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.     

POSITIONAL DISTRIBUTION OF FATTY ACIDS IN TRIACYLGLYCEROLS OF SEAL BLUBBER OIL

The positional distribution of long-chain ω3 polyunsaturated fatty acids namely eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA) in the triacylglycerol (TAG) molecules of seal blubber oil was determined and compared with that of commercially available menhaden oil via stereospecific analysis. In seal blubber oil, EPA, DPA and DHA occurred mainly in the sn-1 and sn-3 positions of the TAG molecules. The amounts were EPA, 8.36 and 11.2; DPA, 3.99 and 8.21; and DHA, 10.5 and 17.9%, respectively. In menhaden oil, DPA and DHA occurred mainly in the sn-2 position of the TAG at 3.11 and 17.2%, respectively. However, EPA was equally distributed in the sn-2 and sn-3 positions and was present only in minute amounts in the sn-1 position.     

胰脂肪酶法测定食用油甘油三酯中脂肪酸的位置分布

本实验采用sn-1,3位专一性脂肪酶(胰脂肪酶)对甘油三酯进行作用,专一水解位于sn-1、sn-3的酯键,将该两个位置上的脂肪酸游离出来,通过薄层层析分离得到游离脂肪酸和sn-2-甘油一酯,甲酯化作用后用气相色谱法对脂肪酸进行测定。用该方法对花生油、大豆油、玉米油、菜籽油、茶油进行了测定。结果表明,这几种油不饱和脂肪酸含量达到了80%,在sn-2位上分布更是超过了90%,而少量的饱和脂肪酸则主要是分布在sn-1,3位上。     

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.     

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.     

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.     

CHANGES IN THE FRYING MEDIUM DURING DEEP-FRYING OF FOOD RICH IN PROTEINS AND CARBOHYDRATES

Changes in the frying medium (rapeseed oil) during controlled deep-frying of sliced potatoes and cod fillets and oil heated without them were compared. Fifteen frying cycles were conducted each day for five days. Results showed that oil after frying of cod fillets contained 10% less triacylglycerol (TAG) polymers and 40% less TAG dimers than other oils. Other parameters (iodine and anisidine values and absorption value at 233nm) showed that less thermooxidative products formed during frying of food rich in proteins. Oil after frying of cod fillets was darker and its acid value was significantly higher than that for other oils. Content of TAG polymers and dimers for oil after frying of sliced potatoes and oil heated without food was equal. The relative loss of C18:2 acid and decrease in iodine value showed that oil after heating without food underwent more intensive thermooxidative transformation than other oils.     

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).     

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     

褐点石斑鱼鱼肉及其副产物的脂肪酸分析

为了评估褐点石斑鱼的脂肪酸组成特点,采用固相萃取和气相色谱技术对鱼肉、头、皮和内脏的脂质含量、总脂及各脂类脂肪酸组成,甘油三酯(triacylglycerols, TAG)和磷脂(phospholipids, PL)中脂肪酸的位置分布进行了较全面的分析。石斑鱼鱼头和内脏中含有丰富的糖脂(saccharolipids, SL)和PL。在各组织总脂中,必需脂肪酸的含量占总脂肪酸的24.17%～24.51%,二十二碳六烯酸(docosahexaenoic acid, DHA)及二十碳五烯酸(eicosapentaenoic acid, EPA)的含量为12.42%～13.73%,动脉粥状硬化指数、血栓形成指数和n-6/n-3的比例处于较低水平。多不饱和脂肪酸(polyunsaturated fatty acids, PUFA),尤其是DHA+EPA(28.43%～37.37%)在PL中的比例要远高于在其它脂类中的占比。饱和脂肪酸主要分布在TAG和PL的sn-2位,单不饱和脂肪酸主要分布在TAG的sn-1,3位和PL的sn-2位,PUFA倾向于分布在TAG的sn-1,3位和PL的sn-1位;T...     

偏甘油酯脂肪酶Lipase G50催化酯化法制备甘油二酯

利用偏甘油酯脂肪酶Lipase G50催化甘油和脂肪酸酯化反应合成甘油二酯.探讨了酶加量、底物摩尔比、反应温度及加水量对酯化反应的影响.结果表明最佳反应条件为:脂肪酶Lipase G50加量为350 U/g,甘油和脂肪酸的摩尔比5∶1,加水量为底物总质量的5％,反应温度30℃,反应时间24h.在最佳反应条件下脂肪酸的酯化率为75.02％,甘油二酯的含量达到44.74％,产物中没有甘油三酯生成.     

Enzyme-Catalyzed Production and Chemical Composition of Diacylglycerols from Corn Oil Deodorizer Distillate

Diacylglycerols (DAG) were enzymatically synthesized by lipase-catalyzed esterification of glycerol with fatty acids from corn oil deodorizer distillate (CrODD). Effects of reaction parameters such as reaction time, temperature, enzyme type, enzyme load, substrate mole ratio, and water content, as well as the effect of molecular sieves as a water adsorbent were investigated. Rhizomucor miehei lipase (Lipozyme RM IM) was found to be most effective among the lipases screened. The following conditions yielded 70.0% (w/w) DAG: 5 h reaction time, 65°C reaction temperature, 10% (w/w) Lipozyme RM IM, 2.5:1 fatty acid to glycerol molar ratio, and 30% (w/w) molecular sieves. DAG synthesis of 12.4% (w/w) was still observed at 10% (w/w) water content. 84.2% (w/w) of DAG was obtained after purification. The DAG oil comprised predominantly of 1-oleoyl-3-linoleoyl-glycerol (28.5%), 1,3-diolein (22.7%), 1-oleoyl-2-linoleoyl-glycerol (17.9%), and 1,2-diolein (10.9%). Fatty acid profile was similar to that of refined, bleached and deodorised (RBD) corn oil. The ratio of 1,3- to 1,2-positional isomers of DAG was at 1.82:1.     

Interpretation of triacylglycerol profiles of palm oil,palm kernel oil and their binary blends

The effects of lipase-catalyzed interesterification (IE) on changes in the chemical composition of palm oil (PO), palm kernel oil (PKO) and their binary blends at 3:1, 1:1 and 1:3 (w/w) ratios, using both 1,3 specific Rhizomucor miehei, (Lipozyme™) and non-specific Pseudomonas sp. lipases were evaluated. IE of the native PO and PKO showed very distinct chemical composition changes. Catalysis of PO, using both lipases, caused synthesis of more medium and long chain triacylglycerols (TAG), with MMM/OLL, MMP, OOO and PPP (M, myristic acid; O, oleic acid; L, linoleic acid; P, palmitic acid) increasing in concentration. In contrast, IE of PKO resulted in the formation of more short and medium chain TAG, with LaLaO and LaMO (La, lauric acid; C, capric acid) experiencing noteworthy increments. Both Rhizomucor miehei and Pseudomonas sp. lipases showed high affinity in hydrolyzing PO fatty acids, resulting in high TAG losses and formation of high percentages of partial glycerides while these lipases were found to enhance the synthesis process in IE of PKO. Catalysis of the three binary blends caused similar TAG compositional changes where the synthesis process focussed on the medium chain TAG, while hydrolysis was observed in the short and long chain TAG that showed corresponding decreases. Catalysis of the three blends was influenced by the major fraction of these blends. Among these blends, PO: PKO at a 1:1 ratio exhibited the highest degree of IE. The diversity and quantity of available TAG are postulated to be the main causes of the different catalytic activities in these binary blends with Pseudomonas sp. lipase showing a higher degree and rate of IE than R. miehei.     

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.     

Lipase-catalyzed Transesterification of Medium-chain Triacylglycerols and a Fully Hydrogenated Soybean Oil

A commercial preparation of medium‐chain TAG (MCT) was enzymatically transesterified with a fully hydrogenated soybean oil (FHSBO) at different weight ratios (40:60, 50:50, 60:40, and 70:30). All reactions were carried out at 70 °C in flasks placed in an orbital shaker (300 rpm). Three different lipase preparations were tested: TLIM (Thermomyces lanuginosus), lipase PS (Burkholderia cepacia), and Chirazyme L2 (Candida antarctica). The progress of the reaction was monitored by following the changes in the triacylglycerol (TAG) profile of the reaction mixture with reversed‐phase high‐performance liquid chromatography (HPLC). The rates of disappearance of the TAG originally present in both the MCT and the FHSBO were fastest when lipase TL IM was used; the slowest rates were observed for lipase PS. Although the relative compositions of the newly formed TAG at equilibrium depended on the particular lipase used and the initial weight ratio of the substrates, the TAG families containing 2 stearic residues and a residue of either capric acid or caprylic acid were the most abundant product species. All samples were analyzed by differential scanning calorimetry (DSC) to determine the effect of this reaction on the melting profile of the resulting products.     

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%。     

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。     

3种海洋鱼油脂肪酸组成及其位置分布

采用米黑根毛霉源固定化脂肪酶(Lipozyme RM IM)对常见的海洋鱼油甘三脂进行水解,分析了凤尾鱼、金枪鱼、三文鱼甘三酯中脂肪酸的组成及位置分布。采用硅胶色谱柱分离3种海洋鱼油甘三脂,利用Lipozyme RM IM的sn-1,3特异性,将酯化在sn-1,3位上的脂肪酸(EFA)水解成游离脂肪酸(FFA),然后通过薄层层析(TLC)分离得到sn-2-单甘酯,再甲酯化后利用气相色谱(GC)测定sn-2位脂肪酸组成,并按照脂肪酸的不饱和程度和n-3,6-多不饱和脂肪酸归类分析海洋鱼油甘三酯中各类脂肪酸位置分布的特点。结果表明:3种海洋鱼油(凤尾鱼油、金枪鱼油、三文鱼油)中不饱和脂肪酸含量达到60%以上,其中单不饱和脂肪酸(MUFA)占24.67%~33.51%,多不饱和脂肪酸(PUFA)占26.89%~36.15%,而且一半以上PUFA分布在sn-2位,有利于其吸收和提高其耐氧化性;而SFA和MUFA倾向于分布在sn-1,3位上。n-3与n-6PUFAs之间的比例均10,均符合FAO/WHO推荐摄入的标准(4),是理想的n-3PUFA天然营养补充剂。     

磷脂酶Lecitase Ultra和磷脂酶C复合对冷榨菜籽油脱胶的影响北大核心CSCD

为了提高脱胶效率,以冷榨菜籽原油为原料,磷脂含量为指标,采用磷脂酶Lecitase Ultra和磷脂酶C复合酶法对冷榨菜籽油进行脱胶。采用单因素试验考察磷脂酶Lecitase Ultra反应时间、磷脂酶C反应时间、加水量、磷脂酶Lecitase Ultra添加量、磷脂酶C添加量、柠檬酸溶液添加量对脱胶油磷脂含量的影响,并通过响应面法优化脱胶条件。对优化的脱胶条件下所得到的脱胶油的理化指标进行了检测,并与国标一级压榨菜籽油进行了比较。结果表明:磷脂酶Lecitase Ultra和磷脂酶C对冷榨菜籽油进行酶法脱胶的最佳工艺条件为磷脂酶Lecitase Ultra添加量33 mg/kg,磷酯酶Lectase Ultra反应时间90 min,磷脂酶C添加量65 mg/kg,磷脂酶C反应时间60 min,加水量33 mL/kg,柠檬酸溶液添加量1.2 mL/kg;在优化条件下脱胶,脱胶油中磷脂含量为2.3 mg/kg,脱胶油的过氧化值和酸值均达到一级压榨菜籽油的国家标准。综上,磷脂酶Lecitase Ultra和磷脂酶C复合脱胶效果较好,所优化的工艺条件可用于菜籽油的脱胶。     

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.