Enzymatic interesterification of triolein and tristearin: Chemical structure and differential scanning calorimetric analysis of the products

The structural composition and thermal properties of the products of enzymatic interesterification of triolein and tristearin were investigated. The biocatalyst for the reaction was an immobilized Candida antarctica lipase, SP435. Enzyme load of 10% (w/w reactants) produced 72% of desired total products. Oleoyl-distearoyl triglycerides (SSO, OSS) had higher melting points than dioleoyl-stearoyl triglycerides (OOS, SOO) because the sample contained larger amounts of stearic acid than oleic acid residues. SOS and OSO were hardly produced (0.2 to 1.2%), which indicates that SP435 acted as a nonspecific lipase when catalyzing the interesterification of triolein and tristearin. The maximal yield of OSS and SSO (46.9%) was achieved with a 1.2 mole ratio of triolein to tristearin. As the proportion of tristearin was increased, the production of SOO and OOS decreased, the melting profile of the interesterified triglycerides shifted toward higher melting forms, and the solid fat content increased, indicating formation of hard fats.     

Enzymatic modification of triolein: Incorporation of caproic and butyric acids to produce reduced-calorie structured lipids

Lipase-catalyzed acidolysis of triolein with caproic and butyric acids was performed to produce reduced-calorie structured lipids (SL). The SL were obtained by incubating a 1:4:4 mole ratio of triolein, caproic acid, and butyric acid, respectively, with 10% of lipase (w/w of total substrates) in 1.5 mL hexane at 55°C for 24 h. Of nine commercially avaialble lipases screened, IM60, which contains the lipase from Rhizomucor miehei, was the most effective and produced 13 mol% unreacted triolein, 49% disubstituted, and 38% monosubstituted triacylglycerols that contained short-chain fatty acids. The products were analyzed by reverse-phase high performance liquid chromatography with an evaporative light-scattering detector. Reaction parameters studied included time course, temperature, enzyme load, and substrate mole ratio. The yields obtained demonstrate that a structured lipid with long-chain and short-chain fatty acids can be synthesized by using IM60 lipase in organic medium.     

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.     

Enzymatic interesterification of palm stearin and palm kernel olein

Palm stearin (POs) and palm kernel olein (PKOo) blends were modified by enzymatic interesterification (IE) to achieve the physical properties of margarine fats. POs and PKOo are both products of the palm oil industry that presently have limited use. Rhizomucor miehei lipase (Lipozyme IM 60) was used to catalyze the interesterification of oil blends at 60°C. The progress of interesterification was monitored by following changes in triacylglyceride composition. At 60°C interesterification can be completed in 5 h. Degrees of hydrolysis obtained through IE for all blends were decreased from 2.9 to 2.0 by use of dry molecular sieves. The solid fat contents of POs/PKOo 30:70 and 70:30 interesterified blends were 9.6 and 18.1 at 20°C, and 0 and 4.1 at 35°C, respectively. The slip melting point (SMP) of POs/PKOo 30:70 was 40.0°C before interesterification and 29.9°C after IE. For POs/PKOs 70:30, SMP was 47.7 before and 37.5°C after IE. These thermal characteristics of interesterified POs/PKOo blend ratios from 30:70 to 70:30 were comparable to those of commercial margarines. Results showed that IE was effective in producing solid fats with less than 0.5% trans.     

Limit of the solid fat content modification of butter

Three ways have been undertaken to modify solid fat content of butter oil: (i) interesterification, (ii) adjunction of high-melting glycerides and (iii) joint effect of adjunction of high-melting glycerides and interesterification. A solvent-free interesterification, carried out with 1,3-specific lipase fromMucor miehei, resulted in an increase of the solid fat content (SFC) by about 114% after 48 h of interesterification. The changes in triglyceride composition induced by this method were followed by quantitative determination of triglycerides of different equivalent carbon number (ECN) and different theoretical carbon number. The major changes in the triglyceride composition occurred mainly in the concentration of three groups of triglycerides with the same ECN (ECN=38). Adding high-melting glycerides trimyristin (MMM) and tripalmitin (PPP) led to an increase of the SFC measured at 20°C as these proportions increased in the mixture. The joint effect of the addition of MMM or PPP and interesterification was quite significant, mainly for triglycerides that included myristic and palmitic acids. As far as the increase of SFC is concerned, the effect of interesterification decreases when both substrate amounts increase.     

Enzymatic synthesis of structured lipids: Transesterification of triolein and caprylic acid ethyl ester

Structured lipids were successfully synthesized by lipase-catalyzed transesterification (ester interchange) of caprylic acid ethyl ester and triolein. The transesterification reaction was carried out in organic solvent as reaction media. Eight commercially-available lipases (10% w/w substrates) were screened for their ability to synthesize structured lipid by incubating with 100 mg triolein and 78.0 mg caprylic acid ethyl ester in 3 mL hexane at 45°C for 24 h. The products were analyzed by reverse-phase high-performance liquid chromatography with evaporative light-scattering detector. Immobilized lipase IM60 fromRhizomucor miehei converted most triolein into structured lipids (41.7% dicapryloolein, 46.0% monocapryloolein, and 12.3% unreacted triolein). However, lipase SP435 fromCandida antarctica had a higher activity at higher temperature. The reaction catalyzed by lipase SP435 yielded 62.0% dicapryloolein, 33.5% monocapryloolein, and 4.5% unreacted triolein at 55°C. Time course, incubation media, added water, and substrate concentration were also investigated in this study. The results suggest that lipase-catalyzed transesterification of long-chain triglycerides and medium-chain fatty acid ethyl ester is feasible to synthesize structured lipids.     

Comparison of acyl donors for lipase-catalyzed production of 1,3-dicapryloyl-2-eicosapentaenoylglycerol

Synthesis of 1,3-dicapryloyl-2-eicosapentaenoylglycerol (CEC) catalyzed by Lipozyme IM (immobilized Rhizomucor miehei lipase) was performed by interesterification of trieicosapentaenoylglycerol (EEE) with caprylic acid (CA) (acidolysis) and EEE with ethyl caprylate (EtC) (interesterification). Both methods involved two steps: (i) transesterification at an optimized water content and temperature for the high yield conversion of the substrate to CEC, 1-capryloyl-2-eicosapentaenoylglycerol (CEOH) and 2-eicosapentaenoylglycerol (OHEOH), and (ii) reesterification of CEOH and OHEOH to CEC by water removal under reduced pressure. Interesterification had clear advantages over acidolysis. The reaction rates for interesterification were higher and the reaction times shorter. The final yield of CEC by interesterification was higher, and the extent of acyl migration, indicated by the tricapryloylglycerol content, was lower. The disadvantage of the higher price of EtC used for interesterification (approximately 10 times higher than the price of CA) was overcome by synthesizing it directly in the same reaction vessel prior to the interesterification step. EtC was rapidly synthesized by esterification of CA with ethanol in high yield (92% obtained in 2.5 h). The amount of water added to the reaction mixture and the reaction temperature influenced the yields of CEC, CEOH, and OHEOH in the transesterification step for both interesterification and acidolysis methods. The regioisomeric purity of CEC was 100% for both methods at temperatures of 40°C or less. The highest yield of CEC (81%) was obtained for the interesterification of EEE with EtC, formed directly in the same reaction vessel, at a CA/EEE molar ratio of 20∶1 and 30°C.     

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.     

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.     

Enzymatic incorporation of docosahexaenoic acid into borage oil

Lipase-catalyzed acidolysis of acylglycerols of borage (Borago officinalis L.) oil with a docosahexaenoic acid (DHA) concentrate, prepared from algal oil, in organic solvents was studied. Seven lipases were used as biocatalysts for the acidolysis reaction. Novozyme 435 from Candida antarctica, as compared to lipases from Mucor miehei and Pseudomonas sp., showed the highest degree of DHA incorporation into borage oil. Other lipases tested, such as those from Aspergillus niger, C. rugosa, Thermomyces lanuginousus and Achromobacter lunatus, were rather ineffective in the incorporation of DHA into borage oil. Effects of variation of reaction parameters, namely, enzyme load, temperature, time course, and type of solvent, were monitored for C. antarctica as the biocatalyst of choice. Incorporation of DHA increased with increasing amount of enzyme, reaching 27.4% at an enzyme concentration of 150 lipase activity units. As incubation time progressed, DHA incorporation also increased. After a reaction time of 24 h, the contents of total n-6 and n-3 polyunsaturated fatty acids in acylglycerols were 44.0 and 27.6%, respectively. The highest degree of DHA incorporation was achieved when hexane was used as the reaction medium. The positional distribution of DHA in modified borage oil was determined using pancreatic lipase hydrolysis. Results showed that DHA was randomly distributed over the sn-1, sn-2, and sn-3 positions of the triacylglycerol. Thus, preparation of modified borage oil acylglycerols containing both DHA (22:6n-3; 27.4%) and γ-linolenic acid (18:3n-6; 17.0%) was successfully achieved and products so obtained may have beneficial effects beyond simple physical mixtures of the two oils. The final oil had a ratio of n-3 to n-6 of 0.42–0.62 which is nutritionally more suitable than the original unaltered borage oil.     

Trans FA and solid fat contents of margarines marketed in Turkey

Trans FA (TFA), solid fat contents (SFC), and slip melting points of 12 different tub and stick margarines marketed in Turkey were examined in this study. No trans isomers were found in four margarines, which suggests they were formulated from interesterified or blended fats and oils. The products with no TFA generally had more short-chain saturated FA, which suggests coconut oil-based oil components. TFA content of the other 10 products varied from 7.7 to 37.8%. Compared to the products formulated in North America, Turkish margarines contain more TFA and have higher SFC.     

TAG composition and solid fat content of palm oil, sunflower oil, and palm kernel olein belends before and after chemical interesterification

Modification of the characteristics of palm oil (PO), sunflower oil, and plam kernel olein (PKOo) according to conventional three-component mixture designs was undertaken by a combination of blending and chemical interesterification (CIE) techniques. TAG composition and solid fat content (SFC) profile of the starting blends were analyzed and compared with those of the interesterified blends. Upon CIE, extensive rearrangement of FA among TAG was evident. Concentrations of several TAG were increased, some were decreased, and several new TAG were formed. The resulting changes in TAG profile were reflected in the SFC of the blends. The SFC values of the chemically interesterified blends, except binary blends of PO/PKOo, revealed that they were softer than their respective starting blends. SFC data also indicated that eutectic interaction occurred between PO and PKOo in the starting blends and that this interaction was diminished after CIE.     

Enzymatic enrichment of conjugated linoleic acid isomers and incorporation into triglycerides

A method was developed for the enrichment of either the cis9,trans11 or the trans10,cis12 isomer of conjugated linoleic acid (CLA) from a synthetic CLA mixture consisting predominantly of these isomers in equal amounts. Lipases were screened for their ability to selectively esterify one isomer at a significantly greater rate than the other isomer. An immobilized lipase from Rhizomucor miehei was nonselective, but a lipase from Geotrichum candidum esterified the cis9,trans11 isomer more rapidly than the trans10,cis12 isomer. This selectivity was exploited at the kilogram scale to prepare an ester fraction with a content of 91% cis9,trans11 CLA and an unreacted free fatty acid fraction consisting of 82% trans10,cis12 CLA, based on total CLA content. The components of the reaction mixture were separated by molecular distillation. Each enriched fraction was then incorporated into palm oil triglycerides by interesterification with the non-selective lipase from R. miehei. Two triglyceride fats resulted, which were enriched in either cis9,trans11 CLA (26.5% cis9,trans11 and 1.7% trans10,cis12) or trans10,cis12 CLA (3.5% cis9,trans11 and 22.9% trans10,cis12).     

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.     

Modelling lipase‐catalysed transesterification of fats containing n‐3 fatty acids monitored by their solid fat content

Transesterification of fat blends rich in n‐3 polyunsaturated fatty acids (n‐3 PUFA), catalysed by a commercial immobilised thermostable lipase from Thermomyces lanuginosa, was carried out batch‐wise. Experiments were performed, following central composite rotatable designs (CCRDs) as a function of reaction time, temperature and media formulation. Mixtures of palm stearin, palm kernel oil and a commercial concentrate of triacylglycerols rich in n‐3 PUFA (“EPAX 2050TG” in CCRD‐1 and “EPAX 4510TG” in CCRD‐2) were used. The time‐course of transesterification was indirectly followed by the solid fat content (SFC) values of the blend at 10 °C, 20 °C, 30 °C and 35 °C. A decrease in all SFC values of the blends at 10 °C, 20 °C, 30 °C and 35°C was observed upon transesterification. The SFC_{10 °C} and SFC_{20 °C} of transesterified blends varied between 18 and 48 and SFC_{35 °C} between 6 and 24. These values fulfil the technological requirements for the production of margarines. Under our conditions, lipid oxidation may be neglected. However, the accumulation up to 8.3% free fatty acids in reaction media is a problem to overcome. The development of response surface models, describing both the final SFC value and the SFC decrease, will allow predicting results for novel proportions of fats and oils and/or a novel combination time‐temperature.     

Alteration of steryl ester content and positional distribution of fatty acids in triacylglycerols by chemical and enzymatic interesterification of plant oils

Steryl ester content of refined and interesterified corn, soybean, and rapeseed oils has been measured via clean-up on a short silica gel column, followed by high performance liquid chromatography with evaporative light-scattering mass detector. Chemical interesterification, catalyzed by sodium methoxide, led to random positional distribution of fatty acids in triacylglycerols and some increase in the steryl ester content of all three oils. Enzymatic interesterification, catalyzed by the immobilized lipase from Rhizomucor miehei (Lipozyme), resulted in a distinct reduction in steryl ester content, but essentially no alteration in positional distribution of fatty acids in triacylglycerols occurred. Formation of steryl esters during chemical and enzymatic interesterification was also examined by radioactive tracer technique with [4-¹⁴C]β-sitosterol added as marker to refined rapeseed oil and measurement of the radioactive steryl esters formed. Chemical interesterification of rapeseed oil resulted in moderate formation (10% of total radioactivity) of radioactive β-sitosteryl esters. Enzymatic interesterification of the oil, catalyzed by Lipozyme, led to little formation of radioactive β-sitosteryl esters, whereas with the lipase from Candida cylindracea high proportions (>90% of total radioactivity) of ¹⁴C-labeled β-sitosteryl esters were formed. Part of doctoral thesis of Roseli Ap. Ferrari to be submitted to Faculdade de Engenharia de Alimentos, Universidade de Campinas, Campinas, Brazil.     

Enzymatic incorporation of stearic acid into a blend of palm olein and palm kernel oil: Optimization by response surface methodology

Response surface methodology was used to model the incorporation of stearic acid into a blend of palm olein and palm kernel oil in hexane using the sn-1,3-regiospecific lipase Lipozyme RM IM. The factors investigated were incubation time, temperature, and substrate molar ratio. A second-order model with interaction was used to fit the experimental data. The coefficients of determination, R ² and Q ², were 0.96 and 0.90, respectively. The adjusted R ² was 0.95. The regression probability was less than 0.001, and the model showed no lack of fit. Also, a linear relationship was observed between the predicted and observed values. All parameters studied had positive effects on incorporation of stearic acid, with substrate molar ratio having the greatest effect. The interaction terms of substrate molar ratio with temperature and time also had positive effects on incorporation, whereas the effect of the squared term of substrate molar ratio was negative. The quadratic terms of temperature and time, as well as their interaction term, had no significant effect on incorporation at α_0.05. Model verification was done by performing a chi-square test, which showed that there was no significant difference between predicted values and a new set of observed responses.     

FA selectivity of lipases in acyl-transfer reactions with acetate esters of polyols in organic media

FA reaction selectivity of Burkholderia cepacia, Rhizomucor miehei, and Candida antarctica fraction B lipases was compared between acyl-transfer and esterification reactions. Multicompetitive reaction mixtures containing a series of n-chain FA (a C₄–C₁₈ series; and a C_18∶x series, where X=0-3 double bonds) and a single acetate ester co-substrate [triacetin, 1,2-propanediol (1,2-PD)diacetate, and 1,3-PD diacetate] were studied in tert-butyl methyl ether at an a _w of 0.69. For B. cepacia lipase, FA optima for C₈, C₁₆, and C_18∶2 were observed in all reactions with 1.0- to 5.9-fold differences in FA selectivity. For R. miehei lipase, an optimum for C₈ FA was observed in all reactions with 1.2- to 6.7-fold differences in FA selectivity. For C. antarctica lipase, FA optima for C₈/C₁₀ were observed in all reactions with 1.0- to 2.8-fold differences in FA selectivity. FA selectivities were broadly modulated upon changing from free polyol to acetate ester co-substrates for B. cepacia and R miehei lipases, whereas FA selectivity modulations were more specific upon this change in reaction configuration for C. antarctica B lipase. For all lipases, reactivity toward unsaturated C_18∶x FA was enhanced in acyl-transfer relative to esterification reactions with these polyol co-substrates.     

High Yield of Wax Ester Synthesized from Cetyl Alcohol and Octanoic Acid by Lipozyme RMIM and Novozym 435

Wax esters are long-chain esters that have been widely applied in premium lubricants, parting agents, antifoaming agents and cosmetics. In this study, the biocatalytic preparation of a specific wax ester, cetyl octanoate, is performed in n-hexane using two commercial immobilized lipases, i.e., Lipozyme^® RMIM (Rhizomucor miehei) and Novozym^® 435 (Candida antarctica). Response surface methodology (RSM) and 5-level-4-factor central composite rotatable design (CCRD) are employed to evaluate the effects of reaction time (1–5 h), reaction temperature (45–65 °C), substrate molar ratio (1–3:1), and enzyme amount (10%–50%) on the yield of cetyl octanoate. Using RSM to optimize the reaction, the maximum yields reached 94% and 98% using Lipozyme^® RMIM and Novozym^® 435, respectively. The optimum conditions for synthesis of cetyl octanoate by both lipases are established and compared. Novozym^® 435 proves to be a more efficient biocatalyst than Lipozyme^® RMIM.     

Parameters affecting diacylglycerol formation during the production of specific-structured lipids by lipase-catalyzed interesterification

Diacylglycerols (DAG) are important intermediates in lipase-catalyzed interesterification, but a high DAG concentration in the reaction mixture results in a high DAG content in the final product. We have previously shown that a high DAG concentration in the reaction mixture increases the degree of acyl migration, thus adding to the formation of by-products. In the present study we examined the influence of water content, reaction temperature, enzyme load, substrate molar ratio (oil/capric acid), and reaction time on the formation of DAG in batch reactors. We used response surface methodology (RSM) to minimize the numbers of experiments. The DAG content of the product was dependent on all parameters examined except reaction time. DAG formation increased with increasing water content, enzyme load, reaction temperature, and substrate ratio. The content of sn-1,3-DAG was higher than that of sn-1,2-DAG under all conditions tested, and the ratio between the contents of the former compounds and the latter increased with increasing temperature and reaction time. The water content, enzyme load, and substrate ratio had no significant effect on this ratio. The DAG content was positively correlated with both the incorporation of acyl donors and the degree of acyl migration.