Strategies for lipase‐catalyzed production and the purification of structured phospholipids

This work provides different strategies for the enzymatic modification of the fatty acid composition in soybean phosphatidylcholine (PC) and the subsequent purification. Enzymatic transesterification reactions with caprylic acid as acyl donor were carried out in continuous enzyme bed reactors with a commercial immobilized lipase (Lipozyme RM IM) as catalyst. Operative stability of the immobilized lipase was examined under solvent and solvent‐free conditions. The long reaction time required to have a high incorporation, combined with rapid deactivation of the enzyme, makes the solvent‐free transesterification reaction unfavorable. Performing the reaction in the presence of solvent (hexane) makes it possible to have high incorporation into PC and deactivation of the lipase is less pronounced as compared to solvent‐free operations. For solvent‐free operation, it is suggested to recycle the reaction mixture through the packed bed reactor, as this would increase incorporation of the desired fatty acids, due to increased contact time between substrate and enzyme in the column. Removal of free fatty acids from the reaction mixture can be done by ultrafiltration; however, parameters need to be selected with care in order to have a feasible process. No changes are observed in the phospholipid (PL) distribution during ultrafiltration, and other techniques as column chromatography may be required if high purity of individual PL species is desired. LC/MS analysis of transesterified PC revealed the presence of 8:0/8:0‐PC, showing that acyl migration takes place during the acidolysis reaction.     

Conjugated linoleic acid formation by hydrogenation/isomerisation of safflower oil over bifunctional structured catalyst Rh/SBA‐15

Directed isomerisation of safflower oil under very low hydrogen partial pressure of 7 psi over a novel bifunctional highly structured rhodium‐based catalyst (Rh/SBA‐15), having narrow pore size distribution ranging from 4 to 8 nm, and BET‐specific surface of ≈1,000 m² g^?1, was investigated as a new chemocatalytic approach for vegetable oil hardening and simultaneously producing health‐beneficial conjugated linoleic acids (CLA). Time course profiles of (cis‐9, trans‐11)‐; (cis‐10, trans‐12)‐; (trans‐10, cis‐12)‐; (cis,cis)‐ and (trans, trans)‐octadecadienoic isomers (CLAs) as well as the other fatty acids traditionally encountered during the hydrogenation of vegetable oils are presented and discussed under selected process conditions. Preliminary results show that it is possible to tailor characteristics of the hydrogenation catalyst in such way to confer its bi‐functional activity: hydrogenation and conjugation isomerisation. © 2011 Canadian Society for Chemical Engineering     

共轭亚油酸锌的合成工艺研究

通过利用共轭亚油酸的弱酸性质,将共轭亚油酸制成盐,以期生理功能的改变,本文利用具有生物功能活性的共轭亚油酸,先与氢氧化钠反应得到共轭亚油酸的钠盐溶液,再加入氯化锌溶液得到共轭亚油酸锌盐,平均收率为67%。为共轭亚油酸锌制成制剂的进一步研究提供了原料。     

Epoxidation of a conjugated linoleic acid isomer

Epoxidation of methyl (9Z, 11E)‐octadecadienoate ( 1 ) with various epoxidizing agents viz. m‐chloroperoxybenzoic acid, dimethyl dioxirane, methyltrioxorhenium/hydrogen peroxide, potassium peroxomonosulfate (Oxone, 2KHSO₅ · KHSO₄ · K₂SO₄)/tetrahydrothiopyran‐4‐one, and Novozyme 435/hydrogen peroxide is described. The reactions furnished the corresponding mono‐epoxy [methyl (11, 12E)‐epoxy‐(9Z)‐octadecenoate ( 2 ) and methyl (9, 10Z)‐epoxy‐(11E)‐octadecenoate ( 3 )] and a mixture of diastereomers of syn‐ and anti‐diepoxy‐stearate [methyl (9, 10Z;11, 12E)‐diepoxystearate ( 4a‐4d )], which were identified by a combination of spectroscopic and spectrometric analyses.     

Long‐term conjugated linoleic acid supplementation in humans – effects on body composition and safety

In our contemporary adipogenic environment even modest improvements in body fat mass could be of relevance. In the last years animal and human studies have investigated the potential benefit of CLA on body composition. However, inconclusive results are often derived from short‐term studies. Long‐term intervention trials with supplemental CLA on body composition have not been reviewed exclusively up to now. Therefore, the objective of this study was to review the evidence of prolonged CLA supplementation as well as its influence on body composition in humans, and to summarize results from safety assessments of CLA intake. A literature search was performed to find intervention trials with CLA supplementation and its effects on body composition, as well as on insulin sensitivity. Only prolonged (≥12 wk) studies on body composition were included. The investigated studies indicate a modest reduction and/or prevention of regain of body fat in overweight/obese subjects. Results on the influence of CLA on insulin sensitivity are inconsistent, with newer data rather adding to the safety of CLA. Impaired insulin sensitivity by CLA remains a safety concern, yet is seemingly restricted to diabetic subjects and single‐isomer application. A meta‐a2 lysis of extended studies only is warranted to quantitatively evaluate the effects of CLA on body composition. Future research may elucidate if CLA should be considered as a marginal missing, semi‐essential nutrient in our present diet.     

Quantum‐chemical treatment of the linoleic acid molecule and two of its conjugated isomers

The structural, vibrational and electronic properties of the linoleic acid molecule and two of its conjugated isomers were investigated theoretically by performing the molecular mechanics (MM+ force field), the semi‐empirical self‐consistent‐field molecular‐orbital (PM3), and the density functional theory (B3LYP) calculations. The geometries of the considered molecules were optimized; the vibrational dynamics and the electronic properties were calculated in their ground states in the gas phase. It was found that the excess charge accumulated on hydrogen atoms bonded to double‐bonded carbon atoms is relatively small, which may cause these hydrogen atoms to be easily abstracted.     

Continuous production of structured phospholipids in a packed bed reactor with lipase from <Emphasis Type="Italic">Thermomyces lanuginosa</Emphasis>

The possibilities of producing structured phospholipids between soybean phospholipids and caprylic acid by lipase-catalyzed acidolysis were examined in continuous packedbed enzyme reactors. Acidolysis reactions were performed in both a solvent system and a solvent-free system with the commercially immobilized lipase from Thermomyces lanuginosa (Lipozyme TL IM) as catalyst. In the packed bed reactors, different parameters for the lipase-catalyzed acidolysis were elucidated, such as solvent ratio (solvent system), temperature, substrate ratio, residence time, water content, and operation stability. The water content was observed to be very crucial for the acidolysis reaction in packed bed reactors. If no water was added to the substrate during reactions under the solvent-free system, very low incorporation corporation of caprylic acid was observed. In both solvent and solvent-free systems, acyl incorporation was favored by a high substrate ratio between acyl donor and phospholipids, a longer residence time, and a higher reaction temperature. Under certain conditions, the incorporation of around 30% caprylic acid can be obtained in continuous operation with hexane as the solvent. Presented at the 95th American Oil Chemists' Society Annual Meeting and Expo in Cincinnati, Ohio, May 10, 2004.     

Enzyme‐catalyzed regioselective synthesis of sucrose‐based esters

Sucrose‐based esters are nonionic biosurfactants, which can be synthesized from the enzyme‐catalyzed esterification/transesterification of sucrose. The multi‐hydroxyl groups of sucrose and the immiscibility of sucrose with the acyl donors are the main bottlenecks in obtaining highly regioselective sucrose‐based esters. In this mini‐review, the effects of reaction conditions such as biocatalysts, design of solvent systems used as reaction media, modification and denaturation of substrates, and coupling with external auxiliary physical fields such as microwave irradiation and ultrasonic waves on the yield of sucrose esters and regioselectivity of products are highlighted. The existing problems in the field of enzymatic synthesis of sucrose esters as well as its future perspectives were pointed out. Copyright © 2011 Society of Chemical Industry     

Enzyme‐catalyzed copolymerization of oxiranes with dicarboxylic acid anhydrides

Ring‐opening copolymerizations of the oxiranes glycidyl phenyl ether (GPE) and diglycidyl ether of bisphenol A (DGEBA) with a dicarboxylic acid anhydride [methyl hexahydrophthalic anhydride, nadic anhydride, maleic anhydride (MA), or itaconic anhydride (IA)] were carried out with the lipases Candida cylindracea (CCL), Lipozyme TL‐IM (LIM), and Novozyme 435 (N435) as catalysts. The CCL‐catalyzed reaction of DGEBA with MA or IA (at a 1:2 molar ratio) at 80°C resulted in only partial curing. We monitored the reactions by Fourier transform infrared spectroscopy and by following the changes in the intensities of carbonyl stretching frequencies of the anhydride and ester groups. The reactivity of the oxirane group in GPE was higher than that in DGEBA; this may have been due to the higher viscosity of DGEBA. The reactivities of the enzymes for the copolymerization of the oxiranes and dicarboxylic acid anhydride were in the order LIM > CCL > N435. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 97: 697–704, 2005     

Lipase‐catalyzed synthesis of structured lipids with fatty acids fractionated from saponified chicken fat and menhaden oil

In this study, free fatty acids (FFA) of chicken fat and menhaden oil, which were obtained by saponification, were dry‐fractionated and solvent fractionated. Using these fractionation processes, FFA fractions enriched in monounsaturated (MUFA) and polyunsaturated fatty acids were obtained. Chicken fat FFA fractions enriched in MUFA were modified further by lipase‐catalyzed esterification with the starting fat to produce structured lipids of high MUFA content.     

Quantitative determination of conjugated linoleic acid isomers in beef fat

The amounts of 14 conjugated linoleic acid (CLA) isomers (t12t14, t11t13, t10t12, t9t11, t8t10, t7t9, t6t8; 12,14 c/t, t11c13, c11t13, t10c12, 9,11 c/t, t8c10, t7c9‐18:2) in 20 beef samples were determined by triple‐column silver‐ion high‐performance liquid chromatography (Ag⁺‐HPLC). Quantitation was performed using an external CLA reference standard consisting of cis9,trans11‐18:2,trans9,trans11‐18:2 and cis9,cis11‐18: 2. Linearity was checked as being r > 0.9999 between 0.02 × 10^‐3 to 2 mg/ml. The determination limit (5‐fold signal/noise ratio) of the CLA reference was estimated to be 0.25, 0.50, 1.0 ng/injection for the cis/trans, trans,trans and cis,cis isomers, respectively. As expected, cis9,trans11‐18:2 was the predominant isomer (1.95 ± 0.54 mg/g fat) in beef, followed by trans7,cis9‐18:2 (0.19 ± 0.04 mg/g fat); cis,cis isomers were below the determination limit in most beef samples. Total CLA amounts determined by Ag⁺‐HPLC were compared to total CLAs determined by gas chromatography (GC, 100 m CPSil^TM 88 column). The amounts obtained by GC were generally higher than those determined by Ag⁺ ‐HPLC due to co‐eluting compounds.     

硫酸氢钠催化合成共轭亚油酸的反应动力学

常压下,以蓖麻油为原料,硫酸氢钠为催化剂,对合成共轭亚油酸的反应动力学进行了研究,分析了搅拌速度、反应时间、催化剂的用量对反应转化率的影响。结果表明,蓖麻油酸异构化反应为一级反应,反应活化能为18.060 kJ/mol,动力学方程为r C=35.943 e-18.060/RT C A。     

N‐3 fatty acids and conjugated linoleic acids of longissimus muscle in beef cattle

The objective of the experiment with cattle was to produce high quality beef under different feeding conditions and to increase the concentration of essential fatty acids in muscle. In total 10 German Simmental (GS) bulls and 9 German Holstein (GH) steers were kept either on pasture (grass feeding) or in stable (concentrate feeding). Despite biohydrogenation in the rumen, linolenic acid (C18:3n‐3) contained in grass was absorbed and deposited into the lipids of muscle. This led to a significantly (p ≤ 0.05) higher content of n‐3 fatty acids in the muscle lipids of grazing cattle. The relative amount of total n‐3 fatty acids increased from 1.4 g/100 g fatty acid methyl ester (%FAME) in the intensively fed Simmental bulls to 5.5 %FAME in grass fed cattle. The n‐6/n‐3 ratio of pasture grazing GS bulls was 1.3 in contrast to 13.7 of the animals kept in the byre. The total n‐3 fatty acid concentration in beef muscle increased from 24.6 mg (concentrate) to 108.6 mg/100 g wet weight (grazing). In GH steers the total n‐3 fatty acid concentration was significantly (p ≤ 0.05) increased up to 86.3 mg/100 g wet weight in pasture grazing steers compared to 28.8 mg/100 g wet weight in animals fed the concentrate. The relative content (%FAME) of CLAcis‐9, trans‐11 (0.6 vs 0.56 %FAME in GS; 0.55 vs 0.52 %FAME in GH) in muscle was not significantly increased by grazing on pasture in comparison to concentrate feeding neither in GS bulls nor in GH steers, respectively.     

Elucidation of acyl migration during lipase-catalyzed production of structured phospholipids

Elucidation of acyl migration was carried out in the Lipozyme RM IM (Rhizomucor miehei)-catalyzed transesterification between soybean phosphatidylcholine (PC) and caprylic acid in solvent-free media. A five-factor response surface design was used to evaluate the influence of five major factors and their relationships. The five factors—enzyme dosage, reaction temperature, water addition, reaction time, and substrate ratio—were varied on three levels together with two star points. Enzyme dosage, reaction temperature, and reaction time showed increased effect on the acyl migration into the sn-2 position of PC, whereas increased water addition and substrate ratio had no significant effect in the ranges tested. The best-fitting quadratic response surface model was determined by regression and backward elimination. The coefficient of determination (R ²) was 0.84, which indicates that the fitted quadratic model has acceptable qualities in expressing acyl migration for the enzymatic transesterification. Correlation was observed between acyl donor in the sn-2 position of PC and incorporation of acyl donor into the intermediate lysophosphatidylcholine. Furthermore, acyl migration into the sn-2 position of PC was confirmed by TLC-FID, as PC with caprylic acid was observed on both positions. Under certain conditions, up to 18% incorporation could be observed in the sn-2 position during the lipase-catalyzed transesterification.     

Production of conjugated linoleic acid isomers by dehydration and isomerization of castor bean oil

The possibility of combining dehydration and isomerization of castor bean oil as a means to obtain CLA as TAG forms was studied. First, dehydration was carried out using various catalysts and reaction parameters. Best results were obtained using phosphoric acid (0.1% w/w) at 280°C for 5 h. Under such conditions, satisfactory proportions of CLA were obtained (54% of total FA) with a majority of 9-cis, 11-trans isomer (61% of total CLA). Other catalysts such as bisulfate-bisulfite, sulfuric acid, tungstic and phosphotungstic acids, or resins and zeolites were also tested. With the exception of resins and zeolites, these catalysts also led to CLA production but in limited amounts in comparison with phosphoric acid. In a second step, an isomerization reaction was carried out to transform the residual nonconjugated linoleic acid also produced during dehydration into CLA. Using Wilkinson catalyst [RhCl(PPh₃)₃] in ethanol solvent, dehydrated castor bean oil was isomerized in high yields (>98%), allowing a complete disappearance of nonconjugated linoleic acids. The resulting dehydrated/isomerized oil contained more than 87% CLA with the 9-cis, 11-trans isomer being predominant (40% of CLA fraction). Finally, urea fractionation was also applied on dehydrated/isomerized castor bean oil FA to obtain FFA products containing about 93% CLA.     

Analysis of conjugated linoleic acid isomers and content in french cheeses

Conjugated linoleic acid (CLA) occurs in food as a result of microbial enzymatic reactions, free radical-type oxidation, and heat treatment. CLA is found in animal products, such as meat and dairy products, especially in cheeses. The CLA composition of 12 different French cheeses was determined by a combination of different analytical methods: reversed-phase high-performance liquid chromatography (RP-HPLC), gas chromatography-mass spectrometry (GC-MS), GC-Fourier transform infrared (GC-FTIR), and silver nitrate thin-layer chromatography (AgNO₃-TLC). New isomers (Δ8,10- and Δ11,13-octadecadienoic acids with all possible cis and trans configurations) that co-eluted with previously identified isomers (Δ9c,11t-; Δ9t,11c-; Δ10c,12t-; Δ10t,12c-; Δ11c,13c-; Δ9c,11c-; Δ10c,12c-; Δ9t,11t-; Δ10t12t-octadecadienoic acids) were detected. Δ9c,11t-Octadecadienoic acid was the major CLA isomer in these cheeses. All isomers were present in each product, whatever the production process. However, CLA content in the cheeses varied from 5.3 to 15.80 mg/g of cheese fat, which depended primarily on the origin of the milk (season, geography) and somewhat on the production process.     

Enzyme‐catalyzed regioselective synthesis of sugar esters and related compounds

In this review, a comprehensive and illustrative survey is made of the regioselective synthesis of esters of sugars and related compounds using lipases. The main emphasis has been given to the screening and use of commercially available lipases for the enzymatic esterification of neutral monosaccharides, disaccharides, sugar alcohols and their selected ether and ester derivatives. The effect of solvents and solubilizing agents in improving the yields of the resultant sugar fatty acid esters has been incorporated. Further, solvent‐free esterification with molten fatty acids, use of ionic liquids and microwave radiations for improvement in the methodology have also been discussed. Copyright © 2006 Society of Chemical Industry