无溶剂体系Lipozyme RM IM催化棕榈酸和山茶油酸解反应的研究

研究了无溶剂体系固定化1,3-特异性脂肪酶Lipozyme RM IM催化棕榈酸和山茶油酸解反应中,棕榈酸在产物甘油三酯中Sn-2位的相对含量分布以及酰基转移规律。考察了酶用量、底物摩尔比、酶水分活度、反应温度和反应时间对酸解反应的影响。结果表明：酶水分活度、反应温度和反应时间对棕榈酸在产物甘油三酯中的含量影响不大,对酰基转移影响较大;在酶水分活度0.550,反应温度60℃,反应时间1 h的反应条件下,酰基转移的程度最小;底物摩尔比和酶用量对酰基转移的程度影响不大。     

溶剂和水分活度对OPO结构脂合成中酰基迁移的影响

以三棕榈酸甘油酯(PPP)和油茶籽油游离脂肪酸(FFA)为底物制备1,3-二油酸-2-棕榈酸甘油酯(OPO)结构脂为反应模型,以专一性脂酶Lipozyme RM IM为催化剂,研究不同有机溶剂(正己烷、丙酮、叔丁醇)、不同反应时间(0、2、4、6、8 h)、不同水分活度(a_w)(0.11、0.53、0.97)对酰基迁移的影响。结果表明:在3种溶剂中,丙酮的反应环境下酰基迁移发生率最低且油酸的键入率最高;在不同溶剂、不同水分活度的条件下,均在a_w为0.53时sn-2位棕榈酸和sn-1,3位油酸的含量较高,说明a_w影响脂肪酶的1,3位置选择性强弱决定于a_w的高低。鉴于结构脂制备过程中的绿色环保安全角度,可以采用无溶剂体系但是应较好地控制反应体系的a_w以抑制酰基迁移。     

响应面优化溶剂体系酶催化阿魏酸乙酯与甘二酯酯交换合成阿魏酰基脂肪酰基结构脂

在异辛烷体系中,通过Novozym 435脂肪酶催化阿魏酸乙酯与甘二酯酯交换反应,以得到阿魏酰基脂肪酰基结构脂。利用Design Expert 8.0软件对响应面进行设计,通过对响应面实验优化和分析得出,3个因素对EF转化率影响大小顺序为:反应时间>加酶量>反应温度。并确定在此体系下酶法催化双硬脂酸甘二酯酯交换合成阿魏酰基脂肪酰基结构脂的最佳工艺条件为:反应温度67℃,反应时间18 h,加酶量11%,在此工艺条件下,可得到阿魏酸乙酯转化率为79.4%,亲脂性产品阿魏酰基脂肪酰基结构脂得率为60.0%。     

水分活度在共轭亚油酸结构酯制备中对酰基迁移的影响

以脂肪酶Lipozyme TLIM催化共轭亚油酸(CLA)和松籽油制备sn-2位含有CLA和松籽油酸(PLA)的结构脂,考察体系水分活度对CLA键入率和酰基迁移的影响。在水分活度0.22~0.80的范围内,松籽油中CLA的键入量随CLA水分活度的增加而增加,而酰基迁移也随着水分活度增加而增加。但是通过研究酰基迁移与CLA键入量的关系,发现当酰基迁移程度一定时(如0.6)水分活度越高总CLA键入量越高。因此,若要抑制酰基迁移则增大水分活度并相应的缩短反应时间,因为高水分活度体系中脂酶催化的反应速率加快;反之,若要促进酰基迁移则选用水分活度低的体系,并相应的延长反应时间,反应时间越长,体系越接近动力学平衡,Sn-2脂肪酸几乎等于总脂肪酸。本实验在反应温度60℃、加酶量10%、松籽油与CLA质量比为4:1、水分活度0.65的条件下反应18 h制备结构脂,在所得结构脂中CLA的含量为10.10%,PLA的含量为15.17%;甘油三酯中sn-2位脂肪酸组成中CLA达到7.41%,PLA含量为9.29%。     

酶法合成1-油酸-2-棕榈酸-3-亚油酸甘油三酯结构脂的研究

在Lipozyme RM IM脂肪酶的催化作用下,将分提后的棕榈硬脂与油酸和亚油酸反应,通过酶法酸解合成富含1-油酸-2-棕榈酸-3-亚油酸甘油三酯(OPL)的结构脂,通过单因素实验对酶法合成反应条件进行了优化。结果表明,合成OPL结构脂的最优反应条件为:棕榈硬脂、油酸、亚油酸摩尔比1∶7∶7,酶添加量8%,反应温度60℃,反应时间6 h。在最优条件下,进行50倍放大实验,合成产物中OPL和sn-2位棕榈酸的含量分别为48.37%和84.70%。     

花色苷酶促酰化反应研究进展

花色苷的功能、稳定性与其结构存在密切关联,特别是其酰基结构。酰化反应是提高花色苷结构稳定性的重要方法。传统化学酰化方法存在许多不足和缺陷,而酶促酰化受到广泛关注,因其反应条件温和,选择性强,产品质量高,可运用在食品,药品,化工合成等领域。总结了酰化反应对提高花色苷稳定性的效用,酶促糖苷酰化反应中催化酶、酰基供体以及溶剂体系方面的研究进展,以期对花色苷结构稳定化技术提供借鉴。     

响应面试验优化Lipozyme TLIM催化制备共轭亚油酸结构脂工艺

以Lipozyme TLIM催化茶油和共轭亚油酸乙酯制备共轭亚油酸(conjugated linoleic acid,CLA)结构脂,并调控酰基迁移反应以制备Sn-2位含CLA的结构脂。通过响应面法研究反应温度、水分活度、反应时间和底物物质的量比对总CLA键入量和酰基迁移的影响,并优化反应条件。本实验得到的结构脂中含有36.97%的总CLA,其中12.54%的CLA分布在Sn-2位,所得总CLA及Sn-2位的CLA键入量均高于文献报道。     

酶催化合成蔗糖酯研究进展

本文对蔗糖酯的酶促合成研究进行了总结,其中主要涉及到酶、反应媒介和酰基供体等影响因素。研究发现,蛋白酶、脂肪酶和抗体酶均能区域选择地酰化蔗糖。酶催化合成蔗糖酯通常在N,N-二甲基甲酰胺(DMF),二甲基亚砜(DMSO)和吡啶等有毒溶剂中进行。近几年来,使用混合溶剂或叔丁醇等毒性较小的溶剂代替毒性较大的溶剂,以及无溶剂条件下酶催化合成蔗糖酯的研究,也越来越受重视。活化酯作为酰基供体得到广泛的应用。     

酸解巴沙鲶鱼油制备富含OPO的人乳替代脂

以巴沙鲶鱼油为原料、高油酸葵花籽油来源的游离脂肪酸为酰基供体,采用Lipozyme RM IM脂肪酶为催化剂催化酸解反应制备富含OPO的人乳替代脂。并采用液质联用对巴沙鲶鱼油、酸解巴沙鲶鱼油、市售Betapol~(TM)的sn-OPO含量进行分析。结果表明,最佳制备条件为:底物摩尔比1∶6,酶用量12%,反应温度60℃,水分含量3.5%(基于酶质量),反应时间3 h。在最佳条件下,酸解产物中sn-2棕榈酸分布和sn-1,3油酸含量分别为73.18%和71.11%。酸解产物中sn-OPO含量从23.35%提高到34.28%,高于市售Betapol~(TM)的33.24%。     

无溶剂体系两步酶法合成阿魏酰基结构脂

通过对不同途径合成阿魏酰基结构脂的研究,确立了减压旋转生物反应器两步酶法催化合成阿魏酰基结构脂的方法.采用减压旋转生物反应器,通过阿魏酸乙酯与甘油酯交换制备1-阿魏酰甘油酯,1-阿魏酰甘油酯再与油酸酯化,两步合成阿魏酰基结构脂,可显著提高反应速率和产物产率,两步反应平衡时,阿魏酸乙酯的转化率和阿魏酰基结构脂的产率分别达到100%和96%.     

Suppression of acyl migration in enzymatic production of structured lipids through temperature programming

Acyl migration in the glycerol backbone often leads to the increase of by-products in the enzymatic production of specific structured lipids. Acyl migration is a thermodynamic process and is very difficult to stop fully in actual reactions. The objective of this study was to investigate the feasibility of suppressing acyl migration by a programmed change of reaction temperature without loss of reaction yield. The model reactions were the acidolysis of tripalmitin with conjugated linoleic acid (CLA) or with caprylic acid (CA) targeted for human milk fat substitutes. Acyl migration was considerably inhibited in the temperature-programmed acidolysis of PPP with CLA or CA, with only slight reduction of acyl incorporation, the reaction leading to the required products. Acyl migration was reduced by 29% (35 h) and 45% (48 h), respectively, in the acidolysis of PPP with CLA under solvent and solvent-free systems, in comparison with 37% (35 h) and 61% (48 h), respectively, for the acidolysis of PPP and CA. Acyl migration in the acidolysis of PPP with CA was, in general, lower than the acidolysis of PPP with CLA in both systems. Temperature programming was more prominent in solvent-free systems for the reduction of acyl migration. Acyl incorporation was not significantly affected by temperature programming. The study suggests that it is feasible to reduce acyl migration by programmed change of acidolysis temperature without significant loss of reaction yield.     

影响填充床酶反应器酸解合成结构脂质因素研究进展

结构脂质因具有独特营养和生理功能而引起人们关注;填充床酶反应器酸解合成结构脂质具有工艺简捷、酶利用率高、副反应较少,适于连续化生产等优点而成为研究热点。该文概述影响填充床酶反应器酸解合成结构脂质主要因素,为其实现工业化生产提供参考。     

COMPARISON OF LINOLEIC AND CONJUGATED LINOLEIC ACIDS IN ENZYMATIC ACIDOLYSIS OF TRISTEARIN

The acyl incorporation and migration of linoleic and conjugated linoleic acids in enzymatic acidolysis were compared in a solvent-free system. Two systems were used in the Lipozyme RM IM-catalyzed acidolysis at 60C temperature and 5% by weight enzyme load (based on substrates). One included tristearin (SSS) and linoleic (L) or conjugated linoleic (cL) acids (1:6, mol/mol). The other was between tristearin and the mixture of linoleic and conjugated linoleic acids (1:3:3, mol/mol/mol). Acyl incorporation and migration together with triacylglycerol composition of the products were monitored with gas chromatography, pancreatic lipase hydrolysis, and high performance liquid chromatography. Both acyl incorporation and migration of linoleic acid were faster than those of conjugated linoleic acid. At 5 h reaction, there were 13.0% LLL, 46.5% LSL, 27.7% LSS, and 5.6% SSS in the product for a system between tristearin and linoleic acid; whereas there were 2.4% cLcLcL, 10.4% cLScL, 50.9% cLSS, and 36.2% SSS in the product for a system between tristearin and conjugated linoleic acid. The results suggest that linoleic acid was more reactive than conjugated linoleic acid in the enzymatic acidolysis probably because of the rigid structure of the latter.     

酶催化酸解生产结构脂质的研究进展

酸解是酯交换的一种,用该法制备结构脂质的主要原料为油脂和脂肪酸,阐述了酶催化酸解制备结构脂质所需的几种重要的脂肪酸,分析了所采用的脂肪酶及酶反应器的特点,指出了当前工业化生产结构脂质的研究重点。     

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.     

Enzymatic Production of ABA-Type Structured Lipids Containing Omega-3 and Medium-Chain Fatty Acids: Effects of Different Acyl Donors on the Acyl Migration Rate

Enzymatic production of ABA-type structured lipids (SLs) containing marine-derived long-chain polyunsaturated fatty acid (n-3 PUFA) and medium-chain fatty acids was investigated. Response surface methodology was applied to optimize the reaction system and evaluate the effects of reaction factors and different acyl donors namely n-3 PUFA and n-3 PUFA ethyl esters (n-3 PUFA-EE). Well-fitting models were obtained by multiple regressions with backward elimination. For both n-3 PUFA and n-3 PUFA-EE systems, both reaction time and enzyme load had significant (P < 0.05) positive effects on incorporation and acyl migration rate. Water content were found to have significant (P < 0.05) negative effects on incorporation but positive effects on acyl migration in the n-3 PUFA-EE system. Although there were no significant difference in terms of incorporation rate, n-3 PUFA were found to produce five times higher acyl migration rate than n-3 PUFA-EE. The optimal reaction conditions to produce high yield of ABA-type SLs of 49.6% with low acyl migration rate of 2.6% were as follows: n-3 PUFA-EE as acyl donors; enzyme load, 6 wt.%; reaction time, 6 h; and water content, 3 wt.%. Thus, n-3 PUFA-EE was found to be a suitable acyl donor to produce high yield of ABA-type SLs with low acyl migration rate.     

Lipase-catalyzed acidolysis of sunflower oil: Kinetic behavior

In the present study, sunflower oil was modified with a palmitic–stearic acids blend by means of an immobilized sn-1,3 specific lipase (Lipozyme RM IM) to produce structured lipids. Products were analyzed to determine fatty acid incorporation (F_M) into triacylglycerol structure and to quantify by-products as monoglycerides (MG) and diglycerides (DG). The effects of the reaction conditions (temperature, time, incorporated water) on enzymatic acidolysis were studied. Nonlinear regression methods were employed to fit experimental data with kinetic models proposed in the literature. The disappearance of reactant fatty acids (RF) over time was successfully modeled by a Ping-Pong Bi-Bi mechanism. F_M was also represented with a lumped parameter model of the enzymatic mechanism. Maximum RF disappearance and F_M onto sunflower oil glycerides increased with increasing reaction temperature. MG and DG concentrations in water-free systems were stabilized in low levels, while the incorporation of water to the reaction mixture produced a considerable increase in DG formation principally. Kinetic and equilibrium parameters showed temperature dependencies.     

酶法合成结构脂质中酯酶催化活性下降机理研究进展

结构脂质是一类将具有特殊生理功能的脂肪酸结合到甘油三酯骨架上特定位置重新组合生成的新型脂质。酶法催化合成结构脂质具有反应条件温和,能耗低,分离纯化工艺简单,以及可以合成特定酰基位置上的功能性脂肪酸而受到重视。工业上随着酯酶使用次数的增加,酯酶催化活力显著下降,导致目标结构脂质产量降低。本文综述了近年来国内外关于酯酶催化合成结构脂质酯酶结构改变、活性变化和催化活性下降机理,酶活性下降主要是因为分子间强相互作用或弱相互作用破坏酶催化活性中心,本研究可为解决酶法合成结构脂质面临的酯酶活力降低的技术瓶颈提供参考。     

无溶剂体系中酶催化合成结构脂质条件初探

以菜籽油和辛酸为原料,在无溶剂体系中用脂肪酶催化酸解合成结构脂质。对6种不同来源的脂肪酶进行筛选,结果表明Lipozyme RMIM催化活性高、Sn-1,3位特异性强。以Lipozyme RMIM为催化用酶,考察了反应时间、反应温度、底物比(菜籽油与辛酸摩尔比)、加酶量、体系水分含量对酸解反应的影响。结果表明,在反应时间15 h,反应温度50℃,底物比1∶4,加酶量10%条件下,辛酸合成率达40%。