Synthesis of structured lipids by two enzymatic steps: Ethanolysis of fish oils and esterification of 2-monoacylglycerols

Process Biochemistry 44 (2009) 723–730

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Synthesis of structured lipids by two enzymatic steps: Ethanolysis of fish oils and esterification of 2-monoacylglycerols ˜ ı´o, Alfonso Robles *, Luis Esteban, Pedro A. Gonza´lez, Emilio Molina Marı´a del Mar Mun Department of Chemical Engineering, University of Almerı´a, Carretera de Sacramento s/n, 04120 Almerı´a, Spain

A R T I C L E I N F O

A B S T R A C T

Article history: Received 28 November 2008 Received in revised form 24 February 2009 Accepted 4 March 2009

This paper studies the synthesis of structured triacylglycerols (STAGs), rich in polyunsaturated fatty acids (PUFAs) by a two-step enzymatic process: (i) alcoholysis of fish oils (cod liver and tuna oils) with ethanol to obtain 2-monoacylglycerols (2-MAGs), catalyzed by 1,3 specific lipases and (ii) esterification of these 2-MAGs with caprylic acid (CA, 8:0), also catalyzed by a 1,3 specific lipase, to produce STAGs of structure CA–PUFA–CA. As regards the alcoholysis reaction, three factors have been studied: the influence of the type of lipase used (lipase D from Rhizopus oryzae, immobilized on Accurel MP1000, and Novozym 435 from Candida antarctica), the operational mode of a stirred tank reactor (STR operating in discontinuous and continuous mode) and the intensity of treatment (IOT = lipase amount  reaction time/oil amount). Although higher 2-MAG yields were obtained with lipase D, Novozym 435 was selected due to its greater stability in the operational conditions. The highest 2-MAG yield (63%) was attained in the STR operating in discontinuous mode at an IOT of 1 g lipase  h g oil1 (at higher IOT the 2-MAGs were degraded to glycerol). This system was scaled up to 100 times the initial volume, achieving a similar yield (65%) at the same IOT. The 2-MAGs in the final alcoholysis reaction mixture were separated from ethyl esters by solvent extraction using solvents of low toxicity (ethanol and hexane); the 2-MAG recovery yield was over 90% and the purity was approximately 87–90%. Regarding the esterification of the 2-MAGs, the following factors were studied: the influence of the lipase type used, the presence or absence of solvent (hexane) and the reaction time or intensity of treatment (IOT = lipase amount  reaction time/2-MAG amount). Of the five lipases tested, the highest STAG percentages (over 90%) were attained with lipases D and DF, immobilized on Accurel MP1000. These STAGs contain 64% CA, of which 98% is at positions 1 and 3. Position 2 contains 5% CA and 45% PUFAs, which means that all the PUFAs that were located at position 2 in the original oil remain in that position in the final STAGs. The lipase D immobilized on Accurel MP1000 is stable in the operational conditions used in the esterification reaction. Finally the purification of STAGs was carried out by neutralization of free fatty acids with hydroethanolic solution of KOH and extraction of STAGs with hexane. By this method purity was over 95% and separation yields were about 80%. ß 2009 Elsevier Ltd. All rights reserved.

Keywords: 2-Monoacylglycerol (2-MAG) Structured lipids Polyunsaturated fatty acids (PUFAs) Docosohexaenoic acid (DHA) Lipase Alcoholysis Esterification Cod liver oil Tuna oil

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), which decreases blood viscosity and the aggregation of platelets and promotes vaso-dilation [1], and docosahexaenoic acid (DHA, 22:6n3) which promotes the sensorial and neuronal maturation in babies, and is therefore often included as a supplement to the diet of pregnant women [2–4].

* Corresponding author. Tel.: +34 950015065; fax: +34 950015484. E-mail address: [email protected] (A. Robles). 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.03.002

The structure and fatty acids composition of triacylglycerols (TAGs) affect their absorption and the distribution of fatty acids in the organism [5]. For nutritional purposes, there is interest in the production of structured triacylglycerols (STAGs) 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 STAGs are claimed to benefit the immune function and to help to improve lipid clearance from the bloodstream [6]. MLM type STAGs with EPA and DHA in position 2 are more readily absorbed sources of these PUFAs than other TAGs with similar fatty acid compositions but with a random fatty acid distribution [7]. Also, the necessary hydrolysis of TAGs for the absorption of lipids is faster in STAGs than in the original oils [8]. STAGs may have the potential to prevent hypertriglyceridemia and obesity caused by a high-fat diet

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[9]. It has been suggested that the daily intake of STAGs could result in weight loss and less accumulation of fats, as well as in a reduction in serum cholesterol [10]. It is also known that whereas cow milk does not contain DHA, mother’s milk contains a small percentage of DHA, all of which is at position 2 of TAGs [11]. The simplest and most direct route for the synthesis of MLM type STAGs is acidolysis between long-chain TAGs and mediumchain free fatty acids, catalyzed by a 1,3 specific lipase [12–16]. A problem of this method is the resistance of PUFAs (especially DHA) occupying positions 1 and 3 to be displaced by the medium-chain fatty acids, which brings about a decrease in the purity of STAGs of the desired MLM structure [17]. In order to avoid this problem, one of the alternatives is a process in two steps [18–21]. First, 2-monoacylglycerols (2-MAGs) are obtained from oils rich in functional fatty acids by enzymatic alcoholysis using 1,3 specific lipases. Then, medium-chain fatty acids are added in the extreme positions of 2-MAG by enzymatic esterification, also using 1,3 specific lipases. According to Irimescu et al. [20] this process leads to higher rates of conversion to STAGs than the one-step process. After the alcoholysis and the esterification steps, the purification of 2-MAGs and final STAGs, respectively, must be carried out. If the lipases used are 1,3 specific, the purity of final STAGs should be higher than that obtained by acidolysis. For example, this procedure was used for the synthesis of MLM type STAGs, with DHA at position 2 (starting from a TAG highly rich in this PUFA), and caprylic acid at positions 1 and 3, using lipases from Candida antarctica (Novozym 435) and Rhizomucor miehei for the alcoholysis and esterification, respectively. Novozym 435 only behaves as 1,3 specific with a great excess of ethanol and presented high activity toward the polyunsaturated TAGs [19]. In previous studies in our laboratories Lipozyme IM and lipase D from Rhizopus oryzae immobilized on Celite 545 were used as catalysts for the alcoholysis of TAGs and the results obtained were both qualitatively and quantitatively dependent on the lipase used. When using Lipozyme the process was controlled by acyl-migration, whereas with lipase D no acyl-migration occurred [22]. The main problems of the alcoholysis step seem to be: (i) the selection of a 1,3 specific lipase and of an immobilization carrier that does not catalyze the acyl-migration; (ii) the lipase–carrier system must be stable to enhance the economic viability of the process; and (iii) the utilization of solvents or reaction means of low toxicity and which are able to restrict acyl-migration. In this step Wongsakul et al. [23] reached high yields (81%) of 2-MAGs highly enriched in PUFAs by using the non-regiospecific lipase Pseudomonas sp. (PS-C). These authors indicated that more important than the positional specificity seems to be the specificity to the fatty acid chain length of the lipase used. This same principle (the specificity toward to a determined fatty acid) it has also been applied for producing STAG by synthesis of 1,3 diacylglycerols (1,3DAG) and the subsequent esterification of the position 2 by using lipases that does not attack to the fatty acids already present in the 1,3-DAG and with high selectivity and activity toward to the fatty acid to be introduced [24]. The esterification step is a very rapid reaction in which we must try to prevent the acyl-migration in the 2-MAGs and eliminate the water formed [25]. The present work outlines the results of the research to synthesize 2-MAGs by alcoholysis and the subsequent production of STAGs by esterification of 2-MAGs using different lipases and cod liver and tuna oils, both highly rich in PUFAs. The alcoholysis reaction was developed in stirred tank reactors (STR) in discontinuous and continuous modes and at small and large scale. The esterification reaction was only studied in STR operating in discontinuous mode. The recovery and purification of 2-MAGs and STAGs were carried out using low toxicity solvents. The goal was to obtain highly pure STAGs with the structure CA–PUFA–CA.

Table 1 Fatty acids composition of cod liver and tuna oils and fatty acids composition in position 2 (% of total fatty acids weight) of TAGs of these oils. Fatty acids

14:0 16:0 16:1n7 16:2n4 18:0 18:1n9 18:1n7 18:2n6 18:4n3 20:1n9 20:4n6 20:5n3 22:1n9 22:5n3 22:6n3 Others

Cod liver oil

Tuna oil

Oila

Position 2b

Oila

Position 2b

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

a

Percentage of each fatty acid with respect to total fatty acids in the TAGs. Percentage of each fatty acid with respect to total fatty acids at position 2 of TAGs determined by ethanolysis with N-435 [33]. b

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 showed that neither of them contained partial acylglycerols. The lipases used were Novozym 435 (N-435) from C. antarctica, Lipozyme RM IM from Mucor miehei (both donated by Novozymes, Denmark), lipases D and DF from R. oryzae (Amano Pharmaceutical Co., Nagoya, Japan) and lipase QLM from Alcaligenes sp. (donated by Meito Sangyo Co., Japan). Lipases N-435 and Lipozyme IM were provided immobilized, whereas lipases D, DF and QLM were provided without immobilizing. Lipases D and DF were immobilized on Accurel MP1000 (Akzo Nobel Faser, Obernburg, Germany) following the procedure ˜ ı´o et al. [26]; this procedure is based on the method of described in Mun Soumanou et al. [18], modified by Hita et al. [16]. Lipase QLM was tested without immobilizing. Analytical grade absolute ethanol (99.5%, v/v), acetone, n-hexane (Panreac S.A., Barcelona, Spain) and caprylic acid (98% purity, Sigma–Aldrich, St Louis, MO, USA) were used as substrates and solvents of alcoholysis and esterification reactions. Molecular sieves of 4-A˚ were obtained from Sigma. All other chemicals (analytical grade or better) were also obtained from commercial sources. 2.2. Ethanolysis reaction In this reaction the oil (TAGs) reacts with ethanol in the presence of acetone (solvent) and a 1,3 specific lipase, to yield 2-MAGs, diacylglycerols (DAGs) and ethyl esters (EEs); in the final reaction mixture there are also ethanol and TAGs that have not reacted. This reaction was catalyzed by lipase D (immobilized on Accurel MP1000) and Novozym 435 [26,27]. The ethanolysis reaction catalyzed by lipase D was carried out in STR operating in discontinuous mode. The ethanolysis catalyzed by Novozym 435 was carried out in STR operating in continuous and discontinuous mode, as described below. 2.2.1. Ethanolysis with lipase D and Novozym 435 in STR operating in discontinuous mode The procedures used with lipases D (MP1000) and N-435 have been described in ˜ ı´o et al. [26] and Esteban et al. [27], respectively. At small scale the reaction Mun mixture consisted of 500 mg oil, 500 mg ethanol, 3 mL acetone (6 mL/g oil) and 60 mg lipase D immobilized on MP1000. This was placed in 50-mL Erlenmeyer flasks with silicone capped stoppers under inert atmosphere. The mixture was incubated at 37 8C and agitated in an orbital shaker, with temperature controlled by air, at 200 rpm (Inkubator 1000, Unimax 1010 Heidolph, Klein, Germany). When lipase N-435 was used the reaction mixture contained 750 mg oil, 3 g dry ethanol (dry ethanol/oil ratio 4:1, w/w) and 375 mg lipase, and it was agitated at 300 rpm at 35 8C. 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 8C until analysis. All reactions and their corresponding analyses were carried out in triplicate; standard deviations were always below 8%.

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Fig. 1. 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.

2.2.2. Ethanolysis with lipase N-435 in STR operating in continuous mode Fig. 1 shows a scheme of this reaction system. The STR was immersed in a thermostatic bath. When working in continuous mode the reactor contains a spillway device in the upper part, equipped with a double grid to prevent the loss of lipase from the reaction mixture (Fig. 1). The peristaltic 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 8C the reactants and lipase were: 75 g of cod liver oil, 300 g dry ethanol (oil/dry ethanol molar ratio 1:79), 6 g lipase N-435. 2.3. Recovery and purification of 2-MAGs Recovery of 2-MAGs from the alcoholysis reaction was performed by solvent ˜ ı´o et al. [26], which is extraction following the methodology specified by Mun summarized in Fig. 2. First, acetone and residual ethanol were eliminated from the reaction mixture 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. Finally the hydroethanolic solution rich in 2-MAGs was stored at 24 8C until analysis. 2.4. Esterification reaction A typical reaction mixture consisted of 66 mg 2-MAG fraction, 78 mg caprylic acid, 3 mL hexane, 0.6 g molecular sieves and 18 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 8C and agitated in an orbital shaker at 200 rpm (Inkubator 1000, Unimax 1010 Heidolph, Klein, Germany). 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 8C until analysis.

2.6. Analysis of reaction products Identification of the reaction products (MAGs, DAGs, TAGs and EEs) was carried out by thin-layer chromatography (TLC). 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 8C 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 [29]. Fractions corresponding to each lipid type were scraped from the plates and methylated according to the original method of Lepage and Roy [30], modified by Rodrı´guez-Ruiz et al. [31]. Methylation and methyl ester analysis by GC have been described elsewhere [32]. Nonadecanoic acid (19:0) (Sigma–Aldrich) was used as an internal standard for quantitative determination of fatty acids. All reactions and their corresponding analyses were carried out in triplicate. Standard deviations were always below 8%. The 2-MAG yield was calculated by determining the weight percentage of lipidic species in the product as equivalent fatty acids. Thus, when 1 mol of initial TAG is completely transformed into 2-MAG (100% yield), the molar percentage of this species in the final reaction mixture is 33.3% (1 mol of 2-MAG and 2 mol of ethyl esters). These percentages could be similarly expressed in weight if we considered a fatty acid type with an average molecular weight to transform moles in weight. This assumption should be made because EEs and TAGs cannot be separated using the usual mobile phase in TLC. The 2-MAG yield can therefore be calculated by multiplying the 2-MAG percentage in the final reaction mixture by 3. The progress of the esterification reaction has been established by the STAG percentage, which has been defined as the percentage of TAGs over total acylglycerols in the final reaction mixture, i.e. STAG ð%Þ ¼

STAG in the final mixture ðwÞ  100 acylglycerols ðMAG þ DAG þ TAGÞ in the final mixture

2.5. Recovery and purification of STAGs Once the lipase and molecular sieves were removed by filtration, the final esterification reaction mixture contained: hexane, STAGs, 1,2-DAGs, free fatty acids and low amounts of 2-MAGs. Recovery of STAGs from this mixture was performed ˜ a [16,28], which is summarized in Fig. 3. First, free following the method of Hita Pen fatty acids were neutralized adding a hydroethanolic solution of KOH and STAGs were then extracted from this solution by hexane.

2.7. Regiospecific analysis of triacylglycerols The fatty acids composition at position 2 of the TAGs and STAGs obtained was analyzed by ethanolysis with lipase Novozym 435 following the method proposed by Shimada et al. [33]. Table 1 shows the fatty acids composition at position 2 of cod liver and tuna oils.

Fig. 2. Experimental procedure for the recovery of 2-MAGs from the final alcoholysis mixture by solvent extraction [26].

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Fig. 3. Experimental procedure for the recovery and purification of STAGs from the final esterification mixture by neutralization of free fatty acids and solvent extraction of STAGs [16,28].

3. Results and discussion 3.1. Synthesis of 2-MAGs by ethanolysis 3.1.1. Selection of the most adequate lipase In previous works [26,27] the lipases selected to produce 2MAGs by ethanolysis of fish oil were: lipase D from R. oryzae, immobilized on Accurel MP1000, and Novozym 435 (N-435) from C. antarctica. Fig. 4 shows the variation of 2-MAG yield obtained by ethanolysis of cod liver oil, catalyzed by these lipases. This figure shows that yields are greater with lipase D (MP1000) than with N435 (72% and 63%, respectively). Moreover, when lipase D is used the 2-MAG yield remains constant once equilibrium is attained; however, with N-435 this yield reaches a maximum and then decreases. This diminution could be due to the transformation of 2-

Fig. 4. Influence of intensity of treatment (IOT) on 2-MAG yield in the alcoholysis of cod liver oil catalyzed by lipases D (MP1000) and Novozym 435 in an STR operating in discontinuous mode. Operational conditions: lipase D: 0.5 g cod liver oil, 0.5 g dry ethanol, 60 mg lipase D (Accurel MP1000), 3 mL acetone, 37 8C, 200 rpm. N-435, low scale: 0.75 g cod liver oil, 3 g dry ethanol, 35 8C and 300 rpm. N-435, large scale: 75 g cod liver oil, 300 g dry ethanol, 3 g lipase N-435, 35 8C and 300 rpm.

MAGs to 1(3)-MAGs by acyl-migration and the posterior degradation to glycerol, by alcoholysis catalyzed by lipase. This behaviour was also observed by Shimada et al. [33]. The analysis of the alcoholysis products by TLC (using a plate activated by immersion in a hydroethanolic solution of boric acid at 1.2% [34]) shows that 97% of the MAGs obtained were 2-MAGs, which confirmed the 1,3 specificity of the lipase in these conditions. Schmid et al. [25] and Soumanou et al. [18] reached 2-MAG yields of 95% and 72%, respectively, after the alcoholysis (by using the lipase RDL from Rhizopus delemar immobilized on Celite and polypropilene EP 100) and purification of 2-MAG by crystallization. These yields are greater than the ones obtained in this work. However, the above results were obtained with tripalmitin [25] and triolein [18] and not with high grade PUFA fish oils. Fig. 5 shows that lipase D (MP1000) quickly losses activity in the operational conditions, with almost no activity after 6 uses. This loss of activity is even more pronounced when no solvent is

Fig. 5. Influence of the number of uses of lipases D (immobilized on Accurel MP1000) and Novozym 435 on their activity, determined as 2-MAG yield obtained in the ethanolysis of cod liver oil. Operational conditions for lipase D: 240 mg lipase D (MP1000) (1:1.5, w/w), 0.5 g tuna oil, 0.5 g dry absolute ethanol, 37 8C, 24 h and 200 rpm. For N-435: 375 mg lipase, 0.75 g cod liver oil, 3 g dry absolute ethanol, 35 8C, 2 h and 300 rpm.

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727

Table 2 Influence of the intensity of treatment (IOT) on 2-MAG yield obtained by ethanolysis of cod liver oil catalyzed by Novozym 435 in a stirred tank reactor operating in continuous mode.

used, which indicates that this deactivation is not caused by acetone (results not shown). To test the influence of ethanol, the lipase was previously treated with ethanol and alcoholysis was then carried out adding oil. The results show that lipase was deactivated due to this previous contact with ethanol, since the 2MAG yields obtained in these alcoholysis are low (4% and 19% with absolute and dry absolute ethanol, respectively). Therefore, the continuous contact of lipase with ethanol deactivates lipase D (MP1000). However, lipase N-435 maintains constant activity over 4 uses (Fig. 5), after which a small reduction (10–13%) was observed. In addition, the stability of N-435 was tested for 300 h in the discontinuous STR without showing loss of activity. Therefore, although lipase D (MP1000) gave greater 2-MAG yields, its rapid deactivation in presence of ethanol does not make it suitable for using it in large-scale processes.

Reaction mixture flow rate (mL/h)

IOT (g lipase h g oil1)

2-MAG yield (%)

62.7 33.4 24.0 11.5

0.59 1.10 1.54 3.20

45.0 50.1 49.5 49.8

Operational conditions: 75 g cod liver oil, oil/absolute ethanol ratio 1:79 mol/mol, 6 g lipase N-435, 35 8C.

on the DAGs, which would then be accumulated in the reaction mixture with the subsequent loss of 2-MAG yield [27]. 3.2. Separation of 2-MAGs

3.1.2. Production of 2-MAGs by alcoholysis catalyzed with N-435 in STR operating in discontinuous and continuous modes Novozym 435 is a lipase that behaves as non-1,3 specific [29,32]; however, when a great excess of ethanol is used this lipase behaves as 1,3 specific (ethanol/oil molar ratios higher than 60:1) [20,26,33]. The small-scale experiment in the STR (Fig. 4) was scaled up to STR operating in discontinuous and continuous modes [27]. The scale-up criterion was to maintain a constant intensity of treatment (IOT) as follows: IOT ¼



mL t V½TG0



 ¼ discontinuous

 mL ¼ constant ½TG0 q continuous

After the alcoholysis reaction the reaction mixture contains 2MAGs, EEs, ethanol and, in lesser proportion, free fatty acids, DAGs and TAGs. From this mixture 2-MAGs must be purified to produce STAGs by esterification. This separation was carried out following the experimental procedure described in Section 2.3 (Fig. 2). Table 3 shows that the content of PUFAs at position 2 of the oils, and of 2MAGs before and after purification are very similar, which shows that neither alcoholysis nor the recovery process change the fatty acids profile at this position. It can also be observed that PUFA content of 2-MAGs obtained from both oils are not very different despite the different total PUFA content of these oils. This is because the PUFA content at position 2 in both oils is similar and, therefore, both oils are equally suitable for producing 2-MAGs rich in PUFAs. The 2-MAG yield is higher than 90% and the purity of 2-MAGs increased from 13–17% to 87–90% by this method. These 2-MAG fractions are therefore good substrates to synthesize STAGs by esterification of 2-MAGs. This yield in the purification of 2-MAG gave rise to an overall 2-MAG yield, after the alcoholysis and purification steps, of about 60% (65% in the alcoholysis step and 90% in the purification step). This yield is lower than the one obtained by Schmid et al. [25] and Soumanou et al. [18], although these authors did not use high grade PUFA fish oils, but tripalmitin (yield 95%) and triolein (yield 72%), respectively. However, with fish oil Schmid et al. [25] attained a maximal 2-MAG yield of 43% after the steps of alcoholysis and purification of 2-MAG by crystallization. They attribute this low yield to that purification of 2-MAG by crystallization is not appropriated when 2-MAG are rich in PUFAs due to their low melting point.

(1)

where mL is the lipase amount, t is the reaction time, V is the reaction mixture volume, [TG]0 is the initial concentration of TAGs (mol L1 or g L1) and q is the substrate mixture flow rate through the continuous STR. Fig. 4 shows that the IOT which gave rise to the highest 2-MAG yield was 1 g lipase h g oil1, and this IOT was taken as the reference to scale up the process. Fig. 4 also shows the results obtained in experiments carried out in a STR of 1 L capacity (100 times greater volume than the original one). It can be observed that the maximum 2-MAG yield (65%) was obtained at the same IOT as in the small-scale experiment, as Eq. (1) indicates. We also produced 2-MAGs in continuous mode (Fig. 1). Table 2 shows that the 2-MAG yield obtained at IOT of around 1 g lipase h g oil1 (the optimum IOT in discontinuous mode) was approximately 50%. It can be observed that this yield was maintained even at IOT of over 3 g lipase h g oil1. This yield is lower than the one obtained in discontinuous mode (63–65%) for a similar IOT. This difference could be explained admitting that the alcoholysis rate of TAGs is higher than that of intermediate DAGs; in discontinuous mode once the TAGs are consumed, the intermediate DAGs continue their transformation to 2-MAGs; in continuous mode, however, the continuous feeding of TAGs determines that the lipase acts preferably on them more than

3.3. Production of STAGs by esterification of 2-MAGs STAGs were synthesized by esterification of the 2-MAGs previously obtained by alcoholysis of fish oil and purified by solvent extraction [26,27]. These 2-MAGs were esterified with

Table 3 Total PUFA contents of the oils and PUFAs in position 2, PUFA contents of 2-MAGs before and after purification, 2-MAG purity before and after its purification and 2-MAG yields obtained after purification. Cod liver oil

PUFA content (%)

PUFA content (%) 2-MAG purity (%) 2-MAG yield (%)

Tuna oil

Oil

Position 2

Oil

Position 2

20.6

39.1

29.6

42.7

Alcoholysis mixture from cod liver oil

Alcoholysis mixture from tuna oil

2-MAG fraction after alcoholysis

2-MAG fraction after purification

2-MAG fraction after alcoholysis

2-MAG fraction after purification

36.2 17.3

39.2 89.9 90.5

45.0 23.3

45.2 87.5 90.7

The separation and purification of 2-MAGs were carried out by the method outlined in Fig. 2, carrying out three extraction steps.

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Fig. 6. Esterification of 2-MAGs with caprylic acid (CA) catalyzed by different lipases: STAG percentage in the final acylglycerol mixture, percentage of CA in STAGs and composition of CA and PUFAs (EPA + EHA) at position 2. Operational conditions: 66 mg 2-MAG, 78 mg CA (CA/2-MAG molar ratio 3:1), 3 mL hexane, 0.6 g molecular sieves, 37 8C, 200 rpm, 18 mg lipase (D/MP1000, DF/MP1000, QLM) or 54 mg (Lipozyme IM) or 30 mg (N-435) and 4 h (D/MP1000) or 24 h (DF/MP1000, Lipozyme IM; N-435, QLM).

caprylic acid [17–19,35] and the reaction was catalyzed with 1,3 specific lipases to obtain STAGs with the desired structure CA– PUFA–CA, where the PUFA at position 2 was mostly DHA. 3.3.1. Selection of the most adequate lipase Fig. 6 shows the results obtained with the five lipases tested. These experiments were carried out in operational conditions valid for all the lipases and based on the conditions used in previous works [17,29,32]. These conditions are also indicated in Fig. 6. This figure shows that the lowest STAG percentages were obtained with lipase QLM, perhaps because it was used without immobilizing. Lipozyme IM gave a high STAG percentage (95.1%) but also a high percentage of CA at position 2 (29%). It is known that Lipozyme IM is 1,3 specific, so it should not catalyze the direct esterification at position 2. However, it is also known that the immobilization support catalyzes the acyl-migration [18,35,36], which could be related with this result. Due to this high CA content at position 2 this lipase is not suitable to obtain STAGs of the desired MLM structure. With lipase N-435 STAGs were obtained with 65.2% of CA at position 2, which is a similar percentage to that of CA in the STAGs. This result indicates that this lipase does not behave as 1,3 specific in these reaction conditions. The highest STAG percentages were obtained with lipases D and DF immobilized on Accurel MP1000. With both lipases STAG percentages of over 97% were attained, with 62% of CA incorporated and only 9% at position 2. PUFA content at this position was 44-47%. These results shown that lipases D and DF immobilized on MP1000 were the most suitable to catalyze the esterification of 2-MAG with caprylic acid to obtain STAGs with the MLM structure. 3.3.2. Optimization of the esterification reaction catalyzed by lipase D (MP1000) 3.3.2.1. Esterification without solvent. Fig. 7 shows the variation in percentages of STAGs and CA incorporated into position 2 of STAGs with the IOT (lipase amount  reaction time/2-MAG amount). This figure shows that although very high IOTs were used, the maximum STAG percentage was only 30%. Moreover, the CA content at position 2 attained was 65%, which is similar to the CA content of STAGs. This result seems to indicate that lipase does not

Fig. 7. Esterification of 2-MAGs and CA catalyzed by lipase D (MP1000) in absence of solvent: influence of the IOT (lipase amount  reaction time/2-MAG amount) on the STAG percentage (%, w/w) and on the CA content at position 2 (%, mol/mol) of STAG.

behave as 1,3 specific in these conditions. The low STAG percentage suggests that without solvent the high viscosity leads to a very low mass transfer rate; as a result the products are not removed from the lipase surface and position 2 is also esterified rather than new 2-MAGs. In this case this result clearly shows that esterification must be carried out with solvent. 3.3.2.2. Influence of reaction time in the STAG percentage. Table 4 shows that for short times (low IOTs) the STAG percentages are high, since at 15 min the STAG percentage is already 88.8%, and, therefore, the initial 2-MAGs have almost been consumed. At that time the incorporation of CA into STAGs is also high (over 60%), whereas at position 2 there is 5% CA and 45% PUFAs. When the reaction time increases, new STAGs are formed from intermediate DAGs, the percentage of CA in STAGs does not vary and the content of CA at position 2 nearly doubles. At 0.5 h (IOT of 0.14 mg lipase  h/mg MAG) an adequate balance was attained between STAG percentage (nearly 95%), CA incorporation (around 61% molar) and a relatively low content of CA at position 2 (5% molar). This means that nearly 98% of the CA incorporated is located at positions 1 and 3 and, therefore, less than 3% is located at position 2. Moreover, the PUFA content of STAGs at 0.5 h is 15.2% molar (3% EPA and 12.2% DHA) and nearly 46% at position 2. This means that 100% of PUFAs are located at position 2 and that in the esterification catalyzed by lipase D (MP1000) all PUFAs that were previously located at position 2 remain in that position in the final STAGs. This rationale can be understood by taking into account the original oil compositions. Cod liver oil is less rich in DHA than tuna oil (11% and 22% molar, respectively), but its DHA distribution is quite different. In cod liver oil 90% of total DHA is located at position 2, whereas in tuna oil only 54% of DHA is located in that position. The STAGs obtained from cod liver oil contains 12% DHA, which means that all the DHA originally occupying position 2 remains in that position in the final STAG, and the composition of this position is not altered during the alcoholysis and esterification reactions due to the 1,3 specificity of the lipases. However, EPA is distributed evenly in the three positions of TAGs of cod liver oil, so after the alcoholysis and esterification reactions its content is reduced to approximately one third of the original content (7.5% in cod liver oil and 2.8% in STAGs). This STAG composition is similar to those obtained by Soumanou et al. [18] in the production of STAG by esterification

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Table 4 Esterification of 2-MAGs and CA catalyzed by lipase D (MP1000): influence of reaction time on STAG percentage and on the composition at position 2 (CA and PUFAs) of STAGs. Time (h)

STAG percentage (%, w/w)

CA in STAGs (%, mol)

CA at position 2 (%, mol)

PUFAs at position 2 (%, mol)

0 0.25 0.50 1.0 2.0 4.0

– 88.8 94.7 95.7 97.4 97.2

0 64.5 61.2 60.1 63.1 61.9

0 5.4 5.3 6.6 8.0 9.9

45.1 45.3 45.9 45.6 45.8 48.2

Operational conditions: 66 mg 2-MAG, 78 mg CA, 3 mL hexane, 18 mg D/MP1000 lipase, 0.6 g molecular sieves, 37 8C and 200 rpm. Table 5 Purification of STAGs by neutralization and separation of free fatty acids (FFA): recovery yield of STAGs and fatty acid composition of STAGs before and after purification. Fatty acids

Initial 2-MAG fractiona

STAGs in final esterification mixture (%)

STAGs after purification (%)

Position 2 of STAGs (%)

8:0 14:0 16:0 16:1n7 16:2n4 18:0 18:1n9 18:1n7 18:2n6 18:4n3 20:1n9 20:4n6 20:5n3 (EPA) 22:1n9 22:5n3 22:6n3 (DHA) Others STAG content (%, w/w) FFA content (%, w/w) Recovery yield (%)

– 6.4 14.0 6.8 0.9 0.3 11.1 0.9 2.0 3.0 3.5 0.5 7.6 0.3 2.0 37.1 3.5

62.6 2.4 5.3 2.6 0.2 0.2 4.5 0.4 0.7 1.2 1.3 0.3 2.9 1.0 0.7 12.3 1.5 71.0 27.4 80

64.0 2.3 5.1 2.5 0.3 0.2 4.5 0.4 0.7 1.2 1.3 0.2 2.7 0.9 0.6 11.6 1.3 95.2 1.5

5.4 5.3 12.4 6.3 0.3 0.2 10.5 0.9 1.9 2.8 3.0 0.3 6.1 2.2 2.0 37.9 2.6

Operational conditions: For 1 g of final esterification mixture without solvent, 8.7 mL of a hydroethanolic solution (80:20, v/v) of KOH 0.5 N and 13 mL of hexane were added; after vigorous shaking, the hexanic phase was extracted and the same volume of hexane as before (13 mL) was added; the mixture was vigorously shaken and the new hexanic phase was extracted and mixed with the previous one. a Purity of 2-MAGs: 91.6%.

of caprylic acid and 2-MAGs (previously obtained from peanut oil), although these 2-MAGs did not contain PUFAs. These STAGs contained 60.1% of caprylic acid in positions 1 and 3 and 32.8% of unsaturated fatty acids (oleic and linoleic acid) in position 2.

PUFA content remained constant in the five esterification reactions catalyzed by the same lipase.

3.3.3. Reuse of lipase Fig. 8 shows that lipase D (MP1000) remains stable over at least 5 uses in the operational conditions used in the esterification of 2MAGs with caprylic acid, since the STAG percentage and the CA and

The elimination of free fatty acids from the final esterification ˜a mixture was performed by the procedure proposed by Hita Pen [16,28] and described in Section 2.7 (Fig. 3). This method consisted in the neutralization of free fatty acids in the mixture (from which the solvent had been previously eliminated) with a hydroethanolic solution of KOH and the extraction of acylglycerols with hexane. Table 5 shows the fatty acids profiles and the purity of STAGs before and after the separation and the final STAG recovery yield obtained (80%). This table shows that the separation of free fatty acids is effective, since its percentage was reduced from 27.4% to 1.5%. In this way the STAG purity was enhanced from 71% to 95.2%, with a recovery yield of 80%. The fatty acids profile of STAGs is the same before and after this separation. This purification has also been carried out by Irimescu et al. [20] by chromatography, using silica gel as stationary phase and hexane/diethyl ether 90:10 (v/v) as mobile phase; by this procedure these authors obtained a STAG yield of 71%.

3.4. Separation of STAGs

4. Conclusions

Fig. 8. Influence of the reuse of lipase D (MP1000) on the STAG percentage and CA and PUFA content in STAGs obtained by esterification of 2-MAGs with caprylic acid. Operational conditions: 66 mg 2-MAG, 78 mg CA, 3 mL hexane, 18 mg D(MP1000) lipase, 0.6 g molecular sieves, 37 8C, 200 rpm and 0.5 h.

 In the alcoholysis step lipase D from R. oryzae, immobilized on Accurel MP1000 and Novozym 435 are suitable to produce 2MAGs. However, lipase D quickly loses activity because of the continued contact with ethanol. Novozym 435 was the most stable because it maintained its activity unaltered for more than

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300 h in the operational conditions. This lipase is suitable to produce 2-MAGs in STR in both continuous and discontinuous mode. The greatest 2-MAG yields were obtained in discontinuous mode (63–65%). The optimum IOT was 1 g lipase  h g oil1, because at higher IOTs 2-MAGs are degraded to glycerol. Separation of 2-MAGs by extraction with hexane and ethanol– water allows 2-MAG purity of about 89% and a recovery yield of 91%. This method uses low toxicity solvents and relatively low amounts of solvents. In the esterification of 2-MAGs with CA, of all the lipases used, lipases D and DF, immobilized on Accurel MP1000, produced the highest STAG percentages (higher than 90%). With lipase D (MP1000) the percentage of STAGs in the final acylglycerol mixture was 95%. These STAGs contain 64% CA, of which 98% is at positions 1 and 3. Position 2 contained 5% CA and 45% PUFAs, which shows that all PUFAs that were located in position 2 in the original oil remain in that position in the final STAGs. These STAGs were obtained at very low IOT (0.14 g lipase  h/g 2MAG). Lipase D immobilized on Accurel MP1000 is stable in the operational conditions used in the esterification reaction, since no significant loss of activity was observed after at least 5 uses. The purification of STAGs was carried out by neutralization of free fatty acids with a hydroethanolic solution of KOH and extraction of STAGs with hexane. By this method STAG purities of over 95% and separation yields of about 80% were achieved.

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