Lipase-catalyzed acidolysis of palm olein and caprylic acid in a continuous bench-scale packed bed bioreactor

Enzymatic acidolysis of refined, bleached and deodorized (RBD) palm olein with caprylic acid was carried out in a continuous packed bed bioreactor to produce structured lipid (SL) that can confer metabolic benefits when consumed. Lipozyme^® IM 60 from Rhizomucor miehei, a 1,3-specific lipase, was used as the biocatalyst in this study. After 24 h of reaction, 30.5% of the total fatty acid content of the modified oil was found to be caprylic acid, indicating its incorporation into the palm olein. The triacylglycerols (TAGs) of palm olein after acidolysis were separated and were characterized by seven clusters of TAG species with equivalent carbon number (ECN), C28, C30, C32, C34, C36, C38 and C40. Caprylic–oleic–caprylic TAGs were predicted in cluster C32, which recorded the highest amount, with 35.3% of the total TAG. Fatty acid composition at the sn-2 position was determined, by pancreatic lipolysis, as C8:0, 9.2%; C12:0, 2.3%; C14:0, 1.8%; C16:0, 21.3%; C18:0, 4.7%; C18:1, 60.7%. Iodine value (IV), slip melting point (SMP) and differential scanning calorimetric (DSC) analyses of SL were also performed. In IV analysis, SL recorded a drop of value from 60.4 to 48.2 while SMP was reduced from 13 to 4.2 °C, in comparison to RBD palm olein. DSC analysis of SL gave a melting profile with two low melting peaks of −15.97 and −11.78 °C and onset temperatures of −18.43 and −14.03 °C, respectively.     

Enzymatic interesterification of tripalmitin with vegetable oil blends for formulation of caprine milk infant formula analogs

The structure of triacylglycerols in vegetable oil blends was enzymatically modified, and the blends were incorporated into skim caprine milk to produce goat milk-based infant formula analogs, homologous to human milk. A modified lipid containing palmitic, oleic, and linoleic acids, resembling the composition of human milk fat, was synthesized by enzymatic interesterification reactions between tripalmitin and a vegetable oil blend containing a 2.5:1.1:0.8 ratio of coconut, safflower, and soybean oils. A commercial sn-1,3-specific lipase obtained from Rhyzomucor miehei, Lipozyme RM IM, was used as the biocatalyst. The effects of substrate molar ratio and reaction time on the incorporation of palmitic, oleic, and linoleic acids at the sn-2 position of the triacylglycerols were investigated. The fatty acid composition and sn-2 position of the experimental formulas were analyzed using gas chromatography. Results showed that the highest incorporation of palmitic acid was obtained at 12 h of incubation at 55°C with a substrate molar ratio of 1:0.4 of tripalmitin to vegetable oil blend. However, the modified milk interesterified for 12 h at a 1:1 molar ratio had a greater resemblance to human milk compared with the other formulas. The level of oleic acid incorporation at the sn-2 position increased with the molar ratio of tripalmitin to vegetable oil blend. It was concluded that, unlike the original goat milk and other formulas, the formulated caprine milk with a molar ratio of 1:1 and a 12-h incubation was similar to the fatty acid composition of human milk.     

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.     

Enzymatic production of human milk fat analogues containing stearidonic acid and optimization of reactions by response surface methodology

Human milk fat analogues containing stearidonic acid (SDA) were produced by enzymatic acidolysis reactions between tripalmitin and free fatty acids (FFA) obtained from hazelnut oil and commercial oil mixture containing Echium oil. Lipozyme^® TL IM, immobilized sn-1,3 specific lipase was used in the enzymatic reactions. Response surface methodology (RSM) was applied by using central composite circumscribed design with five levels and three factors. The factors chosen were: Reaction temperature [55–65 °C], reaction time [4–12 h] and substrate molar ratio [3–5 mol/mol (total FFA/tripalmitin)]. Good quadratic models were obtained for the incorporation of SDA and oleic acid. Optimal conditions generated from the models were determined as 60 °C; 8 h, 4 mol/mol for temperature, time and substrate molar ratio, respectively. The models were verified at optimum conditions and furthermore scale-up synthesis of structured lipids (SLs) was performed. Stearidonic and oleic acid contents of the SL from scale-up production were found to be 2.0 and 22.9 mol/100 mol total FA respectively, with 46.2 mol/100 mol total FA of palmitic acid content located at sn-2 position. Besides, oxidative stability and melting profile of final SL were determined for characterization. SL had 6.1 h of induction time, and also had a wider melting range compared with tripalmitin.     

Production and characterization of α-linolenic acid enriched structured lipids from lipase-catalyzed interesterification

The purpose of this study was to enrich α-linolenic acid (ALA) on the triacylglycerol (TAG) molecules of the selected vegetable oils (soybean and corn oil) in order to synthesized structural lipid (SL) with low ω6 to ω3 ratio. SLs were synthesized by lipase-catalyzed interesterification of soybean (SO) and corn oil (CO) with perilla oil (PO). Lipozyme RM IM from Rhizomucor miehei (5% by weight of total substrate) was used for the reaction. The reaction was performed during 24 hr at 55°C in the stirred-batch type reactor with solvent free system. After 24 hr reaction the ratio of ω6 to ω3 in SL products dramatically decreased comparing to that of substrates (SO and CO) before the reaction. At sn-2 position, the contents of ALA were 25.99% in soybean-SL and 23.25% in corn-SL, respectively. According to the equivalent carbon number (ECN) of TAG profile, newly produced SLs were separated by reverse-phase high performance liquid chromatography (HPLC). These results indicated that lipase-catalyzed interesterification is effective to produce SL with low ω6 to ω3 ratio which might be used as health beneficial oil.     

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.     

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.     

Preparation of palm olein enriched with medium chain fatty acids by lipase acidolysis

Medium chain (MC) fatty acids, caprylic (C8:0) and capric (C10:0) were incorporated into palm olein by 1,3-specific lipase acidolysis, up to 36% and 43%, respectively, when added as mixtures or individually after 24 h. It was found that these acids were incorporated into palm olein at the expense of palmitic and oleic acids, the former being larger in quantity and reduction of 18:2 was negligible. The modified palm olein products showed reduction in higher molecular weight triacylglycerols (TGs) and increase in concentration of lower molecular weight TGs compared to those of palm olein. Fatty acids at sn-2 position in modified products were: C10:0, 4%; C16:0, 13%; C18:1, 66%; and C18:2, 15.4%. DSC results showed that the onset of melting and solids fat content were considerably reduced in modified palm olein products and no solids were found even at and below 10 °C and also the onset of crystallisation was considerably lowered. The cloud point was reduced and iodine value dropped from 55.4 to 38 in modified palm olein. Thus, nutritionally superior palm olein was prepared by introducing MC fatty acids with reduced palmitic acid through lipase acidolysis.     

Enzymatic Synthesis of Refined Olive Oil‐Based Structured Lipid Containing Omega ‐3 and ‐6 Fatty Acids for Potential Application in Infant Formula

Structured lipids (SLs) containing palmitic, docosahexaenoic (DHA), and gamma‐linolenic (GLA) acids were produced using refined olive oil, tripalmitin, and ethyl esters of DHA single cell oil and GLA ethyl esters. Immobilized Lipozyme TL IM lipase was used as the biocatalyst. The SLs were characterized for fatty acid profile, triacylglycerol (TAG) molecular species, solid fat content, oxidative stability index, and melting and crystallization profiles and compared to physical blend of substrates, extracted fat from commercial infant formula (IFF), and milk fat. 49.28 mol% of palmitic acid was found at the sn‐2 position of SL TAG and total DHA and GLA composition were 0.73 and 5.00 mol%, respectively. The total oleic acid content was 36.13 mol% which was very close to the 30.49% present in commercial IFF. Comparable solid fat content profiles were also found between SLs and IFF. The SLs produced have potential for use in infant formulas.     

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 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.     

Characteristics and composition of Washingtonia filifera (Linden ex André) H. Wendl. seed and seed oil

Characteristics of seeds and oil extracted from Washingtonia filifera seeds are evaluated. The percentage composition of the W. filifera seeds is: ash 1.37%, oil 16.30%, protein content 3.46%, total carbohydrate 77.19% and moisture 3.22%. The major nutrients (mg/100 g of seeds) found in the seeds are: potassium (67.33), magnesium (34.35), calcium (187.85) and phosphorus (23.26). Physicochemical properties of the oil include: iodine value 67.33 g/100 g of oil; saponification value, 191.63 mg KOH/g of oil; refractive index (25 °C), 1.469; unsaponifiable matter, 0.83%; acidity, 0.41%; p-anisidine value, 0.87; peroxide value, 7.60 mEq O₂/kg of oil; carotenoid content 14.8 mg/100 g and the chlorophyll content = 0.13 mg/100 g. W. filifera seed oil shows some absorbance in the UV-B and UV-C ranges with potential use as a broad spectrum UV protectant. The oil contains high levels of oleic acid (40.60%) followed by lauric acid (17.87%), linoleic acid (16.26%), myristic acid (11.43%) and palmitic acid (9.23%). The triacylglycerols (TAGs) with equivalent carbon number ECN 44 (20.47%) are dominant, followed by TAGs ECN 46 (16.71%), TAGs ECN 42 (15.43%) and TAGs ECN 48 (15.41%). The DSC melting curves reveal that: melting point = 2.25 °C and melting enthalpy = 82.34 J/g. γ-Tocotrienol is the major tocol (72%) with the rest being δ-tocotrienol and α-tocotrienol. The results of the present analytical study show that W. filifera seed oil could be used in cosmetic, pharmaceutical and food products.     

有机溶剂中脂肪酶催化辛酸辛酯的合成

以猪胰腺脂肪酶为催化剂合成辛酸辛酯。研究有机溶剂、反应温度、加水量、加酶量、反应时间、底物浓度及底物物质的量比7 个因素对酶促酯化反应的影响,优化得到最佳合成条件。结果表明：最佳反应温度45℃、正己烷有机溶剂、加水量29μL、酶质量浓度2.314g/L、反应时间24h、辛酸与辛醇物质的量比为1:1.2～1:1.5、辛酸浓度为0.23～0.33mol/L。在优化条件下,辛酸的转化率达到98.12% 以上,并通过IR、1H NMR、13CNMR 谱表征了产物结构。酶催化法能在温和的反应条件下合成高产率的酯。     

Oxidative and thermal stabilities of genetically modified high oleic sunflower oil

The oxidative and thermal stabilities of genetically modified high oleic sunflower oil (87% oleic acid) were compared with those of regular sunflower (17% oleic acid), soybean, corn, and peanut oils during storage at 55 °C and simulated deep fat frying at 185 °C. Oxidative stability was evaluated by measuring the oxygen content and volatile compounds in the sample bottle headspace and peroxide value. The coefficient variations (CVs) for volatile compound, headspace oxygen, and peroxide value analyses were 2.02%, 1.41%, and 3.18%, respectively. The oxidative stability of high oleic sunflower oil was greater than those of regular sunflower and soybean oil (P < 0.05) and as good as those of corn and peanut oils (P > 0.05). The thermal stabilities of oils during deep fat frying were evaluated by measuring the infrared absorption at 2.9 μm and conjugated diene content. The CV of conjugated diene content was 1.07%. Infrared and conjugated diene results showed that the high oleic sunflower oil had greater thermal stability than had regular sunflower, soybean, corn, and peanut oils (P < 0.05). The genetically modified high oleic sunflower oil, with 5.5% linoleic acid, had better oxidative and thermal stabilities than had the regular sunflower oil with 71.6% linoleic acid.     

Production tactic and physiochemical properties of low ω6/ω3 ratio structured lipid synthesised from perilla and soybean oil

Structured lipid (SL) was synthesised by enzymatic interesterification of soybean oil and perilla oil. Response surface methodology (RSM) was used to determine the effects of three variables on the lipase‐catalysed interesterification. Based on ridge analysis, combination of reaction time (X₁; 18 h), reaction temperature (X₂; 60 °C), and substrate mole ratio (X₃; 1:1) were optimised for higher incorporation of ω3 (alpha (α) linolenic acid (ALA)) (Y). The predictive model was found to be adequate due to no significant lack of fit and satisfactory level of coefficient of determination (R² = 0.97). Experiments were conducted under optimised conditions which were predicted by the model equations, obtained from RSM yielded SL with linoleic (44.01%) and ALA (35.82%) were detected at sn‐2 position. The effects of antioxidants such as catechin, butylated hydroxytoluene (BHT) BHT and rosemary extracted on the oxidative stability in SL were investigated. Among all antioxidants, the highest stability was obtained from catechin.     

Biocatalyzed acidolysis of olive oil triacylglycerols with 9c,11t and 10t,12c isomers of conjugated linoleic acid

Lipase catalyzed acidolysis of triacylglycerols (TAG) of olive oil with 9c,11t and 10t,12c isomers of conjugated linoleic acid (CLA) in an organic solvent was studied. The CLA isomers were first obtained in good yield starting from sunflower oil. The acidolysis reactions were carried out with two immobilized lipase, an sn-1,3-regiospecific one from Rhizomucor miehei and a nonregiospecific one from Candida antartica, in order to valuate not only the total incorporation of CLA isomers in olive TAG but also the distribution of the cited isomers in the three sn positions of TAG molecules. The effect of reaction time was also investigated; in fact two series of reactions, with the two lipases, were carried out for 24, 48 and 72 h. The TAG fractions relative to the starting olive oil TAG (OOTAG) and to the samples obtained from the acidolysis reactions were analyzed to obtain the total and positional fatty acid percentage compositions. The results show that after 24 h of reaction, high levels of CLA isomers were incorporated in OOTAG using Lipozyme IM and that slightly higher values were obtained by increasing the reaction time; Novozym 435 was less effective in catalyzing the incorporation of CLA isomers and CLA isomers were incorporated in OOTAG to a lesser degree. The results of stereospecific analysis of TAG fractions showed that, at every reaction time, the CLA isomers were incorporated mainly in sn-1 and sn-3 positions, as expected on the basis of the enzyme regiospecificity. As regards the TAG sn-2 position, the incorporation of CLA isomers was not observed after 24 h, but after 48 and 72 h; this occurrence was probably due to isomerization phenomena or regiospecificity loss after extended reaction times.     

Enzymatic production of low‐calorie structured lipid from Echium seed oil and lauric acid: optimisation by response surface methodology

Low‐calorie structured lipids (SLs) were produced from Echium seed oil and lauric acid by enzymatic acidolysis reactions. Lipozyme^® RM IM, commercially immobilised sn‐1,3 specific lipase derived from Rhizomucor miehei, was used in the reactions. The effects of substrate molar ratio and reaction time on incorporation of lauric acid were investigated and optimised by response surface technology (RSM) with five‐level, two‐factor central composite design. Good quadratic model was obtained for the response [lauric acid (%) incorporation]. Highest lauric acid incorporation into Echium oil was obtained at 5:1 lauric acid/Echium oil molar ratio and at 4‐h reaction time. The model was verified at these conditions and furthermore scale‐up synthesis of SLs was performed. At these conditions, SL contained predominantly lauric acid (42.8%), oleic acid (9.9%), linoleic acid (10.8%), α‐linolenic acid (15.1%), γ‐linolenic acid (7.5%) and stearidonic acid (8.5%) with% 64.4 of PUFA at sn‐2 position in gram‐scale synthesis.     

国内初榨橄榄油品质特性研究

以国内种植的油橄榄为原料,研究了油橄榄品种、成熟度以及堆放时间对初榨橄榄油脂肪酸组成、酸值、过氧化值及风味的影响。结果表明：不同品种油橄榄的初榨橄榄油中油酸和亚油酸含量差异较大,油酸含量范围65.85%~80.08%,亚油酸含量范围2.61%~17.18%;初榨橄榄油的酸值随油橄榄成熟度的升高而降低,其中鄂植8号酸值（KOH）从0.35 mg/g下降到0.26 mg/g;紫果的初榨橄榄油过氧化值低于青红果和红果;油橄榄堆放时间延长会使初榨橄榄油的过氧化值略有增加,而酸值的增加程度因品种而异。不同形式的油橄榄初榨橄榄油在风味上均能很好地区分。     

食用植物油的高分辨气相色谱分析

为评估食用植物油的营养价值,该文分析了常用食用植物油中的脂肪酸组成和各种油的营养特色。用KOH—CH3OH溶液将植物油皂化后,在三氟化硼作为催化剂的作用下,用CH3OH将样品甲酯化,正己烷提取甲酯化产物。以毛细管柱DB-23作为分离柱,用气相色谱法测定植物油的脂肪酸组成。大豆油、葵花籽油和玉米油中含有50％-60％的亚油酸和20％-30％的油酸,营养均衡合理;花生油和芝麻油中油酸与亚油酸含量相当,有35％-45％,易于人体吸收;菜籽油中含有45％的油酸和15％芥酸,对人体健康有不利作用;橄榄油和茶油中含有75％-80％的油酸,红花籽油中含有约80％的亚油酸,有降低胆固醇功效。