LIPASE-CATALYZED MODIFICATION OF SESAME OIL TO INCORPORATE CAPRIC ACID

Sesame oil was modified to incorporate capric acids (C10:0) with an immobilized lipase, IM60, from Rhizomucor miehei . Transesterification was performed with and without organic solvent. After 24 h incubation in hexane, there was an average of 28.3±3.5 mol% incorporation of C10:0 into sesame oil. The solvent-free reaction produced an average of 25.7p±4.3 mol% capric acid. As enzyme load, substrate mole ratio, and incubation time increased, mol% capric acid incorporation also increased. For the time course reaction, incorporation of C10:0 increased up to 34.3 and 25.3 mol%, at 72 h and 8 h, for the hexane and solvent-free reactions, respectively. The highest C10:0 incorporation (62.2 mol%) occurred at a mole ratio of 1:7 (sesame oil/C10:0) in hexane and for the solventfree reaction (35.7 mol%) was obtained at a mole ratio of 1:5 and 1:7. At a lipase load of 15%, incorporation of C10:0 reached optimal values of 30.0 and 25.2 mol% for the reactions with and without hexane, respectively. There was a decline in mol% incorporation of C10:0 into sesame oil in hexane with the addition of increasing amounts of water ranging from 0–12%. With no added water, C10:0     

Enzyme-catalyzed synthesis of structured lipids via acidolysis of seal (Phoca groenlandica) blubber oil with capric acid

A structured lipid (SL) containing n-3 fatty acids (eicosapentaenoic acid, 20:5n-3; docosahexaenoic acid, 22:6n-3) and capric acid (10:0; a medium chain fatty acid) was prepared using lipase-catalyzed acidolysis of seal blubber oil with capric acid. An immobilized lipase, Lipozyme-IM from Mucor miehei, was used as the biocatalyst. Acidolysis reactions were carried out in hexane and the products were analyzed by gas chromatography. Incorporation of capric acid was affected by mole ratio of substrates, type of organic solvent, reaction temperature, reaction time, water content and the amount of lipase. The optimum reaction mixture and conditions were oil/fatty acid mole ratio of 1:3, hexane, 45 °C, 24 h, 1% (w/w of substrates) water and 10% (w/w of substrates) Lipozyme-IM lipase. Under these conditions, a SL containing 2.3% 20:5n-3, 7.6% 22:6n-3 and 27.1% 10:0 was obtained. Solvents with log P values between 2.5 and 4.5 performed the acidolysis reaction better than those with log P values of less than 2.5. However, in the absence of any organic solvent, Lipozyme-IM afforded a satisfactory incorporation of capric acid into seal blubber oil.     

STRUCTURED LIPIDS: ACIDOLYSIS OF GAMMA-LINOLENIC ACID-RICH OILS WITH n-3 POLYUNSATURATED FATTY ACIDS

Structured lipids were synthesized by acidolysis of γ-linolenic acid-rich oils and n-3 polyunsaturated fatty acids (PUFA), namely eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), using different lipases. Lipase PS-30 from Pseudomonas sp. was chosen over the other enzymes to catalyze the acidolysis reaction owing to higher incorporation of n-3 PUFA. Effects of mole ratio, reaction time, incubation temperature, enzyme load, and solvent type on acidolysis reactions were studied. At 250 enzyme activity units, incorporation of n-3 PUFA reached optimal values of 29.9 and 30.7% for the reactions with borage and evening primrose oils, respectively. For the time course reaction, incorporation of n-3 PUFA increased up to 34.1 and 31.5% (in 30 h), in borage and evening primrose oils, respectively. After 24 h incubation in hexane, n-3 PUFA (EPA+DHA) incorporated into borage and evening primrose oils was 31.8 and 32.7%, respectively. The highest n-3 PUFA incorporation in both oils occurred at a mole ratio of 1:2:2 (oil/EPA/DHA). Among the solvents tested, n-hexane was found to be highly effective; total n-3 PUFA incorporation of 33.3 and 27.8% in borage and evening primrose oils, respectively, was achieved in hexane. However, the solvent-free reaction afforded products with a total of 23.4–28.8% n-3 fatty acids (EPA and DHA).     

MODIFICATION OF FISH OIL BY LIPOZYME TL IM TO PRODUCE STRUCTURED LIPID

Stearic acid methyl esters was enzymatically interesterified with fish oil (EPAX 5500) containing 44.5 and 32.6 mol% eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3), respectively. Lipozyme TL IM from Thermomyces lanuginosus was used to produce structured lipids (SL) that may be suitable for margarine or shortening application. Interesteri-fication was performed in hexane. Fish oil: stearic acid methyl ester levels ranging from 1 to 5 mole ratio was used. The effect of incubation time, substrate ratio and incubation temperature were also studied. Generally, as incubation time and substrate ratio increased, so did the mol % incorporation of stearic acid. After 24 h incubation in hexane, there was a 49.4 mol% incorporation of stearic acid into fish oil, while the mol% of EPA and DHA were reduced to 15.0 and 13.0 mol%, respectively. Time course studies also indicated that the highest stearic acid incorporation occurred at 72 h while 1:Sjish oil to stearic acid mole ratio gave the highest stearic acid incorporation. The data suggests that Lipozyme Ti5 IM could be used to produce SL.     

Lipase-catalysed acidolysis of lard with caprylic acid to produce structured lipid

Enzymatic acidolysis of lard with caprylic acid was investigated. Of the five lipases that were tested in the initial screening, immobilised lipase TL IM from Thermomyces lanuginosus resulted in the highest incorporation of caprylic acid into lard. This enzyme was further studied for the effect of enzyme load, organic solvent, substrate ratio, reaction time and temperature. HPLC was used to analyse the products from the acidolysis reaction. The highest incorporation was attained at 15% enzyme load. Among the solvents tested, n‐hexane was the best reaction medium for the acidolysis of lard with caprylic acid. Time course studied suggests that the incorporation of caprylic acid into lard was increased up to 37.7 mol% after 24 h. Desirable mole ratio of lard to caprylic acid was 1:2, caprylic acid incorporation up to 34.2 mol%. Temperature had no significant effect on enzyme activity in the range of 40–80 °C.     

Production of olive oil enriched with medium chain fatty acids catalysed by commercial immobilised lipases

Structured triacylglycerols, containing medium chain fatty acids, were produced by acidolysis of virgin olive oil with caprylic or capric acid, at a molar ratio of olive oil:fatty acid of 1:2, at 45 °C for 24 h, in solvent-free media or in n-hexane, catalysed by Thermomyces lanuginosa (Lipozyme TL IM), Rhizomucor miehei (Lipozyme RM IM) and Candida antarctica (Novozym 435) immobilised lipases. Incorporations were always greater for capric than for caprylic acid. For both acids, higher incorporations were always attained in solvent-free media: the highest caprylic acid incorporations were obtained with Novozym 435 (25.5 mol%) and Lipozyme RM IM (25.7 mol%), while similar capric acid incorporations were obtained with all biocatalysts (27.1–30.4 mol%).     

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.     

LIPASE-CATALYZED ACIDOLYSIS OF ALGAL OILS WITH CAPRIC ACID: OPTIMIZATION OF REACTION CONDITIONS USING RESPONSE SURFACE METHODOLGY

Lipase-assisted acidolysis of algal oils, arachidoinc acid single cell oil (ARASCO), docosahexaenoic acid single cell oil (DHASCO) and a single cell oil rich in both docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA, n-6) known as OMEGA-GOLD, with a medium-chain fatty acid (capric acid) was studied. Response surface methodology was used to obtain a maximum incorporation of CA into algal oils. The process variables studied were the amount of enzyme (2–6%), reaction temperature (35–55C) and incubation time (12–36 h). The amount of water added and mole ratio of substrate (algal oil to CA) were kept at 2% and 1:3, respectively. All experiments were conducted according to a face-centered cube design. Under optimum conditions (12.3% of enzyme; 45C; 29.4 h), the incorporation of CA was 20.0% into ARASCO, 22.6% into DHASCO (4.2% enzyme; 43.3C; 27.I h) and 20.7% into the OMEGA-GOLD oil (2.5% enzyme, 46.6C; 25.2 h).     

Production of reduced calorie structured lipid by acidolysis of tripalmitin with capric acid: optimisation by response surface methodology

BACKGROUND: Structured lipids (SLs) containing medium‐chain fatty acids (C8:0–C12:0) in the 1‐ and 3‐positions and long‐chain fatty acids in the 2‐position of triacylglycerols have interesting applications as reduced calorie fats. The aim of this study was to produce an SL by inserting capric acid (CA, C8:0) into tripalmitin (TP) and to optimise the reaction conditions by response surface methodology (RSM) with a three‐level, three‐factor face‐centred cubic design. RESULTS: Lipozyme TL IM from Thermomyces lanuginosa was used for the acidolysis of TP with CA in n‐hexane. The effects of three independent parameters, namely substrate molar ratio, enzyme amount and reaction time, on CA incorporation into TP were optimised. The range of each parameter was selected as follows: substrate molar ratio (CA/TP), 4–8; enzyme amount, 9–15 wt% of total substrate; reaction time, 6–10 h. Optimal conditions were determined to be a CA/TP molar ratio of 6.5, an enzyme amount of 11.6 wt% and a reaction time of 8.9 h. Experiments conducted under these optimised conditions yielded an SL containing 35.7 wt% (44.9 mol%) CA. A second‐order polynomial model was obtained for CA incorporation. CONCLUSION: The possibility of enriching CA in TP by enzymatic acidolysis has been established. The SL containing 44.9 mol% CA produced under optimal conditions may be considered a reduced calorie fat. Copyright © 2008 Society of Chemical Industry     

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 Synthesis of Medium- and Long-Chain Triacylglycerols (MLCT): Optimization of Process Parameters Using Response Surface Methodology

The main objective of this study was to understand the effects and relationship amongst four factors, which are reaction temperature, reaction time, enzyme load, and substrate mole ratio with the purpose of producing healthy functional cooking oil for long-term dietary treatment. Lipozyme RM IM lipase-catalyzed esterification of medium- and long-chain triacylglycerols (MLCT) from glycerol and mixtures of capric and oleic acid was optimized using response surface methodology (RSM) with a five-level, four-factorial design. Reaction temperature, reaction time, and substrate mole ratio strongly affected MLCT synthesis (P < 0.01). However, enzyme load did not have a significant (P > 0.01) effect on MLCT yield. Comparison between predicted and experimental value from central composite rotatable design optimization procedures revealed good correlation, implying that the reduced cubic polynomial model with backward elimination statistically expressed the percent MLCT yield obtained. The optimum MLCT yield was 59.76% by using 10 wt% enzyme load, reaction temperature of 70°C, reaction time of 14 h, and substrate mole ratio of 3.5:1. Experiments to confirm the predicted results using the optimal parameters showed an MLCT yield of 56.35% (n = 2). The choice on the types of fatty acids used in MLCT optimization work greatly influenced the physical and chemical properties of MLCT oil produced. The refined MLCT oil characteristics study showed this oil is suitable to be used for cooking/frying purposes as a high-value added product.     

Production of palm oil-based diacylglycerol using Lecitase Ultra-catalyzed glycerolysis and molecular distillation

Diacylglycerol (DAG) was prepared via glycerolysis of palm oil catalyzed by Lecitase Ultra (LU), a novel phospholipase from the fusion of lipase genes from Thermomyces lanuginose and phospholipase genes from Fusarium oxysporum. Glycerolysis was performed in a solvent-free system. The optimized reaction conditions were: a glycerol/palm oil mole ratio of 7.5:1, initial substrate water content of 5%, substrate enzyme load of 2%, reaction temperature of 40°C, and reaction time of 8 h. In a scale-up reaction, a DAG content of 59.5% in the lipid layer was achieved. Through a two-step molecular distillation, the composition of the target product was 88.1% DAG, 2.8% TAG, 9.0% MAG, and 0.1% FFA. The fatty acid composition of the DAG oil, determined using GC-MS, was enriched compared with the original palm oil.     

Response surface modelling of the production of structured lipids from soybean oil using Rhizomucor miehei lipase

The production of structured lipids (SLs) by the acidolysis of soybean oil (SO) with a free fatty acid (FFA) mixture obtained from Brazilian sardine oil, catalysed by Rhizomucor miehei lipase (Lipozyme RM IM) in a solvent-free medium, was optimised by response surface methodology (RSM) using a three-factor central composite rotatable design. The best reaction conditions to achieve an adequate n-6/n-3 FA ratio were: sardine-FFA:SO mole ratio of 3:1, initial water content of the enzyme of 0.87% w/w, reaction time of 12 h, reaction temperature of 40 °C and 10% by weight of the enzyme (% w/w). Under these conditions, the incorporation of eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) into the soybean oil reached 9.2% (% of the total FAs), leading to a significant reduction in the n-6/n-3 FA ratio from 11:1 to 3:1. Analysis of variance (ANOVA) showed that 95% (R² = 0.95) of the observed variation was explained by the model. Lack of fit analysis revealed a non-significant value for the model equation, indicating that the regression equation was adequate for predicting the degree of EPA + DHA incorporation under any combination of values of the variables. Easy ambient sonic-spray ionisation mass spectrometry (EASI-MS) was used for instantaneous characterisation of TAGs. After the enzymatic reaction, a great variety of new TAGs were formed containing EPA, DHA or both in the same molecule.     

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.     

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     

樟树籽油合成富含中链脂肪酸甘油二酯的工艺研究

以固定化脂肪酶Lipozyme RM IM为催化剂,研究无溶剂体系下樟树籽油和甘油合成中链脂肪酸甘油二酯的工艺。采用响应面分析法对甘油酶解反应的工艺条件进行优化,确定最佳条件为底物摩尔比(甘油∶樟树籽油)=1∶2.3,加酶量11.7wt%,反应温度72℃,反应时间15h。在此条件下得到产物油中甘油二酯含量为51.60%。气相色谱法测定合成产物的脂肪酸组成,产物油中癸酸(C10∶0)及月桂酸(C12∶0)含量分别为54.68%和39.79%,与樟树籽油脂肪酸组成基本一致。合成产物中DAG的中链脂肪酸含量虽有一定降低,但总量仍能达到75%左右。      

响应面优化脂肪酶催化合成中长碳链甘三酯

采用响应面设计对脂肪酶Novozym 435在无溶剂体系中催化甘油和中长碳链脂肪酸(辛酸、癸酸和油酸混合物)酯化反应合成中长碳链甘三酯进行了研究.研究发现:反应温度、加酶量和反应时间对中长碳链甘三酯得率具有显著性影响(P＜0.05),而底物摩尔比(脂肪酸与甘油摩尔比)对中长碳链甘三酯得率不具有显著性影响.优化得到的最佳条件为:反应温度90℃,加酶量6.5％(以脂肪酸和甘油的总质量计),底物摩尔比3.5∶1,反应时间12.97 h.在此条件下,平均甘三酯得率为78.5％;产品中甘三酯、甘二酯、甘一酯和游离脂肪酸含量分别为85.6％、0.3％、0.1％和14.0％;产品甘三酯中辛酸、癸酸和长碳链脂肪酸含量分别为25.4％、10.7％和63.9％,与目标中长碳链甘三酯产品指标基本一致.     

Enrichment of Rice Bran Oil with ��-Linolenic Acid by Enzymatic Acidolysis: Optimization of Parameters by Response Surface Methodology

Lipase-catalyzed enrichment of rice bran oil with n-3 fatty acid in order to obtain a structured lipid containing essential fatty acids has been optimized by response surface methodology. In this process, α-linolenic acid was used as an acyl donor using lipase-catalyzed acidolysis in hexane in presence of immobilized lipase from Rhizomucor miehei. The effect of incubation time and temperature, enzyme concentration and substrates mole ratio and their complex interaction on percentage incorporation of n-3 fatty acid, ratios of saturated fatty acid to polyunsaturated fatty acids, monounsaturated fatty acids to polyunsaturated fatty acids and n-6 to n-3 (18:2 to 18:3) fatty acids have been studied using a central composite rotatable design of experiments. The results showed that at the optimum conditions such as reaction time 4.5 h and reaction temperature 37. 5°C, substrate ratio ranging from 1.0 to 1.9, enzyme concentration varying from 1.0% to 2.0% are needed to fulfill the conditions such as percentage incorporation of n-3 fatty acid ≤18%, ratio of saturated fatty acid to poly unsaturated fatty acid ≥0.42, ratio of mono unsaturated fatty acid to poly unsaturated fatty acid ≥0.8, and ratio of n-6 to n-3 ≥1.30.     

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.     

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%。