Lipase-catalyzed incorporation of conjugated linoleic acid into tricaprylin

Three commercially available immobilized lipases, Novozym 435 from Candida antarctica, Lipozyme IM from Rhizomucor miehei, and Lipase PS-C from Pseudomonas cepacia, were used as biocatalysts for the interesterification of conjugated linoleic acid (CLA) ethyl ester and tricaprylin. The reactions were carried out in hexane, and the products were analyzed by gas-liquid chromatography. The effects of molar ratio, enzyme load, incubation time, and temperature on CLA incorporation were investigated. Novozym 435, as compared to Lipozyme IM and Lipase PC-C, showed the highest degree of CLA incorporation into tricaprylin. By hydrolysis with pancreatic lipase, it was found that Lipozyme IM and Lipase PS-C exhibited high selectivity for the sn-1,3 position of the triacylglycerol early in the interesterification, with small extents of incorporation of CLA into the sn-2 position, probably due to acyl migration, at later reaction times. A small extent of sn-1,3 selectivity during interesterification by Novozym 435 was observed.     

Effects of selected substrate forms on the synthesis of structured lipids by two immobilized lipases

Two immobilized lipases, IM 60 from Rhizomucor miehei and SP 435 from Candida antarctica, were used to synthesize structured lipids (SL). Tricaprin and trilinolein were interesterified to produce SL that contained one linoleic acid per triacylglycerol molecule (SL1) and SL with two linoleic acids (SL2). SL1 and SL2 were separated by silver nitrate thin-layer chromatography according to their unsaturation, and the fatty acid at the sn-2 position was determined after pancreatic lipasecatalyzed hydrolysis of SL1 and SL2. With IM 60, 57.7 mol% capric acid and 42.3 mol% linoleic acid were found at the sn-2 position of SL1, while 43.3 mol% capric acid and 56.7 mol% linoleic acid were at the sn-2 position of SL2. The fatty acid at the sn-2 position of SL1 with SP 435 as biocatalyst was 43.6 mol% capric acid and 56.4 mol% linoleic acid, while SL2 contained 56.6 mol% capric acid and 43.4 mol% linoleic acid. Different structural forms of the capric acid-containing substrate (triacylglycerol vs. ethyl ester) and different chainlengths of triacylglycerol were selected to study the substrate selectivity of lipases. Results indicated that SP 435 had some degree of preference for the triacylglycerol form (tricaprin), and IM 60 produced SL more rapidly and reached steady state faster with tricaprin as substrate than with capric acid ethyl ester. For chainlength selectivity, mol% of synthesized SL from tricaprin + trilinolein and tristearin + trilinolein were compared. SP 435 exhibited no apparent preference for either tricaprin or tristearin. However, IM 60 showed a more rapid reaction with tricaprin than with tristearin.     

Enzymatic Synthesis of Structured Triacylglycerols Containing CLA Isomers Starting from <Emphasis Type="Italic">sn</Emphasis>-1,3-Diacylglycerols

The synthesis of structured triacylglycerols (TAG) by the enzymatic reaction between sn-1,3-diacylglycerols (sn-1,3-DAG) and conjugated linoleic acid (CLA) isomers was studied. Both the substrates of the reaction were produced from vegetable oils, the sn-1,3-DAG from extra virgin olive oil and the CLA isomers from sunflower oil. The enzymatic reactions between these substrates were catalyzed for 96 h by an immobilized lipase from Rhizomucor miehei (Lipozyme IM) and the reactions carried out in solvent were monitored every 24 h by using high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). The enzymatic reactions were carried out in different reaction media (hexane, isooctane and solvent free) and with different CLA/sn-1,3-DAG ratios. Total % acidic composition and structural analysis data were evaluated to verify the presence of CLA isomers in sn-2- position of synthesized TAG. The results showed good levels of CLA incorporation in sn-1,3-DAG, from 19.2% of TAG synthesized in solvent free conditions with a 0.5:1 substrate ratio, to 47.5% of TAG synthesized in isooctane with a 2:1 substrate ratio. It was observed that for all the reaction media, the best sn-2- acylic specificity was obtained with a 0.5:1 substrate ratio.     

Use of a fluorescent radiolabeled triacylglycerol as a substrate for lipoprotein lipase and hepatic triglyceride lipase

A fluorescent radiolabeled triacylglycerol has been synthesized by using a fluorescent fatty acid (pyrene decanoic acid) and a radiolabeled oleic acid. This analog of the natural substrate, 1(3)pyrene decanoic-2,3(1,2)-dioleoyl-sn-glycerol, has been tested as substrate for determining lipoprotein lipase and hepatic triacylglycerol lipase activities in post-heparin plasma. Optimal conditions for the determination of the two post-heparin plasma lipases were similar to those using radiolabeled triolein. Using this substrate, both post-heparin lipases exhibited their characteristic properties (pH optimum and effect of inhibitors) and attacked external ester bonds (1 or 3) containing pyrene decanoic and oleic acids at a similar rate     

Synthesis and Structural Analysis of Structured Triacylglycerols with CLA Isomers in the <Emphasis Type="Italic">sn</Emphasis>-2- Position

The present research deals with the chemical esterification of the sn-2- position of sn-1,3-diacylglycerol (sn-1,3-DAG) with 9cis,11trans (c9,t11) and 10trans,12cis (t10,c12) conjugated linoleic acid (CLA) isomers to obtain structured triacylglycerols (TAG); the sn-1,3-DAG substrates were produced from extra virgin olive oil by means of enzymatic reactions while CLA isomers were obtained using a three-step procedure based on alkaline hydrolysis of sunflower oil, urea purification of linoleic acid (LA) and alkaline isomerization of LA. The results showed good levels of CLA incorporation in structured TAG at the tested temperatures: 37.5% at 4 °C and 39.1% at 14 °C. To evaluate the incorporation of CLA isomers in sn-2- position of sn-1,3-DAG structural analysis of the newly synthesized TAG was carried out using an enzymatic and a chemical method. The results of the structural analysis also showed up the occurrence of acyl migration. The pancreatic lipase method allowed the direct determination of the fatty acid composition of TAG sn-2- position but this enzymatic method showed different results (p < 0.05) in respect to the chemical one; this occurrence could be due to an acylic specificity of the lipase. High incorporation of CLA isomers in sn-2- position of TAG was observed, 77.0% at 4 °C and 81.5% at 14 °C, considering the results of the chemical procedure.     

Enzymatic transesterification of triolein and stearic acid and solid fat content of their products

Two systems were investigated and compared as models for making margarine-type fats. Two immobilized lipases, IM60 from Rhizomucor miehei and SP435 from Candida antarctica, were used to catalyze the transesterification of triolein with stearic acid and stearic acid methyl ester, respectively, in n-hexane. The optimal reaction temperature for both enzymes was 55°C at a mole ratio of triolein to acyl donor of 1:2. Equilibria were reached at 18 h for IM60 and 24 h for SP435. Analysis of the overall yield and incorporation of fatty acid at the sn-2 position indicated that the triacylglycerol products contained 38.4 and 16.2% 18:0 for acidolysis and 34.2 and 11.3% for interesterification reactions, respectively, at the 2-position. With SP435, the softest fat was produced after 18 h of incubation, and the hardest after 30 h. For IM60 system, 18 h of incubation gave the most plastic fat.     

<Superscript>13</Superscript>C-NMR Regioisomeric Analysis of EPA and DHA in Fish Oil Derived Triacylglycerol Concentrates

The regio-isomeric distribution of the omega-3 polyunsaturated fatty acids (PUFA) cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in the triacylglycerols (TAG) of anchovy/sardine fish oil was determined by ¹³C nuclear magnetic resonance (NMR) analysis under quantitative conditions. From the measurements of sn-1,3 and sn-2 carbonyl peak areas it was established that EPA was mainly located in the sn-1,3 positions, whereas DHA primarily occupied the sn-2 position. Reconstituted TAG prepared by Candida antarctica lipase-B (CALB) glycerolysis of the ethyl ester (EE) or the free fatty acid (FFA) forms of anchovy/sardine fish oil, displayed a different pattern: EPA was equally distributed, while DHA was preferentially attached to the sn-1,3 positions. TAG concentrates of varying EPA and DHA molar fractions were prepared by CALB-catalyzed glycerolysis of the corresponding EE and FFA. ¹³C-NMR analysis of the purified products revealed a lack of CALB regioselectivity for EPA and a slight sn-1,3 regioselectivity for DHA. Since this pattern was observed in all cases of this study, it was concluded that the lipase regioselectivity during TAG synthesis is independent of both the acyl donor type (carboxylic acid or ester) and the fatty acid content of the oil substrate.     

Regiospecific analysis of fractions of bovine milk fat triacylglycerols with the same partition number

Bovine milk fat was fractionated using preparative reversed-phase high-performance liquid chromatography. The conditions consisted of two successive linear gradients of acetonitrile and tert-butylmethylether, followed by a final isocratic mixture of the two eluants, leading to triacylglycerols grouped by their partition number (PN). Fractions corresponding to partition numbers 32 to 50 were isolated and analyzed for fatty acid distribution between sn-1,3 and sn-2 positions by Grignard degradation. Results showed that the fatty acid distribution in milk fat triacylglycerols is nonrandom. The distribution of short-chain fatty acids, stearic (predominantly at sn-1,3 position) and palmitic (predominantly sn-2 position), did not change with triacylglycerol size. Medium-chain fatty acids were predominantly located at sn-2 position, but their proportion at this position decreased with triacylglycerol size. Oleic acid distribution was also size-dependent in that it was located in high proportions at sn-2 position in smaller triacylglycerols and vice versa. Results also showed that the sn-2 position was more unsaturated than sn-1,3 position in the PN range from 32 to 40, but it was more saturated in triacylglycerols with higher PN.     

Lipase-catalyzed synthesis of structured triacylglycerides from 1,3-diacylglycerides

A new method for the lipase-catalyzed synthesis of structured TAG (ST) is described. First, sn1,3-dilaurin or-dicaprylin were enzymatically synthesized using different published methods. Next, these were esterified at the sn2-position with oleic acid or its vinyl ester using different lipases. Key to successful enzymatic synthesis of ST was the choice of a lipase with appropriate FA specificity, i.e., one that does not act on the FA already present in the sn1,3-DAG, but that at the same time exhibits high selectivity and activity toward the FA to be introduced. Reactions were performed in the presence of organic solvents or in solvent-free systems under reduced pressure. With this strategy, mixed ST containing the desired compounds 1,3-dicaprylol-2-oleyl-glycerol or 1,3-dilauroyl-2-oleyl-glycerol (CyOCy or LaOLa) were obtained at 87 and 78 mol% yield, respectively, using immobilized lipases from Burkholderia cepacia (Amano PS-D) in n-hexane at 60°C. However, regiospecific analysis with porcine pancreatic lipase indicated that in CyOCy, 25.7% caprylic acid and in LaOLa 11.1% lauric acid were located at the sn2-position. Oleic acid vinyl ester was a better acyl donor than oleic acid. Esterification of sn1,3-DAG and free oleic acid gave very low yield (<20%) of ST in a solvent system and moderate yield (>50%) in a solvent-free system under reduced pressure.     

Optimization of the reaction conditions in the lipase-catalyzed synthesis of structured triglycerides

Structured triglycerides of the ABA-type, containing one type of fatty acid (A) in the sn-1 and sn-3 positions and a second type of fatty acid (B) in the sn-2 position of the glycerol, were synthesized using lipases. The highest yields and purities were achieved in a two-step process, where a triglyceride of the B-type was subjected to an alcoholysis reaction in an organic solvent catalyzed by sn-1,3-regiospecific lipases yielding the corresponding 2-monoglyceride (2-MG). Using this strategy, e.g., 2-monopalmitin (2-MP) was obtained in up to 88% yield at >95% purity by crystallization. Esterification of 2-MP with oleic acid resulted in the formation of 1,3-oleyl-2-palmitoyl-glycerol in up to 72% yield containing 94% palmitic acid in the sn-2 position. The best lipases were from Rhizomucor miehei, Rhizopus delemar, and Rhizopus javanicus. Water activity, solvent, and carrier for lipase immobilization strongly influenced the yield and purity of the products in both steps. Furthermore, 2-MG from fish oil were produced by alcoholysis in up to 84% yield at >95% purity.     

Production of Human Milk Fat Substitutes by Interesterification of Tripalmitin with Ethyl Oleate Catalyzed by Candida parapsilosis Lipase/Acyltransferase

In human milk fat, the saturated fatty acids, namely palmitic acid, are located at the sn-2 position of triacylglycerols (TAG) while unsaturated fatty acids (e.g. oleic acid) are esterified at position sn-1,3. Thus, sn-1,3-dioleoyl-2-palmitoylglycerol (OPO) is the target TAG to be used as human milk fat substitutes (HMFS) in infant formulas. In this study, the noncommercial recombinant lipase/acyltransferase from Candida parapsilosis (CpLIP2) was immobilized in Accurel MP1000, and used as a biocatalyst for the interesterification of tripalmitin with ethyl oleate in a solvent-free medium, to obtain structured lipids used as HMFS. Different molar ratios (MR) of ethyl oleate to tripalmitin (2:1–8:1) were used. After 4 h reaction at 60°C, about 30 mol% of oleic acid incorporation was already observed for all tested MR. An apparent equilibrium was reached after 8–24 h, with 32–51 mol% final incorporation, increasing with the MR. The incorporation of oleic acid into TAG was compared with the maximum predicted values when a random or a sn-1,3-regioselective biocatalyst was used. The obtained values are consistent with the maximum incorporation expected for a sn-1,3-regioselective enzyme. In fact, the amount of oleic acid at position sn-2 was approximately 15% for all the MR tested, which is explained by the acyl migration phenomenon. CpLIP2 exhibited higher activity than most commercial immobilized lipases (e.g. faster reaction in solvent-free media, low enzyme load, and low MR needed), and showed a recognized sn-1,3 regioselective behavior.     

Regioselective analysis of the fatty acid composition of triacylglycerols with conventional high-performance liquid chromatography

A new method for regioselective analysis of triacyglycerols via conventional high-performance liquid chromatography (HPLC) has been developed. The method is simple and avoids the time-consuming purification processes normally characteristics of regioselective analyses. The procedure utilizes an sn-1,3-specific lipase from Rhizopus arrhizus to deacylate the fatty acid residues located at the sn-1 and sn-3 positions of triacylglycerols. The fatty acid residues esterified at the sn-2 position are determined by subtraction of the results of the sn-1,3 analysis from an overall composition analysis based on complete saponification of the original sample. The fatty acid mixtures are converted to p-bromophenacyl esters and analyzed using conventional HPLC techniques. The analytical procedure has been verified using a standard structured triacylglycerol. The analytical results for three edible vegetable oils are compared with those obtained via the method proposed by P.J. Williams and co-workers.     

Identification of triacylglycerols in the enzymatic transesterification of rapeseed and butter oil

A blend of rapeseed and butter oil was transesterified using immobilized Thermomyces lanuginosus lipase (Lipozyme® TL IM) as catalyst. The reaction was followed by reversed-phase HPLC and the triacylglycerol peaks were tentatively identified from their elution times by calculating equivalent carbon numbers. Further identification was made using HPLC-electrospray tandem mass spectrometry. A few of the triacylglycerols detected were typical combinations of fatty acids originating from rapeseed oil, such as α-linolenic acid, and short-chain fatty acids from butter oil. Due to the regioselectivity of the lipase, the transesterification reaction involved mainly fatty acids in the sn-1 and sn-3 positions. However, significant changes in the fatty acid composition in the sn-2 position were detected after 6 h.     

Lipase-catalyzed synthesis of structured low-calorie triacylglycerols

Because of their unique fatty acid specificities and regioselectivities, lipases have been found to be effective catalysts for the synthesis of structured lipids that have a predetermined composition and distribution of fatty acyl groups on the glycerol backbone. The prospective plant-derived lipase found in the exudate of Carica papaya is known for its shortchain acyl group specificity, 1,3-glycerol regioselectivity, and sn-3 stereoselectivity. Carica papaya latex (CPL) was therefore examined for its potential ability to synthesize structured lowcalorie short- and long-chain triacylglycerols (SLCT). In this paper, we describe the utility of CPL in the lipase-catalyzed interesterification reaction of triacetin and hydrogenated soybean oil. Normal-phase high-performance liquid chromatography, combined with mass spectrometry, was used to distinguish the structured SLCT synthesized using the lipase from the corresponding SLCT produced by chemical synthesis.     

Lipase-catalyzed modification of borage oil: Incorporation of capric and eicosapentaenoic acids to form structured lipids

Two immobilized lipases, nonspecific SP435 from Candida antarctica and sn-1,3 specific IM60 from Rhizomucor miehei, were used as biocatalysts for the restructuring of borage oil (Borago officinalis L.) to incorporate capric acid (10:0, medium-chain fatty acid) and eicosapentaenoic acid (20:5n-3) with the free fatty acids as acyl donors. Transesterification (acidolysis) reactions were carried out in hexane, and the products were analyzed by gas-liquid chromatography. The fatty acid profiles of the modified borage oil were different from that of unmodified borage oil. Higher incorporation of 20:5n-3 (10.2%) and 10:0 (26.3%) was obtained with IM60 lipase, compared to 8.8 and 15.5%, respectively, with SP435 lipase. However, SP435 lipase was able to incorporate both 10:0 and 20:5n-3 fatty acids at the sn-2 position, but the IM60 lipase did not. Solvents with log P values between 3.5 and 4.5 supported the acidolysis reaction better than those with log P values between −0.33 and 3.0.     

Measuring stereoselectivity in lipase-catalyzed acidolysis reactions by ultra-high resolution 13C nuclear magnetic resonance

Elucidating the stereoselectivity of lipases in synthetic reactions of triacylglycerols has hitherto been carried out using traditional analytical techniques to determine the composition of the reaction products. These methods are laborious and are not always appropriate for analysis of certain triacylglycerol types. A direct method, utilizing a stereospecific deuterium-labeled triacylglycerol substrate, has been developed where the stereoisomeric composition of the reaction product is determined by ultra-high resolution ¹³C nuclear magnetic resonance (NMR) spectroscopy. Through lipase-catalyzed transesterification of deuterium-labeled trilauroylglycerol with oleic acid, chemical shifts were induced in the ¹³C NMR spectrum by the deuterium atom and olefinic double bonds, enabling unambiguous stereospecific assignment of triacylglycerol species. By this method of analysis, we found an effect of the degree of reaction conversion on the extent of stereoisomerism in the triacylglycerol product. Stereoselectivity was greatest (for sn-1) with lipase from Rhizomucor miehei. Lipases from Rhizopus niveus, Candida rugosa, Carica papaya, and the cutinase from Fusarium sp. were also found to exhibit stereoselectivity, with preference for either sn-1 or sn-3 acyl exchange.     

Synthesis of 1,3-dicapryloyl-2-docosahexaenoylglycerol by a combination of nonselective and <Emphasis Type="Italic">sn</Emphasis>-1,3-selective lipase reactions

A two-step consecutive synthetic method for the production of symmetrical, structured lipids by a combination of nonselective and sn-1,3 regioselective ester exchange reactions was investigated. In the first step, TAG with unspecifically substituted DHA were obtained by reacting tricapryloylglycerol (CCC) with ethyl docosahexanoate (EtDHA) using the lipase QLM (from Alcaligenes sp.), followed by removing the ethyl ester and CCC by molecular distillation. In the second step, sn-1,3 regioselective ester exchange was achieved by reacting the resulting TAG with ethyl caprylate (EtC) using the immobilized lipase Novozyme 435 (Candida antarctica lipase), followed by distillation of the ethyl ester and CCC to give sn-1,3-dicapryloyl-sn-2-docosahexaenoylglycerol (CDC). The acylglycerol composition of CDC was analyzed by GLC, which showed that the content of dicapryloyl-docosahexaenoylglycerols (2CD) in the product was 76.4%, and that the ratio of CDC to sn-1,2-dicapryloyl-sn-3-docosahexaenoylglycerol contained in 2CD was 82.7∶17.3 (%). The distillates CCC, EtDHA, and EtC could be recycled repeatedly to produce CDC as the substrate for the consecutive ester exchange reaction. In addition, separation of CCC and EtDHA was unnecessary for reuse. The present method is considered to meet the requirements for industrial utilization, in which simplicity in scaleup, high yields, compact reaction system, and minimal formation of by-products are important factors.     

Study of Some Experimental Parameters in the Synthesis of Triacylglycerols with CLA Isomers and Structural Analysis

The present research deals with the synthesis of structured triacylglycerols (TAG) by enzymatic treatment of sn-1,3-diacylglycerol (sn-1,3-DAG) with conjugated linoleic acid (CLA) isomers using the immobilized lipase from Rhizomucor miehei (Lipozyme^® IM) under different experimental conditions. In particular, the influence of reaction parameters, such as temperature, enzymatic load, reaction time and DAG/CLA ratio has been evaluated using an experimental design software with a screening objective. Two responses have been selected, they are the percentage of CLA isomers in total TAG and in the sn-2- position and a three-level-4-factor fractional factorial experimental design was used to screen the variables. The results showed that the selected experimental variables have an influence on the enzymatic reaction, in particular, the DAG/CLA substrate ratio and the temperature, both of which inversely correlated with CLA incorporation, but also the enzymatic load and the reaction time, both directly correlated with CLA incorporation. The best results for CLA isomer % content both in total TAG (46.3%) and in the sn-2- position (52.2%) were obtained at 40 °C for 96 h, with 20% enzymatic load and a 0.5 reactive ratio.     

Structured lipids: Lipase-catalyzed interesterification of tricaproin and trilinolein

Structured lipids were synthesized by interesterification of trilinolein and tricaproin with sn-1,3-specific (IM 60) and nonspecific (SP 435) lipases. The interesterification reaction was performed by incubating a 1:2 mole ratio of trilinolein and tricaproin in 3 mL hexane at 45°C for the IM 60 lipase from Rhizomucor miehei, and at 55°C for the SP 435 lipase from Candida antarctica. Reaction products were analyzed by reverse-phase high-performance liquid chromatography with an evaporative light-scattering detector. The fatty acids at the sn-2 position were identified after pancreatic lipase hydrolysis and analysis with a gas chromatograph. IM 60 lipase produced 53,5 mol% dicaproyllinolein (total carbon number = C33) and 22.2% monocaproyldilinolein (C45). SP 435 lipase produced 41% C33 and 18% C45. When caproic acid was used in place of tricaproin as the acyl donor, the IM 60 lipase produced 62.9% C33. The effects of variation in mole ratio, temperature, added water, solvent polarity, and time course on the interesterification reaction were also investigated. In the absence of organic solvent, IM 60 lipase produced 52.3% C33.     

Analysis of regiospecific distribution of FA of TAG using the lipase-catalyzed ester-exchange

A very simple and versatile GC method has been developed that can be utilized for quick analysis, in many samples, of the FA compositions at the sn-2- and sn-1,3-positions of TAG. By using the lipase-catalyzed, sn-1,3-regioselective esterexchange reaction of TAG with ethyl acetate, followed by direct injection of the crude reaction mixture into the GC apparatus without any pretreatment, the FA located at the sn-2-position could accurately be analyzed as a TAG derivative in which the sn-1,3-positions were substituted by an acetate residue. Furthermore, the FA located at the sn-1,3-positions could simultaneously be analyzed as the corresponding ethyl ester derivatives using this method. The reliability of the analytical method was compared with conventional methods by analyzing the TAG consisting of caprylic acid (C) at the sn-2-position and oleic acid (O) at the sn-1,3-positions, giving comparable analytical results. The present method was applied to the analysis of the structured lipids CCD and CCE, consisting of TAG as a major component in which C and the highly unsaturated FA, DHA (D) or EPA (E), were specifically bound at the sn-2- and the sn-1,3-positions, respectively.