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.     

LIPASE-CATALYZED ACIDOLYSIS OF CORN OIL WITH CONJUGATED LINOLEIC ACID IN HEXANE

Lipase-mediated acidolysis of corn oil with conjugated linoleic acid (CLA) was studied in hexane. The largest extent of incorporation was obtained using a weight ratio of CLA to corn oil of 1:7 (1:2.22 mole ratio). Commercial immobilized lipases from Rhizomucor miehei and Candida antarctica effectively catalyzed interesterification. Of the total acylglycerols in the products, triacylglycerols constituted >90%. For both immobilized enzymes, CLA residues were esterified mainly at the sn-1,3 positions.     

Lipase-catalyzed modification of lard to produce human milk fat substitutes

The objective of the present work was to modify lard into human milk fat substitutes (HMFS) by Lipozyme RM IM-catalyzed acidolysis. Lard and soybean fatty acids were esterified in a solvent-free system. The reaction substrates for HMFS production were specially chosen to mimic human milk fats. Factors such as temperature, time, water content, enzyme load, substrate ratio, and enzyme reusability were investigated. The relationships between initial incorporation rate (Inc/h) and temperature (T, K) were set up, based on the Arrhenius law for both linoleic and for linolenic acids. Scale-up trials were carried out to confirm the feasibility of enzymatic modification for the production of HMFS. The characteristics of the product, produced in the scale-up acidolysis under selected conditions (temperature 61 °C, water content 3.5%, lard:fatty acids 1/2.4 (mol/mol), Lipozyme RM IM load 13.7%, and time 1.0 h), were similar to the fat in Chinese mothers' milk. The results showed that it was possible to produce human milk fat substitutes from lard through enzymatic acidolysis with soybean fatty acids.     

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。     

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。     

无溶剂体系酶法催化酸解合成共轭亚油酸甘油酯

采用商业化固定化酶Novozym 435作为生物催化剂,催化共轭亚油酸(CLA)和葵花籽油的酸解反应合成富含CLA的结构脂质(CLA-SL).研究了在无溶剂体系中,底物摩尔比、酶用量、体系含水量、反应温度和反应时间对产物中CLA含量和Sn-2位CLA含量的影响.结果表明,最佳反应条件为:CLA与葵花籽油摩尔比3 :1,酶用量10%,体系含水量1%,反应温度55 ℃,反应时间36 h.在最佳反应条件下,产物中的CLA含量和Sn-2位CLA含量分别为15.7%和2.73%.     

气相色谱-质谱联用法分析苏尼特羔羊肉中共轭亚油酸含量

共轭亚油酸是亚油酸的一组几何和位置异构体，具有多种生理功能。作为瘤胃微生物的产物，反刍动物肉制品是共轭亚油酸异构体较丰富的天然来源，本研究利用气相色谱-质谱联用法分离测定了5月龄苏尼特羔羊肌肉和腹部脂肪中共轭亚油酸总含量，研究结果表明，背最长肌间脂肪中CLA相对百分含量平均为0．83％，腹部脂肪中CLA相对百分含量平均为0．99％。     

响应面优化脂肪酶催化酸解合成共轭亚油酸磷脂

采用商业化脂肪酶Lipozyme RM IM作为生物催化剂,催化共轭亚油酸(CLA,80％)和大豆磷脂( PC90)酸解反应合成富含CLA的结构磷脂.利用响应面分析方法研究了在正己烷溶剂体系中,底物摩尔比、酶用量、反应温度和反应时间对产物中CLA含量的影响.通过分析验证得到最佳反应条件为∶CLA与大豆磷脂的摩尔比6∶1,酶用量30％(以底物总质量计),反应温度48℃,反应时间64 h.在最佳反应条件下,产物中CLA的含量为24.18％.     

酶促酸解合成结构脂反应中酰基迁移的研究进展

酶促酸解合成结构脂具有显著的优越性,其中酰基迁移副反应是结构脂合成中亟需研究和控制的关键。对酶催化酸解反应中酰基迁移的理论和实践研究进行综述,归纳总结温度、时间、酶、载体、水分活度、溶剂、底物比、甘油酯结构等因素对酰基迁移的影响。目前,对酰基迁移的研究主要在酶促反应水平上,机理尚不深入,且优化酶促酸解条件提高接入率、产率与减少酰基迁移是不可兼得的矛盾关系。今后需要进行酰基迁移在复杂体系下的影响机制和评测方法的深入研究,以期更高效地开发特殊用途的结构脂产品。     

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.     

Production and structural analysis of triacylglycerols containing capric acid and conjugated linoleic acid isomers obtained by enzymatic acidolysis

BACKGROUND: Structured lipids containing medium‐chain fatty acids have interesting applications as reduced‐calorie fats; moreover, conjugated linoleic acid (CLA) isomers have shown interesting biological properties. The aim of this study was to synthesize triacylglycerols (TAGs) containing capric acid in the sn‐1‐ and sn‐3‐ positions and CLA isomers in the sn‐2‐ position, using different commercial available lipases. RESULTS: The homogeneous CLA‐TAGs (Tri‐CLA) were chemically synthesized starting from glycerol and CLA isomers, 9‐cis,11‐trans and 10‐trans,12‐cis CLA. The acidolysis reactions of Tri‐CLA with capric acid were carried out at 55 °C for different times in hexane; after 96 h the acidolysis average yield was 65%. The best capric acid incorporation in total TAGs was obtained after 96 h with Lipozyme IM (56.6%). The results of structural analysis carried out on the obtained TAGs showed that both Novozyme 435 and anhydrous Lipozyme IM gave the best incorporation of capric acid in sn‐1(3)‐ positions (61.8%). However, anhydrous Lipozyme IM gave also the highest CLA percent content in sn‐2‐ position (73.2%). CONCLUSION: Anhydrous Lipozyme IM appears to be the more effective enzyme in acidolysis reactions to obtain structured TAGs containing CLA isomers in the central position and capric acid at external positions. Copyright © 2009 Society of Chemical Industry     

Transesterification of conjugated linoleic acid and tricaprylin by lipases in organic solvents

Lipase-catalyzed transesterification of tricaprylin with conjugated linoleic acid (CLA) ethyl ester was performed to produce triacylglycerols containing conjugated linoleic acid. Six commercially available lipases, and seven solvents were screened for their ability to incorporate CLA into tricaprylin. The transesterification reaction was performed by incubating a 1:2 mole ratio of tricaprylin and CLA ethyl ester in 3 ml solvents at 55°C, and the products were analyzed by gas chromatography. Three lipases, Novozym 435, Lipozym IM and lipase PS-C were chosen to allow a comparison of transesterification activity in our model reaction. The highest CLA incorporation with Novozym 435 and Lipozym IM was achieved in hexane while isooctane produced the highest CLA incorporation with lipase PS-C. Lipase PS-C gave higher CLA incorporation into tricaprylin when fatty acid was used as the acyl donor than other lipases did. Lipozym IM and lipase PS-C had not restrict specificity to sn-1, 3 positions, even though they had high specificity at sn-1, 3 positions. Novozym 435 among lipases tested was the most effective on the incorporation of CLA into tricaprylin.     

嗜酸乳杆菌发酵生产共轭亚油酸工艺研究

共轭亚油酸是一种具有多种生理活性的天然脂肪酸。以盐生植物紫花苜蓿的种籽油为底物，用嗜酸乳酸杆菌（Lactobacillus acidophilus）1．1854催化紫花苜蓿籽油中的亚油酸转化为共轭亚油酸。通过单因孝实验分析了发酵过程中pH、温度、预培养和苜蓿籽油的浓度对生产共轭亚油酸的影响。     

共轭亚油酸生物合成的研究

共轭亚油酸是80年代末才被发现的一种具有多种生理功能的天然不饱和脂肪酸，具有抗癌、抗动脉粥样硬化、减肥、促进生长、缓和免疫反应副作用等许多重要生理功能，在医药、食品、保健品、化妆品等中具有广阔的应用前景。目前，工业化生产共轭亚油酸的方法是碱并构化法，但共轭亚油酸的生物合成法是未来的发展趋势。本文对近年来共轭亚油酸生物合成方面的研究进展进行了综述。     

Production of structured lipids containing conjugated linolenic acid: optimisation by response surface methodology

Structured lipids containing conjugated linolenic acid (CLNA) were produced separately by enzymatic acidolysis reaction of corn and canola oils (CAO) with bitter gourd (Momordica charantia L) seed oil fatty acids [bitter gourd seed oil fatty acids (BGFA)]. Reactions were conducted using a commercial immobilised sn‐1,3‐specific lipase from Thermomyces lanuginosa (Lipozyme TL IM) in hexane. The effects of reaction time, substrate molar ratio, temperature and enzyme amount on incorporation yield of CLNA were investigated and optimised by response surface methodology with three‐level, two‐factor face‐centred cube design. When reactions were conducted using 10% enzyme for 3 h, the optimum reaction conditions were found for corn oil (CO) as 53.5 °C and 5.9:1 BGFA/CO molar ratio. At these conditions, the incorporation of CLNA into CO was determined as 41.4%. However, CLNA incorporation into CAO was resulted as 37% at optimum conditions which were 54.2 °C and 6.8:1 BGFA/CAO molar ratio.     

VARIATION IN THE CONJUGATED LINOLEIC ACID CONTENT OF THREE TRADITIONAL GREEK CHEESES DURING A 1-YEAR PERIOD

The fat and fatty acid content of three traditional Greek cheeses were determined monthly for a period of 1 year. The analyzed cheeses, feta, graviera and manouri, are traditional Greek dairy products made exclusively or primarily from sheep milk. In all cheeses, the content of the fatty acids varies during several months, especially the conjugated linoleic acid (CLA) content of feta, graviera and manouri ranged between 0.5 and 0.9, 0.5 and 1.0 and 0.4 and 0.8 g/100 g total fatty acids, respectively. The average content of CLA in graviera and feta was similar (0.75 and 0.72 g/100 g fatty acids, respectively). On the other hand, the CLA content of manouri (0.54 g/100 g fatty acids) was less than that of the other two cheeses. In every case, the analyzed cheeses can be characterized as good or very good CLA sources.

PRACTICAL APPLICATIONS

No data exist about the variation in the fatty acid composition of standardized cheeses during the various months of the year. Several investigations were carried out in order to determine the fatty acid composition and especially the conjugated linoleic acid (CLA) content of various kinds of cheese. However, cheeses were produced from milk and it is suggested that the milk composition is affected by seasonal factors. In the present investigation, we tried to find out if the beneficial fatty acid content of cheeses, which were previously found as rich CLA sources, is season dependent. The results of this research are a potential tool for food technologist in order to provide CLA-enriched diets.     

EFFECTS OF LINOLEIC ACID SUPPLEMENTS ON THE SYNTHESIS BY YEAST OF LIPIDS AND ACETATE ESTERS

The concentrations of acetate esters in beer were reduced by up to 85% by addition of linoleic acid to the fermentation or by pitching with yeast previously enriched with this unsaturated fatty acid. Linoleic acid was rapidly incorporated into yeast lipids and was effective in reducing the rate of ethyl acetate formation within 2 h. Addition of linoleic acid altered the pattern of synthesis of fatty acids by yeast, causing a shift from medium toward long chain acids. Secondly, the amount of squalene in yeast was reduced by up to 70% whereas that of lanosterol was increased threefold. Since total yeast lipid synthesis was reduced by up to 40%, we conclude that less acetylCoA is synthesized in the presence of linoleic acid. Further, high concentrations of linoleic acid decreased the proportion of acetylCoA consumed by the synthesis of acetate esters. Therefore linoleic acid may directly decrease acetate ester synthesis in addition to its effect via reduction of acetylCoA availability.     

Interesterification (acidolysis) of butterfat with conjugated linoleic acid in a batch reactor

Six commercial lipases, in free or immobilized form, were tested for their ability to catalyze acyl exchange between conjugated linoleic acid and anhydrous butterfat under solvent-free conditions. Immobilized Candida antarctica lipase exhibited the best activity. Experiments were conducted for this lipase in butterfat to conjugated linoleic acid ratios of 10:1 (vol/vol), temperatures from 30 to 70 degrees C, enzyme concentrations of 50 to 200 mg/g of reaction mixture, and water contents of 0.15 to 2% (wt/wt). At the maximum enzyme concentration used, equilibrium was reached within the first 24 h of reaction. The optimum temperature was 50 degrees C. The triacylglycerol profile of the product butterfat reflected changes in the relative proportions of fatty acid residues as the reaction proceeded, with increases in those triacylglycerols containing 46 to 54 carbon atoms and concomitant decreases in those triacylglycerols containing 34 to 42 carbon atoms.     

ANTIOXIDANT EFFICACY OF EXTRACTS OF AN EDIBLE RED ALGA (GRATELOUPIA FILICINA) IN LINOLEIC ACID AND FISH OIL

The antioxidant activity of an extract of a marine red alga (Grateloupia filicina) was evaluated in linoleic acid and fish oil induct period oil at 65C. Oxidative stability of the oils was evaluated by employing peroxide value (PV), 2-thiobarbituric acid reactive substances (TBARS), conjugated diene (CD) and weight gain experiments. The algal extract was applied to linoleic acid and fish oil at 0.01, 0.03 and 0.05% and results were compared with those of commercial antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisol (BHA) and α-tocopherol at 0.01 %. Results indicated the ability of the alga extract to inhibit oxidation of linoleic acid and fish oil; at α 0.05%     

FATTY ACID COMPOSITION IN TISSUES OF MICE FED DIETS CONTAINING CONJUGATED LINOLENIC ACID AND CONJUGATED LINOLEIC ACID

The influence of 1% alpha-eleostearic acid (α-ESA, cis9,trans11,trans 13-18:3) and 1% punicic acid (PA, cis9,trans11,cis13-18:3) on fatty acid composition in mouse tissues was compared with conjugated linoleic acid (CLA, mixture of primarily cis9,trans11- and trans10,cis12-18:2) in the present study. The content (% total fatty acids) of 18:2n-6 was significantly reduced in the heart and adipose tissues, and total polyunsaturated fatty acids (PUFAs) and n-6 PUFA were significantly reduced in adipose tissue by α-ESA, PA and CLA feeding. The content of 22:6n-3 and total n-3 PUFA were significantly increased in the liver, kidney and heart by PA feeding, but not by α-ESA. In contrast to PA, supplementation with CLA significantly decreased 22:6n-3 in the liver, kidney and heart. The content of 20:4n-6 was significantly decreased in the liver and kidney by CLA feeding, but not by α-ESA and PA. The present results indicate that α-ESA, PA and CLA have differential effects on 22:6n-3 and 20:4n-6 content in mouse tissues.

PRACTICAL APPLICATIONS

Conjugated linolenic acid (CLnA), a group of octadecatrienoic acid isomers with a conjugated triene system, has been reported to exhibit favorable physiological effects, including anticancer properties and regulation of lipid metabolism. Punicic acid and alpha-eleostearic acid, two isomers of CLnA, have been shown to convert into cis 9, trans 11-18:2 in vivo . The effect of CLnA on fatty acid composition in mouse tissues was investigated in comparison with CLA mixtures in the present study. The data obtained here could provide information for the potential application of CLnA-containing seeds as functional food ingredients, a natural source of endogenously formed cis 9, trans 11-18:2 and a dietary feeding strategy to beneficially modify the fatty acid composition of animal tissues.