Synthesis of 2-monoacylglycerols (2-MAG) by enzymatic alcoholysis of fish oils using different reactor types

Biochemical Engineering Journal 44 (2009) 271–279

Contents lists available at ScienceDirect

Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej

Synthesis of 2-monoacylglycerols (2-MAG) by enzymatic alcoholysis of fish oils using different reactor types ˜ Luis Esteban, María del Mar Munío, Alfonso Robles ∗ , Estrella Hita, María J. Jiménez, Pedro A. González, Belén Camacho, Emilio Molina Departamento de Ingeniería Química, Universidad de Almería, 04120 Almería, Spain

a r t i c l e

i n f o

Article history: Received 30 July 2008 Received in revised form 8 January 2009 Accepted 13 January 2009 Keywords: Bioreactors Enzyme Packed bed reactor (PBR) Stirred tank reactor (STR) 2-Monoacylglycerol (2-MAG) Alcoholysis Extraction

a b s t r a c t This paper studies the synthesis of 2-monoacylglycerols (2-MAGs) by alcoholysis of cod liver oil and tuna oil, catalyzed by lipases, in stirred tank (STR) and packed bed (PBR) type reactors, operating in discontinuous and continuous modes. Firstly, several lipases were tested (DF from Rhizopus oryzae, Palatase 20000L from Mucor miehei and Novozym 435 from Candida antarctica), and although the highest 2-MAG yield was obtained with lipase DF, Novozym 435 was selected due to its greater stability. 2-MAGs were then produced with this lipase in the above-mentioned reactors. Using Novozym 435 2-MAGs can be obtained by ethanolysis of TAGs, and the major operational variable is the treatment intensity (TI, lipase amount × reaction time/oil amount). The highest 2-MAG yields (63–65%) were obtained in the STR operated in discontinuous mode. For TI of over approximately 1 g lipase × h/g oil, the 2-MAGs were degraded to glycerol. This system was scaled up to 100 times the initial volume, achieving the same yield at the same TI (1 g lipase × h/g oil). Operating in continuous mode, the 2-MAG yields obtained (53–54%) were roughly 15% less in both reactors at this TI. 2-MAGs in the final reaction mixture were separated from the ethyl esters by solvent extraction using solvents of low toxicity (ethanol and hexane); 2-MAG recovery yield (g 2-MAGs extracted/100 g of 2-MAGs in the reaction mixture) and 2-MAG purity in the target product (g 2-MAGs/100 g of total product) were approximately 90%. The fatty acid profile of the 2-MAGs produced was similar to the fatty acid profile in position 2 of the original oils. The two major n-3 polyunsaturated fatty acids (n-3 PUFAs) of 2-MAGs produced were eicosapentaenoic acid (EPA) and docosohexaenoic acid (DHA), and their combined contents were about 40% and 45% for cod liver oil and tuna oil, respectively. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The beneficial effects of n-3 polyunsaturated fatty acids (n-3 PUFAs) on human health have been widely recognized. Particular attention has been paid to eicosapentaenoic acid (EPA, 20:5n3), mainly for preventing arteriosclerosis and thrombosis [1], and docosohexaenoic acid (DHA, 22:6n-3) for brain development [2]. The structure and fatty acids composition of triacylglycerol (TAG) affect their absorption and the distribution of fatty acids in the organism [3]. For nutritional purposes, there is increasing interest in the production of structured triacylglycerols (STAG) containing medium chain fatty acids (M) located at positions 1 and 3 of the glycerol backbone and functional long chain polyunsaturated fatty acids (L) located at position 2 (MLM). These STAG are claimed to benefit the immune function and to help improve lipid clearance from the bloodstream [4].

∗ Corresponding author. Tel.: +34 950015065; fax: +34 950015484. E-mail address: [email protected] (A. Robles). 1369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2009.01.004

The simplest and most direct route for the synthesis of MLM type STAG is by acidolysis between a long chain fatty acid TAG and medium chain fatty acids, catalyzed by a 1,3 specific lipase [5–10]. Nevertheless, acyl-migration is a major problem in the synthesis of STAG in batch reactors, causing a decrease in the yield of the target STAG. To prevent this problem, one of the alternatives is a process in two steps [11–14]. First, 2-monoacylglycerol (2-MAGs) can be produced from high grade functional fatty acid oils by enzymatic alcoholysis, and then medium chain fatty acids are added in the extreme positions of 2-MAGs by enzymatic esterification using 1,3 specific lipases. The bottleneck in this process is the alcoholysis of the TAGs to obtain 2-MAGs and the subsequent purification of the 2-MAGs produced. Major problems concerning the alcoholysis reaction are: (i) the deactivation of lipase, caused by the ethanol added or the glycerol formed by hydrolysis of 2-MAG, and (ii) the acyl-migration, caused by the lipase immobilization support, solvents, high temperatures, etc. [15]. To prevent lipase deactivation, sequential addition of the alcohol and a reduction in the reaction time to avoid the formation of glycerol has been used [16,17]. The major operational strate-

272

L. Esteban et al. / Biochemical Engineering Journal 44 (2009) 271–279

Nomenclature DAG DHA e0 EE EPA EtOh FFA GC [i] k1ap

diacylglycerol docosohexaenoic acid, 22:6n-3 lipase concentration (g L−1 ) ethyl ester eicosapentaenoic acid, 20:5n-3 ethanol free fatty acid gas chromatography concentration of specie i (g L−1 or mol L−1 ) apparent kinetic constant for the formation of 2MAG (g oil h−1 g lipase−1 ) k2ap apparent kinetic constant for the degradation of 2MAG (g oil h−1 g lipase−1 ) L long chain polyunsaturated fatty acid M medium chain fatty acid MAG monoacylglycerol mL lipase amount (g) initial oil amount (g) mTAG0 MLM structured triacylglycerol containing medium chain fatty acid at positions 1 and 3 of the glycerol backbone and functional long chain polyunsaturated fatty acids at position 2 PBR packed bed reactor PUFA polyunsaturated fatty acid q reaction mixture flow rate (mL h−1 ) STAG structured triacylglycerol STR stirred tank reactor t reaction time (h) TAG triacylglycerol [TG]0 initial concentration of TAGs (g L−1 or mol L−1 ) TI treatment intensity, lipase amount (g) × reaction time (h)/oil amount (g) in discontinuous mode, and lipase amount (g)/(reaction mixture flow rate (mL h−1 ) × oil concentration (g mL−1 ) in continuous mode, Eq. (2) TLC thin-layer chromatography V reaction mixture volume, mL, and volume of solvent free mixture in Fig. 3 1(3)-MAG 1(3) monoacylglycerol 2-MAG 2-monoacylglycerol

gies used to avoid acyl-migration are: (i) immobilization of lipases on support such as Accurel EP-100 or Celite (washed with acids) [15], (ii) utilization of solvents that restrict acyl-migration, such as ethers or ketones [11] and (iii) carrying out the reactions at relatively low temperatures [15]. Among the lipases used, good results were obtained with Novozym 435, which behaves as 1,3 specific when an excess of ethanol is used [13]. In a previous work [18] lipase D, immobilized on Accurel MP1000 (equivalent to EP-100), was selected for synthesizing 2-MAGs by alcoholysis of fish oils with dry ethanol, using acetone as solvent. Although high 2-MAG yields were attained (about 70%) and no acylmigration was detected, later attempts to scale up the method to a large packed bed reactor (PBR) failed, mainly due to the deactivation of lipase. The aim of this work was to provide a robust and scaleable alternative for the synthesis of 2-MAGs when using different reactors and operational modes. To this end several lipases were tested, the stability and reutilization of lipases being the key operational parameters along with the recovery and purification of the 2-MAGs produced.

Table 1 Fatty acid composition of cod liver and tuna oils and fatty acid composition in position 2 (% of total fatty acids weight) of TAGs of these oils determined by the Shimada et al. (2003) method [24]. Fatty acids

14:0 16:0 16:1n-7 16:2n-4 18:0 18:1n-9 18:1n-7 18:2n-6 18:4n-3 20:1n-9 20:4n-6 20:5n-3 22:1n-9 22:5n-3 22:6n-3 Others

Cod liver oil

Tuna oil

Oil

Position 2

Oil

Position 2

3.1 10.0 7.5 0.5 2.3 17.3 5.4 1.3 1.6 12.9 0.4 9.5 9.5 1.3 11.1 6.3

6.9 16.1 7.5 1.0 n.d. 9.6 1.2 1.5 2.3 6.3 n.d. 9.0 5.3 2.3 30.1 1.2

4.6 19.5 6.8 1.2 5.6 14.6 2.8 1.8 0.9 3.1 2.1 7.5 1.9 1.4 22.1 4.1

6.9 19.3 6.9 1.4 1.6 9.3 1.7 1.8 1.5 1.6 2.1 6.8 n.d. 2.3 35.9 1.5

2. Materials and methods 2.1. Oils, lipases and chemicals Cod liver oil (provided by Acofarma, Barcelona, Spain) and tuna oil (donated by Brudy Technologies, S.L., Barcelona, Spain) were used as substrates for the enzymatic reactions. Table 1 shows the fatty acid composition of these oils. Verification by thin-layer chromatography (TLC) showed that neither of them contained partial acylglycerols. The lipases used were Novozym 435 (N-435) from Candida antarctica, Palatase 20000L from Mucor miehei (both donated by Novozymes, Denmark) and lipase DF from Rhizopus oryzae (Amano Pharmaceutical Co., Nagoya, Japan). Novozym 435 (N-435) was provided immobilized, whereas lipases DF and Palatase were provided non immobilized, the former as powder and the latter as liquid. Analytical grade ethanol (96%, v/v), absolute ethanol (99.5%, v/v) and absolute dry ethanol (99.8%, v/v) were used as substrates of alcoholysis and analytical grade acetone (Panreac S.A., Barcelona, Spain) was used as solvent. All other chemicals (analytical grade or better) were also obtained from commercial sources.

2.2. Immobilization of lipases Lipases DF and Palatase were immobilized on Accurel MP-1000 (Akzo Nobel Faser, Obernburg, Germany) following the procedure described by Soumanou et al. [12], modified by Hita et al. [10]. For the immobilization of Palatase the following procedure was applied: between 1 and 15 g of liquid lipase was dissolved in 25 mL of phosphate buffer (pH 6.0, 20 mM); the solution was added at room temperature to a mixture of 1.5 g support and 5 mL ethanol; after shaking for 8 h at 150 rpm (Inkubator 1000, Unimax 1010 Heidolph, Klein, Germany), 5 mL acetone at −24 ◦ C was added. The immobilized lipase was recovered by filtration, washed three times with phosphate buffer (pH 6.0, 20 mM) at 0 ◦ C, dried under vacuum for 48 h and stored at 5 ◦ C until use. The best result was obtained with a lipase/support ratio 15:1.5 (w/w) for Palatase. The lipase DF was immobilized following the same procedure and the best result was obtained with a lipase/support ratio 1:1.5 (w/w).

L. Esteban et al. / Biochemical Engineering Journal 44 (2009) 271–279

273

bath. This reaction mixture was pumped upward into the column by a peristaltic pump (Watson-Marlow 520S) at flow rates between 13.2 and 15.1 mL h−1 . The column also had a jacket to control the reaction temperature and the column and substrate mixture temperature was maintained constant at 35 ◦ C by the circulation of water from the water bath. A three-way valve was placed at the exit of the lipase bed that allowed samples to be taken and operation in two modes: by recirculating to the substrate reservoir the mixture coming out from the bed (discontinuous operation), or directing the product mixture from the bed to an additional reservoir (continuous operation). The pipelines of 2 mm of internal diameter were made of Teflon, except for the one in contact with the peristaltic pump, which was made of tigon (i.d. 3.2 mm) (Saint-Gobain Performance Plastics, Cleveland, Ohio, USA) or viton (i.d. 2 mm) (Masterflex, Illinois, USA). Tigon or viton were used depending on the substances, since viton, for example, is not resistant to acetone.

Fig. 1. Immobilized lipase packed bed reactor (PBR). (1) Magnetic stirrer, (2) substrate reservoir, (3) thermostated water bath, (4) peristaltic pump, (5) bed of immobilized enzyme, (6) water jacket, (7) three-way valve, (8) product reservoir, (9) recirculation current (discontinuous operation), (10) product current (continuous operation), (11 and 12) cooling/heating water, (13) sampling (continuous operation) and (14) sampling (discontinuous operation).

2.3. Alcoholysis reaction In the alcoholysis reaction the oil (TAGs) reacts with ethanol in the presence of solvent and a 1,3 specific lipase, to produce 2-MAGs, diacylglycerols (DAGs) and ethyl esters (EEs). In the final reaction mixture there are also ethanol and TAGs that have not reacted. sn-1,3 specific lipase

oil (TAGs) + ethanol

−→

solvent

MAG + DAG + EE

2.3.1. STR operating in discontinuous mode A typical reaction mixture consisted of 500 mg oil, 500 mg ethanol, 3 mL acetone and 240 mg immobilized lipase. This mixture was placed in a 50-mL Erlenmeyer flask with silicone capped stoppers under inert atmosphere. The mixture was incubated at 37 ◦ C and agitated in an orbital shaker at 200 rpm (Inkubator 1000, Unimax 1010 Heidolph, Klein, Germany). However, when lipase N435 was used the reaction mixture, at 35 ◦ C, containing 750 mg oil, 3 g dry ethanol and 375 mg lipase, was agitated at 300 rpm. The reaction was stopped at different times by removing the lipase by filtration. The volume was then adjusted to 25 mL by addition of hexane. This final mixture was stored under inert atmosphere at −24 ◦ C until analysis. All reactions and their corresponding analyses were carried out in triplicate. Standard deviations were always below 8%. The alcoholysis reaction with lipase N-435 was scaled up 100 times (75 g cod liver oil, 300 g dry ethanol and 3 g lipase N-435) maintaining the substrate ratio constant. This reaction was carried out in a 1 L jacket reactor, thermostated at 35 ◦ C, with mechanical agitation, protected from light and under argon atmosphere. 2.3.2. PBR in discontinuous and continuous operational mode Fig. 1 shows a scheme of the reaction system. The immobilized lipase (2 or 6 g) was packed into a glass column (6 mm internal diameter × 235 mm length), and covered with aluminium foil to prevent photo-induced oxidation. The column had two mobile perforated disks, which allowed the volume of the column to be adjusted to that of the packed lipase. The substrate mixture (5–60 g cod liver oil and ethanol with an oil/absolute ethanol molar ratio 1:79) was kept in a reservoir submerged in a thermostated water

2.3.3. STR operating in continuous mode Fig. 2 shows a scheme of this reaction system. Feeding system and pipelines were the same as in the packed bed reactor. The stirred tank reactor (STR) (670 mL of utilizable volume) was immersed in a thermostatic bath. For working in continuous mode the reactor contains a spillway device in the upper part, equipped with a double grid to prevent the withdrawal of lipase from the reaction mixture (Fig. 2). Before carrying out each experiment the reactor 5 (Fig. 2) was completely filled of substrates (ethanol/oil 4:1, w/w) and the experiment began by adding the lipase and by switching on the peristaltic pump 3 that fed the reaction mixture. This pump allowed the reaction mixture to enter continuously, while the product mixture leaving the reactor was conducted to a product reservoir. In a typical reaction at 35 ◦ C the reactants and lipase were put in the proportion: 75 g of cod liver oil, 300 g dry ethanol (oil/dry ethanol molar ratio 1:79), 6 g lipase Novozym 435. 2.4. Analysis of reaction products Identification of the reaction products (MAGs, DAGs, TAGs and ethyl esters) was carried out by thin-layer chromatography. The fatty acids profile in the acylglycerols was identified and determined quantitatively by gas chromatography (GC). The identification of acylglycerols by TLC was carried out on silica-gel plates (Precoated TLC plates, SIL G-25; Macherey-Nagel, Sigma–Aldrich) activated by heating at 105 ◦ C for 30 min. The samples were spotted directly on the plate by adding 0.2 mL of product mixture. The plate was then developed in chloroform/acetone/methanol (95:4.5:0.5, v/v/v). Spots of each lipid were visualized by spraying the plate with iodine vapor in a nitrogen stream [19]. Fractions corresponding to each lipid type were scraped from the

Fig. 2. Dispersion reactor (STR) operating in continuous mode. (1) Magnetic stirrer, (2) substrate reservoir, (3) peristaltic pump, (4) stirrer vane, (5) reaction tank, (6) spillway with grid for solid retention, (7) thermostated water bath and (8) product reservoir.

274

L. Esteban et al. / Biochemical Engineering Journal 44 (2009) 271–279

Fig. 3. Experimental procedure for the recovery of 2-MAGs from the final alcoholysis mixture by solvent extraction [18].

plates and methylated according to the original method of Lepage and Roy [20], modified by Rodríguez-Ruiz et al. [21]. Methylation and methyl ester analysis by GC have been described elsewhere [22]. Nonadecanoic acid (19:0) (Sigma–Aldrich) was used as an internal standard for quantitative determination of fatty acids. To detect the presence of 1(3)-MAG isomers of the desired product (2-MAG), which can be eventually formed by acyl-migration, a modified TLC was carried out; in this case the plate was activated after immersion in a hydroethanolic solution (50%, v/v) of boric acid (1.2% in weight) [23]. This method allows us to separate, identify and quantify by posterior GC the 2-MAG and 1(3)-MAG.

The 2-MAG yields were calculated determining the weight percentage of lipidic species in the product as equivalent fatty acids. Thus, when 1 mol of initial TAGs is completely transformed in 2MAGs (100% yield), the molar percentage of this species in the final reaction mixture is 33.3% (1 mol of 2-MAGs and 2 mol of ethyl esters). The 2-MAG yield can therefore be calculated by multiplying the 2-MAG percentage in the final reaction mixture by three. These percentages would equally be expressed in weight if a fatty acid type, with a mean molecular weight, is considered to transform moles in weight. This assumption should be made because ethyl esters and TAGs cannot be separated using the usual mobile phase in TLC.

Table 2 Fatty acid composition (% (w/w) of total fatty acids) of 2-MAGs and 2-MAG yield (% (w/w) on initial TAGs) obtained by ethanolysis of cod liver and tuna oils, catalyzed by lipases DF, Palatase and N-435. Fatty acids

DF non immobilizeda

DF (MP-1000)b

Palatase (MP-1000)b

N-435c

N-435d

14:0 16:0 16:1n-7 16:2n-4 18:0 18:1n-9 18:1n-7 18:2n-6 18:4n-3 20:1n-9 20:4n-6 20:5n-3 22:1n-9 22:5n-3 22:6n-3

7.9 24.9 8.5 1.7 2.7 12.9 2.7 2.5 n.d. n.d. n.d. 6.7 n.d. n.d. 29.4

6.3 20.2 6.3 1.6 1.7 8.4 1.9 1.7 1.6 1.7 2.9 8.4 n.d. 2.0 35.5

6.6 19.2 6.6 1.6 1.7 9.5 1.8 2.0 1.5 1.9 3.3 8.3 n.d. 2.2 33.7

6.7 16.6 7.8 0.8 n.d. 9.6 1.1 1.4 2.4 5.6 0.4 9.0 5.3 2.1 29.3

9.9 26.1 9.6 n.d. n.d. 10.9 n.d. 0.8 n.d. n.d. n.d. 7.7 n.d. n.d. 35.0

2-MAG yield (w/w)

30.3

68.2

48.0

63.9

48.9

n.d.: undetected. a Operational conditions: Similar conditions to footnote b but with 100 mg of lipase DF non-immobilized. b Operational conditions: 500 mg tuna oil, 500 mg dry ethanol, 3 mL acetone, 240 mg lipase immobilized on Accurel MP-1000 (lipase/support ratio 1:1.5 for lipase DF and 15:1.5 (w/w) for Palatase), 37 ◦ C, 24 h and 200 rpm. c Operational conditions: 750 mg cod liver oil, 3 g dry ethanol, 375 mg N-435, 35 ◦ C, 2 h and 300 rpm. d Operational conditions: Similar conditions to footnote c but with 750 mg tuna oil instead of cod liver oil.

L. Esteban et al. / Biochemical Engineering Journal 44 (2009) 271–279

2.5. Regiospecific analysis of triacylglycerols The fatty acid composition at position 2 of TAGs was analyzed by the method proposed by Shimada et al. [24] using the lipase Novozym 435. Table 1 shows the fatty acid composition at position 2 of cod liver and tuna oils. These profiles are reasonably consistent with the results obtained in our laboratory carrying out alcoholysis reactions of cod liver and tuna oils with the 1,3 specific lipases D (R. oryzae) and R. delemar. In addition, these profiles are also consistent with the ones referenced when these oils were analyzed using NMR and Grignard reagents [25]. 2.6. Separation-purification of 2-MAGs The alcoholysis final mixture contains mainly 2-MAGs and EEs, but also minor amounts of DAGs and residual TAGs, and all these products were mixed with ethanol and acetone (when this solvent was used). Purification of 2-MAGs from the alcoholysis reaction was performed by solvent extraction, following a two-step procedure ˜ et al. [18] and summarized in Fig. 3. In a first described in Munío step, acetone and residual ethanol were eliminated from the reaction mixture in a rotary evaporator and the resulting 2-MAG–ethyl ester mixture was dissolved in an ethanol/water mixture (90:10, v/v) in the proportion 1:9 (v/v); for extracting ethyl esters this mixture was extracted three times with an equal volume of hexane. The hexanic phases were mixed and its volume adjusted to a fixed value. Finally this solution was stored at −24 ◦ C until analysis. A second purification step was then applied, adding ethanol/water 90:10 (v/v) to eliminate the glycerol eventually formed. In this work several trials were carried out both with and without this second step. The 2-MAG fraction was used to synthesize structured triacylglycerols. As foreseen (results not shown), no significant differences were observed between the results obtained with the 2-MAGs subjected to this second purification step and those which were not. We believe that the formation of glycerol is minimal under our experimental conditions, and, therefore, the second separation step is not necessary. 3. Results and discussion 3.1. Selection of lipase Lipases DF, Palatase and Novozym 435 were tested. Lipase DF is similar to lipase D, which we had already used for producing 2-MAGs [18], and it was provided to replace the lipase D by the same manufacturer. Palatase is similar to Lipozyme IM, because it comes from the same microorganism, M. miehei, and it is 1,3 specific. However, unlike Lipozyme IM, Palatase is supplied without immobilizing. Novozym 435 is not 1,3 specific, but in presence of a great excess of ethanol it behaves as 1,3 specific in the alcoholysis of TAG [13,14,18]. In this work the 2-MAG yields obtained with these lipases are compared, and we have also studied their stability in the operational conditions and the possible presence of 1(3)-MAG, formed by acyl-migration from the 2-MAG. Table 2 shows the 2-MAG yields and the fatty acid profiles of the products obtained with these lipases. Lipase DF immobilized on Accurel MP-1000 and Novozym 435 gave rise to the highest 2-MAG yields. The TLC analysis with boric acid did not detect 1(3)-MAGs in the final product mixtures from the experiments. These results may be due to the following circumstances: i) the three lipases behaved as 1,3 specific, and the acyl-migration of the fatty acids in position 2 is negligible or (ii) if acyl-migration occurs, the 1(3)MAGs formed were rapidly hydrolyzed to glycerol and free fatty acids (FFAs), which would be immediately esterified with ethanol to ethyl esters.

275

Table 3 Influence of the number of uses of lipases DF, Palatase and Novozym 435 on their activity, determined as the percentage of 2-MAGs obtained in the ethanolysis of cod liver and tuna oils. Number of uses

2-MAG yield (%) DF/MP-1000a

1 2 3 4 5 6 7

68.2 65.1 54.6 47.4 39.3 29.7 16.8

Palatase/MP-1000a 48.0 23.4 12.6 – – – –

N-435b 63.9 61.5 60.0 64.8 54.0 55.5 51.0

a Operational conditions: 500 mg tuna oil, 500 mg dry ethanol, 3 mL acetone, 37 ◦ C, 24 h, 200 rpm and 240 mg lipase DF/MP-1000 (1:1.5, w/w) or Palatase/MP-1000 (15:1.5, w/w). b Operational conditions: 750 mg cod liver oil, 3 g dry ethanol, 375 mg lipase N-435, 35 ◦ C, 2 h and 300 rpm.

Table 2 also shows that Novozym 435 is more active with cod liver oil than with tuna oil. This behaviour has been observed previously, and it seems to be due to the different DHA content of these oils at positions 1 and 3 and to the resistance of DHA to displacement from the glyceride by the lipase [18]. It can also be observed that the DHA and EPA contents of 2-MAGs are quite similar to those of the original oil at position 2 (Table 1), which shows that this lipase only modifies the external positions 1 and 3. An important aspect for the selection of the most adequate lipase is its stability in the operational conditions. For this reason, experiments were carried out to determine how many times the lipases can be used while maintaining their activity constant. Table 3 shows that Palatase lost activity very quickly; lipase DF is more active, but it also lost activity gradually with use. Only Novozym 435 maintained its activity constant for four uses, after which a small reduction (10–13%) was observed. In addition, the stability of Novozym 435 was tested for 300 h in the discontinuous STR without loss of activity, and also for 160 h in an experiment with the same reactor but operating in continuous mode. The 2MAG yield in these experiments remained constant. On completion of these two experiments the lipase was washed with hexane and dried and it was then ready to be used again. Therefore, due to its consistently higher stability and its activity, Novozym 435 was selected for producing 2-MAGs in both STR and PBR. We think that lipases DF and Palatase are deactivated because of the prolonged contact with ethanol, since lipase D (which is similar to lipase DF) was used in STR and PBR in acidolysis reactions between oils and caprylic acid in presence of hexane, and, in these conditions no deactivation was observed. 3.2. Alcoholysis catalyzed by Novozym 435 3.2.1. STR in discontinuous mode: influence of treatment intensity (TI) The influence of TI (amount of lipase × time/amount of oil) in the STR was first studied on a low scale using Novozym 435; then experiments were carried out on a higher scale. Fig. 4 shows that on a low scale 2-MAG yield attained a maximum of 63% for a TI of approximately 1 g lipase h/g oil; however, at higher TI yield decreased sharply. This behaviour was also observed by Shimada et al. [24] ˜ et al. [18] and may be due to the degradation of 2-MAGs and Munío in two steps: (i) transformation of 2-MAG to 1(3)-MAG by acylmigration, and (ii) the subsequent alcoholysis of these 1(3)-MAGs to glycerol and ethyl esters. An alternative explanation is that when TAG concentration is low and 2-MAG concentration high, Novozym 435 might hydrolyze the acyl group at position 2 directly forming glycerol; indeed this lipase may not be 1,3 specific in other

276

L. Esteban et al. / Biochemical Engineering Journal 44 (2009) 271–279



 [2-MAG]  [TAG]0

= max

k1ap k2ap

k2ap /(k2ap −k1ap ) (5)

replacing in these expressions the values of the apparent kinetic constants that fit the experimental results (Table 4), it can be calculated the best TI and the maximum yields in each of the systems that operate in discontinuous mode. Thus for example, for the dispersion reactor at low scale (TI)max = 1.12 g h g−1 and a maximum yield of 57% were obtained. These values coincide acceptably whit the experimental values observed in Fig. 4 (around (TI)max = 2 h × 0.375 g lipase/0.75 g oil = 1.0 g h g−1 and 62.7%, respectively). We have already demonstrated [9,27] that an appropriate procedure to change the reactor type and operational mode and to scale up the enzymatic reaction without loss of yield is to maintain a constant TI as follows:

 mt  L Fig. 4. Influence of treatment intensity (TI) and reactor type on 2-MAG yield obtained by ethanolysis of cod liver oil catalyzed by Novozym 435 in discontinuous mode. (䊉) Low-scale dispersion reactor (operational conditions: 750 mg oil, 3 g dry ethanol, 35 ◦ C, 300 rpm); () large-scale dispersion reactor (operational conditions: 75 g oil, 300 g dry ethanol, 3 g lipase, 35 ◦ C, 300 rpm); () lipase immobilized in a PBR (operational conditions: 15 g oil, 60 g dry ethanol, 2 g lipase, 35 ◦ C and flow rate 15.1 mL h−1 ). Fitting of experimental results to a kinetic model of two first order reactions in series.

conditions and this behaviour may only occur when the majority substrate is 2-MAGs. In any case the following lipase-dependent process must occur in the reactor: k1

k2

TAG−→2-MAG−→glycerol

(1)

The variation of the 2-MAG yield with the TI (Fig. 4) corresponds to the usual way in which the concentration of the intermediate product varies in a series reaction system according to Eq. (1). If both are first order reactions and, as in this case, the reaction is carried out in a batch reactor, the 2-MAG formation rate is given by equation [26]: d[2-MAG] = k1 [TAG] − k2 [2-MAG] dt

(2)

where [TAG] and [2-MAG] are the TAG and 2-MAG concentrations, respectively, and k1 and k2 are the first order kinetics constants of the first and second reaction, respectively. Multiplying all terms of Eq. (2) by the initial triacylglycerol concentration/lipase concentration ratio ([TAG]0 /e0 ), the time may be replaced by the treatment intensity e0 t/[TAG]0 = mL t/mTAG0 (TI, lipase amount × time/initial amount of oil). For TI = 0, [2-MAG] = 0, [TAG] = [TAG]0 and the 2-MAG yield at a determined TI, [2-MAG]/[TAG]0 , is given by equation: k1ap [2-MAG] = [TAG]0 k2ap − k1ap





−exp



exp

−k2ap

−k1ap

mL t mTAG0



mL t mTAG0



(3)

where k1ap and k2ap are the apparent kinetic constants, g oil h−1 g lipase−1 . Fig. 4 shows that Eq. (3) fits the experimental results for the values of the apparent kinetic constants show in Table 4. Derivating Eq. (3) and equating to zero, it can be calculated the TI value for which the maximum 2-MAG is attained, (TI)max , and the value of this maximum yield, ([2-MAG]/[TAG]0 )max , according with the following equations: (TI)max

ln(k1ap /k2ap ) = k1ap − k2ap

(4)

V [TG]0

= discontinuous

 m  L q[TG]0

= constant

(6)

continuous

where mL is the lipase amount, t the reaction time, V the reaction mixture volume, [TG]0 the initial concentration of TAGs (mol L−1 or g L−1 ) and q the reaction mixture flow rate. Fig. 4 shows that the TI that gave rise to the highest 2-MAG yield was 1 g lipase h g oil−1 . This TI was taken as reference to scale up the process. Fig. 4 also shows the results obtained in experiments carried out in a jacketed STR of 1 L capacity (100 times greater volume than the original one). It can be observed that the maximum 2-MAG yields (65%) were obtained at the same TI as in the small-scale experiment (0.96–1 g h g−1 ). These results also have been acceptably fitted to the model of two reactions in series (Fig. 4) and the values of the apparent kinetic constants are very similar to the ones obtained at low scale (Table 4). The form of the curves in Fig. 4 seems to indicate that at low TIs (or low reaction times) the preferred substrate of lipase is TAG, and that only when the TAG concentration is low and the 2MAG concentration high (even greater than the TAG one) does the degradation of 2-MAG to glycerol predominate. This result can be observed in Fig. 5, which shows the evolution with time of the relative amounts of all the lipidic species involved in the alcoholysis reaction. It can be seen that the maximum 2-MAG concentration coincides with the point at which the TAG concentration is almost negligible (i.e. the 2-MAGs are degraded when virtually no TAGs exist). 3.2.2. PBR operating in discontinuous mode Taking as reference the TI that gave rise to the maximum 2-MAG yield in the STR operating in batch mode (1 g h g−1 ), the process was scaled up using a packed bed column where the lipase was immobilized. In a column of 6 mm i.d. × 235 mm length, 2 g of Novozym 435 was immobilized, while the reaction mixture consisted of 15 g of cod liver oil and 60 g of ethanol. In these conditions the maximum 2-MAG yield should be obtained at a reaction time of 7.5 h (TI of 1 g h g−1 Eq. (6)). Fig. 4 shows that the maximum 2-MAG yield was indeed attained at a TI similar to the one used in the STR (although a lower global rate was observed).

Table 4 Values of the apparent kinetic constants, k1ap and k2ap , obtained for the STR and PBR operating in discontinuous mode in the ethanolysis of cod liver oil with Novozym 435. Reactor type

Scale

k1ap (g oil h−1 g lipase−1 )

k2ap (g oil h−1 g lipase−1 )

STR

Low scale Large scale

1.44 1.72

0.50 0.50

PBR

–

0.51

0.67

L. Esteban et al. / Biochemical Engineering Journal 44 (2009) 271–279

277

Table 5 Influence of the mean reaction mixture flow rate on the 2-MAG yield in the ethanolysis of cod liver oil, catalyzed by Novozym 435 immobilized in a PBR operating in continuous mode. Mean flow rate (mL/h)

Treatment intensity (g lipase × h/g oil)

2-MAG yield (%)

7.9 9.3 13.2

1.54 1.31 0.92

26.9 ± 11.1 39.0 ± 6.1 52.6 ± 6.9

Operational conditions: 15 g cod liver oil, ethanol/oil ratio 4:1 (w/w), 2 g lipase N435, 35 ◦ C, column of 6 mm × 235 mm.

Fig. 5. Evolution with time of the relative amounts of the lipidic species during the ethanolysis of cod liver oil catalyzed by Novozym 435 in a large-scale STR. Operational conditions: ethanol/oil ratio 4:1 (w/w), 75 g oil, 3 g N-435 lipase, and 35 ◦ C.

Fig. 4 also shows that the fitting of the experimental results to the model of two reactions in series (Eq. (3)) can be considered acceptable. Table 4 shows that while the apparent kinetic constant of formation of 2-MAG, k1ap , is lower in this system than in the STRs, the degradation constant, k2ap , is higher. Fig. 4 shows that the most important difference between the results obtained in the STR and in the PBR is that in the latter the maximum 2-MAG yield is much lower (36% in the PBR and 63% in the STR). This lower yield agrees with the lower values of the apparent kinetic constants in both reactors and could be due to: (i) the lower global reaction rate due to the lower agitation rate in the PBR, which could consequently have a high influence of the external mass transfer in the PBR [28]; (ii) as has already been indicated, the results in the STR (maximum 2-MAG yield of 63%) reveal that TAG seems to be the preferred substrate for the lipase, and that only when the TAG concentration is very low and the 2-MAG concentration attains a certain value (Fig. 5) does the lipase begin to transform them to glycerol, (iii) when operating with PBR a lot of lipase receives a limited amount of TAGs and 2-MAGs (most of them remain in the reservoir tank of reaction mixture; see Fig. 2 for details), and both substrates are degraded because there are not enough TAGs for the lipase to act only on them; i.e. the lipase/TAGs ratio is higher than the one in Eq. (6); this means that the 2-MAG concentration declines quickly. In addition, this phenomenon is accentuated because at low flow rates, in order to prevent compression of the lipase against the upper support, the fluid-dynamic behaviour of a PBR is far from that of the dispersion reactor [27]. This fact is also reflected in the lower value of k2ap in the PBR with respect to its value in the STR (Table 4). It is clear that PBR is not appropriate with this lipase. The first hypothesis (control of the external mass transfer in the PBR) can be tested by calculating the values of the Thiele module in both systems (STR and PBR). This Thiele module is given by equation: ˚e =

R robs 3 ks cAb

(7)

where R is the catalyst particle radii, robs the observable reaction rate per unit of volume of catalyst particle (mol/(s m3 )), ks the external mass transfer coefficient and cAb is the substrate concentration into the reaction mixture. The relation between this concentration and the one on the catalyst surface, cAs , is: cAs = 1 − ˚e cAb

(8)

Thus, when ˚e is small, cAs = cAb and the influence of the external mass transfer can be considered negligible. ˚e has been calculated for both systems and it has been observed that the most important difference corresponds to the reaction rate, robs . The initial reaction rates are 0.20 and 0.070 mol h−1 L−1 , for the STR and PBR, respectively. These values have been estimated from the proposed kinetic model and from the apparent kinetic constants shown in Table 4. In any case, the values of the Thiele modulus calculated are much lower than the unit, and therefore the influence of the external mass transfer can be considered negligible in both systems. 3.2.3. PBR operating in continuous mode The change from a discontinuous to a continuous reactor can also be carried out maintaining a constant TI, as indicated in Eq. (6), using 2 g of immobilized lipase and a reaction mixture with an oil concentration of 164 g L−1 (the ethanol/oil ratio was always 4:1, w/w). In these conditions a flow rate of 12.2 mL h−1 should be used to maintain a TI of 1 g h g−1 (Eq. (6)). Table 5 shows the results obtained at three flow rates close to 12.2 mL h−1 (7.9–13.2 mL h−1 ). The maximum 2-MAG yield was 52.6% achieved at a TI of 0.92 g h g−1 (similar to 1 g h g−1 ). Table 5 shows that at a lower flow rate (or higher residence time and TI, Eq. (6)) the 2-MAG yield decreased. This result can be explained by the fact that in these conditions the residence time, and therefore the TI, is excessive (higher than 1 g h g−1 , Table 4) and the 2-MAGs formed previously are being degraded to glycerol (Eq. (6)), as also occurs in the discontinuous mode at high TIs (Fig. 4). The maximum 2-MAG yield (52.6%) at a similar TI is placed between those obtained in the STR (63–65%) and PBR (36%) operating in discontinuous mode. The fact that it is lower than the one obtained in the STR may be due to the highest degradation rate of 2MAG due to the low lipase/TAG ratios and to the high lipase/2-MAG ratios that can be achieved in the column. However, when operating in continuous mode, this latter phenomenon occurs with less intensity, as it is evidenced by the higher yield reached with respect to the one attained when it is operated in discontinuous mode; in continuous mode the 2-MAGs leaving the lipase bed does not go into contact with the lipase again, which happens when it is operated with recirculation (i.e. discontinuous mode). 3.2.4. STR operating in continuous mode As the STR provided the highest 2-MAG yield operating in discontinuous mode, it was decided to design a STR operating in continuous mode (Fig. 2). The TI selected was the one that gave rise to the maximum 2-MAG yield in the discontinuous dispersion reactor, i.e. 0.96–1 g h g−1 . For these experiments 6 g of immobilized lipase was used and a reaction mixture with an oil concentration of 162.4 g L−1 (the ethanol/oil ratio was always 4:1, w/w). Thus, the reaction mixture flow rate used (calculated from Eq. (2)) was 37.7 mL h−1 . Fig. 6 shows the 2-MAG yields obtained in the alcoholysis carried out at flow rates similar to 37.7 mL h−1 . These results are represented as a function of the time elapsed since starting to pump the reaction mixture. It can be observed that when the reactor starts to operate the response time at the outlet suffers a

278

L. Esteban et al. / Biochemical Engineering Journal 44 (2009) 271–279 Table 6 Recovery of 2-MAGs by solvent extraction: lipid profile and 2-MAG yield obtained after successive extractions. Lipid profile (%) 2-MAG FFAa 1,2-DAGb TAGc + EEd 2-MAG yield (%) Initial mixture

19.5

0.1

6.6

73.6

First extraction step 2-MAG fraction EE fraction

90.5 8.7

0.1 0.1

0.9 4.0

8.6 87.4

Second extraction step to the EE fraction of the first extraction step 2-MAG fraction 90.9 0.1 1.0 8.1 EE fraction 1.7 0.1 3.8 94.4

57.0

Third extraction step to the EE fraction of the second extraction step 2-MAG fraction 84.3 0.3 1.7 13.7 EE fraction 1.6 0.0 3.7 94.7

54.5

Global purity and yield of 2-MAGs 90.0

90.3

a

Fig. 6. Production of 2-MAGs by ethanolysis of cod liver oil catalyzed by Novozym 435 in a STR operating in continuous mode: influence of time and reaction mixture flow rate on 2-MAG yield. Operational conditions: 75 g cod liver oil, oil/absolute ethanol ratio 1:79 mol/mol, 6 g lipase N-435, 35 ◦ C.

transitory state; however at longer time the outlet yield becomes stationary. It can be observed that 2-MAG yield is about 50% upon reaching the steady-state for the flow rates up to 33.4 mL h−1 . The 2MAG yield at the steady-state is a little less for 62.7 mL h−1 . These yields are lower than those obtained in the STR operating in discontinuous mode (65%, see Fig. 4) for similar TIs. The difference could be explained according to the following rationale: in discontinuous mode at the same time as the TAGs are consumed, the intermediate diacylglycerols (DAGs) continue their transformation to 2-MAGs; in continuous mode, the continuous feeding of TAGs determines that the lipase preferably acts on them rather than on the intermediate DAGs. These DAGs will then be accumulated in the reaction mixture and therefore the 2-MAG yield will decrease. This effect can be observed in Fig. 7 in which at the highest flow rate assayed, the greater entrance flow rate of TAGs determines a higher concentration in the intermediate DAGs and consequently a lower transformation of them into 2-MAGs. Fig. 6 also shows that at the lowest flow rates tested (11.5 and 24.0 mL h−1 ) maximum 2-MAG yields were obtained at approximately 20 h. After this time 2-MAG yield decreased due to the

58.8

b c d

0.3

3.0

7.1

Free fatty acids. 1,2-diacylglycerols. Triacylglycerols. Ethyl esters.

degradation of 2-MAGs, as in the discontinuous STR (Fig. 4). However, at longer times the 2-MAG yield did not decrease, because in this case the 2-MAGs produced were continuously leaving the reactor, thus avoiding potential degradation. Fig. 6 also shows that at greater flow rates (>24.0 mL h−1 ) this maximum was not observed, possibly because the lower residence time prevented sufficiently high concentrations of 2-MAGs (and sufficiently low TAG concentrations) to observe an appreciable 2-MAG degradation. 3.3. Separation of 2-MAGs The possible presence of 1(3)-MAGs in the 2-MAG mixtures obtained was systematically checked by boric acid TLC. This species was detected in very few samples, and when it was detected, 2MAGs represented over 95% of total MAGs. 2-MAGs were recovered following the procedure already ˜ et al. [18]. This method relies on the formation described by Munío of the biphasic system ethanol–water 90:10 (v/v) and hexane, in which the MAGs remain in the hydroalcoholic phase and esters and TAGs are extracted toward the hexanic phase. Table 6 shows that with three extraction steps 2-MAG recovery yields and purities of about 90% were achieved. Table 7 shows the fatty acid profile of the Table 7 Fatty acid composition of 2-MAGs (% (w/w) of total fatty acids) obtained by ethanolysis of cod liver and tuna oils, catalyzed by lipase Novozym 435 and separated from the product mixture by solvent extraction. Fatty acids

Fig. 7. Influence of time and reaction mixture flow rate on percentage of intermediate DAG on total lipids in the production of 2-MAGs by ethanolysis of cod liver oil catalyzed by Novozym 435 in a STR operating in continuous mode. Operational conditions: 75 g cod liver oil, oil/absolute ethanol ratio 1:79 mol/mol, 6 g lipase N-435, 35 ◦ C.

14:0 16:0 16:1n-7 16:2n-4 18:0 18:1n-9 18:1n-7 18:2n-6 18:4n-3 20:1n-9 20:4n-6 20:5n-3 22:1n-9 22:5n-3 22:6n-3 Others

2-MAG final fraction Cod liver oil

Tuna oil

6.8 17.5 7.4 0.9 0.1 9.9 1.1 2.3 3.3 4.3 n.d. 10.3 2.5 1.4 30.5 1.8

7.5 21.5 7.7 0.7 1.5 10.2 0.9 0.9 0.8 0.6 1.1 9.7 n.d. 1.2 35.0 1.0

L. Esteban et al. / Biochemical Engineering Journal 44 (2009) 271–279

2-MAGs produced by alcoholysis and separation with the two fish oils used. It can be observed that these profiles are similar to the fatty acid content at position 2 of the original oils (Table 1), which confirms that the fatty acids that occupied that position remain there in the final 2-MAGs produced. This confirms the 1,3 specificity of lipase Novozym 435 in the operational conditions used. 4. Concluding remarks a) The three lipases tested are effective to synthesize 2-MAGs. However Novozym 435 is the most stable, since it maintains activity unaltered for more than 300 h in the operational conditions used in this work. b) The feasibility of producing 2-MAGs catalyzed by Novozym 435 has been demonstrated when using both STR and PBR, operating in discontinuous as well as continuous modes. However, the highest 2-MAG yield was obtained in the STR operating in discontinuous mode, since in this system the reaction rate is higher and 2-MAG degradation starts at higher times. In addition, this system is easy to scale up. c) 2-MAG yields of about 65% were obtained in STR by alcoholysis of fish oils with dry ethanol, catalyzed by Novozym 435, at a treatment intensity close to 1 g h g−1 . These 2-MAGs do not contain 1(3)-MAG isomers or free fatty acids, which facilitates their separation from ethyl esters. d) The variation of the 2-MAG yield with the TI in the alcoholysis catalyzed by Novozym 435 in the discontinuous reactors corresponds to the way in which varies the concentration of the intermediate product in a system of two first order reactions in series, both reactions being catalyzed by lipase. e) The fatty acid profiles of the 2-MAGs produced are similar to the fatty acid content at position 2 of the original oils, and these 2MAGs have a PUFA (EPA + DHA) content of about 40–41%, for cod liver oil and 44–45% for tuna oil. f) The recovery of 2-MAGs from the final alcoholysis mixture can be carried out by low toxicity solvents (ethanol and hexane) with yields and purities of the final 2-MAGs produced of around 90%. Acknowledgments This research was supported by grants from the Ministerio de Educación y Ciencia (Spain), Projects AGL2003-03335 and CTQ2007-64079. Both projects are co-funded with the FEDER (European Fund for Regional Development). References [1] A.P. Simopoulos, Omega-3 fatty acids in health and disease and in growth and development, Am. J. Clin. Nutr. 54 (1991) 438–463. [2] J.A. Nettleton, Are n-3 fatty acids essential nutrients for fetal and infant development? J. Am. Dietetic Assoc. 93 (1993) 58–64. [3] H. Mu, T. Porsgaard, The metabolism of structured triacylglycerols, Prog. Lipid Res. 44 (2005) 430–448. [4] X. Xu, C.-E. Hoy, S. Balchen, J. Adler-Nissen, Specific-structured lipid: nutritional perspectives and production potentials, in: Proceedings of International Symposium on the Approach to Functional Cereals and Oils, CCOA, Beijing, 1997, pp. 806–813. [5] C.C. Akoh, K.H. Huang, Enzymatic synthesis of structured lipids: transesterification of triolein and caprylic acid, J. Food Lipids 2 (1995) 219–230.

279

[6] Y. Shimada, A. Sugihara, K. Maruyama, T. Nagao, S. Nakayama, H. Nakano, Y. Tominaga, Production of structured lipids containing docosahexaenoic and caprylic acids using immobilized Rhizopus delemar lipase, J. Ferment. Bioeng. 81 (1996) 299–303. [7] Y. Shimada, M. Suenaga, A. Sugihara, S. Nakai, Y. Tominaga, Continuous production of structured lipid containing ␥-linolenic and caprylic acids by immobilised Rhizopus delemar lipase, J. Am. Oil Chem. Soc. 76 (1999) 189–193. [8] X. Xu, S. Balchen, C.-E. Hoy, J. Adler-Nissen, Pilot batch production of specific-structured triacylglycerols by lipase-catalyzed interesterification in a laboratory-scale continuous reactor, J. Am. Oil Chem. Soc. 75 (1998) 301–308. [9] B. Camacho Páez, A. Robles Medina, F. Camacho Rubio, P. González Moreno, E. Molina Grima, Production of structured triglycerides rich in n-3 polyunsaturated fatty acids by the acidolysis of cod liver oil and caprylic acid in a packed-bed reactor: equilibrium and kinetics, Chem. Eng. Sci. 57 (2002) 1237–1249. ˜ [10] E. Hita, A. Robles, B. Camacho, A. Ramírez, L. Esteban, M. Jiménez, M.M. Munío, P.A. González, E. Molina, Production of structured triacylglycerols (STAG) rich in docosahexaenoic acid (DHA) in position 2 by acidolysis of tuna oil catalyzed by lipases, Process Biochem. 42 (2007) 415–422. [11] U. Schmid, U.T. Bornscheuer, M.M. Soumanou, F.P. McNeill, R.D. Schmid, Optimization of the reaction conditions in the lipase-catalyzed synthesis of structured triglycerides, J. Am. Oil Chem. Soc. 75 (1998) 1527–1531. [12] M.M. Soumanou, U.T. Bornscheuer, R.D. Schmid, Two-step enzymatic reaction for the synthesis of pure structured triacylglycerides, J. Am. Oil Chem. Soc. 75 (1998) 703–710. [13] R. Irimescu, K. Furihata, Y. Hata, Y. Iwasaki, T. Yamane, Utilization of reaction medium-dependent regiospecificity of C. antarctica lipase (Novozym 435) for the synthesis of 1 3-dicapryloyl-2-docosahexanoyl (or eicosapentanoyl) glycerol, J. Am. Oil Chem. Soc. 78 (3) (2001) 285–289. [14] R. Irimescu, K. Furihata, Y. Hata, Y. Iwasaki, T. Yamane, Two-step enzymatic synthesis of docosahexaenoic acid-rich symmetrically structured triacylglycerols via 2-monoacylglycerols, J. Am. Oil Chem. Soc. 78 (7) (2001) 743–748. [15] A. Millqvist Fureby, C. Virton, P. Adlercreutz, B. Mattiasson, Acyl group migration in 2-monoolein, Biocatal. Biotransform. 14 (1996) 89–111. [16] Y. Watanabe, Y. Shimada, A. Sugihara, Y. Tominaga, Stepwise ethanolysis of tuna oil using immobilized Candida antarctica lipase, J. Biosci. Bioeng. 88 (6) (1999) 622–626. [17] L. Deng, X. Xu, G. Haraldsson, T. Tan, F. Wang, Enzymatic production of alkyl esters through alcoholysis: a critical evaluation of lipases and alcohols, J. Am. Oil Chem. Soc. 82 (5) (2005) 341–347. ˜ L. Esteban, A. Robles, E. Hita, M.J. Jiménez, P.A. González, B. Cama[18] M.M. Munío, cho, E. Molina, Synthesis of 2-monoacylglycerols rich in polyunsaturated fatty acids by ethanolysis of fish oil catalyzed by 1,3 specific lipases, Process Biochem. 43 (2008) 1033–1039. ˜ González, [19] L. Esteban Cerdán, A. Robles Medina, A. Giménez Giménez, M.J. Ibánez E. Molina Grima, Synthesis of polyunsaturated fatty acid-enriched triglycerides by lipase-catalyzed esterification, J. Am. Oil Chem. Soc. 75 (1998) 1329–1337. [20] G. Lepage, C. Roy, Improved recovery of fatty acid through direct transesterification without prior extraction or purification, J. Lipid Res. 25 (1984) 1391–1396. [21] J. Rodríguez-Ruiz, J.L. El Hassan Belarbi, D. García Sánchez, López Alonso, Rapid simultaneous lipid extraction and transesterification for fatty acid analysis, Biotechnol. Tech. 12 (9) (1999) 689–691. [22] A. Robles Medina, L. Esteban Cerdán, A. Giménez Giménez, B. Camacho Páez, M.J. ˜ Ibánez González, E. Molina Grima, Lipase-catalyzed esterification of glycerol and polyunsaturated fatty acids from fish and microalgae oils, J. Biotechnol. 70 (1999) 379–391. [23] R.J. Henderson, D.R. Tocher, in: R.J. Hamilton, S. Hamilton (Eds.), Thin-layer Chromatography. Lipids Analysis: A Practical Approach, IRL Press at Oxford University Press, 1993. [24] Y. Shimada, J. Ogawa, Y. Watanabe, T. Nagao, A. Kawashima, T. Kobayashi, S. Shimizu, Regiospecific analysis by ethanolysis of oil with immobilized Candida antarctica lipase, Lipids 38 (12) (2003) 1281–1286. [25] Z. Shen, Ch. Wijasundera, Evaluation of ethanolysis with immobilized Candida antarctica lipase for regiospecific analysis of triacylglycerols containing highly unsaturated fatty acids, J. Am. Oil Chem. Soc. 83 (2006) 923–927. [26] J.M. Coulson, J.F. Richardson, Ingeniería Química, Tomo III, Ed. Reverté, Barcelona, 1984, pp. 87–88. [27] P.A. González Moreno, A. Robles Medina, F. Camacho Rubio, B. Camacho Páez, E. Molina Grima, Production of structured lipids by acidolysis of an EPA-enriched fish oil and caprylic acid in a packed bed reactor: analysis of three different operation modes, Biotechnol. Prog. 20 (2004) 1044–1052. [28] P.A. González Moreno, A. Robles Medina, F. Camacho Rubio, B. Camacho Páez, L. Esteban Cerdán, E. Molina Grima, Production of structured triacylglycerols in an immobilized packed-bed reactor: batch mode operation, J. Chem. Biotechnol. 80 (2005) 35–43.