The production of monoacylglycerol rich in polyunsaturated fatty acids (PUFA) via enzymatic glycerolysis of sardine oil in a homogeneous system was evaluated. Reactions were conducted in two different tert-alcohols. Based on the phase equilibrium data, the amount of solvent added to create a homogeneous system has been calculated and optimized. The immobilized lipase used in this work was Lipozyme RM IM from
Marine raw materials are rich sources of omega-3 PUFA, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The health benefits of omega-3 PUFA have been demonstrated (Nichols
The overall glycerolysis reaction is given by TAG+2 GLY ⇆ 3 MAG [1] TAG+GLY ⇆ MAG+DAG [2] DAG+GLY ⇆ 2 MAG [3]
To avoid external mass transfer limitations due to the immiscibility between reactants, oil and glycerol, a solvent can be added to the system to create a homogenous phase. In the literature, other studies about solvent-free glycerolysis have been found (Fregolente
According to Damstrup
The most widely used lipases to carry out enzymatic glycerolysis reactions are from
Several authors use Novozym 435 as biocatalyst to carry out the glycerolysis reaction due to its highest activity (Pawongrat
Some commercially available 1,3 specific lipases are from
In this work the enzymatic glycerolysis of sardine oil in two different tert-alcohols (tert-pentanol and tert-butanol) as reaction media has been studied. The commercial 1,3-specific lipase Lipozyme RM IM (from
The sn-1,3 specific lipase Lipozyme RM IM (immobilized
The standards of MAG, DAG, FFA and fatty acid methyl ester (FAME) for the chromatography analysis were purchased from Sigma. All other solvents and reagents were of analytical or chromatographic grades from VWR.
The mixture of sardine oil, glycerol, water and tert-alcohol (TB or TP) was incubated at 50 °C in a water bath with orbital agitation in different vials. The reaction was initiated by the addition of the lipase. The enzyme loading was 10 wt%, based on the total amount of substrates. At different time intervals, the corresponding vial was withdrawn and filtered through a microfilter (0.45 μm, Sartorius RC) to stop the reaction by removing the lipase. All samples were stored at −18 °C prior to analysis. The samples were analyzed at least in duplicate.
The conditions of the kinetic experiments performed in this work are presented in
Glycerolysis reaction conditions (mole ratio of substrates, water content and amount of tert-alcohol added to the system) at 50 °C (Solaesa
Reactants mole ratio (glycerol:oil) | Tert-alcohol (wt%) | Amount of water (wt% of glycerol) | ||||
---|---|---|---|---|---|---|
1:1 | 50 | 4 | 7 | 10 | 20 | 30 |
3:1 | 60 | – | 7 | 10 | 12 | 24 |
5:1 | 65 | – | 10 | 12 | 17 | 28 |
Amount of tert-alcohol added in other glycerolysis systems found in the literature.
Oil | Mole ratio (glycerol:oil) | T (° C) | % wt of tert-alcohol | Reference |
---|---|---|---|---|
Palm | 8:1 | 45 | 82% of TB | (Majid et al., |
Olive | 6:1, 3:1, 0.8:1, 0.5:1.5 | 40, 55, 70 | 44% of TB | (Voll et al., |
Olive | 9:1, 6:1, 3:1, 1:1 | 40, 55, 70 | 44% and 80% of TB | (Krüger et al., |
Triolein | 2:1 | 40 | 93% of TP | (Rendón et al., |
The neutral lipid profile (MAG, DAG, TAG and FFA) was analyzed by a normal-phase high performance liquid chromatography (NP-HPLC). The chromatographic apparatus consisted of an HPLC system (Agilent 1200) formed by a quaternary pump and an auto-injector. The chromatographic separation of the compounds was carried out at room temperature with a Lichrospher Diol column (5 μm, 4 mm×250 mm) and detection was performed in an evaporative light scattering detector (Agilent 1200 series) at 35 °C and 0.35 MPa. Gradient elution was achieved by mobile phases A (isooctane) and B (methyl tert-butyl ether:acetic acid =99.9:0.1, v/v). The course of the gradient was as follows: first, solvent A was flowing for 1 min, after that, solvent B was added in three steps, at up to 10% in 10 min, to 44% in 22 min and to 100% in 30 min. Subsequently, solvent B was decreased to 0% in 17 min to return to the initial conditions. Finally, the stationary phase was rinsed with solvent A for 2 min. Injection volumes of 10 μl and the elution flow-rate of 1 ml/min were used in all experiments. The different family of compounds were identified and quantified by using calibration curves of the corresponding standard compounds of DAG, a mixture of dipalmitin (26657-95-4) and diolein (25637-84-7), MAG, a mixture of 1-monopalmitin (542-44-9), 1-monoolein (111-03-05), 2-monoolein (3443-84-3) and monodocosahexaenoin and FFA, a mixture of oleic acid (112-80-1) and palmitic acid (57-10-3) in tert-pentanol. In the case of TAG, the calibration curve used was made with the refined sardine oil (TAG ≥99%) because the response factor of the oil as a complex mixture of TAG was very different than the pure standards of TAG. The calibration method has been validated by analyzing two different samples formed by MAG, DAG and FFA standards and sardine oil in the concentration range of the corresponding calibration curves to obtain good reliability.
The analysis of remaining glycerol was performed using High-Temperature Gas Chromatography (HT-GC). The method and the calibration procedure were previously developed (Solaesa
The MAG fraction separated by NP-HPLC under the optimal reaction conditions with both solvents was collected according to its retention time. Separations were repeated at least six times to obtain enough sample for the fatty acid profile analysis. The fractions were stored in special flasks to evaporate the solvent under vacuum using a rotary evaporator (Heibolph VV2000) at 40 °C. Then the samples were transferred to screw-capped tubes to carry out the derivatization for conversion to methyl esters by the AOAC method (AOAC Official Method 991.39,
The separation of the different acylglycerols was performed by TLC on silica gel plates (Silica gel 60 F254, Merck). A similar method of Jin
In a first step, the glycerolysis of sardine oil using both tert-alcohols as reaction media, but without any enzyme as catalyst was performed. The experimental conditions were the following: 50 °C, initial mole ratio glycerol to oil 3:1 and solvent to substrates ratio of 1.2:1 w/w. It was observed that the glycerolysis reaction does not present an auto-catalytic behavior since under these experimental conditions the observed reaction rate was almost zero. Yang
In this work, further glycerolysis experiments were performed at 50 °C, solvent to substrates ratio of 1.2:1 wt/wt and 10 wt% of Lipozyme RM IM loading at different glycerol to oil mole ratios (1:1, 3:1 and 5:1). In these experiments no water was added and it was observed that the glycerolysis reaction did not occur. This might be because the carrier of Lipozyme RM IM is hydrophilic and the glycerol, as a hydrophilic substrate, can be preferably adsorbed onto the support depraving the essential water from the enzyme. Therefore, the water content in the reaction medium must be an essential factor in the activity of Lipozyme RM IM. In the next section the effect of water addition in the glycerolysis system catalyzed by Lipozyme RM IM will be presented and discussed.
As has been already mentioned, the carrier support of Lipozyme RM IM is hydrophilic. In the presence of a hydrophilic support, the tert-alcohol and mainly glycerol (μ=2.617, log P=−1.84 and ε (23 °C) =41.01) (Riddick
First, some preliminary experiments were carried out to study the effect of the polarity medium in the retention of titrable water by the enzyme. The moisture of different reaction media (tert-alcohol+glycerol+sardine oil) at different reactant mole ratio glycerol:oil, from 1:1 to 6:1 was first determined. Then, Lipozyme RM IM with a known amount of titrable water was added at 10% w/w based on the weight of the reactants. After 24 hours of contact time at 50 °C (time enough to reach sorption equilibrium) the water content of the reaction medium was again determined. Total titrable water retention by Lipozyme RM IM was evaluated according to
Titrable water retention by Lipozyme RM IM at different initial molar ratio glycerol:oil in both tert-alcohols. Lines are to guide the eye.
The effect of water content in the reaction medium on MAG and FFA yield at three different initial reactant mole ratios (glycerol:oil), 1:1, 3:1 and 5:1, was studied at 50 °C by varying the amount of water added to the reactive system in the range of 4–30 wt% of water based on glycerol weight (
Effect of water content, based on glycerol weight, on glycerolysis of sardine oil at different initial mole ratio glycerol:oil (a) 1:1 (b) 3:1 and (c) 5:1. Reaction conditions: 10 wt% Lipozyme RM IM (based on substrates), 50 °C at 20 h.
Substrate mole ratio, glycerol:oil, has different effects on lipase-catalyzed glycerolysis. According to the equilibrium law, an increase in the glycerol content will shift the equilibrium towards MAG production. In the literature, it has been also described that glycerol can act as an effective stabilizer against thermal and solvent de-activation (Zhong
The effect of glycerol:oil mole ratio on the reaction rate was studied at 50 °C, 10 wt% of lipase loading, by varying the mole ratio from 1:1 to 5:1.
Effect of substrate mole ratio on the time course of glycerolysis of sardine oil (a) TB, (b) TP at different initial mole ratio glycerol:oil (-○-) 1:1, (-□-) 3:1, (-Δ-) 5:1. Reaction conditions: 10 wt% Lipozyme RM IM (based on oil and glycerol), 50 °C and 24–30 wt% of added water based on glycerol weight (see
Solvents can have different effects on reaction systems. They help to create a homogeneous reaction system and improve mass transfer by reducing the viscosity of the reaction medium. When polar solvents are used, essential water from the lipase can be removed leading to a decrease in the lipase activity. In this work two tertiary alcohols, tert-butanol and tert-pentanol, have been considered as reaction media. Log P is one of the most widely used parameters to correlate the solvent properties to the enzyme activity. Taking into account the log P of TB and TP (0.35 and 0.89 respectively), TB is slightly more hydrophilic than TP; therefore water retention by the lipase will be lower when using TB as reaction medium and slower reaction rates can be expected.
Effect of solvent type and water content on the time course of glycerolysis of sardine oil at different amounts of water in TP (grey lines) and in TB (black lines). (a) 7 wt%, (b) 10 wt%, (c) 12 wt%, (d) 24 wt%. (-●-) TAG in TB, (-○-) MAG in TB, (-
Based on the results presented in this work, the optimal conditions for MAG production with 10 wt% loading of Lipozyme RM IM, based on substrate weight, at 50 °C were the following: initial mole ratio glycerol:oil 3:1, water content in glycerol 12 wt% and 60 wt% of the corresponding tert-alcohol to provide a homogeneous medium. Mole ratio higher than 3:1 does not lead to an increase in MAG yield (
Time course of glycerolysis of sardine oil under optimal conditions: glycerol/oil=3:1 (mol/mol), 10 wt% Lipozyme RM IM (based on oil and glycerol), 50 °C and 12% of water content in the glycerol. (a) in TB and (b) in TP. TAG (-□-), MAG (-○-), DAG (-◊-) and FFA (-x-).
From
The FA profile of the different MAG fractions obtained under the optimal conditions was analyzed by GC. MAG fractions at two different reaction times (4 h and 20 h) were separated by HPLC and collected to determine their fatty acid profiles (
Fatty acid composition of sardine oil and at the sn-2a position of TAG (% mol) (Solaesa
Fatty acid | Sardine oil | TP medium | TB medium | ||||
---|---|---|---|---|---|---|---|
TAG |
|
4h (70%) | 20h (77%) | 4h (22%) | 20h (66%) | ||
Myristic (M) | 14:0 | 12.4 | 41.8 | 12.2±0.3 | 12.0±0.2 | 12.3±0.5 | 11.9±0.5 |
Palmitic (P) | 16:0 | 22.8 | 41.9 | 25.2±0.3 | 25.1±0.1 | 27.1±0.6 | 25.6±0.2 |
Palmitoleic (Po) | 16:1n-7 | 12.5 | 38.1 | 13.4±0.3 | 12.6±0.2 | 13.5±0.1 | 13.3±0.2 |
Stearic (S) | 18:0 | 3.6 | 6.7 | 4.4±0.4 | 4.3±0.1 | 6.0±0.2 | 4.6±0.1 |
Oleic (O) | 18:1n-9 | 9.8 | 16.6 | 12.3±0.3 | 11.5±0.3 | 15.3±0.3 | 12.4±0.5 |
Vaccenic (V) | 18:1n-7 | 3.7 | 10.1 | 4.3±0.2 | 4.1±0.1 | 5.0±0.1 | 4.3±0.1 |
Linoleic (Lo) | 18:2n-6 | 2.5 | 32.7 | 3.0±0.2 | 2.7±0.1 | 3.6±0.7 | 3.0±0.1 |
Linolenic (Ln) | 18:3n-3 | 1.0 | 29.0 | 1.1±0.1 | 1.0±0.1 | 1.2±0.1 | 1.1±0.1 |
Steriadonic (St) | 18:4n-3 | 3.3 | 26.4 | 1.9±0.2 | 2.5±0.2 | 1.1±0.1 | 1.8±0.1 |
Eicosatrienoic (Et) | 20:3n-3 | 1.3 | 19.6 | 1.0±0.1 | 1.2±0.1 | 0.7±0.1 | 1.0±0.1 |
Eicosapentaenoic (Ep) | 20:5n-3 | 18.3 | 12.1 | 14.4±0.3 | 15.5±0.2 | 10.1±0.2 | 14.3±0.2 |
Docosapentaenoic (Dp) | 22:5n-3 | 1.8 | 76.4 | 1.7±0.1 | 1.8±0.1 | 1.1±0.1 | 1.7±0.1 |
Docosahexaenoic (Dh) | 22:6n-3 | 7.0 | 82.8 | 5.1±0.2 | 5.7±0.2 | 3.0±0.1 | 5.0±0.2 |
(a)%
From
Evolution of the different types of FA in the MAG fraction according to the conversion degree. Black symbols represent reaction with TB and white symbols with TP. SFA (-●-) or (-○-), MUFA (-◆-) or (-⃟-) and PUFA (-■-) or (-□-).
TLC analyses have been performed at certain reaction times (from 0.5 to 20 h) to check the formation of 1(3)-MAG and 2-MAG isomers. The FA profile of 1(3)-MAG and 2-MAG fractions at 8 h of reaction time was known by extraction of the appropriate band and the subsequent methylation for the FAME analysis in GC (data not shown). The results showed that the majority of EPA is found as 1(3)-MAG, while DHA is found mainly in 2-MAG. These outcomes are in agreement with the FA at the sn-2 position in the TAG (
In this work, the glycerolysis of sardine oil in two different tert-alcohols (TP and TB) as reaction medium has been performed. The amount of solvent added has been optimized to ensure the homogeneity of the reaction medium. The 1,3 specific lipase Lipozyme RM IM has been used as catalyst. This enzyme has been proved to be water dependent in this glycerolysis system, showing no activity without water and a maximum in the MAG production when adding around 12% water, based on glycerol, at a mole ratio glycerol:oil of 3:1. A high content of MAG, up to 70 wt%, can be reached for both tert-alcohols, with a low FFA content. Furthermore, the fatty acid profile of 1(3)-MAG and 2-MAG fractions show that the original FA at the sn-2 position in the TAG is preserved, finding the majority of DHA as 2-MAG.
This research could be an economical alternative to take advantage of by-products of marine oils rich in PUFA. However, further work must be done to purify the MAG derivatives rich in PUFA due to their health benefits to be used in the pharmaceutical and food industries.
The authors acknowledge the Spanish Government through MINECO and the European Regional Development Fund (ERDF) for financial support to the project CTQ2012-39131-C02-01. They wish to thank Novozymes A/S for kindly supplying the enzymes. RM acknowledges MINECO for an FPI grant (reference BES-2013-063937). SLB acknowledges Mexican Secretariat of Public Education and Technological University of Morelia for a fellowship through PROMEP program. AGS acknowledges University of Burgos for a fellowship.