and equation of velocity
was deduced as dcB/dt = 0.71 67cA. The apparent energy of activation (Ea) is quantified as Ea = 67.22kJ/mol. The constant of reaction rate (k) vs the temperature formula is that
67220
k = 1. 47456 104s-1e.
The strains of bacteria identified as Lactobacillus were isolated from yoghourt, pickle and intestine of domestic animal such as chook, duck and rabbit. 0.1 percent of linoleic acid was added to MRS medium, strains of Lactobacillus isolated and strain of Lactobacillus casei were cultured under microaerobic conditions at 37 for 24h in MRS medium. CLA were extracted from the culture by hexane. The levels of CLA formation were determined by spectrophotometric analysis. The result showed that four strains can produce CLA from linoleic acid, and yield of CLA produced from strain isolated from yoghourt is maximal, which is 0.853%.
引文
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[2] Pariza M W, Hargraves W A. A beef-derived mutagenesis modulator inhibits initiation of mouse epidermal tumors by 7,12-dimethibenz[a]anthracene. Carcinogenesis, 1985, 6:591-593
[3] Ha Y L, Grimn N K, Pariza M W, et al. Anticarcinogens from fried ground beef: heat-altered derivatives of linoleic acid. Carcinogenesis, 1987,8:1881-1887
[4] Elin D. Scientific forum explores CLA knowledge. Inform., 1998,8(1):69-72
[5] Fritsche J, Yurawecz M P, Pawlosky R, et al. Spectroscopic characterization of unusual conjugated l inoleic acid (CLA) isomers. J. Separation Sci., 2001, 24:59-61
[6] Lin H, Boylston T D, Chang M J, et al. Survey of the conjugated linoleic acid contents of dairy products. J. Dairy Sci., 1995, 78:2358-2365
[7] Kepler C IL Hirons K P, McNeill J J, et al. Intermediates and products of the biohydrogenation of linoleic acid by Butyrivibrio fibrisolvens. Biol. Chem., 1966,41: 350-1354
[8] Belury M A. Conjugated dienoic linoleate: a polyunsaturated fatty acid with unique chemoprotective properties. Nutr. Rev., 1995,53:83-89
[9] Chin S F, Liu W, Storkson J M. et al. Dietary sources of conjugated dienoic isomers of linoleic acid, a newly recognized class of anticarcinogens. J. Food Comp. Anal., 1992,5:185-197
[10] Ha Y L, Grimn N K, Pariza M W. et al. Newly recognized anticarcinogenic fatty acids: identification and quantification in natural and processed cheeses. J. Agr. Food Chem., 1989,37:75-81
[11] Shantha N C, Crum A D, Decker E A. et al. Evaluation of conjugated linoleic acid concentrations in cooked beef. J. Agric. Food Chem., 1994,42(8):1757-1760
[12] Oshea M, Stanton C, Devery R. et al. Antioxidant enzyme defense response of human MCF-7 and Sw480 cancer cells to conjugated linoleic acid. Anticaner Res., 1999,19:1953-1959
[13] Choi Y, Park Y, Storkson J M. et al. Inhibition of stearoyl-CoA desaturase activity by the cis-9,trans-11 isomer and the trans-10,cis-12 isomer of conjugated linoleic acid in MDA-MB-231 and MCF-7 human breast cancer cells. Biochem. Biophys. Res. Commun.,2002, 294(4):785-790
[14] Masso-Welch P A, Zangani D, Ip C, et al. Inhibition of angiogenesis by the cancer chemopreventive agent conjugated linoleic acid. Cancer Res.,2002,62(15): 4383-4389
[15] O'Connor A, McDonnell S, Devery IL et al. The effect of Conjugated Linoleic Acid (CLA) and its isomers on the proliferation, migration and invasion of a metastatic murine mammary cell line. Brit. J. Cancer,2002,86:S82-S82 Suppl.
[16] Palombo J D, Ganguly A, Bistrian B IL et al. The antiproliferative effects of biologically active isomers of conjugated linoleic acid on human colorectal and prostatic cancer cells. Cancer Lett., 2002,177:163-I 72
[17] Kim E J. Jun J G, Park H S, et al. Conjugated linoleic acid (CLA) inhibits growth of Caco-2 colon cancer cells: Possible mediation by oleamide. Anticancer Res., 2002,22(4):2193-2197
[18] Schonberg S. Krokan H E. The inhibitory effect of conjugated dienoic derivatives (cla) of linoleic acid on the growth of human tumor cell lines is in part due to increased lipid peroxidation. Anticancer. Res., 1995,15:1241-1246
[19] Parodi P W, Conjugated linoleic acid and other anticarcinogenic agents of Bovine milk fat. J. Dairy Sci.,1999, 82(6):1339-1349
[20] 刘家仁，陈炳卿，刘瑞海等，共轭亚油酸对人胃腺癌细胞的抑制作用[J]卫生研究，1999，28(6)：353-355
[21] 薛英本，陈炳卿，郑玉梅等，共轭亚油酸对鼠黑色素瘤细胞转移特性的影响[J]．卫生研究，2001，30(1)：37-39
[22] Lee K N, Pariza M W, Kritchevsky D, et al. Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis, 1994,108:19-25
[23] Kritchevsky D, Tepper S A, Wright S, et al, Influence of graded levels of conjugated linoleic acid (CLA) on experimental atherosclerosis in rabbits. Nutr. Res., 2002,22(11):1275-1279
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