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.     

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.     

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%。     

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.     

Lipase catalyzed modification of fish oil to incorporate capric acid

Immobilized lipase, IM60, from Rhizomucor miehei, was used as a biocatalyst for the incorporation of capric acid (C10:0) into menhaden fish oil concentrate containing 34.7 mol% eicosapentaenoic acid (20:5n-3) and 34.4 mol% docosahexaenoic acid (22:6n-3). Transesterification (acidolysis) was performed in hexane and solvent-free media. Tocopherol content was analyzed before and after enzymatic modification. Products were analyzed by gas liquid chromatography. After 24 h incubation in hexane, there was an average of 31.1±4.6 mol% incorporation of C10:0 into fish oil, while 20:5 and 22:6 were reduced to 12.6±3.1 and 13.7±4.4, respectively. The solvent-free reaction produced an average of 28.8±4.7 mol% capric acid incorporation; 20:5 and 22:6 decreased to 16.1±5.7 and 13.5±3.0 mol%. The effect of incubation time, substrate mole ratio, enzyme load, and added water were also studied. Generally, as enzyme load, mole ratio, and incubation time increased, mol% capric acid incorporation also increased. Time course of reaction indicated that the highest C10:0 incorporation occurred at 72 h, for both the reaction in hexane (33.5 mol%) and the solvent-free reaction (36.0 mol%). The highest C10:0 incorporation for the substrate mole ratio reaction occurred at a mole ratio of 1:8 in hexane (50.7 mol%) and the solvent-free reaction (36.7 mol%). Although the highest C10:0 incorporation (31.8 and 48.6 mol%) occurred at an enzyme load of 15% in hexane and 20% for the solvent-free reaction respectively, the values were not significantly different (P<0.05) after 5% enzyme load. Mol% incorporation of C10:0 declined with increasing amounts of water. At 1% added water, high C10:0 incorporation was achieved for the reaction in hexane (39.3 mol%) and the solvent-free reaction (26.0 mol%). Pancreatic lipase catalyzed sn-2 positional analysis was performed on the fish oil before and after enzymatic modification. Fish oil containing capric acid was successfully produced and may be beneficial in certain food and nutritional applications.     

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-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).     

菜籽油酶催化改性反应参数及产物性质研究

在非水相酶催化改性菜籽油制备富含辛酸与油酸的结构脂质工艺中,采用Box-behnken设计研究了酶添加量、反应底物比、反应时间、反应温度及其交互作用对辛酸合成率的影响,并分析了产物物化性质.经过回归分析建立了辛酸合成率对反应参数的二次回归模型,回归方程的决定系数为0.994.改性后产物中Sn-1,3位辛酸质量分数为60...     

MLM型结构脂的酶法合成及其理化性质分析

以醇解反应制得单甘酯和辛酸为底物,1,3-特异性固定化脂肪酶为催化用酶,在单因素试验的基础上,通过正交试验获得酯化反应制备MLM型结构脂质的最适条件：Lipozyme RM IM酶加入量9%(以底物质量计)、反应时间14h、反应温度40℃、单甘酯与辛酸(底物)物质的量比1:6;该条件下辛酸插入率达60.48%,其中92.84%的辛酸分布在甘油结构的1,3位上。经二级分子蒸馏纯化后得到的MLM型结构脂质,辛酸插入率达到73.34%;对其进行理化指标检测分析表明,该产品最低可达国家四级菜籽油的标准。     

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     

Enzymatic synthesis of structured lipids using a novel cold-active lipase from Pichia lynferdii NRRL Y-7723

Structured lipids (SL) were synthesized by the acidolysis of borage oil with caprylic acid using lipases. Six commercial lipases from different sources and a novel lipase from Pichia lynferdii NRRL Y-7723 were screened for their acidolysis activities and Lipozyme RM IM and NRRL Y-7723 lipase were selected to synthesize symmetrical SL since recently NRRL Y-7723 lipase was identified as a novel cold-active lipase. Both lipases showed 1,3-regiospecifity toward the glycerol backbone of borage oil. The effects of enzyme loading and temperature on caprylic acid incorporation into the borage oil were investigated. For Lipozyme RM IM and NRRL Y-7723 lipase, the incorporation of caprylic acid increased as enzyme loading increased up to 4% of total weight of the substrate, but significant increases were not observed when enzyme loading was further increased. The activity of NRRL Y-7723 lipase was higher than that of Lipozyme RM IM in the temperature range between 10 and 20 °C.     

酶催化菜籽油酸解制备结构脂质工艺

以菜籽油和辛酸为原料,用来自Thermomyces lanuginosa固定化脂肪酶TL IM作为催化剂,采用单因素试验结合响应曲面(Box-Behnken设计)优化得到酶法制备结构脂质的工艺条件,最佳制备条件为底物(菜籽油:辛酸)物质的量比1:3.57、加酶量10.57%(以底物质量计)、加水量5.2%(以酶质量计)、反应温度48.6℃、反应时间12h。该条件下辛酸插入率可达23.19%。对提高油菜籽油附加值、延长油菜加工产业链具有重要意义。     

ENZYMATIC SYNTHESIS OF STRUCTURED LIPIDS: TRANSESTERIFICATION OF TRIOLEIN AND CAPRYLIC ACID

Structured lipids were successfully synthesized by lipase-catalyzed trans-esterification (acidolysis) of caprylic acid and triolein in nonaqueous medium. Twelve commercially available lipases (10%, w/w substrates) were screened for their ability to form structured lipid by incubating 100 mg triolein and 65.3 mg caprylic acid in 3 ml hexane at 55C for 24 h. The products were analyzed by reverse-phase high performance liquid chromatography (HPLC) with evaporative light scattering detection. Monocapryloolein was the major component of the products (57.4 mol %) and IM60 lipase from Rhizomucor miehei was the best biocatalyst. Dicapryloolein and triolein contents were 35.4% and 5.3%, respectively. Temperature, mole ratio, time course, incubation media, added water, enzyme load, and substrate concentration were also investigated in this study. The results suggest that it is possible to synthesize structured lipids with lipase as biocatalyst.     

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.     

BATCH ENZYMATIC SYNTHESIS, CHARACTERIZATION AND OXIDATIVE STABILITY OF DHA-CONTAINING STRUCTURED LIPIDS

Fortification of processed foods with n-3 polyunsaturated fatty acids (n-3 PUFA) is rarely practiced in North America. This study utilized, DHA single cell oil (DHASCO), an algal source of PUFA (docosahexaenoic acid, DHA), for the synthesis of structured lipids (SL) and compared the oxidative stability and melting characteristics of the products with those of native DHASCO as control. Immobilized lipase, IM60 from Rhizomucor miehei was the biocatalyst. DHASCO was modified with caprylic, oleic, or stearic acids as acyl donors, in a stirred-batch reactor, to produce three different SL. The reactions were performed at 55C for 48 h in n-hexane for caprylic and oleic acid SL, and at 60C for stearic acid-SL. Mole ratio of substrates were 1:6 for DHASCO-C8:0, 1:2 for DHASCO-C18:1, and 1:1 for DHASCO-C18:0. Mol% incorporation and the fatty acids at the sn-2 position of the SL were determined by gas chromatography (GC).
After DHASCO oil modification, mol% of the incorporated fatty acids were 47.6, 46.3 and 31.2 for C8:0, C18:1, and C18:0-containing SL, respectively. Alkaline extraction was a better deacidification method than short-path distillation, and reduced free fatty acid levels to 0.3% in all SL. DHA was the predominant fatty acid at the sn-2 position in all the SL. DHASCO melting peak was -10.6C. The melting peaks for the SL were -10 and -6.2C for oleic-SL, -8.1 and -0.7C for caprylic-SL, and 16.0, 20.4, and 34.4C for stearic-SL. Oxidative stability studies showed that SL were less stable to oxidation than DHASCO, with DHASCO-18:0 being the most susceptible. With the addition of adequate antioxidants, such SL products synthesized from DHASCO will be stabilized and may be useful in DHA fortification of processed foods such as nutrition bars, dressings, infant formula or in functional foods.     

Optimization of omega-3 enriched-diacylglycerol production by enzymatic esterification using a response surface methodology

Incorporation of an eicosapentaenoic acid/docosahexaenoic acid (EPA/DHA) moiety into diacylglycerol (DAG) oil using lipase-catalyzed esterification was optimized using an ethyl ester form of EPA/DHA. A response surface methodology (RSM) was used to optimize reaction parameters (time, temperature, and substrate mole ratio) for incorporation of DHA and EPA into DAG oil. Predictive models for DHA+EPA contents of DAG and the amount of DAG produced after esterification were adequate and reproducible. DHA+EPA contents of DAG significantly increased with reaction time and substrate mole ratio (p<0.05). In contrast, the reaction temperature negatively affected the amount of DAG after esterification. Synthesis of DHA+EPA-enriched DAG was optimized for a maximum DAG content with the highest DHA+EPA content, in which 630.0 mg of DAG containing 34.8% DHA and EPA was predicted using the RSM model. The optimal reaction conditions were predicted at 20.6 h, 57.9 and a DHA/EPAenriched ethyl ester: DAG oil ratio of 2.5:1.     

Lipase-catalyzed acidolysis of olive oil and caprylic acid in a bench-scale packed bed bioreactor

Lipase-catalyzed acidolysis of olive oil and caprylic acid was performed in a bench-scale packed bed bioreactor to produce structured lipids (SL). A 1,3-specific lipase, IM 60 from Rhizomucor miehei was used as the biocatalyst. Reaction products were analyzed by reversed phase high-performance liquid chromatography, with an evaporative light scattering detector. Olive oil is characterized by four major clusters of triacylglycerol species with equivalent carbon number (ECN), C44, C46, C48, and C50. Three monosubstituted products and two disubstituted products were detected after the reaction. Monosubstituted products had ECN of C36, C38, and C40, and disubstituted products had ECN of C30 and C32. The effect of solvent, temperature, substrate mol ratio, and flow rate/residence time were studied. Optimal solvent-free production of SL was obtained at a substrate flow rate of 1 ml/min, residence time 2.7 h, temperature 60°C, and mol ratio 1:5 (olive oil/caprylic acid). Fatty acid distribution at the sn-2 position of olive oil was determined by pancreatic lipase hydrolysis as 74.8% oleic acid and 25.2% linoleic acid. SL produced at optimal conditions had 7.2% caprylic acid, 69.6% oleic acid, 21.7% linoleic acid and 1.5% palmitic acid at the sn-2 position.     

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.     

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.