Lipase-Catalyzed Interesterification of Schizochytrium sp. Oil and Medium-Chain Triacylglycerols for Preparation of DHA-Rich Medium and Long-Chain Structured Lipids

DHA-rich medium and long-chain structured lipids (MLSL) were successfully synthesized by lipase-catalyzed interesterification of microbial oil from Schizochytrium sp. with medium-chain triacylglycerols (MCT) containing 99% of caprylic acid. Parameters that affected the reaction process were investigated and the conditions were selected as follows: lipase from Aspergillus oryzae, NS40086; reaction time, 8 hours; substrate molar ratio (MCT/microbial oil), 1:1; lipase load, 8 wt%; reaction temperature, 60 °C. Under these conditions, the proportions of MCT, MLSL, and long-chain triacylglycerols (TAG) in the final products were 12.5%, 62.8%, and 24.6%, respectively. The final product was then subjected to UPLC-MS/MS. Eighty-three types of TAG were identified, in which 54 types contained MCFA and MLSL species with relatively high contents were 22:6–8:0–8:0 (6.8%), 8:0–8:0–16:0 (7.5%), and 16:0–16:0–8:0 (7.5%). This product rich in MLSL with DHA and MCFA in the same TAG molecule is beneficial for fat digestion and absorption in infant and thus can increase the bioavailability of DHA at the molecular level.     

Medium- and Long-Chain Triacylglycerols Reduce Body Fat and Blood Triacylglycerols in Hypertriacylglycerolemic, Overweight but not Obese, Chinese Individuals

In contrast to the consumption of long-chain triacylglycerols (LCT), consumption of medium- and long-chain triacylglycerols (MLCT) reduces the body fat and blood triacylglycerols (TAG) level in hypertriacylglycerolemic Chinese individuals. These responses may be affected by BMI because of obesity-induced insulin resistance. We aimed to compare the effects of consuming MLCT or LCT on reducing body fat and blood TAG level in hypertriacylglycerolemic Chinese subjects with different ranges of BMI. Employing a double-blind, randomized and controlled protocol, 101 hypertriacylglycerolemic subjects (including 67 men and 34 women) were randomly allocated to ingest 25–30 g/day MLCT or LCT oil as the only cooking oil for 8 consecutive weeks. Anthropometric measurements of body weight, BMI, body fat, WC, HC, blood biochemical variables, and subcutaneous fat area and visceral fat area in the abdomen were measured at week 0 and 8. As compared to subjects with BMI 24–28 kg/m² in the LCT group, corresponding subjects in the MLCT group showed significantly greater decrease in body weight, BMI, body fat, WC, ratio of WC to HC, total fat area and subcutaneous fat area in the abdomen, as well as blood TAG and LDL-C levels at week 8. Based upon our results, consumption of MLCT oil may reduce body weight, body fat, and blood TAG and LDL-C levels in overweight hypertriacylglycerolemic Chinese subjects but may not induce these changes in normal or obese hypertriacylglycerolemic subjects.     

Interesterification of sesame oil and a fully hydrogenated fat using an immobilized lipase catalyst in both batch and continuous‐flow processes

Lipase‐mediated interesterification of sesame oil and a fully hydrogenated soybean oil was studied at 70 °C in both a batch reactor (BR) and a continuous‐flow packed‐bed reactor (PBR) using four different initial weight ratios of substrates (90 : 10, 80 : 20, 70 : 30 and 60 : 40) with Lipozyme TL IM (Thermomyces lanuginosa) as the biocatalyst. Reaction rates were determined by following the dependence of the profile of the product triacylglycerols (TAG) on the reaction time (BR) or the space time (PBR) via RP‐HPLC‐ELSD. Product TAG identities were confirmed by HPLC‐APCI‐MS. Primary differences between the performances of the two reactors were the maximum level of net hydrolysis (ca. 3 and 10 wt‐% lower acylglycerols at equilibrium for the PBR and BR, respectively), the time or space time required to approach quasi‐equilibrium conditions, and less migration of acyl groups in the PBR trials. For the BR trials, quasi‐equilibrium conditions were approached in 4–6 h, while for the PBR trials short space times (15 min to 2 h) were sufficient to produce effluent compositions similar to equilibrium BR compositions. The predominant TAG families formed by interesterification were LLS, PSO, PSL, SSL, and SSO (L = linoleic; S = stearic; P = palmitic; O = oleic). Oxidative stabilities, melting profiles and solid fat contents were determined for selected reaction products.     

Comparison of the Structures of Triacylglycerols from Native and Transgenic Medium-Chain Fatty Acid-Enriched Rape Seed Oil by Liquid Chromatography–Atmospheric Pressure Chemical Ionization Ion-Trap Mass Spectrometry (LC–APCI-ITMS)

The sn position of fatty acids in seed oil lipids affects physiological function in pharmaceutical and dietary applications. In this study the composition of acyl-chain substituents in the sn positions of glycerol backbones in triacylglycerols (TAG) have been compared. TAG from native and transgenic medium-chain fatty acid-enriched rape seed oil were analyzed by reversed-phase high performance liquid chromatography coupled with online atmospheric-pressure chemical ionization ion-trap mass spectrometry. The transformation of summer rape with thioesterase and 3-ketoacyl-[ACP]-synthase genes of Cuphea lanceolata led to increased expression of 1.5% (w/w) caprylic acid (8:0), 6.7% (w/w) capric acid (10:0), 0.9% (w/w) lauric acid (12:0), and 0.2% (w/w) myristic acid (14:0). In contrast, linoleic (18:2n6) and alpha-linolenic acid (18:3n3) levels decreased compared with the original seed oil. The TAG sn position distribution of fatty acids was also modified. The original oil included eleven unique TAG species whereas the transgenic oil contained sixty. Twenty species were common to both oils. The transgenic oil included trioctadecenoyl-glycerol (18:1/18:1/18:1) and trioctadecatrienoyl-glycerol (18:3/18:3/18:3) whereas the native oil included only the latter. The transgenic TAG were dominated by combinations of caprylic, capric, lauric, myrisitic, palmitic (16:0), stearic (18:0), oleic (18:1n9), linoleic, arachidic (20:0), behenic (22:0), and lignoceric acids (24:0), which accounted for 52% of the total fat. In the original TAG palmitic, stearic, oleic, and linoleic acids accounted for 50% of the total fat. Medium-chain triacylglycerols with capric and lauric acids combined with stearic, oleic, linoleic, alpha-linolenic, arachidic, and gondoic acids (20:1n9) accounted for 25% of the transgenic oil. The medium-chain fatty acids were mainly integrated into the sn-1/3 position combined with the essential linoleic and alpha-linolenic acids at the sn-2 position. Eight species contained caprylic, capric, and lauric acids in the sn-2 position. The appearance of new TAG in the transgenic oil illustrates the extensive effect of genetic modification on fat metabolism by transformed plants and offers interesting possibilities for improved enteral applications.     

Enzymatic Synthesis of Infant Formula Fat Analog Enriched with Capric Acid

A structured lipid (SL) with a substantial amount of palmitic acid at the sn‐2 position and enriched with capric acid (C), was produced in two enzymatic interesterification stages by using immobilized lipase, Lipozyme^® TL IM (Novozymes North America Inc., Franklinton, NC, USA). The substrates for the reactions were high melting point palm stearin, high oleic sunflower oil and tricaprin. The SL was characterized for total and positional fatty acid profiles, triacylglycerol (TAG) molecular species, free fatty acid content, melting and crystallization profiles. The final SL contained 20.13 mol% of total palmitic acid, of which nearly 40 % was located at the sn‐2 position. The total capric acid content was 21.22 mol%, mostly at the sn‐1 and sn‐3 positions. The predominant TAGs in the SL were oleic–palmitic–oleic, POP and CLC. The melting completion and crystallization onset temperatures of the SL were 27.7 and 6.1 °C, respectively. The yield for the overall reaction was 90 wt%. This SL might be totally or partially used in commercial fat blends for infant formula.     

Synthesis of Structured Lipids Containing Pinolenic Acid at the <Emphasis Type="Italic">sn</Emphasis>-2 Position via Lipase-Catalyzed Acidolysis

Lipase-catalyzed acidolysis of a modified pine nut oil (MPNO)—the pine nut oil was obtained from Pinus koraiensis Siebold &; Zucch.—with capric acid was studied in a continuous packed bed reactor (PBR) using Lipozyme RM IM from Rhizomucor miehei as a biocatalyst. The MPNO containing pinolenic acid (PLA) at the sn-2 position of the triacylglycerol (TAG) backbone was prepared by lipase-catalyzed redistribution of pine nut oil using Novozym 435 from Candida antarctica. The effects of the water content in the reaction mixture and the molar ratio of substrates on the extent of the acidolysis reaction as a function of residence time in a PBR were investigated. The water content of the reaction mixture significantly influenced both the rate of acidolysis and the degree of acyl migration, but the molar ratio of substrates affected only the rate of acidolysis. The optimum water content and molar ratio for synthesis of the structured lipid containing PLA at the sn-2 position and capric acid at the sn-1,3 positions of the TAG backbone were 0.04%, and 1:5 (MPNO to capric acid), respectively.     

Preparation of regioisomers of structured TAG consisting of one mole of CLA and two moles of caprylic acid

TAG (MLM) with medium-chain FA (MCFA) at the 1,3-positions and long-chain FA (LCFA) at the 2-position, and TAG (LMM) with LCFA at the 1(3)-position and MCFA at 2,3(1)-positions are a pair of TAG regioisomers. Large-scale preparation of the two TAG regioisomers was attempted. A commercially available FFA mixture (FFA-CLA) containing 9-cis, 11-trans (9c, 11t)- and 10t,12c-CLA was selected as LCFA, and caprylic acid (C₈FA) was selected as MCFA. The MLM isomer was synthesized by acidolysis of acyglycerols (AG) containing two CLA isomers with C₈FA: A mixture of AG-CLA/C₈ FA (1∶10, mol/mol) and 4 wt% immobilized Rhizomucor miehei lipase was agitated at 30°C for 72 h. The ratio of MLM to total AG was 51.1 wt%. Meanwhile, LMM isomer was synthesized by acidolysis of tricaprylin with FFA-CLA: A mixture of tricaprylin/FFA-CLA (1∶2, mol/mol) and 4 wt% immobilized R. miehei lipase was agitated at 30°C for 24 h. The ratio of LMM to total AG was 51.8 wt%. MLM and LMM were purified from 1,968 and 813 g reaction mixtures by stepwise short-path distillation, respectively. Consequently, MLM was purified to 92.3% with 49.1% recovery, and LMM was purified to 93.2% with 52.3% recovery. Regiospecific analyses of MLM and LMM indicated that the 2-positions of MLM and LMM were 95.1 mol% LCFA and 98.3 mol% C₈ FA, respectively. The results showed that a process comprising lipase reaction and short-path distillation is effective for large-scale preparation of high-purity regiospecific TAG isomers.     

Assessment of lipase- and chemically catalyzed lipid modification strategies for the production of structured lipids

The purpose of the present study was to devise a two-step reaction to produce partial glycerides, which would subsequently be used as substrates in both lipase-catalyzed and chemically catalyzed esterification reactions with caprylic acid. The yields and kinetics of these two-step reactions were compared to established lipase-catalyzed acidolysis and transesterification as well as to chemical transesterification reactions. Acyl migration did not occur during the hydrolysis or short-path distillation steps in the preparation of free fatty acid-free partial glycerides for esterification reactions. No significant differences in final yields (59.9% to 82.8% w/w of total triacylglycerols) of new structured lipids were detected among lipase-catalyzed (24 h) and chemically catalyzed (5 h) reactions; however, the yield of new structured triacylglycerols (TAG) after 2 h was lower for acidolysis than for the other lipase-catalyzed reactions (P≤0.05). Since no differences in final yields were detected among the reactions, chemical esterification using hydrolyzed oil could represent the best synthetic option, since it offers the advantage of positional distribution control associated with lipase-catalyzed reactions as well as rapid reaction times associated with chemically catalyzed reactions. Attempts to evaluate the positional distribution of caprylic acid using allyl magnesium bromide (Grignard reagent) were hampered by production of unknown species, which prevented accurate determination of the concentration of some key fatty acids.     

Separation of Triacylglycerol Species from Interesterified Oils by High-Performance Liquid Chromatography

Using a 1,3-regioselective lipase as a catalyst, soybean oil and olive oil were interesterified with the short-chain triacylglycerol tributyrin (1,2,3-tributyrylglycerol) to produce mixtures of structured triacylglycerols (SL-TAG). The SL-TAG were purified by column chromatography and analyzed by both normal-phase (silica column; NP_SIL) and reversed-phase [octadecyl silane (ODS) column] high-performance liquid chromatography (HPLC). Individual SL-TAG molecular species were detected by evaporative light-scattering detection, and characterized by mass spectrometry. NP_SIL HPLC successfully separated the newly synthesized SL-TAG into two groups of TAG: one composed of one butyryl group and two long-chain fatty acyl groups (from soybean or olive oil); the second was composed of two butyryl groups and one long-chain fatty acyl group. The SL-TAG species were further analyzed by reversed-phase HPLC which gave a more detailed separation of the TAG species present in the two SL-TAG.     

Increasing Stearidonic Acid (SDA) in Modified Soybean Oil by Lipase-Mediated Acidolysis

The objective of this work was to synthesize a structured lipid (SL) enriched in stearidonic acid (SDA, C18:4 ω-3), from modified soybean oil (MSO) originally containing ~25% SDA. Low temperature crystallization (LTC) of MSO triacylglycerols (TAG) and free fatty acids (FFA) was performed. The TAG and FFA crystallization products (LTC-TAG and LTC-FFA, respectively) had SDA contents of 48.72 and 60.78%, respectively. Enzymatic acidolysis between MSO and LTC-FFA was studied utilizing Novozym 435 and Lipozyme TL IM as biocatalysts. Substrate molar ratio, incubation time, solvent, and enzyme load were explored. Equilibrium was reached at 96 and 48 h for Novozym 435 and Lipozyme TL IM-catalyzed reactions, respectively. The best conditions from these studies were also applied to the acidolysis of LTC-TAG and LTC-FFA. Utilizing Lipozyme TL IM and solvent free conditions, SLs with SDA contents of 37.61 ± 1.00% (20.86 ± 6.48% at sn-2 position) and 53.46 ± 1.85% SDA (36.37 ± 3.14% at sn-2 position) were obtained from the acidolysis reaction between MSO and LTC-FFA, and LTC-TAG and LTC-FFA, respectively. Compared to the original SDA content of MSO, this process leads to a 52 and 116% increase in SDA content, respectively.     

Dietary medium- and long-chain triacylglycerols accelerate diet-induced thermogenesis in humans

This study investigated the effects of a liquid meal containing medium- and long-chain triacylglycerols (MLCT) on diet-induced thermogenesis (DIT) and was conducted in double-blind cross-over manner. Twenty subjects participated in this examination. The subjects consumed the liquid meal, which was made with 14 g of canola oil (LCT, long-chain triacylglycerols) or MLCT containing about 12% medium-chain fatty acids (MCFA). Oxygen consumption and carbon dioxide production were measured by indirect calorimetry. Resting energy expenditure (REE) was determined based on there parameters, applying the equation of Weir. Increase in DIT after ingesting the liquid meal with MLCT during 6h was significantly greater than with LCT (P<0.05). The results suggest that the substitution of MLCT for cooking oil is useful to control body weigh and fat in subjects.     

Production of structured TAG rich in 1,3-dicapryloyl-2-γ-linolenoyl glycerol from borage oil

γ-Linolenic acid (GLA) has the physiological functions of modulating immune and inflammatory responses. We produced structured TAG rich in 1,3-dicapryloyl-2-γ-linolenoyl glycerol (CGC) from GLA-rich oil (GLA45 oil; GLA content, 45.4 wt%), which was prepared by hydrolysis of borage oil with Candida rugosa lipase having weak activity on GLA. A mixture of GLA45 oil/caprylic acid (CA) (1∶2, w/w) was continuously fed into a fixed-bed bioreactor (18×180 mm) packed with 15 g immobilized Rhizopus oryzae lipase at 30°C, and a flow rate of 4 g/h. The acidolysis proceeded efficiently, and a significant decrease of lipase activity was not observed in full-time operation for 1 mon. GLA45 oil contained 10.2 mol% MAG and 27.2 mol% DAG. However, the reaction converted the partial acylglycerols to structured TAG and tricaprylin and produced 44.5 mol% CGC based on the content of total acylglycerols. Not only FFA in the reaction mixture but also part of the tricaprylin and partial acylglycerols were removed by molecular distillation. The distillation resulted in an increase of the CGC content in the purified product to 52.6 mol%. The results showed that CGC-rich structured TAG can efficiently be produced by a two-step process comprising selective hydrolysis of borage oil using C. rugosa lipase (first step) and acidolysis of the resulting GLA-rich oil with CA using immobilized R. oryzae lipase (second step).     

Lipase-catalyzed acidolysis of perilla oil with caprylic acid to produce structured lipids

Structured lipids were synthesized by acidolysis of perilla oil and caprylic acid using two lipases, Lipozyme RM IM from Rhizomucor miehei and Lipozyme TL IM from Thermomyces lanuginosa. Effects of molar ratio, reaction time, reaction temperature, enzyme load, and solvent content on acidolysis reactions were studied. The solvent content ranged from 0.0 (solvent-free) to 85.3%. The results showed that the incorporation increased in parallel with solvent content to 49.0% with Lipozyme RM IM and to 63.8% with Lipozyme TL IM. After 24 h incubation in n-hexane, caprylic acids were incorporated to 48.5 mol% with Lipozyme RM IM and to 51.4 mol% with Lipozyme TL IM, respectively, whereas linolenic acid content was reduced from 61.4 to 31.5 mol% with Lipozyme RM IM and to 28.4 mol% with Lipozyme TL IM, respectively. Lipozyme TL IM showed a higher acyl migration rate than Lipozyme RM IM when acidolysis was performed in the reaction system containing n-hexane as a solvent, whereas the difference in acyl migration between the two lipases in the solvent-free system was negligible.     

Acidolysis Reaction of Terebinth Fruit Oil with Palmitic and Caprylic Acids to Produce Low Caloric Spreadable Structured Lipid

The lipase (Lipozyme IM from Rhizomucor miehei) catalyzed acidolysis reaction of terebinth (Pistacia terebinthus L.) fruit oil with caprylic and palmitic acid in hexane was investigated in a batch system. The effect of reaction conditions and relationship among them were analyzed and optimized by response surface methodology with a four-factor five-level central composite rotatable experimental design. The four major factors chosen were enzyme load (10–20 wt%), reaction time (12–20 h), reaction temperature (45–60 °C) and substrate mol ratio (TO:PA:CA, 1:2.3–4.1:1.15–2.05). Optimum reaction conditions for reaction time, temperature, enzyme load and substrate mole ratio were 12 h, 45 °C, 10 wt% and 1:4.1:2.05, respectively. The maximum yield of desired triacylglycerols (TAG) obtained at these optimum conditions was 50.87 %. Produced structured lipid had a caloric value which was 1.5 % lower than that of terebinth fruit oil. Its solid fat content was found comparable with commercially available margarines. The relative activity of lipase was well maintained in up to 10 repeated cycles.     

Optimization of the synthesis of lower glycerides rich in unsaturated fatty acid residues obtained via enzymatic ethanolysis of sesame oil

The lipases Novozym 435, Lipozyme TL IM and Lipozyme RM IM were employed in the production of lower acylglycerols (LG), i.e. mono‐ (MAG) and diacylglycerols (DAG), rich in unsaturated fatty acids from sesame oil in batch reactors. The effect of the molar ratio of ethanol to fatty acids on the reusability of these immobilized lipases was studied in detail. The effects of pretreatment on lipase activity for ethanolysis were investigated. Glycerol had a strong product inhibition effect on the ethanolysis reaction, and a relatively large excess of ethanol was necessary to remove the glycerol adsorbed on these biocatalysts. The enzymatic activity was drastically reduced by addition of water to the reaction medium. The presence of organic solvents (hexane and acetone) did not favor the production of LG. For the Novozym 435‐catalyzed reaction, optimum conditions were a molar ratio of ethanol to fatty acid residues of 5 : 1, 15 wt‐% lipase and 50 °C. For Lipozyme TL IM, the optimum conditions were a molar ratio of ethanol to fatty acid residues of 5 : 1, 20 wt‐% biocatalyst, and 30 °C. Novozym 435 and Lipozyme TL IM produced LG with molar ratios of unsaturated to saturated fatty acids of 20.4 in 1 h and 25.3 in 5 h, respectively. In the original oil, this ratio was 5. For trials conducted under optimum conditions, the products from the Novozym 435 trials contained 21.8 wt‐% triacylglycerols (TAG), 24 wt‐% DAG and 54.2 wt‐% MAG. The products of the Lipozyme TL IM trials consisted of 12.9 wt‐% DAG and 87.1 wt‐% MAG. No TAG species were detected.     

Regioselective Synthesis of Palm-Based Sorbitol Esters as Biobased Surfactant by Lipase from Thermomyces lanuginosus in Nonaqueous Media

This paper describes the regioselective production of palm-based sorbitol monoesters via esterification catalyzed by Lipozyme^® TL IM (Thermomyces lanuginosus lipase adsorbed onto silica gel, Novozymes, Inc., Franklington, NC, USA). Effects of various reaction parameters including types of solvent, substrate molar ratio, molecular sieve and lipase concentration, temperature, reaction time, and fatty acid chain length were investigated. Approximately 76% conversion of sorbitol to sorbitol esters was achieved within 24 h under optimal conditions: sorbitol (0.4 M), fatty acid (0.8 M), 20 wt% Lipozyme^® TL IM in 100 mL tert-butanol at 55 °C for 24 h in the presence of 25 wt% 3 Å molecular sieve as water absorbent. The reactions were conducted in an orbital incubator shaker at a shaking rate of 200 rpm. Lipozyme^® TL IM was highly regioselective, esterifying exclusively at sorbitol's primary hydroxyl groups, producing 1-O- and 6-O-sorbitol monoesters. The biocatalyst also exhibited substrate selectivity toward shorter chain acyl donors, with caprylic acid exhibiting the highest conversion of sorbitol. In addition, Lipozyme^® TL IM was reused up to four successive reaction cycles without significant loss of activity. The biocatalytic process reported in this paper is a one-step process to produce biobased surfactants that does not involve the use of toxic or expensive solvents that are commonly employed for derivatization of sugars, or pre-derivatization of the substrates molecules.     

Two-step enzymatic synthesis of docosahexaenoic acid-rich symmetrically structured triacylglycerols via 2-monoacylglycerols

Symmetrically structured triacylglycerols (TG) rich in docosahexaenoic acid (DHA) with caprylic acid (CA) at the outer positions were synthesized enzymatically form bonito oil in a two-step process: (i) ethanolysis of bonito oil TG to 2-monoacylglycerols (2-MG) and fatty acid ethyl esters, and (ii) reesterification of 2-MG with ethyl caprylate. Ethanolysis catalyzed by immobilized Candida antarctica lipase (Novozym 435) yielded 92.5% 2-MG with 43.5% DHA content in 2 h. The 2-MG formed were reesterified with ethyl caprylate by immobilized Rhizomucor miehei lipase (Lipozyme IM) to give structured TG with 44.9% DHA content [based on fatty acid composition with caprylic acid (CA) excluded] in 1 h. The final structured lipids comprised 85.3% TG with two CA residues and one original fatty acid residue, 13% TG with one CA residue and two original fatty acid residues, and 1.7% tricaprylolglycerol (weight percent). The amount of TG with two CA residues and one C₂₂ residue (22∶6=DHA, 22∶5, and 22∶4) was 51 wt%. The 1,3-dicapryloyl-2-docosahexaenoylglycerol to 1,2(2,3)-dicapryloyl-3 (1)-docosahexaenoylglycerol ratio (based on high-performance liquid chromatography peak area percentages) was greater than 50∶1. The recovery of TG as structured lipids after silica gel column purification was approximately 71%. Ethyl esters and 2-MG formed at 2 h of ethanolysis could be used to determine the positional distribution of fatty acids in the intial TG owing to the high 1,3-regiospecificity of Novozym 435 and the reduced acyl migration in the system.     

Optimized Production of MLM Triacylglycerols Catalyzed by Immobilized Heterologous <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> Lipase

Response surface methodology was used to model and optimize the acidolysis of virgin olive oil with caprylic (C8:0) or capric (C10:0) acids, aimed at the production of low caloric triacylglycerols (TAG) of MLM type, in solvent free media, catalyzed by the heterologous Rhizopus oryzae lipase (r-ROL) immobilized in Eupergit^® C. This lipase was produced in the methylotrophic yeast Pichia pastoris Mut^s phenotype (experiments with C10:0) or a Mut⁺ phenotype (experiments with C8:0), under different operational conditions. The r-ROL used in experiments with C10:0 presented a hydrolytic activity about 5 times of that presented by r-ROL used in acidolysis with C8:0. The experiments were carried out following a central composite rotatable design, as a function of the molar ratio (MR) medium chain fatty acid/TAG (1.6–4.4) and temperature (25–55 °C). Convex surfaces described by second order polynomials as a function of MR and temperature were well fitted to fatty acid incorporation values. After 24-h reaction, the predicted maximum incorporation of caprylic (15.5 mol%) or capric (33.3 mol%) acids in olive oil occurs at 37 and 35 °C, respectively, and at C8:0/TAG of 2.8:1 or C10:0/TAG of 3:1. These predicted optima were experimentally validated. Fermentation conditions used in r-ROL production highly affected hydrolytic activity and to a lesser extent interesterification activity.     

Aspergillus oryzae Lipase-Catalyzed Synthesis of Dioleoyl; Palmitoyl-Rich Triacylglycerols in Two Reactors

Dioleoyl; palmitoyl‐rich triacylglycerols (OPO‐rich TAG) were synthesized through Aspergillus oryzae lipase (AOL)‐catalyzed acidolysis of palm stearin with commercial oleic acid by a one‐step process in a stirred tank reactor and continuous packed bed reactor to evaluate the feasibility of using immobilized AOL. AOL was found to be valuable for the synthesis of OPO‐rich TAG when compared with commercial lipase from Thermomyces lanuginose (Lipozyme^® TL IM; Novozymes A/S, Bagsvaerd, Denmark). The C52 (triglycerides with a carbon number of 52, stands for OPO, OPL, LPL and their isomers) content of AOL was higher (45.65 %), and the intensity of treatment (IOT: lipase amount × reaction time/TAG amount) of AOL was just 6.25 % of that of Lipozyme^® TL IM under similar reaction conditions in the stirred tank reactor. Response surface methodology were used to optimize the reaction conditions of the AOL‐catalyzed acidolysis is reaction in the packed bed reactor. The optimal point for the set of experimental conditions generated were as follows: residence time 3.0 h; temperature 62.09 °C; substrate molar ratio 7.13 mol/mol. The highest C52 content obtained was 48.60 ± 2.36 %, with 57.46 ± 1.72 % total palmitic acid at the sn‐2 position and 74.21 ± 2.45 % oleic acid at the sn‐1,3 positions. The half‐life of AOL was 24 h in the stirred tank reactor and 140 h in the packed bed reactor. The immobilized AOL achieved similar conversion and selectivity to commercial lipases for the catalyzed synthesis of OPO‐rich TAG and may offer a cheaper alternative.     

Production of structured TAG rich in 1,3-capryloyl-2-arachidonoyl glycerol from <Emphasis Type="Italic">Mortierella</Emphasis> single-cell oil

Two oils containing a large amount of 2-arachidonoyl-TAG were selected to produce structured TAG rich in 1,3-capryloyl-2-arachidonoyl glycerol (CAC). An oil (TGA58F oil) was prepared by fermentation of Mortierella alpina, in which the 2-arachidonyoyl-TAG content was 67 mol%. Another oil (TGA55E oil) was prepared by selective hydrolysis of a commercially available oil (TGA40 oil) with Candida rugosa lipase. The 2-arachidonoyl-TAG content in the latter was 68 mol%. Acidolysis of the two oils with caprylic acid (CA) using immobilized Rhizopus oryzae lipase showed that TGA55E oil was more suitable than TGA58F oil for the production of structured TAG containing a higher concentration of CAC. Hence, a continuous-flow acidolysis of TGA55E oil was performed using a column (18×125 mm) packed with 10 g immobilized R. oryzae lipase. When a mixture of TGA55E oil/CA (1∶2, w/w) was fed at 35°C into the fixed-bed reactor at a flow rate of 4.0 mL (3.6 g)/h, the degree of acidolysis initially reached 53%, and still achieved 48% even after continuous operation for 90 d. The reaction mixture that flowed from the reactor contained small amounts of partial acylglycerols and tricaprylin in addition to FFA. Molecular distillation was used for purification of the structured TAG, and removed not only FFA but also part of the partial acylglycerols and tricaprylin, resulting in an increase in the CAC content in acylglycerols from 44.0 to 45.8 mol%. These results showed that a process composed of selective hydrolysis, acidolysis, and molecular distillation is effective for the production of CAC-rich structured TAG.