Reaction selectivity of <Emphasis Type="Italic">rhizomucor miehei</Emphasis> lipase as influenced by monoacylation of <Emphasis Type="Italic">sn</Emphasis>-glycerol

Reaction selectivities were determined in multicompetitive reactions mediated by Rhizomucor miehei (RM) lipase at water activity of 0.19 in hexane. Saturated FA (C4–C18 even chain) and oleic acid (C18∶1) were reacted with a single alcohol, glycerol, or α-or β-MAG containing C4, C10, C16, or C18∶1 individually as alcohol cosubstrate. Similar patterns of broad FA selectivity toward C8–C18 FA were generally observed for esterification into specific acylglycerol (AG) pools with the different α/β-CX-MAG cosubstrates. Exceptions were enrichment of C18 in the MAG pool with α-C16-MAG substrate, and a general suppression of C4/C6 FA reactivity and a specific discrimination toward >C8 FA incorporation into the TAG pool, both for reactions with α-C10- and α-C16-MAG. RM lipase selectivity toward MAG was in descending order: β-C18∶1-MAG>α/β-C4-MAG∼β-C10-MAG∼β-C16-MAG>α-C18∶1-MAG >α-C10-MAG∼α-C16-MAG. Selectivity in channeling CX of the original CX-MAG substrates into higher AG species was in descending order: α-C10-MAG∼α-C16-MAG>β-C10-MAGβ-C16-MAG>α-C18∶1-MAG>β-C18∶1-MAG∼ α/β-C4-MAG. Aside from their characteristic FA selectivity, Burkholderia cepacia (PS-30) and RM lipases behaved similarly in terms of MAG selectivity as well as a general conservation of FA selectivity throughout the sequential steps of TAG assembly from FA and glycerol for processes designed to yield specifically structured TAG.     

Reaction selectivity of <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> (PS-30) lipase as influenced by monoacylation of <Emphasis Type="Italic">sn</Emphasis>-glycerol

Reaction selectivities were determined in multicompetitive reactions mediated by Burkholderia cepacia lipase (Amano PS-30) at a water activity of 0.19 in hexane. Saturated FA (C4–C18 even chain) and oleic acid (C18∶1) were reacted with a single alcohol, glycerol, α-or β-MAG, containing C4, C10, C16, or C18∶1 individually as alcohol cosubstrate. Similar odrinal patterns of FA selectivity, with C8, C10, and C16 preferred over others, were generally observed for incorporation of FA into specific acylglycerol (AG) pools of the 24 specific cases evaluated. The three exceptions were enrichment of C14 and C18 in the MAG pool with α-C16-MAG, substrate, and a general suppression of >C8 incorporation into the TAG pool for reactions with α-C10- and α-C16-MAG. PS-30 lipase selectivity toward MAG was in descending order: α/β-C4-MAG>β-C10-MAG>β-C16-MAG>α/β-C18∶1-MAG>α-C10-MAG>α-C16-MAG. Selectivity in channeling CX of the original CX-MAG substrates into higher AG species was in descending order: α-C10-MAG∼α-C16-MAG>α-C18∶1-MAG>β-C10-MAG∼β-C16-MAG∼β-C18∶1-MAG >α/β-C4-MAG. Generally, MAG were better acyl donors than FA for esterification reactions leading to DAG formation. These observations are relevant to the design of biocatalytic processes intended to yield specifically structured TAG.     

Enzymatic acidolysis in hexane to produce n−3 or n−6 FA-enriched structured lipids from coconut oil: Optimization of reactions by response surface methodology

Response surface methodology is a statistical design that helps one to determine optimal conditions for an enzyme-catalyzed reaction by performing a minimal number of experiments. This methodology was adapted for modifying coconut oil TAG by using lipase-catalyzed acidolysis in hexane to incorporate n−3 or n−6 PUFA. FFA obtained after hydrolysis of cod liver oil and safflower oil were used as acyl donors. Immobilized lipase, Lipozyme IM60, from Rhizomucor miehei was used for catalyzing the reaction. The reaction conditions—substrate molar ratio, incubation time, and temperature—were optimized. The experimental data were fitted to a response function based on the central composite rotatable design. The optimal conditions generated from models indicated that maximal incorporation of n−3 PUFA occurred at a 1∶4 molar ratio of TAG/FFA when incubation was carried out for 34 h at 54°C. Similarly, maximal incorporation of n−6 FA was predicted at a 1∶3 molar ratio of TAG/FFA when incubated for 48.5 h at 39°C. Experiments conducted at optimized conditions predicted by the equation obtained from response surface methodology yielded structured lipids with 13.65 and 45.5% of n−3 and n−6 FA, respectively. These values agreed well with that predicted by the model. The reactions were also scaled up to 100 g levels in batch reactors with the incorporation level of n−3 and n−6 fatty acids agreeing closely with that observed when the reactions were carried out at lab scale (100 mg). These studies indicated that response surface methodology is a useful tool in predicting the conditions for incorporating desired levels of specific FA during the synthesis of structured lipids.     

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).     

Effect of triacylglycerol structures on the thermal oxidative stability of edible oil

Different molecular species of TAG were assessed to determine the influence of TAG structure on the thermal oxidative stability of edible oil. TAG containing palmitic acid (16∶0, P) as saturated FA (SFA) and oleic acid (18∶1, O), linoleic acid (18∶2, L), or linolenic acid (18∶3, Ln) as unsaturated FA (UFA) were chemically synthesized and then heated at 180 or 150°C. Thermal oxidative stability of TAG was determined by evaluating the resultant UFA, polar compound, FFA, carbonyl compound, polymerized compound, and tocopherol contents. When TAG containing 16∶0 and 18∶2 in the ratio of 2∶1 (mol/mol) were heated at 180°C, a 2∶1 (mol/mol) mixture of saturated TAG (PPP) and unsaturated TAG (LLL) was found to be more susceptible to thermal oxidation than PPP/PLL (1∶1) and PPL. Similarly, a 2∶1 mixture of PPP and OOO or LnLnLn was more unstable toward thermal oxidation than PPO or PPLn, respectively. Thermal oxidative stability of TAG containing SFA and UFA (2∶1) was negatively correlated with the moles of UFA in a single TAG molecule. This tendency was also observed at 150°C. From these results, it is suggested that the TAG structure could be one of the factors determining the thermal oxidative stability of edible oil.     

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.     

Fatty acids and oil content in white lupin seed as affected by production practices

Characteristics of the seed oil of white lupin (Lupinus albus L.), a potential alternative winter crop in the mid-Atlantic region of the United States, are not well established. Replicated experiments were conducted during the 1998–1999 and 1999–2000 growing seasons with a determinate and an indeterminate cultivar to characterize oil and FA in lupin seed in relation to production practices. The experiments were planted in early October, late October, and mid-November using row spacings of 0.3, 0.6, and 0.9 m at each planting time. Seeds from the planting date of early October had significantly (P<0.05) higher oil content than the later plantings (late October and mid-November). A closer row spacing (0.3 m) also had significantly (P<0.05) higher oil content than the wider row spacing (0.9 m). Planting data effects on FA content were significant for some FA, but row spacing did not affect FA contents. Oil content in the seed varied from 7.2 to 8.2% (w/w). The oil from white lupin seed contained FA in the order of 18∶1>18∶2> 18∶3>16∶0>20∶1>22∶1>22∶0>18∶0>24∶0>20∶0. The saturated FA/unsaturated FA ratio in lupin oil was 0.14. White lupin seed contained higher contents of oil and FA than literature values for seed of navy, kidney, and pinto beans.     

Enzymatic preparation of enantiomerically pure sn-2,3-diacylglycerols: A stereoselective ethanolysis approach

Stereoselective ethanolysis of monoacid TAG by immobilized Rhizomucor miehei lipase (RML) was studied for preparation of optically pure sn-2,3-DAG. Trioctanoylglycerol (TO) was used as a model substrate. The enantiomeric purity of the product, sn-2,3-dioctanoylglycerol (sn-2,3-DO), was very high (percent enantiomeric excess >99%) when an excess of ethanol was used. The result indicated that RML was highly stereoselective toward the sn-1 position of TO under conditions of excess ethanol. The stereoselectivity of RML depended on the amount of ethanol. The larger the amount of ethanol was, the higher the stereoselectivity became. After optimizing the parameters such as reactant molar ratio, water content, and temperature, (ethanol/TO molar ratio =31∶1 and water content =7.5 wt% of the reactants at 25°C), optically pure sn-2,3-DO was obtained at 61.1 mol% in the glyceride fraction in 20 min. The above conditions were further applied for ethanolysis of monoacid TAG with different acyl groups such as tridecanoylglycerol (C10∶0), tridodecanoylglycerol (C12∶0), tritetradecanoylglycerol (C14∶0) and trioctadecenoylglycerol [triolein, (C18∶1)]. The yields and enantiomeric purities of 1,2(2,3)-DAG were dramatically reduced when TAG with FA longer than decanoic acid were used.     

Phospholipid synthesis by chick retinal microsomes: Fatty acid preference and effect of fatty acid binding protein

The acylation of 1-palmitoyl-sn-glycerophosphocholine (1-16∶0-GPC) or 1-palmitoyl-sn-glycerophosphoethanolamine (1-16∶0-GPE) was measured using the microsomal fraction prepared from retinas of 14–15-day-old chick embryos. Rates of incorporation of exogenously supplied fatty acids into diacyl-GPC were generally 5–7 times greater than into diacyl-GPE. Substrate preferences for incorporation into diacyl-GPC and diacyl-GPE were, respectively, 18∶2>18∶3=20∶5>20∶4>18∶1>22∶6=18∶0 and 18∶2>22∶6≽18∶3=18∶0≽20∶4=18∶1>20∶5. The apparent selectivities were not consistent with the reported fatty acid compositions of these lipid classes. The addition of partially purified fatty acid binding protein (FABP) to the reaction had no effect either on overall rates of incorporation or on the substrate preference. When fatty acyl-CoA substrates were used, rates of incorporation of the 18∶0 derivative were much higher than with the fatty acid, while rates with other fatty acyl-CoA were similar to those with the respective fatty acid. Substrate preferences for CoA derivatives incorporated into diacyl-GPC were: 18∶0>20∶4>18∶2≽22∶6, and into diacyl-GPE: 20∶4=22∶6>18∶0>18∶2. Polyunsaturated fatty acyl CoA (PUFA-CoA) were thus favored for incorporation into diacyl-GPE, and to a lesser extent into diacyl-GPC, a result that is consistent with composition data. When purified FABP was added to the reactions, there was an increase in the incorporation of 18∶0-CoA and a decrease or no change in the incorporation of PUFA-CoA. The deacylation/reacylation cycle thus appears to play a role in the modification of phospholipid composition. The data are not consistent, however, with a role for FABP in directing PUFA toward membrane lipid synthesis.     

Lipase-assisted acidolysis of high-laurate canola oil with eicosapentaenoic acid

The production of structured lipids via acidolysis of high-laurate canola oil (Laurical 15) with EPA in hexane was carried out using lipase from Pseudomonas sp. The optimal reaction conditions used 4% lipase, at a mole ratio of oil to EPA of 1∶3 at 45°C over 36 h. The positional distribution of FA on the glycerol backbone of unmodified oil indicated that lauric acid was mainly located at the sn-1,3 positions. Stereospecific analysis of the oil modified with EPA showed that lauric acid remained mostly esterified to the sn-1,3 positions of the TAG molecules and that EPA was also primarily in the sn-1,3 positions of the TAG molecules. Thus, the resultant structured lipids may have optimal value for use in applications where quick energy release and EPA supplementation are required.     

Enzyme-assisted acidolysis of menhaden and seal blubber oils with γ-linolenic acid

Oils containing both n−3 and n−6 fatty acids have important clinical and nutritional applications. Lipase-catalyzed acidolysis of seal blubber (SBO) and menhaden oils (MO) with γ-linolenic acid (GLA) was carried out in hexane. The process variables studied for lipase-catalyzed reaction were concentration of enzyme (100–700 units/g of oil), reaction temperature (30–60°C), reaction time (0–48 h), and mole ratio of GLA to triacylglycerols (TAG) (1∶1 to 5∶1). Two lipases chosen for acidolysis reaction were from Pseudomonas species (PS-30) and Mucor miehei. Lipase PS-30 was chosen over Mucor (also known as Rhizomucor) miehei to catalyze the acidolysis reaction owing to higher incorporation of GLA. For the acidolysis reaction, optimal conditions were a 3∶1 mole ratio of GLA to TAG, reaction temperature of 40°C, reaction time of 24 h, and an enzyme concentration of 500 units/g of oil. Under these conditions, incorporation of GLA was 37.1% for SBO and 39.6% for MO.     

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.     

Distribution of medium-chain FA in different lipid classes after administration of specific structured TAG in rats

Structured TAG (STAG) containing medium-chain FA (MCFA) in the sn-1,3 positions and essential FA in the sn-2 position were synthesized by lipase-catalyzed acidolysis. In our previous studies we found that part of the MCFA from STAG could be absorbed in the small intestine; however, it was unclear how they were absorbed. In order to get a better understanding of the metabolism of STAG to improve future design and application of STAG, in the present study lymph lipids collected after feeding STAG were fractionated into different classes and the FA composition of each lipid class was studied by GC after methylation to FAME. Caprylic acid was detected in the fraction of TAG only after administration of 1,3-dioctanoyl-2-linoleyl-sn-glycerol (8∶0/18∶2/8∶0), whereas lauric acid was detected in TAG, DAG, and FFA as well as phospholipids after administration of 1,3-didodecanoyl-2-linoleyl-sn-glycerol (12∶0/18∶2/12∶0). We conclude that the enterocyte has the ability to reacylate the MCFA into TAG and that the intestinal absorption of MCFA from STAG mainly occurs by resynthesis of TAG. Caprylic acid from STAG is not incorporated into phospholipids, whereas lauric acid from STAG can be incorporated into phospholipids.     

Acidolysis of several vegetable oils by mycelium-bound lipase of Aspergillus flavus link

The ability of mycelium-bound lipase of a locally isolated Aspergillus flavus to modify the triglyceride structure of vegetables oils was studied. The catalysis involved the acidolysis of vegetable oils, such as palm olein, coconut oil, cotton-seed oil, rapeseed oil, corn oil and soybean oil, with selected fatty acids (FA). The reactions were followed against time, and the percentages of FA incorporated were determined by gas chromatography. Percentage of FA incorporated after 20-h reaction was in the range of 13 to 18%. Reaction between cottonseed oil with lauric acid gave the highest percentage of incorporation (18%), followed by soybean oil with lauric acid (16%) and coconut oil with oleic acid (16%). The results indicated that the hydrolytic affinity of A. flavus lipase demonstrates an acyl group specificity toward short-chain FA (C₈–C₁₀). Changes in triglyceride profiles of each oil were also monitored by reverse-phase high-pressure liquid chromatography. In all products, there were increases in the concentrations of several existing triglycerides and formation of new triglycerides. The melting points of all acidolyzed vegetable oils were determined by differential scanning calorimetry, and significant changes in melting profiles were noted.     

Lipase-catalyzed acidolysis of tristearin with oleic or caprylic acids to produce structured lipids

Two different structured lipids (SL) were synthesized by transesterifying tristearin with caprylic acid (C8∶0) or oleic acid (C18∶1). The objective was to synthesize SL containing stearic acid (C18∶0) at the sn-2 position as possible nutritional and low-calorie fats. The reaction was catalyzed by IM60 lipase from Rhizomucor miehei in the presence of n-hexane. The effects of reaction parameters affecting the incorporation of caprylic acid into tristearin were compared with those for incorporating oleic acid into tristearin. For all parameters studied, oleic acid incorporation was higher than caprylic acid. The range of conditions favorable for synthesizing high yields of C8∶0-containing SL was narrower than for oleic acid. An incubation time of 12–24 h and an enzyme content of 5% (w/w total substrates) favored C8∶0 incorporation. The mole percentage of incorporated C18∶1 did not increase further at enzyme additions greater than 10%. C18∶1 incorporation decreased with the addition of more than 10% water (w/w total substrates) to the tristearin-oleic acid reaction mixture. Increasing the mole ratio of fatty acid (FA) to triacylglycerol increased oleic acid incorporation. The highest C8∶0 incorporation was obtained at a 1∶6 mole ratio of tristearin to FA. Positional analysis confirmed that C18∶0 remained at the sn-2 position of the synthesized SL. The melting profiles of tristearin-caprylic acid and tristearin-oleic acid SL displayed peaks between −20 to 30°C and −20 to 40°C, respectively. Their solid fat contents (∼25%) at 25°C suggest possible use in spreads or for inclusion with other fats in specialized blends.     

FA composition of crude oil recovered from catfish viscera

The FA composition of crude catfish oil recovered from whole viscera, digestive tract, liver, gallbladder, and visceral storage fat was determined and compared with that of fillet and nugget (abdominal portion). About 34% crude fat (wet basis) could be recovered from the whole catfish viscera. FA found in crude catfish visceral oil were C14∶0, C16∶0, C16∶1, C18∶0, C18∶1, C18∶2, C18∶3, C20∶0, C20∶1, C20∶2, C20∶3, C20∶4, and C22∶6, the predominant FA being C18∶1, C16∶0, C18∶2, and C18∶0. Catfish visceral oil was characterized by a high level of unsaturated FA, which was similarly found in fillet and nugget. Total unsaturated FA in visceral oil amounted to 261.3 mg/g (dry basis) compared to that of fillet (259.3 mg/g) and nugget, (307.6 mg/g). The whole viscera contained 4.2 mg/g DHA compared to that of gallbladder (9.2 mg/g), fillet (9.3 mg/g), and nugget, (10.7 mg/g). The total n−3 FA in the whole and/or portioned visceral ranged from 4.3 to 20.9 mg/g.     

Production of structured lipids containing essential fatty acids by immobilizedRhizopus delemar lipase

An attempt was made to produce structured lipids containing essential fatty acid by acidolysis with 1,3-positional specificRhizopus delemar lipase. The lipase was immobilized on a ceramic carrier by coprecipitation with acetone and then was activated by shaking for 2 d at 30°C in a mixture of 5 g safflower or linseed oil, 10 g caprylic acid, 0.3 g water and 0.6 g of the immobilized enzyme. The activated enzyme was transferred into the same amount of oil/caprylic acid mixture without water, and the mixture was shaken under the same conditions as for the activation. By this reaction, 45–50 mol% of the fatty acids in oils were exchanged for caprylic acid, and the immobilized enzyme could be reused 45 and 55 times for safflower and linseed oils, respectively, without any significant loss of activity. The triglycerides were extracted withn-hexane after the acidolysis and then were allowed to react again with caprylic acid under the same conditions as mentioned above. When acidolysis was repeated three times with safflower oil as a starting material, the only products obtained were 1,3-capryloyl-2-linoleoylglycerol and 1,3-capryloyl-2-oleoyl-glycerol, with a ratio of 86∶14 (w/w). Equally, the products from linseed oil were 1,3-capryloyl-2-α-linolenoyl-glycerol, 1,3-caprylol-2-linoleoyl-glycerol, and 1,3-capryloyl-2-oleoly-glycerol (60∶22∶18, w/w/w). All fatty acids at the 1,3-positions in the original oils were exchanged for caprylic acid by the repeated acidolyses, and the positional specificity ofRhizopus lipase was also confirmed to be strict.     

Enzymatic synthesis of high-purity structured lipids with caprylic acid at 1,3-positions and polyunsaturated fatty acid at 2-position

We attempted to synthesize high-purity structured triacylglycerols (TAG) with caprylic acid (CA) at the 1,3-positions and a polyunsaturated fatty acid (PUFA) at the 2-position by a two-step enzymatic method. The first step was synthesis of TAG of PUFA (TriP), and the second step was acidolysis of TriP with CA. Candida antarctica lipase was effective for the first reaction. When a reaction medium of PUFA/glycerol (3∶1, mol/mol) and 5% immobilized Candida lipase was mixed for 24 h at 40°C and 15 mm Hg, syntheses of TAG of γ-linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acids reached 89, 89, 88, and 83%, respectively. In these reactions, the lipase could be used for at least 10 cycles without significant loss of activity. In the second step, the resulting trieicosapentaenoin was acidolyzed at 30°C for 48h with 15 mol parts CA using 7% of immobilized Rhizopus delemar lipase. The CA content in the acylglycerol fraction reached 40 mol%. To increase the content further, the acylglycerols were extracted from the reaction mixture with n-hexane and were allowed to react again with CA under conditions similar to those of the first acidolysis. After three successive acidolysis reactions, the CA content reached 66 mol%. The content of dicapryloyl-eicosapentaenoyl-glycerol reached 86 wt% of acylglycerols, and the ratio of 1,3-dicapryloyl-2-eicosapentaenoyl-glycerol to 1(3),2-dicapryloyl-3(1)-eicosapentaenoyl-glycerol was 98∶2 (w/w). In this reaction, the lipase could be used for at least 20 cycles without significant loss of activity. Repeated acidolysis of the other TriP with CA under similar conditions synthesized 1,3-dicapryloyl-2-γ-linolenoyl-glycerol, 1,3-dicapryloyl-2-arachidonoyl-glycerol, and 1,3-dicapryloyl-2-docosahexaenoyl-glycerol in yields of 58, 87, and 19 wt%, respectively.     

Incorporation of n−3 fatty acids into plasma and liver lipids of rats: Importance of background dietary fat

The health benefits of long-chain n−3 PUFA (20∶5n−3 and 22∶6n−3) depend on the extent of incorporation of these FA into plasma and tissue lipids. This study aimed to investigate the effect of the background dietary fat (saturated, monounsaturated, or n−6 polyunsaturated) on the quantitative incorporation of dietary 18∶3n−3 and its elongated and desaturated products into the plasma and the liver lipids of rats. Female weanling Wistar rats (n=54) were randomly assigned to six diet groups (n=9). The fat added to the semipurified diets was tallow (SFA), tallow plus linseed oil (SFA-LNA), sunola oil (MUFA), sunola oil plus linseed oil (MUFA-LNA), sunflower oil (PUFA), or sunflower oil plus linseed oil (PUFA-LNA). At the completion of the 4-wk feeding period, quantitative FA analysis of the liver and plasma was undertaken by GC. The inclusion of linseed oil in the rat diets increased the level of 18∶3n−3, 20∶5n−3, and, to a smaller degree, 22∶6n−3 in plasma and liver lipids regardless of the background dietary fat. The extent of incorporation of 18∶3n−3, 20∶5n−3, and 22∶5n−3 followed the order SFA-LNA>MUFA-LNA>PUFA-LNA. Levels of 22∶6n−3 were increased to a similar extent regardless of the type of major fat in the rat diets. This indicates that the background diet affects the incorporation in liver and plasma FA pools of the n−3 PUFA with the exception of 22∶6n−3 and therefore the background diet has the potential to influence the already established health benefits of long-chain n−3 fatty acids.     

The regiospecific position of 18∶1 cis and trans monoenoic fatty acids in milk fat triacylglycerols

TAG of butterfat were fractionated according to the type and degree of unsaturation into six fractions by silver-ion HPLC. The fractions containing TAG with either cis-or trans-monoenoic FA were collected and fractionated further by reversed-phase HPLC to obtain fractions containing cis TAG of ACN:DB (acyl carbon number:double bonds) 48∶1, 50∶1, and 52∶1 as well as trans 48∶1, 50∶1, and 52∶1. The FA compositions of these fractions were elucidated by GC. The MW distribution of each fraction was determined by ammonia negative-ion CI-MS. Each of the [M-H]⁻ parent ions was fractionated further by collision-induced dissociation with argon, which gave information on the location of cis-and trans-FA between the primary and secondary positions of TAG. The results suggest that the sn-positions of the monoenoic cis-and trans-FA depend on the two other FA present in the molecule. With 14∶0 FA in the TAG molecule, the 18∶1 FA in the sn-2 position are mostly present as cis-isomers. When there is no 14∶0 in the TAG molecule, the trans-18∶1 isomers seem to be more common in the sn-2 position. Also when other long-chain FA are present, the trans-isomers are more likely to be located in the secondary (sn-2) position.