胰脂肪酶法测定食用油甘油三酯中脂肪酸的位置分布

本实验采用sn-1,3位专一性脂肪酶(胰脂肪酶)对甘油三酯进行作用,专一水解位于sn-1、sn-3的酯键,将该两个位置上的脂肪酸游离出来,通过薄层层析分离得到游离脂肪酸和sn-2-甘油一酯,甲酯化作用后用气相色谱法对脂肪酸进行测定。用该方法对花生油、大豆油、玉米油、菜籽油、茶油进行了测定。结果表明,这几种油不饱和脂肪酸含量达到了80%,在sn-2位上分布更是超过了90%,而少量的饱和脂肪酸则主要是分布在sn-1,3位上。     

米曲霉脂肪酶富集浓缩裂殖壶藻油脂中DHA的机理研究

本研究采用了米曲霉(Aspergillus oryzae, AO)脂肪酶水解法对裂殖壶藻油脂中的二十二碳六烯酸(Docosahexaenoic Acid, DHA)进行了浓缩。在AO脂肪酶最适反应条件下,水解12 h后,反应达到了平衡,产物中甘三酯含量降至40.2%,DHA含量升至55.43%,浓缩了1.58倍,回收率高达68.2%。通过气相色谱结合核磁共振碳谱(13C-NMR)对产物中甘油三酯脂肪酸组成及含量进行了分析,结果显示在酶解反应过程中,DHA倾向于留在甘三酯甘油骨架上,且sn-1,3位上的饱和脂肪酸易被水解而含量下降,表明AO脂肪酶水解反应对甘油骨架上的脂肪酸具有选择性,可将DHA富集在甘油三酯中。AO脂肪酶价格低廉,基于酶解反应的DHA富集工艺简单,深入了解反应的作用机制,将为后续的理论研究和实际应用提供依据。     

利用月桂酸和山茶油的酸解反应分析脂肪酶的位置专一性

脂肪酶的位置专一性在结构酯的合成和油脂改性中具有重要意义。由于水体系和非水体系的差异,传统的水解判定法得出的专一性与该酶在合成反应中的表现可能并不一致。本研究利用月桂酸和山茶油的酸解反应来达到直接评估酶在无溶剂体系中位置专一性的目的。反应产物的脂肪酸组成和位置分布由气相色谱测定。通过此方法,Lipozyme RM IM、L02、L03和L04被鉴定为sn-1(3)位专一性,L01为弱专一性,Novozym 435近似无专一性。通过替换酶的底物,模型反应的可预测性得到验证。根据酸解法和水解法结果的对比分析,2种条件下酶的位置专一性通常是相同的,除了易受到溶剂体系影响的Novozym 435。因此,新方法能够避免水解判定结果在合成反应中应用的局限性。此外,它还降低了对底物纯度的要求,这意味着其能在低成本下用于脂肪酶的大规模筛选。     

人乳脂替代品的酶法合成及其评价的研究进展

由于经济、社会和个人因素方面,我国及其他一些发展中国家的婴儿纯母乳喂养率普遍偏低。与植物油和反刍动物乳脂相比,人乳脂中棕榈酸分布在甘油骨架的sn-2位更有利于婴儿对脂肪酸和钙的吸收,减少婴儿便秘、上火等。合成与人乳脂相似的人乳脂替代品,将其作为婴儿配方奶粉的脂肪来源具有着重要的市场潜力。酶法催化具有反应条件温和、高度位置专一性和脂肪酸专一性,因此,可以通过sn-1,3位专一性脂肪酶催化合成人乳脂替代品。本文介绍了人乳脂替代品的定义及发展,并就近年来的酶法合成人乳脂替代品的底物选择、脂肪酶筛选、合成方法与技术、检测分析方法、相似度评价以及氧化稳定性等方面的研究状况进行系统阐述,并对今后人乳脂替代品研究做了展望。     

酶促油脂水解和酯交换

生物催化反应具有许多优于物理,化学催化反应的特点。在油脂工业中,可以利用脂肪酶催化油脂的水解反应与酯交换反应,从而获得一般催化反应难以得到的产品。不同的脂肪酶具有不同的特性,按其专一性可分为:(1)无专一性酶,(2)作用于甘三酯1,3位的酶,(3)作用于一定结构脂肪酸的酶。酶促油脂水解反应主要用于获得甘油和脂肪酸,特别在从稳定性差的油脂制取不饱和度高的或共轭脂肪酸等更显特色。酶促油脂酶交换反应主要用于油脂的改性,这是利用较便宜的油脂制取价值较高的特殊专用油脂的一条极好途径,尤其在制取类可可脂等产品方面,效益更为显著。目前,发达国家在此方面的研究不断深入,有望酶催化技术将成为油脂工业将来发展的先进技术之一。     

重组米根霉脂肪酶的酶学性质及其催化水解鳀鱼油富集DHA的研究

米根霉脂肪酶(Rhizopus oryzae lipase)是一种具有广泛用途的工业化脂肪酶,对重组米根霉脂肪酶(r ProROL)的酶学性质进行考察,发现其水解甘油三酯时具有明显的sn-1,3位置专一性;催化二十碳五烯酸(EPA)和二十二碳六烯酸(DHA)酯化时其活力分别为催化油酸酯化活力的37.42%和8%。鳀鱼油是天然EPA/DHA主要来源之一,在鳀鱼油中DHA含量为12.1%。13C核磁共振波谱分析表明,鳀鱼油中DHA主要分布在甘油酯的sn-2位,利用重组米根霉脂肪酶(r ProROL)催化鳀鱼油部分水解富集其中的DHA时,得到的1,2-甘油二酯中DHA的含量可以达到19.7%,甘油单酯中DHA的含量可以达到25.2%,DHA的回收率为65.6%。表明r ProROL脂肪酶具有水解鳀鱼油富集DHA的应用潜能。     

脂肪酶催化单油酸甘油酯制备功能性1,3-甘油二酯

研究固定化脂肪酶TLIM催化单油酸甘油酯(glycerol monooleate,GMO)制备1,3-甘油二酯(sn-1,3-diacylglyerol,sn-1,3-DAG)。比较了游离脂肪酸(共轭亚油酸)和脂肪酸乙酯(共轭亚油酸乙酯)两种不同类型酰基供体、反应时间、底物物质的量比对酰基迁移和sn-1,3-DAG的影响。通过对实验结果的判定及分析得到最佳反应条件为采用20%(质量分数)脂肪酶TLIM、底物物质的量比(共轭亚油酸乙酯和GMO)3∶1、在50?℃的220 r/min水浴摇床中反应2 h,最后得到sn-1,3-DAG转化率为65%。本研究利用GMO而不是常规的甘油或者甘油三酯来制备sn-1,3-DAG,并比较了不同酰基供体对酰基迁移和sn-1,3-DAG转化率的影响,旨在为脂肪酶催化法制备功能性sn-1,3-DAG的研究提供一定参考。     

水活度对全细胞脂肪酶GZUF36催化合成1,3-甘油二酯中的影响

1,3-甘油二酯是一种健康油脂,并且可用来合成药物的中间材料,但其在天然油脂中含量有限。所以本文用黑曲霉GZUF36全细胞脂肪酶法甘油解合成了1,3-甘油二酯,重点探讨了影响其合成的关键因素:水活度。控制水活度的方法包括用饱和盐溶液平衡酶粉、水合盐平衡反应体介质、水合盐控制水活度水合盐分别预平衡酶粉和反应介质。结果表明,水活度显著地影响1,3-甘油二酯的产量和脂肪酶对1,3-甘油二酯的选择性。过高或过低的水活度都不利于全细胞脂肪酶GZUF36催化甘油解反应合成1,3-甘油二酯,水活度过低会导致酶活力不高,水活度过高则易使催化反应向水解方向进行。当用aw=0.58的水合盐分别预平衡酶粉和反应介质时,1,3-甘油二酯的得率和脂肪酶对1.3-甘油二酯的选择性都为最优,分别是26.100%和83.056%。本文为进一步优化全细胞脂肪酶GZUF36催化的选择性合成1,3-甘油二酯中的反应体系奠定了基础。     

酶促生产结构化脂质

结构化脂质系指具有特定功能的并且是用人工方法生产的三酰基甘油,这种三酰基甘油含有附着在甘油主链上的多种短链、中链和长链脂肪酸。根据与三酰基甘油的甘油主链酰化的各种脂肪酸的链的长度,结构化脂质主要分为MLM型、SLS型、LML型、MLS型等类型。L代表长链脂肪酸（≥C14）M代表中链脂肪酸（C8-C10）,S代表短链脂肪酸（≤C6）。胰和胃的脂酶多半在三酰基甘油的sn-1和sn-3位置上有酯键,而且与长链脂肪酸相比,尤其是与长链多不饱和脂肪酸相比,在靠近短链和中链脂肪酸处,胰和胃的脂酶显出更高的活性。因此,膳食中的三酰基甘油被脂酶水解为肠道中的sn-2单酰基甘油和脂肪酸。水解产物被人体吸收,并在肠道的粘膜细胞中转化成新的三酰基甘油。这样,如果膳食中的油脂是MLM或SLS型三酰基甘油,那么位于sn-2处的长链多不饱和脂肪酸就可以提供sn-2单酰基甘油,这是粘膜细胞中三酰基甘油合成的起始物质。Sn-1和sn-3处的短链或中链脂肪酸,被门静脉吸收并且快速在肝脏中氧化成能源以取代葡萄糖。在sn-1和sn-3两处都含有短链或中链脂肪酸,并且在sn-2处含有长链脂肪酸的各种型号的结构化脂质可以有效地提供易于人体吸收的各种脂肪酸,作为营养素、功能性类脂物和药物,治疗特殊的疾病及代谢病症。     

富含亚油酸硬脂的研制初报

用高亚油酸型的红花籽油和硬脂酸,经1,3位专一性固定化脂肪酶Lipozyme的催化,通过有方向性的酯交换,制备出了以1,3——二硬脂酰—2—亚油酰甘油酯为主要组分的硬脂。这种富含亚油酸的硬脂具有可塑性范围小的特性,同时其营养价值也得到了提高。这为充分利用大宗油脂资源、开发有营养性等特色的硬脂提出了新的研究课题。     

PREPARATION OF STRUCTURED LIPIDS WITH SPECIAL FUNCTIONAL PROPERTIES

Enzymatic acidolysis of rapeseed oil with capric acid was carried out to obtain structured lipids. The reaction was catalyzed by Lipozyme IM lipase from Rhizomucor miehei. The enzyme preparations contained 2.8 and 10% water. The reaction conditions were enzyme load of 8% (w/w total substrates), substrate mole ratio of 1:6 (rapeseed oil:capric acid), and reaction temperature of 65C. The results showed that triacylglycerols (TAG) after transesterification contained mainly oleic, linoleic and linolenic acids (about 90%) in the internal sn-2 position, whereas capric acid was mostly in the external sn-1,3 positions (approximately 40%). The quantity of water in the reaction medium had a significant influence on the yield and quality of the TAG fraction.     

3种海洋鱼油脂肪酸组成及其位置分布

采用米黑根毛霉源固定化脂肪酶(Lipozyme RM IM)对常见的海洋鱼油甘三脂进行水解,分析了凤尾鱼、金枪鱼、三文鱼甘三酯中脂肪酸的组成及位置分布。采用硅胶色谱柱分离3种海洋鱼油甘三脂,利用Lipozyme RM IM的sn-1,3特异性,将酯化在sn-1,3位上的脂肪酸(EFA)水解成游离脂肪酸(FFA),然后通过薄层层析(TLC)分离得到sn-2-单甘酯,再甲酯化后利用气相色谱(GC)测定sn-2位脂肪酸组成,并按照脂肪酸的不饱和程度和n-3,6-多不饱和脂肪酸归类分析海洋鱼油甘三酯中各类脂肪酸位置分布的特点。结果表明:3种海洋鱼油(凤尾鱼油、金枪鱼油、三文鱼油)中不饱和脂肪酸含量达到60%以上,其中单不饱和脂肪酸(MUFA)占24.67%~33.51%,多不饱和脂肪酸(PUFA)占26.89%~36.15%,而且一半以上PUFA分布在sn-2位,有利于其吸收和提高其耐氧化性;而SFA和MUFA倾向于分布在sn-1,3位上。n-3与n-6PUFAs之间的比例均10,均符合FAO/WHO推荐摄入的标准(4),是理想的n-3PUFA天然营养补充剂。     

Biodiesel fuel production from plant oil catalyzed by Rhizopus oryzae lipase in a water-containing system without an organic solvent

A new enzymatic method of synthesizing methyl esters from plant oil and methanol in a solvent-free reaction system was developed. It is anticipated that such plant oil methyl esters can be used as a biodiesel fuel in the future. Lipase from Rhizopus oryzae efficiently catalyzed the methanolysis of soybean oil in the presence of 4-30 wt% water in the starting materials; however the lipase was nearly inactive in the absence of water. The methyl ester (ME) content in the reaction mixture reached 80-90 wt% by stepwise additions of methanol to the reaction mixture. The kinetics of the reaction appears to be in accordance with the successive reaction mechanism. That is, the oil is first hydrolyzed to free fatty acids and partial glycerides, and the fatty acids produced are then esterified with methanol. Although R. oryzae lipase is considered to exhibit 1(3)-regiospecificity, a certain amount of 1,3-diglyceride was obtained during the methanolysis and hydrolysis of soybean oil by R. oryzae lipase solution. Therefore, the high ME content in the reaction mixture is probably attributable to the acyl migration from the sn-2 position to the sn-1 or sn-3 position in partial glycerides.     

脂肪酶在食用油脂工业上的应用

脂肪酶按其对底物的特异性不同可分为非特异性酶、１,３—定向酶和脂肪酸特异性酶,在食用油脂工业上具有较大应用价值的是１,３—定向脂肪酶。介绍了脂肪酶（主要是１,３—定向脂肪酶）在油脂水解、酯交换和生物精炼三个方面的应用原理、特点及实例,从而展示出了脂肪酶催化技术在食用油脂工业上应用的前景     

LIPASE-CATALYZED PRODUCTION OF STRUCTURED LIPIDS VIA ACIDOLYSIS OF FISH OIL WITH CAPRYLIC ACID

Structured lipids containing eicosapentaenoic and docosahexaenoic acids were manufactured in a batch reactor by lipase-catalyzed acidolysis of fish oil with caprylic acid. The following free lipases (Lipase AP, Aspergillus niger ; Lipase P, Pseudomonus sp. ; Lipase AY, Candida rugosa ; Lipase AK, Pseudomonas fluoresescens ; Lipase F, Rhizopus oryzae ; Lipase D, Rhizopus delemar ) were screened under selected reaction conditions. The conditions were enzyme load 5%, substrate mole ratio 1:6 (fish oil: caprylic acid), and reaction temperature of 50C. Lipase AK had the highest activity and was suitable for production of structured lipids from fish oil. The optimal mole substrate ratio of fish oil to caprylic acid for Lipase AK was 1:6 to 1:8. The time course of the reaction at different enzyme loads demonstrated that 40% incorporation of caprylic acid could be obtained for Lipase AK in 5 h with 10% enzyme load. Addition of water had little effect on the activity of the lipase. Lipase AK and Lipozyme IM were further compared under the same conditions, in which Lipase AK had a slightly higher incorporation of caprylic acid, similar acyl migration of caprylic acid from sn-1,3 positions to the sn-2 position, and a slightly lower selectivity towards docosahexaenoic acid.     

OPTIMIZATION OF LIPASE-CATALYZED ACIDOLYSIS OF SOYBEAN OIL TO PRODUCE STRUCTURED LIPIDS

The condition optimization of lipase-catalyzed acidolysis of soybean oil was conducted by using the response surface methodology (RSM). The results showed that when decision coefficient was 98.34%, the optimal conditions for highest caprylic acid (CA) incorporation was: reaction temperature 35.8C, lipase dosage 11.9% (w/w of substrate), substrate ratio 5.7 (oil/CA, mol/mol), water content 15.4% (w/w of enzyme) and reaction time 20.4 h, and under which conditions, the actually measured incorporation of CA was up to 44.9 mol%, very close to the predicted value 45.7 mol%. This indicates that with RSM, we can effectively optimize the reaction conditions of lipase-catalyzed acidolysis of soybean oil to produce structured lipids.

PRACTICAL APPLICATIONS

In digestion procedure, pancreatic and gastric lipases both have preference for ester bonds located at sn-1 and sn-3 position in triacylglycerols (TGs), and show higher activity towards short- and medium-chain fatty acids (FAs). Dietary TGs are hydrolyzed to sn-2 monoacylglycerols and FAs in the intestine by lipases. Therefore, long-chain polyunsaturated FAs located at sn-2 position may provide sn-2 monoacylglycerols, which are the raw materials in synthesis of TGs in mucosal cells if the dietary fats or oils are MLM- or SLS-type TGs (SLS or MLM are the short-chain or medium-chain FAs located at sn-1 and sn-3 position, while the long-chain FAs are located at sn-2). The short-chain or medium-chain FAs located at sn-1 and sn-3 position are absorbed by the portal vein and rapidly oxidized in the liver as quick source of energy instead of glucose.     

Milk lipoprotein lipases: a review.

Lipoprotein lipase activity has been found in the milks from severals species where it is assumed to result from leakage from the mammary gland into milk. The function of the enzyme in the gland is apparently to assist in the transfer of blood lipoprotein triacylglycerol fatty acids into milk triacylglycerols. Bovine skim milk is one of the richest sources of lipoprotein lipase and this enzyme has been purified extensively (7000 fold) by affinity chromatography. The lipase has a molecular weight of about 62000, is inhibited by protamine sulfate, 1.0 M sodium chloride, apolipoprotein C-I (apolipoprotein-serine), and apolipoprotein C-III (apolipoprotein-alanine). The enzyme is activated by apolipoprotein C-II (apolipoprotein-glutamic acid), serum, and by heparin to which it also binds. The lipase is highly specific for the primary esters of acylglycerols and exhibits a slight stereospecificity for the sn-1 ester in preference to the sn-3-ester. Bovine milk also has separate activity toward 1-monoacylglycerols. Human milk contains a serum stimulated lipoprotein lipase with many of the characteristics of the enzyme in bovine milk, as well as an enzyme stimulated by bile salts which resembles the sterol ester hydrolase of rat pancreatic juice. The assay, function, purification, characteristics, and substrate specificities of these enzyme are discussed.     

Enzymatic production of infant milk fat analogs containing palmitic acid: optimization of reactions by response surface methodology

Infant milk fat analogs resembling human milk fat were synthesized by an enzymatic interesterification between tripalmitin, coconut oil, safflower oil, and soybean oil in hexane. A commercially immobilized 1,3-specific lipase, Lipozyme RM IM, obtained from Rhizomucor miehei was used as a biocatalyst. The effects of substrate molar ratio, reaction time, and incubation temperature on the incorporation of palmitic acid at the sn-2 position of the triacylglycerols were investigated. A central composite design with 5 levels and 3 factors consisting of substrate ratio, reaction temperature, and incubation time was used to model and optimize the reaction conditions using response surface methodology. A quadratic model using multiple regressions was then obtained for the incorporation of palmitic acid at the sn-2 positions of glycerols as the response. The coefficient of determination (R²) value for the model was 0.845. The incorporation of palmitic acid appeared to increase with the decrease in substrate molar ratio and increase in reaction temperature, and optimum incubation time occurred at 18 h. The optimal conditions generated from the model for the targeted 40% palmitic acid incorporation at the sn-2 position were 3 mol/mol, 14.4 h, and 55°C; and 2.8 mol/mol, 19.6 h, and 55°C for substrate ratio (moles of total fatty acid/moles of tripalmitin), time, and temperature, respectively. Infant milk fat containing fatty acid composition and sn-2 fatty acid profile similar to human milk fat was successfully produced. The fat analogs produced under optimal conditions had total and sn-2 positional palmitic acid levels comparable to that of human milk fat.