Optimization of reaction conditions for the production of DAG using immobilized 1,3-regiospecific lipase lipozyme RM IM

Oils with a high DAG (1,3-DAG) content have attracted considerable attention as a healthful food oil component. In this study, we report on the synthesis of 1,3-DAG from a mixture of FA, constituted largely of oleic and linoleic acids, using an immobilized 1,3-regioselective lipase from Rhizomucor miehei in a solvent-free system. The kinetics of 1,3-DAG production from FA and glycerol were investigated on the basis of a simplified model, taking into consideration the acyl migration reaction, the removal of water, and glycerol dissolution in the oil phase in addition to the esterification reactions. Both the yield of 1,3-DAG and the purity of DAG were evaluated under a variety of experimental conditions, including reaction temperature, pressure, and amount of enzyme present. When either the reaction temperature or the amount of enzyme used was increased, the 1,3-DAG production rate increased, but yield remained relatively constant. The 1,3-DAG yield as well as the purity of DAG gradually decreased because of the enhancement of acyl migration at later stages of the reaction after the 1,3-DAG concentration reached a maximum. Vacuum was important for attaining high yields of 1,3-DAG. Under conditions of a high vacuum (1 mm Hg) at 50°C, 1.09 M 1,3-DAG was produced from 1.29 M glycerol and 2.59 MFA in an 84% yield and in 90% purity.     

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.     

Elucidation of acyl migration during lipase-catalyzed production of structured phospholipids

Elucidation of acyl migration was carried out in the Lipozyme RM IM (Rhizomucor miehei)-catalyzed transesterification between soybean phosphatidylcholine (PC) and caprylic acid in solvent-free media. A five-factor response surface design was used to evaluate the influence of five major factors and their relationships. The five factors—enzyme dosage, reaction temperature, water addition, reaction time, and substrate ratio—were varied on three levels together with two star points. Enzyme dosage, reaction temperature, and reaction time showed increased effect on the acyl migration into the sn-2 position of PC, whereas increased water addition and substrate ratio had no significant effect in the ranges tested. The best-fitting quadratic response surface model was determined by regression and backward elimination. The coefficient of determination (R ²) was 0.84, which indicates that the fitted quadratic model has acceptable qualities in expressing acyl migration for the enzymatic transesterification. Correlation was observed between acyl donor in the sn-2 position of PC and incorporation of acyl donor into the intermediate lysophosphatidylcholine. Furthermore, acyl migration into the sn-2 position of PC was confirmed by TLC-FID, as PC with caprylic acid was observed on both positions. Under certain conditions, up to 18% incorporation could be observed in the sn-2 position during the lipase-catalyzed transesterification.     

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.     

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.     

Production of specific-structured lipids by enzymatic interesterification: Elucidation of Acyl migration by response surface design

Production of specific-structured lipids (SSL) by lipase-catalyzed interesterification has been attracting more and more attention recently. However, it was found that acyl migration occurs during the reaction and causes the production of by-products. In this paper, the elucidation of acyl migration by response surface design was carried out in the Lipozyme IM (Rhizomucor miehei)-catalyzed interesterification between rapeseed oil and capric acid in solvent-free media. A five-factor response surface design was used to evaluate the influence of five major factors and their relationships. The five factors, water content, reaction temperature, enzyme load, reaction time and substrate ratio, were varied at three levels together with two star points. All parameters besides substrate ratio had strong positive influences on acyl migration, and reaction temperature was most significant. The contour plots clearly show the interactions between the parameters. The migration rates of different fatty acids were also compared from three different sets of experiments during the lipase-catalyzed reaction. The best-fitting quadratic response surface model was determined by regression and backward elimination. The coefficients of determination (R ²) of the model were 0.996 and 0.981 for Q ² value. The results show that the fitted quadratic model satisfactorily expresses acyl migration for the enzymatic interesterification in the batch reactor used.     

Production of specific-structured lipids by enzymatic interesterification: Elucidation of acyl migration by response surface design

Production of specific-structured lipids (SSL) by lipase-catalyzed interesterification has been attracting more and more attention recently. However, it was found that acyl migration occurs during the reaction and causes the production of byproducts. In this paper, the elucidation of acyl migration by response surface design was carried out in the Lipozyme IM (Rhizomucor miehei)-catalyzed interesterification between rapeseed oil and capric acid in solvent-free media. A five-factor response surface design was used to evaluate the influence of five major factors and their relationships. The five factors, water content, reaction temperature, enzyme load, reaction time and substrate ratio, were varied at three levels together with two star points. All parameters besides substrate ratio had strong positive influences on acyl migration, and reaction temperature was most significant. The contour plots clearly show the interactions between the parameters. The migration rates of different fatty acids were also compared from three different sets of experiments during the lipase-catalyzed reaction. The best-fitting quadratic response surface model was determined by regression and backward elimination. The coefficients of determination (R ²) of the model were 0.996 and 0.981 for Q ² value. The results show that the fitted quadratic model satisfactorily expresses acyl migration for the enzymatic interesterification in the batch reactor used.     

Enzymatic Synthesis of Diacylglycerols Enriched with Conjugated Linoleic Acid by a Novel Lipase from <Emphasis Type="Italic">Malassezia globosa</Emphasis>

Diacylglycerols (DAG) of conjugated linoleic acid (CLA) were prepared by esterification of glycerol with fatty acids enriched with CLA (FFA–CLA, >95%) in the presence of a novel lipase from Malassezia globosa (SMG1). Lipase SMG1 is strictly specific to mono- and diacylglycerols but not triacylglycerols, which is similar to the properties of lipase from Penicillium camembertii (lipase G 50), but lipase SMG1 showed preference on the production of DAG with the reaction proceeding. Low temperature was beneficial for the conversion of FFA–CLA into acylglycerols, the degree of esterification reached 93.0% when the temperature was 5 °C. The maximum DAG content (53.4%) was achieved at 25 °C. The rate of DAG synthesis increased as the enzyme loading increased. However, at lipase amounts above 240 U/g mixtures, no significant increases in DAG concentration were observed. The molar ratio of FFA–CLA to glycerol and initial water content were optimized to be 1:3 (mol/mol) and 3%. Lipase SMG1 showed no regioselectivity because the contents of 1,3-DAG and 1,2-DAG were 43.1% and 21.2% based on total content of acylglycerols. By calculating the ratio of 9c, 11t-CLA to 10t, 12c-CLA, it was indicated that lipase SMG1 showed a little preference to 10t, 12c-CLA at the sn-1(3) position of monoacylglycerols (MAG), while no selectivity for 9c, 11t-CLA at the sn-2 position of DAG was obviously found.     

Production of specific-structured triacylglycerols by lipase-catalyzed interesterification in a laboratory-scale continuous reactor

A laboratory-scale continuous reactor was constructed for production of specific structured triacylglycerols containing essential fatty acids and medium-chain fatty acids (MCFA) in the sn-2 and sn-1,3 positions, respectively. Different parameters in the lipase-catalyzed interesterification were elucidated. The reaction time was the most critical factor. Longer reaction time resulted in higher yield, but was accompanied by increased acyl migration. The concentration of the desired triacylglycerol (TAG) in the interesterification product increased significantly with reaction time, even though there was only a slight increase in the incorporation of MCFA. Increased reactor temperature and content of MCFA in the initial reaction substrate improved the incorporation of MCFA and the yield of the desired TAG in the products. Little increase of acyl migration was observed. Increasing the water content from 0.03 to 0.11% (w/w substrate) in the reaction substrate had almost no effect on either the incorporation or the migration of MCFA, or on the resulting composition of TAG products and their free fatty acid content. Therefore, we conclude that the water in the original reaction substrate is sufficient to maintain the enzyme activity in this continuous reactor. Since the substrates were contacted with a large amount of lipase, the reaction time was shorter compared with a batch reactor, resulting in reduced acyl migration. Consequently, the purity of the specific structured TAG produced was improved. Interesterification of various vegetable oils and caprylic acid also demonstrated that the incorporation was affected by the reaction media. Reaction conditions for lipase-catalyzed synthesis of specific structured TAG should be optimized according to the oil in use. Presented in part at Food Science Conference, Copenhagen, Denmark, January 30–31, 1997.     

Production of specific-structured triacylglycerols by lipase-catalyzed interesterification in a laboratory-scale continuous reactor

A laboratory-scale continuous reactor was constructed for production of specific structured triacylglycerols containing essential fatty acids and medium-chain fatty acids (MCFA) in the sn-2 and sn-1,3 positions, respectively. Different parameters in the lipase-catalyzed interesterification were elucidated. The reaction time was the most critical factor. Longer reaction time resulted in higher yield, but was accompanied by increased acyl migration. The concentration of the desired triacylglycerol (TAG) in the interesterification product increased significantly with reaction time, even though there was only a slight increase in the incorporation of MCFA. Increased reactor temperature and content of MCFA in the initial reaction substrate improved the incorporation of MCFA and the yield of the desired TAG in the products. Little increase of acyl migration was observed. Increasing the water content from 0.03 to 0.11% (w/w substrate) in the reaction substrate had almost no effect on either the incorporation or the migration of MCFA, or on the resulting composition of TAG products and their free fatty acid content. Therefore, we conclude that the water in the original reaction substrate is sufficient to maintain the enzyme activity in this continuous reactor. Since the substrates were contacted with a large amount of lipase, the reaction time was shorter compared with a batch reactor, resulting in reduced acyl migration. Consequently, the purity of the specific structured TAG produced was improved. Interesterification of various vegetable oils and caprylic acid also demonstrated that the incorporation was affected by the reaction media. Reaction conditions for lipase-catalyzed synthesis of specific structured TAG should be optimized according to the oil in use. Presented in part at Food Science Conference, Copenhagen, Denmark, January 30–31, 1997.     

Diacylglycerol acyltransferases from Vernonia and Stokesia prefer substrates with vernolic acid

Genetic engineering of common oil crops for industrially valuable epoxy FA production by expressing epoxygenase genes alone had limited success. Identifying other key genes responsible for the selective incorporation of epoxy FA into seed oil in natural high accumulators appears to be an important next step. We investigated the substrate preferences of acyl CoA: diacylglycerol acyltransferases (DGAT) of two natural high accumulators of vernolic acid, Vernonia galamensis and Stokesia laevis, as compared with a common oilseed crop soybean. Developing seed microsomes were fed with either [¹⁴C]oleoyl CoA or [¹⁴C]vernoloyl CoA in combinations with no exogenous DAG or with 1,2-dioleoyl-sn-glycerol, 1-palmitoyl-2-vernoloyl-sn-glycerol, 1,2-divernoloyl-sn-glycerol, 1,2-dioleoyl-rac-glycerol, or 1,2-divernoloyl-rac-glycerol to determine their relative incorporation into TAG. The results showed that in using sn-1,2-DAG, the highest DGAT activity was from the substrate combination of vernoloyl CoA with 1,2-divernoloyl-sn-glycerol, and the lowest was from vernoloyl CoA or oleoyl CoA with 1,2-dioleoyl-sn-glycerol in both V. galamensis and S. laevis. Soybean DGAT was more active with oleoyl CoA than vernoloyl CoA, and more active with 1,2-dioleoyl-sn-glycerol when oleoyl CoA was fed. DGAT assays without exogenous DAG, or with exogenous sn-1,2-DAG fed individually or simultaneously showed consistent results. In combinations with either oleoyl CoA or vernoloyl CoA, DGAT had much higher activity with rac-1,2-DAG than with their corresponding sn-1,2-DAG, and the substrate selectivity was diminished when rac-1,2-DAG were used instead of sn-1,2-DAG. These studies suggest that DGAT action might be an important step for selective incorporation of vernolic acid into TAG in V. galamensis and S. laevis.     

Acyl Migration Kinetics of Vegetable Oil 1,2-Diacylglycerols

The acyl migration kinetics of long-chain 1,2-diacylglycerol (1,2-DAG) to form 1,3-diacylglycerol (1,3-DAG) over the temperature range of 25–80 °C were examined using ¹H-NMR spectroscopy. Lipase-catalyzed ethanolysis of high-oleic sunflower oil, followed by a series of solvent extraction steps, generated high purity 1,2-DAG (0.93 mol fraction of the DAG content). The 1,2-DAG mole fraction of 0.32 at equilibrium was found to be insensitive to temperature, indicating that long-chain acyl group migration is neither endothermic nor exothermic. Determination of the temperature-dependent, first-order reaction kinetic parameters revealed a 1,2-DAG half life (t _1/2) of 3,425 h and 15.8 h at 25 and 80 °C, respectively. A comparison of 1,2-DAG with 2-monoacylglycerol indicated that there is no difference between the two in the potential energy state (ΔG ^‡) of their respective transitions states or cyclic intermediates. Product names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may be suitable.     

Production of specific structured lipids by enzymatic interesterification: optimization of the reaction by response surface design

Rapeseed oil and capric acid were interesterified in solvent-free media catalyzed by Lipozyme IM from Rhizomucor miehei to produce specific-structured lipids (SSLs). The process was optimized by response surface design concerning the effects of acyl migration and the byproducts of diacylglycerols (DAGs). A five-factor response surface design was used to evaluate the influences of five major factors and their relationships. The five factors were water content (W_c, wt-% based on enzyme used), reaction temperature (T_e, °C), enzyme load (E_l, wt-% based on substrates), reaction time (T_r, h) and substrate ratio (S_r, rapeseed oil/capric acid, mol/mol), varied at three levels together with two star point levels. The net incorporation [Δ(I_f–M_f), in which I_f represents incorporation (1,3-positions) and M_f acyl migration (2-position), and the contents of DAGs were analyzed and calculated. All parameters had strong influence on the net incorporation, and the experimental and predicted values were close. The best fitting quadratic model was determined by regression and backward elimination. The coefficients of determination (R²) of the models were 0.971 for net incorporation and 0.938 for DAG content. Thus, we conclude that the quadratic response models adequately expressed the reaction. Based on the models, the reaction was optimized for the maximum net incorporation and minimum DAG content. The reaction and the control of water content or water activity (Aw) was also discussed.     

Diacylglycerols from butterfat: Production by glycerolysis and short-path distillation and analysis of physical properties

The aim of this paper was to develop a process for the production of DAG from butterfat through glycerolysis and short-path distillation and to evaluate the physical properties of the DAG in comparison with the original butterfat. Chemical glycerolysis produced a mixture of acylglycerols containing DAG together with MAG and TAG. From the mixture of glycerolysis products, MAG were removed through three consecutive distillations (vacuum <0.001 mbar) at 150°C. TAG were separated from DAG by distillation at 210°C, which gave a product with more than 80% DAG in the distillates. Distillation temperatures had significant effects on acyl migration. The formation of desirable 1,3-DAG was favored at higher temperatures. Under 210°C distillation, the equilibrium ratio of 6∶4 was obtained between 1,3-DAG and 1,2(2,3)-DAG. The FA profile of the DAG product was relatively similar to the original butterfat. The total DAG recovery was around 77% in the pilot-scale production. The different patterns of m.p. were observed between butterfat and the DAG fraction produced as well as the MAG fraction collected. Solid fat content profiles of the DAG fraction and its mixtures with rapeseed oil possessed trends similar to those of the corresponding butterfat and its mixtures with rapeseed oil. Compared with butterfat, the DAG fraction behaved differently in its thermal profiles, crystallization patterns, and rheological properties; for example, the dropping point was 13°C higher for the latter than for the former, and the crystal pattern was mostly β form for the latter, whereas the former was the β′ form.     

Pilot batch production of specific-structured lipids by lipase-catalyzed interesterification: Preliminary study on incorporation and acyl migration

Effects of water content, reaction time, and their relationships in the production of two types of specific-structured lipids (sn-MLM- and sn-LML-types: L-long chain fatty acids; M-medium chain fatty acids) by lipase-catalyzed interesterification in a solvent-free system were studied. The biocatalyst used was Lipozyme IM (commercial immobilized lipase). The substrates used for sn-MLM-type were fish oil and capric acid, and medium chain triacylglycerols and sunflower free fatty acids for sn-LML-type. The observed incorporation with the time course agrees well with the Michaelis-Menten equation, while the acyl migration is proportional to time within the range of 20 mol% acyl migration (MLM-type: M _f =0.2225 T, R²=0.98; LML-type: M _f =0.5618 T, R²=0.99). As water content (wt%, on the enzyme basis) increased from 3.0 to 11.6% for MLM-type and from 3.0 to 7.2% for LML-type in the solvent-free systems, the incorporation rates in the first 5 h increased from 3.34 to 10.30%/h, and from 7.29 to 11.12%/h, respectively. However, the acyl migration rates also increased from 0.22 to 1.12%/h and from 0.56 to 1.37%/h, respectively. Different effects in the production of two totally position-opposed lipids can be observed. Presumably these are caused by the different chain length of the fatty acids. The relationships between reaction time and water content are inverse and give a quantitative prediction of incorporation and acyl migration in selected reaction conditions and vice versa. The acyl migration can not be totally avoided in present systems, but can be reduced to a relatively low level. Acyl migration during the downstream processing has also been observed and other factors influencing the acyl migration are briefly discussed.     

Acidity and DAG Content of Olive Oils Recently Produced on the Island of Mallorca

In this study, the quality characteristics, i.e., the acidity and acylglycerols, of currently produced Mallorcan oil was analyzed by titration and gas-chromatographic technique, respectively, in approximately 400 samples of monovarietal olive oil made from three genetic varieties (Arbequina, Empeltre, and Picual) on the island of Mallorca during the 2003/2004 and 2005/2006 seasons, and the differences in the compositions were discussed. Composition analysis showed that free fatty acids (FFAs) and DAGs were produced mainly by hydrolysis of triacylglycerols (TAGs). Fruit storage tests revealed that hydrolysis occurred during storage of olive fruits. The DAG content was higher in oil with higher acidity, but the maximal DAG content was only about 10 with 30% acidity, and the primary isomer found in Mallorcan oil was 1,3-DAG. However, the chiral-chromatographic study on the ratio of sn-1,2-DAG to the sum of sn-1,2-DAG and sn-2,3-DAG in a slightly hydrolyzed oil sample indicated that Empeltre and Picual fruits possess an sn-3-enantioselective lipase that leads to accumulation of sn-1,2-DAG. In currently produced Mallorcan oil, significant isomerization appears to occur and hydrolysis of the resulting 1,3-DAGs seems to lessen DAG accumulation.     

Continuous production of structured phospholipids in a packed bed reactor with lipase from <Emphasis Type="Italic">Thermomyces lanuginosa</Emphasis>

The possibilities of producing structured phospholipids between soybean phospholipids and caprylic acid by lipase-catalyzed acidolysis were examined in continuous packedbed enzyme reactors. Acidolysis reactions were performed in both a solvent system and a solvent-free system with the commercially immobilized lipase from Thermomyces lanuginosa (Lipozyme TL IM) as catalyst. In the packed bed reactors, different parameters for the lipase-catalyzed acidolysis were elucidated, such as solvent ratio (solvent system), temperature, substrate ratio, residence time, water content, and operation stability. The water content was observed to be very crucial for the acidolysis reaction in packed bed reactors. If no water was added to the substrate during reactions under the solvent-free system, very low incorporation corporation of caprylic acid was observed. In both solvent and solvent-free systems, acyl incorporation was favored by a high substrate ratio between acyl donor and phospholipids, a longer residence time, and a higher reaction temperature. Under certain conditions, the incorporation of around 30% caprylic acid can be obtained in continuous operation with hexane as the solvent. Presented at the 95th American Oil Chemists' Society Annual Meeting and Expo in Cincinnati, Ohio, May 10, 2004.     

Production of structured lipids in a packed-bed reactor with <Emphasis Type="Italic">thermomyces lanuginosa</Emphasis> lipase

Lipase-catalyzed interesterification between fish oil and medium-chain TAG has been investigated in a packedbed reactor with a commercially immobilized enzyme. The enzyme, a Thermomyces lanuginosa lipase immobilized on silica by granulation (lipozyme TL IM; Novozymes A/S, Bagsvaerd, Denmark), has recently been developed for fat modification. This study focuses on the new characteristics of the lipase in a packed-bed reactor when applied to interesterification of TAG. The degree of reaction was strongly related to the flow rate (residence time) and temperature, whereas formation of hydrolysis by-products (DAG and FFA) were only slightly affected by reaction conditions. The degree of reaction reached equilibrium at 30–40 min residence time, and the most suitable temperature was 60°C or higher with respect to the maximal degree of reaction. The lipase was stable in a 2-wk continuous operation without adjustment of water content or activity of the column and the substrate mixture.     

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.     

Production of Diacylglycerol-Mixture of Regioisomers with High Purity by Two-Step Enzymatic Reactions Combined with Molecular Distillation

Pure diacylglycerol (DAG) is of vital importance for the biomedical and dietetic properties research of DAG. In this study, we aimed to develop an effective process to produce DAG-mixture of regioisomers with high purity. Firstly, DAGs and monoacylglycerols (MAGs) were synthesized by enzymatic esterification of glycerol and free fatty acids (FFAs) from camellia oil with catalysis of Penicillium camembertii lipase, and the obtained reaction mixture was composed of 49.9 % DAG [33.4 % for 1,3-DAG and 16.5 % for 1,2 (2,3)-DAG], 31.6 % MAG and 18.5 % FFA. Secondly, a monoacylglycerol lipase (lipase CBD-MGLP), which was produced by recombinant Escherichia coli in our laboratory, was employed to hydrolyze MAG in the above reaction mixture, and the MAG content decreased to 1.9 % under the optimal conditions with 375 U/g (U/w, with respect to the mass of MAG in the mixture) of CBD-MGLP loading, temperature of 45 °C, mass ratio of esterification mixture to Tris–HCl buffer (w/w) 10:10, and pH of Tris–HCl buffer 9.0. Then, the hydrolytic products were further purified by molecular distillation at low temperature of 130 °C under a pressure of 10 Pa [equivalent to 377 °C at 101.325 kPa (1 atm)], and the DAG purity was up to 98.0 % (66.6 % for 1,3-DAG and 31.4 % for 1,2-DAG) in the final products. This indicated that two-step enzymatic reactions combined with molecular distillation at low temperature could be a feasible and prospective process to produce DAG-mixture of regioisomers with high purity.