功能性甘油二酯的酶促酯化合成及其减肥功能的研究
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摘要
甘油二酯(DAG)是常见的表面活性剂,其优良的可生物降解特性以及无臭、无味、无毒、无腐蚀及非离子特性是其广泛应用的主要原因。同时DAG也是天然植物油脂的微量成分及体内脂肪代谢的内源中间产物。它是公认安全(GRAS)的食品成分,可用于面团增强剂、润滑剂、脱模剂、表面装饰剂、增稠剂、稳定剂及组织形成剂等(FDA,2000)。此外近年来发现膳食DAG具有降低内脏脂肪、抑制体重增加、减少血脂的作用,因而受到广泛的关注。然而,长期以来DAG的大规模生产还主要依赖于有机化学、化工过程。化学方法通常是利用碱性催化剂在高温下进行反应,其缺点是耗能高,产品易褪色,常会出现苦味及反应缺乏专一性,而且由于化学催化剂不易降解、有毒使得产品在食品、化妆品及制药方面的应用受到限制。酶法由于具有专一性强、反应条件温和及环境友好等优点日益受到人们的重视,因此为DAG的合成提供了有效的替代方法。
研究开发了一个新颖、有效的酶法合成DAG的生产过程。生产中直接以非吸附的甘油、脂肪酸为起始原料,操作方便,时间短,产率高,而且产物组成简单,利于下游纯化工艺。合成反应中,生物催化剂的选择是至关重要的。酶筛选结果表明Lipozyme RMIM、Novo435不仅酯化活力高,而且可反复利用,用于生产DAG是较合适的。结合考虑位置专一性及成本经济性,实验选择Lipozyme RMIM作为优化的对象。酯化特性研究表明Lipozyme RMIM在40℃优先作用SCFA、MCFA,而60℃对LCFA选择性更强。醇底物选择性研究发现Lipozyme RMIM对伯羟基要优于仲羟基,而对叔羟基或芳环上的羟基活力很低或不起作用。无溶剂体系中,最适水分活度为0.11,当Aw≥0.54时,酯化活力明显降低。非水相中酶活力研究表明,异辛烷是较佳的反应介质,然后依次为正庚烷、正己烷、丁酮、丙酮。为监测酯化反应中间过程及分析终产品,分别采用TLC、HPLC/ELSD、HPLC/RID对甘油酯产物进行分离、定性定量分析。结果表明1,3-DAG与1,2-DAG异构体分离良好。
实验详细研究了间歇反应条件下,无溶剂体系中Lipozyme RMIM催化亚油酸、甘油酯化反应,以及各因素对酯化产物组成的影响。并采用二次旋转组合设计对最适条件进行了优化,对因素的重要性作了适当的评价,并给出了拟合良好、回归显著、可靠性具佳的经验性模型方程。
研究表明真空脱水可有效增加亚油酸的转化率,使反应平衡向合成方向移动。为减少能耗、降低脱水成本,成功地采用离子交换树脂吸附替代真空减压脱水,效果良好。
不同的反应温度、底物摩尔比、酶添加量等热力学因素条件下的酯化进程曲线对比研究表明,温度、底物摩尔比、酶添加量不仅影响酯化反应的初速度、转化率,还与产物组成密切相关。响应面优化结果表明63℃、亚油酸甘油摩尔比为2.1,固定化酶添加量为18EU/g底物,0.01MPa真空脱水,反应8hr亚油酸酯化率最高可接近100%。三因子对亚油酸转化率、1,3-DAG/1,2-DAG比例的影响大小依次分别为:酶添加量>温度>底物比例;温度>底物比例>酶添加量。
动力学因素研究表明,多相酯化反应中传质至关重要。添加适当的乳化剂,可明显增加底物接触面积,改善传质,提高反应初速度,减少反应达到平衡所需时间,但不会改变反应平衡。本实验中单甘酯是最佳的乳化剂,添加量为0.3%。适当的搅拌会改善传质,增加脂肪酸、甘油两相的接触,加快反应速度。试验发现雷诺准数小于10000时,外部传质是主要的限速步骤;当雷诺准数大于10000后,内扩散及酶催化反应速度是主要限速步骤。而且搅拌速度过高,可能会对固化酶造成机械损伤,影响酶活。在雷诺准数为10000左右时,内外传质及催化反应速度达到统一,因而反应速度最快。
通过对实验条件下酶操作稳定性的研究,给出了Lipozyme RMIM的失活动力学方程及时间、产量关系模型。采用Matlab对实验数据进行拟合,求得Lipozyme RMIM的半衰期为594小时,酶失活常数Kd为0.001062hr-1,生产能力为2000-2500g产物/g酶。两次采用不同的方法拟合误差很小,仅为2.26%,而且根据模型求得预测值与真实值拟合良好,模型是极为有效的。
实验进一步研究了填充柱反应器中酶催化亚油酸、甘油连续酯化反应的影响因素。发现填充柱内径,亚油酸、甘油的比例,反应温度,进料速度不仅影响酯化反应的转化率、反应速度,还影响酯化产物中各组分的比例。通过对数据的分析确定固定化酶填充柱长径比7.8(id16mm, L125mm),亚油酸、甘油摩尔比1:2,进料速度1.5ml/min(保留时间12min),65℃条件下酯化反应可实现最终的产量、产品纯度及生产效率三者的统一。
填充床反应器中固定化酶连续催化酯化反应的一个主要问题即体系水分清除困难。实验研究了采用过量甘油吸附脱水的可行性,模拟试验表明甘油的干燥脱水作用良好。稳定性研究确定65℃条件下,亚油酸、甘油摩尔比为1:2,进料速度1.2ml/min,可明显提高酯化反应的转化率,改善固定化酶的稳定性,增加Lipozyme RMIM的使用寿命。连续运行10天,残余酶活仍保持在80%以上,而对照组则仅为52%。
对酯化的产品,尝试了采用液液萃取方式清除单甘酯的研究。实验发现乙醇水溶液可方便地萃取产品中的单甘酯,萃取的选择性随温度增加、载荷量的减少、水比例的加大而增加。载荷量增大,萃取效率增加但选择性下降。多段萃取可实现萃取效率
Diacylglycerols (DAGs) are well known as bio-surfactants. Their excellent biodegradability as well as the behaviors of tasteless, odorless, nontoxic, non-irritant and non-ionic has been paid more and more attraction in numerous industrial fields. It is found that, DAGs are a minor natural component in vegetable oils and fats, as well as an endogenous product of triglyceride metabolism. DAGs are generally recognized as safe as direct human food ingredients and are approved for use in food as dough strengtheners, formulation acid, lubricant and release agents, vehicles, surface finishing agents, stabilizers and thickeners, texturizers (FDA, 2000). In addition, the long-term ingestion of dietary DAG, in contrast to TAG, prevented the accumulation of visceral fat and body weight gain in obesity-prone mice, and decreased postprandial blood lipid levels of rats administration with lipid emulsion. On the other hand, for a long time, large scale’s production of DAG remained mainly in the realms of organic chemistry and chemical processing. Chemical methods are mainly performed at high temperatures in the presence of alkaline catalysts. High energy’s consumption, coloring of products and low selectivity are major disadvantages of these methods. Moreover, some chemicals are toxic and not readily biodegradable, thus causing their limited application in cosmetics, food industry and pharmaceutics. In this case, the enzymatic synthesis offers an alternative way.
In the present work, a novel and effective enzymatic method which could eliminate most of the above-mentioned problems, was developed for the production of DAGs. Non-adsorptive glycerol and free fatty acids were directly used as starting materials in order to increase production yield. The selection of biocatalyst is very crucial. It turned out that Rhizopusmucor miehei lipase (Lipozyme RMIM) and Candida antarictica lipase (Novo 435) are the only suitable biocatalysts for the production of 1,3-DAG, not only for their higher esterification activities, but also for the easy reuse. In view of regiospecificity (1,3-specific) and economics, Rhizopusmucor miehei lipase is more suitable than Candida antarictica lipase. Studies on esterification characteristics of Lipozyme RMIM suggested that the selectivity for LCFA is higher than for MCFA and SCFA at 60 degree, but the situation is reversed at 40 degree. The optimum alcoholic substrates for Lipozyme RMIM are primary alcohols, followed by the secondary alcohols, and then tertiary alcohols. However, Rhizopusmucor
miehei lipase hardly catalyst phenol hydroxyl group. The optimum water activity is 0.11. When water activity is more than 0.54, the enzymatic activity decrease significantly. Studies on non-aqueous phase suggested that isooctane is the optimum medium, followed by heptane and hexane, and then EMK and acetone, as their polarities are strong enough to peel off essential water for enzyme activity.
For monitoring of intermediate process and analysis of products, thin-layer chromatography, normal-, reverse-phase high-performance liquid chromatography were adopted to isolate neutral lipid. Mono-, di-, and tri-acylglycerol were determined with density colorimetric, evaporative light-scatting detector (ELSD) and refractive index detector (RID) individually. It was found that the 1,3-DAGs were evidently resolved from the 1,2-DAG position isomers. Intermediate and final product can be assayed according to methods mentioned above.
Esterification reaction of linoleic acid and glycerol catalyzed by Lipozyme RMIM in solvent-free batch system were studied in detail. The conversion rate of fatty acid was obviously affected by reaction conditions such temperature, molar ratio of substrate, enzyme load, emulsifier, agitation and water-removal methods, and so was the composition of esterification products. In order to get high conversion yield and concentration of 1,3-DAG, some important parameters such as substrate ratio (R, acyl donor to glycerol), reaction temperature (T), enzyme load ([E], based on s
研究开发了一个新颖、有效的酶法合成DAG的生产过程。生产中直接以非吸附的甘油、脂肪酸为起始原料,操作方便,时间短,产率高,而且产物组成简单,利于下游纯化工艺。合成反应中,生物催化剂的选择是至关重要的。酶筛选结果表明Lipozyme RMIM、Novo435不仅酯化活力高,而且可反复利用,用于生产DAG是较合适的。结合考虑位置专一性及成本经济性,实验选择Lipozyme RMIM作为优化的对象。酯化特性研究表明Lipozyme RMIM在40℃优先作用SCFA、MCFA,而60℃对LCFA选择性更强。醇底物选择性研究发现Lipozyme RMIM对伯羟基要优于仲羟基,而对叔羟基或芳环上的羟基活力很低或不起作用。无溶剂体系中,最适水分活度为0.11,当Aw≥0.54时,酯化活力明显降低。非水相中酶活力研究表明,异辛烷是较佳的反应介质,然后依次为正庚烷、正己烷、丁酮、丙酮。为监测酯化反应中间过程及分析终产品,分别采用TLC、HPLC/ELSD、HPLC/RID对甘油酯产物进行分离、定性定量分析。结果表明1,3-DAG与1,2-DAG异构体分离良好。
实验详细研究了间歇反应条件下,无溶剂体系中Lipozyme RMIM催化亚油酸、甘油酯化反应,以及各因素对酯化产物组成的影响。并采用二次旋转组合设计对最适条件进行了优化,对因素的重要性作了适当的评价,并给出了拟合良好、回归显著、可靠性具佳的经验性模型方程。
研究表明真空脱水可有效增加亚油酸的转化率,使反应平衡向合成方向移动。为减少能耗、降低脱水成本,成功地采用离子交换树脂吸附替代真空减压脱水,效果良好。
不同的反应温度、底物摩尔比、酶添加量等热力学因素条件下的酯化进程曲线对比研究表明,温度、底物摩尔比、酶添加量不仅影响酯化反应的初速度、转化率,还与产物组成密切相关。响应面优化结果表明63℃、亚油酸甘油摩尔比为2.1,固定化酶添加量为18EU/g底物,0.01MPa真空脱水,反应8hr亚油酸酯化率最高可接近100%。三因子对亚油酸转化率、1,3-DAG/1,2-DAG比例的影响大小依次分别为:酶添加量>温度>底物比例;温度>底物比例>酶添加量。
动力学因素研究表明,多相酯化反应中传质至关重要。添加适当的乳化剂,可明显增加底物接触面积,改善传质,提高反应初速度,减少反应达到平衡所需时间,但不会改变反应平衡。本实验中单甘酯是最佳的乳化剂,添加量为0.3%。适当的搅拌会改善传质,增加脂肪酸、甘油两相的接触,加快反应速度。试验发现雷诺准数小于10000时,外部传质是主要的限速步骤;当雷诺准数大于10000后,内扩散及酶催化反应速度是主要限速步骤。而且搅拌速度过高,可能会对固化酶造成机械损伤,影响酶活。在雷诺准数为10000左右时,内外传质及催化反应速度达到统一,因而反应速度最快。
通过对实验条件下酶操作稳定性的研究,给出了Lipozyme RMIM的失活动力学方程及时间、产量关系模型。采用Matlab对实验数据进行拟合,求得Lipozyme RMIM的半衰期为594小时,酶失活常数Kd为0.001062hr-1,生产能力为2000-2500g产物/g酶。两次采用不同的方法拟合误差很小,仅为2.26%,而且根据模型求得预测值与真实值拟合良好,模型是极为有效的。
实验进一步研究了填充柱反应器中酶催化亚油酸、甘油连续酯化反应的影响因素。发现填充柱内径,亚油酸、甘油的比例,反应温度,进料速度不仅影响酯化反应的转化率、反应速度,还影响酯化产物中各组分的比例。通过对数据的分析确定固定化酶填充柱长径比7.8(id16mm, L125mm),亚油酸、甘油摩尔比1:2,进料速度1.5ml/min(保留时间12min),65℃条件下酯化反应可实现最终的产量、产品纯度及生产效率三者的统一。
填充床反应器中固定化酶连续催化酯化反应的一个主要问题即体系水分清除困难。实验研究了采用过量甘油吸附脱水的可行性,模拟试验表明甘油的干燥脱水作用良好。稳定性研究确定65℃条件下,亚油酸、甘油摩尔比为1:2,进料速度1.2ml/min,可明显提高酯化反应的转化率,改善固定化酶的稳定性,增加Lipozyme RMIM的使用寿命。连续运行10天,残余酶活仍保持在80%以上,而对照组则仅为52%。
对酯化的产品,尝试了采用液液萃取方式清除单甘酯的研究。实验发现乙醇水溶液可方便地萃取产品中的单甘酯,萃取的选择性随温度增加、载荷量的减少、水比例的加大而增加。载荷量增大,萃取效率增加但选择性下降。多段萃取可实现萃取效率
Diacylglycerols (DAGs) are well known as bio-surfactants. Their excellent biodegradability as well as the behaviors of tasteless, odorless, nontoxic, non-irritant and non-ionic has been paid more and more attraction in numerous industrial fields. It is found that, DAGs are a minor natural component in vegetable oils and fats, as well as an endogenous product of triglyceride metabolism. DAGs are generally recognized as safe as direct human food ingredients and are approved for use in food as dough strengtheners, formulation acid, lubricant and release agents, vehicles, surface finishing agents, stabilizers and thickeners, texturizers (FDA, 2000). In addition, the long-term ingestion of dietary DAG, in contrast to TAG, prevented the accumulation of visceral fat and body weight gain in obesity-prone mice, and decreased postprandial blood lipid levels of rats administration with lipid emulsion. On the other hand, for a long time, large scale’s production of DAG remained mainly in the realms of organic chemistry and chemical processing. Chemical methods are mainly performed at high temperatures in the presence of alkaline catalysts. High energy’s consumption, coloring of products and low selectivity are major disadvantages of these methods. Moreover, some chemicals are toxic and not readily biodegradable, thus causing their limited application in cosmetics, food industry and pharmaceutics. In this case, the enzymatic synthesis offers an alternative way.
In the present work, a novel and effective enzymatic method which could eliminate most of the above-mentioned problems, was developed for the production of DAGs. Non-adsorptive glycerol and free fatty acids were directly used as starting materials in order to increase production yield. The selection of biocatalyst is very crucial. It turned out that Rhizopusmucor miehei lipase (Lipozyme RMIM) and Candida antarictica lipase (Novo 435) are the only suitable biocatalysts for the production of 1,3-DAG, not only for their higher esterification activities, but also for the easy reuse. In view of regiospecificity (1,3-specific) and economics, Rhizopusmucor miehei lipase is more suitable than Candida antarictica lipase. Studies on esterification characteristics of Lipozyme RMIM suggested that the selectivity for LCFA is higher than for MCFA and SCFA at 60 degree, but the situation is reversed at 40 degree. The optimum alcoholic substrates for Lipozyme RMIM are primary alcohols, followed by the secondary alcohols, and then tertiary alcohols. However, Rhizopusmucor
miehei lipase hardly catalyst phenol hydroxyl group. The optimum water activity is 0.11. When water activity is more than 0.54, the enzymatic activity decrease significantly. Studies on non-aqueous phase suggested that isooctane is the optimum medium, followed by heptane and hexane, and then EMK and acetone, as their polarities are strong enough to peel off essential water for enzyme activity.
For monitoring of intermediate process and analysis of products, thin-layer chromatography, normal-, reverse-phase high-performance liquid chromatography were adopted to isolate neutral lipid. Mono-, di-, and tri-acylglycerol were determined with density colorimetric, evaporative light-scatting detector (ELSD) and refractive index detector (RID) individually. It was found that the 1,3-DAGs were evidently resolved from the 1,2-DAG position isomers. Intermediate and final product can be assayed according to methods mentioned above.
Esterification reaction of linoleic acid and glycerol catalyzed by Lipozyme RMIM in solvent-free batch system were studied in detail. The conversion rate of fatty acid was obviously affected by reaction conditions such temperature, molar ratio of substrate, enzyme load, emulsifier, agitation and water-removal methods, and so was the composition of esterification products. In order to get high conversion yield and concentration of 1,3-DAG, some important parameters such as substrate ratio (R, acyl donor to glycerol), reaction temperature (T), enzyme load ([E], based on s
引文
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20 董建林,曹万新,郜顺成. 低热量油脂 (脂肪代用品 )的市场需求及研究开发现状.中国油脂 2000, 25(3):5-9.
21 郑健仙, 李璇. 论低能量食品的开发. 食品工业. 1999, 5: 39-42
22 Takuji Yasukawa and Koichi Yasunaga. Nutritional functions of dietary diacylglycerol. J. Oleo. 2001,50(5): 161-166.
23 Hiroyuki Taguchi, Toshiko Omachi,Tomonori Nagao, Noboru Matsuo, Ichiro Tokimitsu &Hiroshige Itakura. Dietary diacylglycerol suppresss high fat diet-induced hepatic fat accumulation and microsomal triacylglycerol transfer protein activity in rats. J. Nutr Biochem.2002, 13: 678-683.
24 F. B. Padley. The role of fats in human nutrition. London: Society of Chemical Industry, c1985.
25 Tomonorri Nagao,Hiroyuki, watanabe, Naohiro, Goto Dietary DG supresses accumulationof body fat compared to TG J. Nutr. 2000,130:792-797 .
26 Hiroyuki Taguchi, Tonmonon,Nagao, Hiroyuki Watanabe. et.al energy value and digestibility of dietairy oil containing 1,3-DG . Lipid 2001, 36(4): 379-382
27 Takatoshhi Murase Tomohito, Mizuno, Joshiko Omachi et.al. Diacyglycerol supress high fat dietary induced body fat accumulation in c57bl/6j mice. J. of Lipid Res., 2001, 42:372-380.
28 孟祥河,毛忠贵. 甘油二酯的应用现状. 中国食品添加剂. 2001, 53(4):58-61.
29 Doucet; Jim (Olathe, KS). Shortening system, products there with and methods for making and using the same. US 5908655, 1997.
30 Christian Heine, Monhein, Reinhold wust Kaarst.B Neuss; Brigitte Kamp. Processes for preserving the fressiness of fresh meat using acetylate fatty acid mono, di and triglyceride. US 4137334,1979.
31 Yang; Angel A. (Yorba Linda, CA); Druz; Loren L. (Yorba Linda, CA); Berman; Terry (Orange, CA). Process for making a shelf-stable, ready-to-eat rice. US 5702745, 1997.
32 Yukitaka Tanaka; Minoru Nakamura, Johshin Okada. et al. Suppositiory and base thereof. US4837214, 1989.
33 Clandia Waldinger and Manfred Schneider. Enzymatic esterification of glycerol. JOACS 1996,73 (11): 1513-1518.
34 张树政,徐家立. 酶制剂工业. 北京, 科学出版社. pp 655-670, 1984.
35 Kosaku Daimon. Characteristics of immobilized lipase in oils and fats industry. J. Oleic Soc. 2001,1 (8): 31-37.
36 Ryouichi Minoshima. Production and use of lipase in oil and fats industry. J.Oleic Soc.2001,1 (8):37-42.
37. De, D.R, CRc. Handbook of chemistry and physics 73rd 1992-1993, pp5-84, 5-86.
38 D.L. Ingle, A. Dietary energy value of medium-chain triglycerides J. of Food Sci., 1999, 64 (60): 960-964.
Math, J.R Dietary MCFA raise and (n-3) polyunsaturated fatty acids Lower hepatic TG synthesis. J. Nutr .1995,125:2449-2456.
Yu.J. Continuous production of structured lipid containing–linolenic and caprylic acids by immobilized Rhizopous, delemar lipase. JAOCS 1999, 76(2): 189-195.
41 Ryozo Takada, Mamoru Saiton and Toru Mori. Dietary γ-Linolenic Acid-Enriched Oil Reduces
Body Fat Content and Induces Liver Enzyme Activities Relating to Fatty Acid β-Oxidation in Rats. J. Nutr. 124:469-474,1994.
42 David W.L. Preparation of conjugated linolenic acid from safflower oil. JAOCS., 1999, 76(6):1999.
43 Sugivca Masakatsu. Preparition of diglyceride. EP 0307154, 1997.
44 Gotoh. et al. General-purpose oils composition. US 6004611,1999.
45 Gain Frederick, Willian., Moore Stephen., Raymond Peilow., Anne Cythia, Quinlan., Paul Thomas. Fat blends, based on diglyceride, US 5879735,1996.
46 Matsuzaki; Narihide; Kurashige; Jun mase; Tamio; Yamaguchi; Shotaro. Method for reforming fats and oils with enzymes, US 5061498, 1991.
47 高修功, 章克昌. 微生物脂肪酶的生产及其在非水相中催化特性应用的研究.无锡轻工大学博士学位论文, 1997.
48 徐岩 章克昌. 溶剂相中微生物脂肪酶催化脂肪酸酯的合成.无锡轻工大学博士学位论文. 1998.
49 蒋惠亮,章克昌.有机相中酶催化油脂甘油解合成单甘酯的研究. 无锡轻工大学硕士学位论文, 1999.
50 夏咏梅,章克昌.微水体系中酶促合成脂肪酸偏甘油酯的研究.无锡轻工大学博士学位论文, 2000.
51 2002中国统计年鉴,中华人民共和国国家统计局. 中国统计出版社:北京,pp 782-783, 2002.
52 张印俊. 降血脂药物的研究进展. 国外医药抗生素分册2003, 24(60):242-245.
53 陈灏珠. 调脂药物治疗的进展. China Pharmacist. 2002,5(7): 424.
54 赵瑞祥. 降血脂药的临床应用. 中国医院药学杂志. 2001, 21(7): 423-424.
55 唐传核, 彭志英. 一种新型功能性食品—植物甾醇酯. 中国油脂 2001,26(3),60-63.
56 Maarit A. Hallikainen, Essi S. Sarkkinen and Matti I.J. Uusitupa. Plant stanol esters affect serum cholesterol concentration of hperocholesterolemic men and women in a dose-dependent manner. J. Nutr. 2000, 130: 767-776.
57 Marie-Pierre St-Onge and Peter J.H. Jones. Phytosterols and human lipid metabolism:efficacy, safety, and novel foods. Lipids 2003,38:367-375.
58 Lee, A.M., Mok, H.Y., Lee. R.S Mccluskey, M.A&Grundy, S.M. Plant sterols as cholesterol-lowing agents: Clinical trials in patients with hypercholesterolemia and studies of sterol balance. Atherosclerosis 1977, 28:325-338,
59 Grundy, S.M & Mok, H.Y.Effect of low dose phytosterols on cholesterol absorption in men. Lipoprotein Metabolism. Pp112-118, Springer-Verlag, Berlin, Germany.
Heinemann. T., Kullak-Ublick G.A. Pietruck.B, Von Bergmann .K . Mechanisms of action of plant sterols on inhibition od cholesterol absorption composition of sitosterol and sitostanol. Eur. J. Clin pharmacol .1991,40: 59-63.
61 亦森. 植物甾醇和甾烷醇的生理功能. 粮食与油脂 1999,1:46-47.
62 Vanhanen,H.T., Blomqvist,S., Ehnholm,C., Hyvonen,M., Jauhiainen,M., Torstila,I. & Miettinen. Stitostanol ester in dietary oil reduces serum cholesterol. Effect on serum plant sterols and cholesterol precursors. J. Lipid res.1993,34:1535-1544.
63 Miettinen,T.A., Puska,P., Gylling, H.Vanhanen, H.&Vartiaine, E. Reduction of serum cholesterol with sitostanol-ester margarine in a mildly hypercholosterolemic population. N. England . J. Med., 1995, 333:1308-1312.
64 Bruce; Richard D. (Rydal, PA); Burruano; Brid (King of Prussia, PA); Hoy; Michael R. (Sellersville, PA); Paquette; Nicholas R. (Danbury, CT). Method for producing dispersible sterol and stanol compounds. US 6387411, 2002.
Akashe; Ahmad (Mundelein, IL); Miller; Miranda (Arlington Heights, IL). Plant sterol-emulsifier complexes , United States Patent Application 6267963, 2001.
66 Akashe; Ahmad (Mundelein, IL); Miller; Miranda (Arlington Heights, IL). Use of mesophase-stabilized compositions for delivery of cholesterol-reducing sterols and stanols in food products. US 6274574, 2001.
67 Roden,Allan; Williams, James L., Bruce, Ruey; Detrano, Frank; Boyer, Marie H. Preparation of sterol and Stanol-esters. US 20020010349, 2002.
68 Kuczmarski R.J., Flwgal K.M, Campbell S.M. Increasing prevalence of overweight among United States. JAMA. 1994, 272:205-211.
69 Wolf A.N., Colditz G.A. Correct estimates of the economics cost of obesity in the United States. Obes. Res. 1998, 6:97-105.
rian W., Tobin & Gregory D. miller. Nutritional and metabolitic diversity: understanding the basis of biological variance in the obesity,diabetes cardiovascular disease connection. J. Nutr. 2001,131(2):333-335
2 Mokdad A, Marks J, Stroup D, Gerberding J. Actual Causes of Death in the United States, 2000. Journal of the American Medical Association. 2004, 291: 1238-1245.
Kannel, W.B., Cupples, L.A., Ramaswami, R., Stokes, J. 3rd, Kreger, B.E. and Higgins, M. Regional Obesity and Risk of Cardiovascular Dieases, the Framingham Study. J. Clin. Epidemiol. 1991, 44:183-190.
4 杨全海. 肥胖及减肥药的现状及评价. 首都医药 2001, 8(9):63-64.
5 Chow, Moses S.S. Focus On Orlistat: A Nonsystemic Inhibitor Of Gastrointestinal Lipase For Weight Reduction In The Management Of Obesity. Formulary, 1998, 33(10): pp943.
周咏明, 邓文, 邹大茎. 新型减肥药β3-肾上腺素受体激动剂.中国新药杂志2001,10(1): 497-500
Loftus TM. Jaworsky DE, Frehywot GL. et al. Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors. Science.2000, 288(30): 2379-2381.
吉清,何风田. 胖胖基因的研究现状. 生命的化学2002, 22(3):200-204.
何天培. 肥胖相关蛋白的研究进展. 国外医学卫生学分册.1999, 26(6): 327-331.
10 戴经铨. 肥胖与减肥药研究的新动向. 国外医药—合成药、生化药、制剂分册 2002,23(3): 142-146.
11 邹大进. 肥胖治疗研究的现状与展望. 药学服务与研究. 2002, 2 (2): 73-78.
12 Yoshiko, Y., Terue, K., Osamu, T., Masanobu, K., Kyoko, H.&Yasuo, Kagawa. Improvement in blood lipid levels by dietary sn-1,3-diacylglycerol in young women with variants of lipid transporters 54T-FABP2 and –493g-MTP. Biochem. Biophy. Res .Com . 2003, 302:743-748
13 Sakurai T. 0rexins and orexin receptors:implication infeeding behavior. Regul Pept. 1999, 85(1): 25-30.
杨明. 解偶联蛋白及其与肥胖的关系窃究进展. 国外医学内学. 2002, 22(3):169-173.
15 Bray GA. Tartaglia LA. Medicinial strategies in the treatment of obesity. Nature 2000, 404:672-677.
16 T.J. Mangos, K.C. Jones, and T.A. Foglia. Lipase-Catalyzed Synthesis of Structured Low-Calorie Triacylglycerols. JAOCS. 1999,76(10):1127-1132.
17 Lydia B.Fomuso and Casimir C.Akoh. Enzymatic Modification of Triolein: Incorporation of Caproic and Butyric Acids to Produce Reduced-Calorie Structured Lipids. JAOCS, 1997,74(3):269-272.
18 Xuebing Xu,Anja Skands,Gunnar Jonsson& Jens Adler-Nissen. Production of structured lipids by lipase-catalysed interesterification in an ultrafiltration membrane reactor. Biotechnology Letters 2000, 22:1667-1671.
19 刘永,周家华,曾颢. 碳水化合物型脂肪替代品的研究进展. 食品科技2000, 1:15-17.
20 董建林,曹万新,郜顺成. 低热量油脂 (脂肪代用品 )的市场需求及研究开发现状.中国油脂 2000, 25(3):5-9.
21 郑健仙, 李璇. 论低能量食品的开发. 食品工业. 1999, 5: 39-42
22 Takuji Yasukawa and Koichi Yasunaga. Nutritional functions of dietary diacylglycerol. J. Oleo. 2001,50(5): 161-166.
23 Hiroyuki Taguchi, Toshiko Omachi,Tomonori Nagao, Noboru Matsuo, Ichiro Tokimitsu &Hiroshige Itakura. Dietary diacylglycerol suppresss high fat diet-induced hepatic fat accumulation and microsomal triacylglycerol transfer protein activity in rats. J. Nutr Biochem.2002, 13: 678-683.
24 F. B. Padley. The role of fats in human nutrition. London: Society of Chemical Industry, c1985.
25 Tomonorri Nagao,Hiroyuki, watanabe, Naohiro, Goto Dietary DG supresses accumulationof body fat compared to TG J. Nutr. 2000,130:792-797 .
26 Hiroyuki Taguchi, Tonmonon,Nagao, Hiroyuki Watanabe. et.al energy value and digestibility of dietairy oil containing 1,3-DG . Lipid 2001, 36(4): 379-382
27 Takatoshhi Murase Tomohito, Mizuno, Joshiko Omachi et.al. Diacyglycerol supress high fat dietary induced body fat accumulation in c57bl/6j mice. J. of Lipid Res., 2001, 42:372-380.
28 孟祥河,毛忠贵. 甘油二酯的应用现状. 中国食品添加剂. 2001, 53(4):58-61.
29 Doucet; Jim (Olathe, KS). Shortening system, products there with and methods for making and using the same. US 5908655, 1997.
30 Christian Heine, Monhein, Reinhold wust Kaarst.B Neuss; Brigitte Kamp. Processes for preserving the fressiness of fresh meat using acetylate fatty acid mono, di and triglyceride. US 4137334,1979.
31 Yang; Angel A. (Yorba Linda, CA); Druz; Loren L. (Yorba Linda, CA); Berman; Terry (Orange, CA). Process for making a shelf-stable, ready-to-eat rice. US 5702745, 1997.
32 Yukitaka Tanaka; Minoru Nakamura, Johshin Okada. et al. Suppositiory and base thereof. US4837214, 1989.
33 Clandia Waldinger and Manfred Schneider. Enzymatic esterification of glycerol. JOACS 1996,73 (11): 1513-1518.
34 张树政,徐家立. 酶制剂工业. 北京, 科学出版社. pp 655-670, 1984.
35 Kosaku Daimon. Characteristics of immobilized lipase in oils and fats industry. J. Oleic Soc. 2001,1 (8): 31-37.
36 Ryouichi Minoshima. Production and use of lipase in oil and fats industry. J.Oleic Soc.2001,1 (8):37-42.
37. De, D.R, CRc. Handbook of chemistry and physics 73rd 1992-1993, pp5-84, 5-86.
38 D.L. Ingle, A. Dietary energy value of medium-chain triglycerides J. of Food Sci., 1999, 64 (60): 960-964.
Math, J.R Dietary MCFA raise and (n-3) polyunsaturated fatty acids Lower hepatic TG synthesis. J. Nutr .1995,125:2449-2456.
Yu.J. Continuous production of structured lipid containing–linolenic and caprylic acids by immobilized Rhizopous, delemar lipase. JAOCS 1999, 76(2): 189-195.
41 Ryozo Takada, Mamoru Saiton and Toru Mori. Dietary γ-Linolenic Acid-Enriched Oil Reduces
Body Fat Content and Induces Liver Enzyme Activities Relating to Fatty Acid β-Oxidation in Rats. J. Nutr. 124:469-474,1994.
42 David W.L. Preparation of conjugated linolenic acid from safflower oil. JAOCS., 1999, 76(6):1999.
43 Sugivca Masakatsu. Preparition of diglyceride. EP 0307154, 1997.
44 Gotoh. et al. General-purpose oils composition. US 6004611,1999.
45 Gain Frederick, Willian., Moore Stephen., Raymond Peilow., Anne Cythia, Quinlan., Paul Thomas. Fat blends, based on diglyceride, US 5879735,1996.
46 Matsuzaki; Narihide; Kurashige; Jun mase; Tamio; Yamaguchi; Shotaro. Method for reforming fats and oils with enzymes, US 5061498, 1991.
47 高修功, 章克昌. 微生物脂肪酶的生产及其在非水相中催化特性应用的研究.无锡轻工大学博士学位论文, 1997.
48 徐岩 章克昌. 溶剂相中微生物脂肪酶催化脂肪酸酯的合成.无锡轻工大学博士学位论文. 1998.
49 蒋惠亮,章克昌.有机相中酶催化油脂甘油解合成单甘酯的研究. 无锡轻工大学硕士学位论文, 1999.
50 夏咏梅,章克昌.微水体系中酶促合成脂肪酸偏甘油酯的研究.无锡轻工大学博士学位论文, 2000.
51 2002中国统计年鉴,中华人民共和国国家统计局. 中国统计出版社:北京,pp 782-783, 2002.
52 张印俊. 降血脂药物的研究进展. 国外医药抗生素分册2003, 24(60):242-245.
53 陈灏珠. 调脂药物治疗的进展. China Pharmacist. 2002,5(7): 424.
54 赵瑞祥. 降血脂药的临床应用. 中国医院药学杂志. 2001, 21(7): 423-424.
55 唐传核, 彭志英. 一种新型功能性食品—植物甾醇酯. 中国油脂 2001,26(3),60-63.
56 Maarit A. Hallikainen, Essi S. Sarkkinen and Matti I.J. Uusitupa. Plant stanol esters affect serum cholesterol concentration of hperocholesterolemic men and women in a dose-dependent manner. J. Nutr. 2000, 130: 767-776.
57 Marie-Pierre St-Onge and Peter J.H. Jones. Phytosterols and human lipid metabolism:efficacy, safety, and novel foods. Lipids 2003,38:367-375.
58 Lee, A.M., Mok, H.Y., Lee. R.S Mccluskey, M.A&Grundy, S.M. Plant sterols as cholesterol-lowing agents: Clinical trials in patients with hypercholesterolemia and studies of sterol balance. Atherosclerosis 1977, 28:325-338,
59 Grundy, S.M & Mok, H.Y.Effect of low dose phytosterols on cholesterol absorption in men. Lipoprotein Metabolism. Pp112-118, Springer-Verlag, Berlin, Germany.
Heinemann. T., Kullak-Ublick G.A. Pietruck.B, Von Bergmann .K . Mechanisms of action of plant sterols on inhibition od cholesterol absorption composition of sitosterol and sitostanol. Eur. J. Clin pharmacol .1991,40: 59-63.
61 亦森. 植物甾醇和甾烷醇的生理功能. 粮食与油脂 1999,1:46-47.
62 Vanhanen,H.T., Blomqvist,S., Ehnholm,C., Hyvonen,M., Jauhiainen,M., Torstila,I. & Miettinen. Stitostanol ester in dietary oil reduces serum cholesterol. Effect on serum plant sterols and cholesterol precursors. J. Lipid res.1993,34:1535-1544.
63 Miettinen,T.A., Puska,P., Gylling, H.Vanhanen, H.&Vartiaine, E. Reduction of serum cholesterol with sitostanol-ester margarine in a mildly hypercholosterolemic population. N. England . J. Med., 1995, 333:1308-1312.
64 Bruce; Richard D. (Rydal, PA); Burruano; Brid (King of Prussia, PA); Hoy; Michael R. (Sellersville, PA); Paquette; Nicholas R. (Danbury, CT). Method for producing dispersible sterol and stanol compounds. US 6387411, 2002.
Akashe; Ahmad (Mundelein, IL); Miller; Miranda (Arlington Heights, IL). Plant sterol-emulsifier complexes , United States Patent Application 6267963, 2001.
66 Akashe; Ahmad (Mundelein, IL); Miller; Miranda (Arlington Heights, IL). Use of mesophase-stabilized compositions for delivery of cholesterol-reducing sterols and stanols in food products. US 6274574, 2001.
67 Roden,Allan; Williams, James L., Bruce, Ruey; Detrano, Frank; Boyer, Marie H. Preparation of sterol and Stanol-esters. US 20020010349, 2002.
68 Kuczmarski R.J., Flwgal K.M, Campbell S.M. Increasing prevalence of overweight among United States. JAMA. 1994, 272:205-211.
69 Wolf A.N., Colditz G.A. Correct estimates of the economics cost of obesity in the United States. Obes. Res. 1998, 6:97-105.
rian W., Tobin & Gregory D. miller. Nutritional and metabolitic diversity: understanding the basis of biological variance in the obesity,diabetes cardiovascular disease connection. J. Nutr. 2001,131(2):333-335