Kinetic study and kinetic parameters of lipase‐catalyzed glycerolysis of sardine oil in a homogeneous medium

1 DOI: 10.1016/S1872-2067(15)61040-3 CJC-2015-11-042 编辑润色稿 Kinetic study and kinetic parameters of lipase-catalyzed glycerolysis of sardine oil in a homogeneous medium. Ángela García Solaesa, María Teresa Sanz*, Sagrario Beltrán, Rodrigo Melgosa Department of Biotechnology and Food Science (Chemical Engineering Section), University of Burgos, 09001 Burgos. Spain Abstract The production of polyunsaturated fatty acids (PUFAs) concentrates by enzymatic catalysis has gained interest due to their stereospecificity and the milder conditions needed compared to the use of inorganic catalysts. The enzymatic glycerolysis of sardine oil by Lipozyme 435 to get PUFA concentrates in the forms of diand monoacylglycerols (DAGs, MAGs) in an optimized amount of tert-butanol as the organic solvent was studied. First, mass transfer limitation of the reaction system was analyzed. The effect of different operating variables such as lipase loading, temperature and feed composition was investigated. A semi-empirical kinetic model based on the reversible elementary reactions of glycerolysis and hydrolysis of the glycerides was employed to correlate the experimental kinetic data. A mole ratio glycerol:oil of 3:1 was the optimum, which produced more than 84 wt% of MAG at 50ºC. A comparison with other glycerolysis systems was performed using MAG yield, reaction rate and significance of kinetic parameters. Keywords: lipase-catalyzed; glycerolysis; tert-butanol; mass transfer; kinetic model. Received 18 November 2015. Accepted 4 January 2016. ∗ Corresponding author. Tel.: +34 947 258810. Fax: ++34947258831. E-mail address: [email protected] 1. Introduction Fish oil is rich in omega-3 (n-3) polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid. The health benefits of n-3 fatty acids have been widely established in the literature [1-3]. Among the different types of lipid derivatives containing PUFA concentrates, MAG and DAG has good bioavailability [4, 5]. In addition, MAG or its mixtures with DAG account for 75 % of worldwide emulsifier production [6]. The process currently used in industry to obtain MAG is glycerolysis using an inorganic alkaline catalyst at high temperature (220 - 260ºC). This method has several disadvantages such as it gives a dark color and burnt taste as well as high energy consumption. Furthermore, chemical glycerolysis is not suitable for producing MAG rich in PUFA due to oxidization problems. Enzymatic glycerolysis is an attractive alternative for the production of MAG rich in PUFA since the reaction can be carried out under mild conditions [7] and structured products are obtained. The immiscibility of the reactants, glycerol and oil leads to mass transfer limitation in the glycerolysis of oils. Different approaches have been used in the literature to improve the contact between the reactants and hence reduce mass transfer limitation. Lipase-catalyzed glycerolysis has been carried out in different reaction media such as organic solvents [8], compressed fluids [9], and ionic liquids [10] in order to improve the mass transfer. 2 Recently the use of different surfactants to increase the interfacial area [11] and ultrasound irradiation [12] have also been proposed to reduce mass transfer limitation. This paper is part of a wider project for the optimization of MAG production by enzymatic glycerolysis of sardine oil. First, different tert-alcohols were evaluated as the solvent used to create a homogeneous phase [13]. Tertiary alcohols enhance the enzyme activity and accelerate the reaction rate as compared to the solvent-free system [14]. In a previous work, tert-pentanol was selected as the solvent and the effect of the glycerol:oil mole ratio was evaluated for its effect on kinetic behavior and MAG yield. The glycerolysis product was subsequently fractionated by a two-step molecular distillation to obtain a concentrated product of MAG and DAG rich in PUFA [15]. In this work, a different tertiary alcohol, tert-butanol was used as the solvent. Tertbutanol has been used in different glycerolysis systems of vegetable oils such as olive oil [16, 17], palm oil [18], camellia oil [19] and sunflower oil [8, 20]. The main objective of this work is to present a detailed kinetic study of enzymatic glycerolysis of refined sardine oil in tert-butanol as the solvent catalyzed by a commercial lipase Lipozyme 435. The amount of tertbutanol added to create a monophasic system has been optimized based on liquid-liquid equilibrium (LLE) data previously determined [13]. This value was compared with the amount of tert-butanol added to other glycerolysis systems. The results in terms of MAG and DAG yields were compared with literature data reported for different type of oils and related to the high activity of the lipase for short and medium chain length fatty acids. First, the external and internal mass transfer resistances were analyzed in the heterogeneous system of the immobilized lipase. Mass transfer limitation can play an important role in the reaction. However, in most glycerolysis studies reported in the literature, no mass transfer studies were performed. Mathematical models are needed to predict and optimize the industrial process. However, not many works in the literature deal with the kinetic modeling of glycerolysis. One of the first works was carried out by Moquin et al. [9]. In that work, the kinetics of the non-catalyzed glycerolysis of soybean oil in SCCO2 medium were correlated by a sequence of reversible reactions to take into account the parallel hydrolysis reaction. The same model was used by Valerio et al. [11] in the kinetic study of solvent-free lipase-catalyzed glycerolysis of olive oil by Novozym 435 with Triton X-100 as surfactant. Although glycerolysis and hydrolysis reactions were proposed, no information on the experimental FFA production and rate of change of glycerol were provided and only the TAG, MAG and DAG concentrations were used in the fitting procedure to obtain the kinetic parameters. The mechanism of glycerolysis and hydrolysis of pure POP (1,3-palmitin-2-olein) by Rhizopus arrhizus lipase was studied by Tan and Yin [21] by including hydrolysis, esterification and isomerization of MAG and DAG. Cheirsilp et al. [22] proposed a Ping-Pong Bi Bi model that focused on the kinetics of the hydrolysis and esterification steps involved in the glycerolysis of palm oil in an acetone/isooctane mixture (3:1 v/v). Water was dissolved in glycerol (10 % w/v of water added to glycerol) and therefore a large amount of 3 water was present in the reaction medium. Recently, Voll et al. [17] proposed a kinetic model based on the ordered-sequential Bi Bi mechanism for a lipase-catalyzed glycerolysis system of olive oil in tert-butanol as the solvent. In that work, the reaction products were expressed as total amount of MAG, DAG, TAG and FFA by weight percentage on a solvent-free basis composition. No experimental information on the glycerol concentration rate of change was provided. Fiametti et al. [12] used a similar model to the one proposed by Voll et al. [17] in the glycerolysis of olive oil by ultrasound irradiation. However, the parameters were not provided in the open literature although they could be available upon request to the authors. In this work, a similar approach to that previously proposed by Moquin et al. [9] was used. The kinetic parameters were compared when possible with previous values reported in the literature. This model was able to consider the concentration of all the compounds involved in the glycerolysis system: TAG, DAG, MAG, FFA, glycerol and water. 2. Experimental 2.1 Materials Refined sardine oil was provided by Industrias Afines S.L. (Spain) with a water content of 0.19 ± 0.03%. Glycerol was purchased from Sigma Aldrich with a purity of ≥ 99.5% and a water content of 0.18 ± 0.04%. Tert-butanol (TB) was purchased from Merck with a purity of ≥ 99% and a water content of 0.20 ± 0.03%. The products were stored over activated 3 Å molecular sieve to keep them dry. The food grade lipase Lipozyme 435 from Candida antarctica (immobilized on a macroporous hydrophobic acrylic resin) was donated by Novozymes A/S (Bagsvaerd, Denmark). The water content of this lipase was 3.5 ± 0.3% as determined in triplicate by Karl-Fisher titration with a Mitsubishi CA-20 moisture meter. According to Novozymes A/S, the specific activity of the lipase is ≥ 8000 propyl laurate units/g. No additional water was added to the system. Therefore, water present in the reaction medium came only from the reactants. 2.2 Enzymatic Glycerolysis of Sardine Oil Different vials containing a mixture of sardine oil, glycerol and TB were incubated at different temperatures from 303 to 333 K in a water bath with stirring. Different mole ratios of substrate and enzyme dosage were also studied. The amount of TB added was fixed at a mass ratio of 1.5:1 (TB:substrates) on the basis of previous studies on LLE [13]. At selected time intervals (from five minutes up to eight hours), a sample of the reaction mixture 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 ᵒC prior to analysis. The reusability of Lipozyme 435 in this process was tested by recycling the immobilized enzyme in six batches. After each run, the lipase was washed once with TB, and then twice with hexane in order to eliminate the remaining compounds. Afterwards, the lipase was dried at 303 K and stored in a desiccator under vacuum. No significant reduction in enzyme activity was found. In any event, a fresh biocatalyst was used in each run. Tert-butanol was evaporated under vacuum using a rotary evaporator (Heibolph VV2000) at 333 K. In this way, 4 TB can be reused by using the molecular sieve to eliminate the water content. 2.3 Analysis of the reaction products The neutral lipid profile (TAG, DAG, MAG and FFA) was analyzed by a normal phase high performance liquid chromatography (NP-HPLC). The chromatographic apparatus consisted of a 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 by an evaporative light scattering detector (Agilent 1200 series) at 35 ºC 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 method and calibration procedure were previously reported [23]. The regioisomers of DAG and MAG could not be distinguished by the applied analytical procedure. Therefore the total amount of MAG and DAG was reported for the kinetic experiments. The analysis of the remaining glycerol was performed by a high temperature gas chromatograph (HT-GC) system (HP 6890 Series GC System) equipped with a flame ionization detector (FID), a fused silica capillary column of 30 m × 0.25 mm i.d. coated with a 0.25 µm film thickness of 65% phenyl methylpolisiloxane (65HT) as the stationary phase and an Agilent Technologies 7683B Series automatic injector. The method and calibration procedure were previously reported [13]. 2.4 Kinetic modeling The overall glycerolysis reaction can be described by: TAG + 2 Gly 3 MAG [1] However, glycerolysis is believed to follow a two-step reaction. First, one molecule of glycerol reacts with one molecule of TAG to yield one molecule of DAG and another molecule of MAG. The reaction of one molecule of DAG with one molecule of glycerol can also take place to yield two molecules of MAG: TAG + Gly DAG + MAG [2] DAG + Gly 2 MAG [3] The breakdown of TAG due to reaction with MAG can also occur to produce two molecules of DAG [9]: TAG + MAG 2 DAG [4] Even in the presence of small amounts of water in the glycerolysis reaction medium, unwanted hydrolysis reactions must be considered: TAG + H2O DAG + FFA [5] DAG + H2O MAG + FFA [6] k 1 k5 k3 k 2 k4 k 6 k 7 k9 k 8 k 10 k 1 k7 5 MAG + H2O GLY + FFA [7] Kinetic models are needed to predict and simulate the reaction. By formulating the mass balance equation for all the species of the reaction system, the concentration profile versus time can be obtained. In this way, the process can be optimized. The rate of change in concentration for each of the reaction components are described by the following differential equations: 𝑑𝑑𝑛𝑛𝑇𝑇𝑇𝑇𝑇𝑇 𝑛𝑛𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 ⁄ 𝑑𝑑𝑑𝑑 =−𝑘𝑘1𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝑇𝑇𝑡𝑡𝐺𝐺+𝑘𝑘2𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇−𝑘𝑘5𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇+ 𝑘𝑘6(𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇)2−𝑘𝑘7𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂+𝑘𝑘8𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇 [8] 𝑑𝑑𝑛𝑛𝐷𝐷𝐷𝐷𝐷𝐷 𝑛𝑛𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 ⁄ 𝑑𝑑𝑡𝑡 =𝑘𝑘1𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝑇𝑇𝑡𝑡𝐺𝐺-𝑘𝑘2𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇−𝑘𝑘3𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝑇𝑇𝑡𝑡𝐺𝐺+𝑘𝑘4(𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇)2+ 2𝑘𝑘5𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇−2𝑘𝑘6(𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇)2+𝑘𝑘7𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂−𝑘𝑘8𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇−𝑘𝑘9𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂+𝑘𝑘10𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇 [9] 𝑑𝑑𝑛𝑛𝑀𝑀𝐷𝐷𝐷𝐷 𝑛𝑛𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 ⁄ 𝑑𝑑𝑡𝑡 =𝑘𝑘1𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝑇𝑇𝑡𝑡𝐺𝐺-𝑘𝑘2𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇+2𝑘𝑘3𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝑇𝑇𝑡𝑡𝐺𝐺−2𝑘𝑘4(𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇)2− 𝑘𝑘5𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇+𝑘𝑘6(𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇)2+𝑘𝑘9𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂−𝑘𝑘10𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇−𝑘𝑘11𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂+𝑘𝑘12𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇 [10] 𝑑𝑑𝑛𝑛𝐷𝐷𝑡𝑡𝐺𝐺 𝑛𝑛𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 ⁄ 𝑑𝑑𝑡𝑡 = −𝑘𝑘1𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝑇𝑇𝑡𝑡𝐺𝐺 +𝑘𝑘2𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇−𝑘𝑘3𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝑇𝑇𝑡𝑡𝐺𝐺+𝑘𝑘4(𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇)2+𝑘𝑘11𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂−𝑘𝑘12𝑥𝑥𝑇𝑇𝑡𝑡𝐺𝐺𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇 [11] 𝑑𝑑𝑛𝑛𝐹𝐹𝐹𝐹𝐷𝐷 𝑛𝑛𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 ⁄ 𝑑𝑑𝑡𝑡 = 𝑘𝑘7𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂−𝑘𝑘8𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇+𝑘𝑘9𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂−𝑘𝑘10𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇+𝑘𝑘11𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂−𝑘𝑘12𝑥𝑥𝑇𝑇𝑡𝑡𝐺𝐺𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇 [12] 𝑑𝑑𝑛𝑛𝐻𝐻2𝑂𝑂𝑛𝑛𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 ⁄ 𝑑𝑑𝑡𝑡 =−𝑘𝑘7𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂+𝑘𝑘8𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇−𝑘𝑘9𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂+𝑘𝑘10𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇−𝑘𝑘11𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂+𝑘𝑘12𝑥𝑥𝑇𝑇𝑡𝑡𝐺𝐺𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇 [13] As explained above in the analytical procedure, the stereoisomers of DAG and MAG could not be distinguished and no difference was made between them in the model. The concentrations of the reaction products were expressed on a solvent-free basis. TAG, DAG, MAG, FFA and glycerol concentrations were experimentally determined. The water concentration could not be measured versus reaction time. According to Moquin et al. [9], it is possible to estimate the change in water concentration by subtracting the experimental FFA concentration from the initial water concentration since the formation of one mole FFA requires one mole of k11 k 12 6 water (Equations 5-7). The rate constants for the six kinetic equations were obtained by solving the set of differential equations simultaneously. The differential equations were solved numerically with a fourth order Runge-Kutta method and the parameters were optimized by minimizing the following objective function (O.F.): 𝑂𝑂. F. = ∑ ∑ �xi,exp−xi,calc�2 n i=1 all samplesnsamples ·100 [14] using the simplex Nelder-Mead method. The subscript “i” refers to the different components in the glycerolysis system: TAG, DAG, MAG, FFA, glycerol and water. The subscripts “exp” and “calc” refer to the experimental and calculated mole fraction of the different components for each experimental kinetic data point (nsamples) The root mean square deviation (rmsd) was calculated to evaluate the quality of the fitting: 𝑟𝑟𝑟𝑟𝑟𝑟𝑑𝑑=�∑�𝑤𝑤𝑖𝑖𝑒𝑒𝑒𝑒𝑒𝑒−𝑤𝑤𝑖𝑖𝑐𝑐𝑡𝑡𝑡𝑡𝑐𝑐�2 𝑁𝑁𝑂𝑂𝑁𝑁𝑁𝑁 𝑖𝑖=1 𝑁𝑁𝑂𝑂𝑁𝑁𝑁𝑁 [15] where NOBS is the total number of kinetic data points for all the kinetic experiments and 𝑤𝑤𝑖𝑖𝑒𝑒𝑒𝑒𝑒𝑒 and 𝑤𝑤𝑖𝑖𝑐𝑐𝑡𝑡𝑡𝑡𝑐𝑐 are the experimental and calculated weight fractions for the reaction compounds. 3. Results and discussion 3.1 Mass transfer analysis External and intraparticle mass transfer resistance can influence the observed reaction rate in heterogeneous catalytic processes such as immobilized lipase biocatalysis. Before the study of the effect of the kinetic variables, the mass transfer rate was analyzed. Tert-butanol was used as the organic solvent to provide an environment where oil and glycerol can interact since both reactants are completely immiscible. Tert-butanol helps to create a homogeneous phase and also decreases the viscosity of the reaction medium since both reactants are highly viscous, especially glycerol (Table 1). To evaluate the external mass transfer resistance, the glycerolysis reaction was carried out at different stirring speeds, from 120 to 200 rpm, while keeping constant the rest of the reaction conditions. The results are presented in Table 1. From these results, it can be concluded that there was no increase in the initial reaction rate of MAG formation in the speed range studied. This result was expected since external diffusion does not usually control the overall rate unless the stirring speed is very low or the reaction mixture is very viscous [24]. Tert-butanol helps to decrease the viscosity of the reaction medium since its viscosity is 100 time smaller than the viscosity of glycerol (Table 1), resulting in a low external mass transfer resistance and it acts as an inert carrier for the reactants to the active site of the enzyme. Hence, 170 rpm was chosen for all the glycerolysis reactions. Slow intraparticle diffusion can reduce the overall reaction rate, especially if the reactant molecules are large [25] and have a low mobility in the lipase support. Chesterfield et al. [26] analyzed the relative magnitude of the 7 external liquid mass transfer resistance to the combined internal resistances (intraparticle diffusion and reaction resistances) in the ethanolysis of waste cooking oil using Novozym 435 by plotting the reciprocal initial reaction rate (1/ro) as a function of inverse lipase loading (1/m). This plot should be a straight line, with a slope proportional to the combined internal resistances, and the intercept is proportional to the interphase mass transfer resistance. Figure 1 illustrates this linear dependence in the glycerolysis of sardine oil. The linear fit proved that the rate controlling step is the combined internal resistances since the intercept can be considered negligible. To evaluate the intraparticle diffusion effect, the lipase Lipozyme 435 was separated into two fractions by a 400 µm sieve (46 wt % of Lipozyme 435 particles with ϕp > 400 µm). Kinetic experiments were carried out with each of the fractions obtained and compared with the results obtained with unsieved lipase. Figure 2 shows that the initial reaction rate of MAG formation was increased by decreasing the particle size of Lipozyme 435. This may indicate internal mass transfer limitation for the larger particles, although the same MAG yield was achieved at long reaction time. A significant pore diffusion resistance was also found by Chesterfield et al. [26] in the ethanolysis study with Novozym 435 (technical grade of Candida antartica). The experimental Thiele modulus, φexp, was calculated to evaluate the intraparticle resistance [27]: 𝜙𝜙𝑒𝑒𝑒𝑒𝑒𝑒=�𝑑𝑑𝑒𝑒 6�2𝑟𝑟𝑒𝑒𝑒𝑒𝑒𝑒,𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑡𝑡𝑠𝑠𝑡𝑡𝑡𝑡𝑒𝑒 𝐷𝐷𝑒𝑒𝑒𝑒𝑒𝑒𝐶𝐶𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑡𝑡𝑠𝑠𝑡𝑡𝑡𝑡𝑒𝑒,𝑡𝑡 [16] dp is the mean particle diameter of Lipozyme 435 (dp = 383 µm, [26]). The effective diffusivity, Deff, was evaluated using [28]: 𝐷𝐷𝑒𝑒𝑒𝑒𝑒𝑒=𝐷𝐷𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑡𝑡𝑠𝑠𝑡𝑡𝑡𝑡𝑒𝑒−𝑠𝑠𝑡𝑡𝑡𝑡𝑠𝑠𝑒𝑒𝑠𝑠𝑡𝑡𝜀𝜀𝑒𝑒𝜎𝜎 𝜏𝜏 [17] where εp, τ and σ are Lipozyme 435 porosity, tortuosity and constriction factor. These values were taken from Chesterfield [26] for Novozym 435 (εp = 0.5, τ = 6 and σ = 1). Dsubstrate-solvent is the molecular diffusivity of the reactants (glycerol and fish oil) in the reaction medium (tert-butanol in this work). It was estimated using the Wilke-Chang equation [29]: 𝐷𝐷𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑡𝑡𝑟𝑟𝑡𝑡𝑡𝑡𝑒𝑒−𝑠𝑠𝑡𝑡𝑡𝑡𝑠𝑠𝑒𝑒𝑛𝑛𝑡𝑡=7.4·10−8𝑇𝑇(𝑀𝑀𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑡𝑡𝑠𝑠𝑡𝑡𝑡𝑡𝑒𝑒𝜓𝜓𝑠𝑠𝑡𝑡𝑡𝑡𝑠𝑠𝑒𝑒𝑠𝑠𝑡𝑡) 𝜂𝜂𝑠𝑠𝑡𝑡𝑡𝑡𝑠𝑠𝑒𝑒𝑠𝑠𝑡𝑡𝑉𝑉𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑡𝑡𝑠𝑠𝑡𝑡𝑡𝑡𝑒𝑒 0.6 [18] where Dsubstrate-solvent is the diffusion coefficient of the substrate in the solvent (cm2·s-1), Msubstrate is the molecular weight of the solvent (g/mol), T is the temperature (K), ηsolvent is the viscosity of the solvent, cP, Vsustrate is the molar volume of the substrate at its normal boiling temperature, cm3/mol and ψ the association factor of the solvent (dimensionless, ψ = 1 for non-associated compounds). The parameters values used in the calculation of φ are listed in Table 2. Molar volumes at the normal boiling point were estimated by the Tyn and Calus method [29]: 8 𝑉𝑉= 0.285𝑉𝑉𝑐𝑐1.048 [19] where Vc is the critical volume in cm3/mol. Vc for glycerol was 255 cm3/mol [29]. No data of Vc for fish oil was found in the literature. The corresponding estimated value for triolein (Vc = 3235.65 cm3/mol) was used [30]. Φ was evaluated for both substrates, glycerol and sardine oil, at 323 K for rexp,glycerol = 0.0173 mmol·L-1·s-1, Cglycerol,o = 47.5 mmol·L-1 rexp,fish oil = 0.023 mmol·L-1·s-1 Cfish oil,o = 47.5 mmol·L-1. According to Bailey [31], when Φ is sufficiently large (Φ ≥ 3), diffusion of substrate is slow relative to its consumption. When Φ < 0.3 the limiting rate process is the chemical reaction. Φ for diffusion of glycerol in the reaction medium was found to be 1.9·10-2. However, a value of 0.36 was obtained for the diffusion of fish oil in tert-butanol, probably due to the bigger oil molecules that can lead to more diffusional limitation (Table 2). In any case, the value of Φ was close to the limit of 0.3 and the observed rate can be considered kinetically controlled. Based on the Φ values, the lipase was used in its commercially available size without sieving for further kinetic experiments. Yang et al. [20] studied the effect of the loading of Novozym 435 on the glycerolysis of sunflower oil. They found that an enzyme loading of more than 10% resulted in only a small increase in MAG yield. Therefore they suggested that 10-15% of enzyme loading was enough to obtain the maximum reaction performance. Moreover, other authors as Valerio et al. and Fiametti et al. [11, 12] have shown that high enzyme concentrations can lead to the formation of aggregates, making the enzyme active site unavailable to the substrates. Based on this and the results shown in Figure 1, further glycerolysis kinetics were performed with 10 wt% of Lipozyme 435 based on reactant weight. 3.2 Glycerolysis reaction system The presence of a catalyst is necessary since it has been shown in the literature [16] that under 70ºC the observed reaction rate without a catalyst is nearly zero. Figure 3c shows a typical glycerolysis profile of fish oil at the mole ratio of glycerol:sardine oil of 3:1 at 323 K with 10% of lipase loading in tert-butanol (68 % of tertbutanol). The main reaction product at the above conditions was MAG (around 51 % mole percentage), but DAG and FFA production were also observed although the mole percent was around 3 % for both compounds. The initial water content in the reaction medium was less than 1 % by weight but it was nearly 10 % of the mole content of water in the reaction medium. Therefore FFA production can be observed. TAG consumption was nearly complete with a mole percent at equilibrium conditions lower than 2 %. 3.2.1 Effect of reactant mole ratio The initial mole reactant ratio (MR) was varied between 1 and 9. Figures 3a-3d show the glycerolysis product profile expressed in mole fraction on a solvent-free basis. The reaction rate of formation of MAG was always higher than that of DAG and FFA. The presence of a solvent, tert-butanol, helped both reactants to diffuse to the active sites of the enzyme and MAG formation was favored. Valerio et al. [11] studied the kinetics of 9 glycerolysis of olive oil in a surfactant system (with Triton X-100 as surfactant) as an alternative to the use of organic solvents, and found that the DAG initial reaction rate was higher than that of MAG even with an excess of glycerol (MR=9:1). This behavior could be due to mass transfer limitation and can be compared to a situation of low glycerol concentration in the reaction medium. The optimal MR glycerol:oil must consider the MAG yield as well as the excess of glycerol employed in the glycerolysis reaction. The equilibrium yield of MAG was calculated as: 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝑟𝑟𝐸𝐸𝐸𝐸𝑟𝑟 𝑀𝑀𝑀𝑀𝑀𝑀 𝑦𝑦𝐸𝐸𝑦𝑦𝐸𝐸𝑑𝑑 (%)=𝑀𝑀𝑡𝑡𝑡𝑡𝑒𝑒𝑠𝑠 𝑡𝑡𝑒𝑒 𝑀𝑀𝑇𝑇𝑇𝑇 𝑖𝑖𝑛𝑛 𝑡𝑡ℎ𝑒𝑒 𝑒𝑒𝑒𝑒𝑠𝑠𝑖𝑖𝑡𝑡𝑖𝑖𝑠𝑠𝑟𝑟𝑖𝑖𝑠𝑠𝑒𝑒 𝐼𝐼𝑛𝑛𝑖𝑖𝑡𝑡𝑖𝑖𝑡𝑡𝑡𝑡 𝑒𝑒𝑡𝑡𝑡𝑡𝑒𝑒𝑠𝑠 𝑡𝑡𝑒𝑒 𝑇𝑇𝑇𝑇𝑇𝑇·3·100 [20] Figure 4 shows that the MAG equilibrium yield remained practically constant at a MR higher than 5:1. A similar behavior was observed by Chesterfield et al [26] in the ethanolysis of waste cottonseed cooking oil by Novozym 435. These authors proposed the following relationship for the equilibrium yield: 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝑟𝑟𝐸𝐸𝐸𝐸𝑟𝑟 𝑀𝑀𝑀𝑀𝑀𝑀 𝑦𝑦𝐸𝐸𝑦𝑦𝐸𝐸𝑑𝑑 (%)=𝑡𝑡 1+𝑒𝑒𝑒𝑒𝑒𝑒�𝑅𝑅𝑀𝑀𝑡𝑡−𝑅𝑅𝑀𝑀 𝑠𝑠� [21] Non-linear regression was performed by using the Marquardt algorithm (Statgraphics) giving a = 89.285 defined as the limiting normalized MAG equilibrium [26], b = 0.922 and RMo = 1.35 with r2 = 0.999. McNeil (1990) also found that the MAG equilibrium yield was independent of the glycerol:oil mole ratio from mole ratio higher than 5:1. To take into account the excess of glycerol employed, Figure 4 also shows the MAG composition (expressed as mole percentage) on a solvent-free basis and on a solvent and glycerol-free basis. In the lipid basis (no glycerol), on increasing the MR, the MAG content increased sharply from a MR of 1:0 to 3:1 and then the MAG content slightly increased in the lipid fraction. On a solvent-free basis, when glycerol was considered in the global composition, a maximum was observed in the MAG content at a MR of 3:1, due to the excess of glycerol employed that was not consumed. Table 3 summarizes the glyceride equilibrium composition found in this work, as well as for other glycerolysis systems in the literature that use tert-butanol as solvent and immobilized Candida antarctica as the biocatalyst. The results are expressed in weight percentage on a lipid basis since in most studies, the composition was usually expressed this way. Although different lipase loadings were used in Table 3, the data listed in this table corresponded in most cases to equilibrium conditions and the comparison of the MAG yield can be established as valid. Table 3 shows the different results in terms of the MAG and DAG yields at the same initial MR (as will be explained in Section 3.2.2, the effect of temperature on the MAG equilibrium yield was not important). For instance, at the MR glycerol:oil of 4:1, the MAG percentage on a lipid basis ranged from 70% for sunflower oil to 91% for tuna oil. Regarding the type of oil, fish oils gave a higher MAG yield than vegetable oils. According to the shape and properties of the scissile fatty acid binding sites of Candida antarctica lipase, in the literature, it has been reported that this lipase has high activity for short and medium chain length fatty acids [32]. Table 4 presents the fatty acid composition of the oils listed in Table 3. It can be observed that fish oils 16 Table 3. Equilibrium composition of glycerolysis reaction found in this work and for other glycerolysis systems found in the literature that use tert-butanol as solvent and immobilized Candida antarctica as biocatalyst. Oil T, K % E MR % TB % MAG % DAG % TAG % FFA Reference Sardine 323 10 1:1 3:1 5:1 9:1 63 68 68 74 43.0 ± 1.5 83.3 ± 2.1 89.1 ± 1.8 92.9 ± 1.5 25.8 ± 1.9 6.9 ± 1.1 3.7 ± 0.8 2.0 ± 0.7 24.6 ± 1.5 5.9 ± 1.0 3.0 ± 1.0 2.4 ± 0.8 6.6 ± 1.1 3.8 ± 1.1 4.0 ± 1.3 2.8 ± 1.1 This work Sunflower 323 21 4:1 73 71.3 22.1 0.6 5.2 [8] Sunflower a 313 15 4.5:1 60 70 25 1 4 [20] Tuna 318 15 4:1 58.6 90.8 2.5 5.5 1.2 [37] Camelia 323 5 4:1 66 74.1 ± 2.7 24.6 ± 0.1 1.3 ± 0.1 - -b [19] Olive a, c 328 328 328 343 343 10 2.5 2.5 2.5 2.5 6:1 3:1 3:1 9:1 9:1 45 45 80 45 80 67 34 42 53 60 17 15 19 11 14 12 50 36 33 23 4 1 3 3 3 [16] Olive a 328 10 6:1 45 ∼62 ∼19 ∼15 ∼4 [17] (a) Graphical lecture (b) No reference to FFA formation (c) Data at 720 min of reaction time. 17 Table 4. Composition of medium chain length fatty acids in the oils used in the glycerolysis systems listed in Table 3. Oil Medium chain length fatty acids (%) Reference C14:0 C16:0 C16:1 Sardine 12.4 ± 0.4 22.8 ± 0.2 12.5 ± 0.1 This work Tuna 4.2 30.6 4.7 [38] Sunflower 0.1 6.7 0.2 [8] Olive 0.1 – 1.2 7.0 – 16.0 - [16, 17] Camellia - 8.2 - [19] 18 Table 5. Calculated kinetic parameters at different glycerol:oil mole ratios (T = 323.15 K, 10 wt% Lipozyme 435 based on substrate weight). Objective function and root mean square deviation (wt %) for the glycerolysis products. Model parameter Mole ratio 1:1 3:1 5:1 9:1 k 1 0.0350 0.0264 0.0263 0.0285 k 2 0.0023 0.0248 0.0292 0.0276 k 3 0.7638 0.9167 0.9411 0.9411 k 4 0.0749 0.0400 0.0348 0.0338 k 5 0.0108 0.0096 0.0047 -- k 6 -- -- -- -- k 7 -- -- -- -- k 8 0.0711 0.0046 -- -- k 9 1.8168 1.9017 1.9022 1.9019 k 10 0.4052 0.0234 0.0202 0.0209 k 11 0.5853 0.8911 0.8942 0.8983 k 12 1.9714 1.8052 1.8033 1.8008 O.F. 0.0019 0.0013 0.0009 0.0012 Root mean squared deviation (wt %) TAG 4.7 3.4 5.6 4.6 DAG 1.0 0.9 0.4 0.2 MAG 3.8 2.7 5.8 5.6 FFA 1.1 0.7 0.5 0.3 Glycerol 0.4 1.3 1.7 2.2 Water 0.2 0.3 0.1 0.3 Table 6. Calculated kinetic parameters at different reaction temperatures (MR = 3:1, 10 wt% Lipozyme 435 based on substrate weight). Objective function and root mean square deviation (wt %) for the glycerolysis products. Model parameter Reaction Temperature, K 303 313 323 333 k 1 0.0104 0.0164 0.0264 0.0357 k 2 0.0106 0.0201 0.0248 0.0494 19 k 3 0.9132 0.9132 0.9167 0.9194 k 4 0.0380 0.0395 0.0400 0.0436 k 5 0.0072 0.0114 0.0096 0.0174 k 6 -- -- -- -- k 7 -- -- -- -- k 8 0.0041 0.0049 0.0046 0.0049 k 9 1.9020 1.9017 1.9017 1.9021 k 10 0.0207 0.0223 0.0234 0.0241 k 11 0.8967 0.8923 0.8911 0.8970 k 12 1.8017 1.8041 1.8052 1.8069 O.F. 0.0027 0.0021 0.0013 0.0030 Root mean squared deviation (wt %) TAG 4.7 4.7 3.4 5.5 DAG 0.6 0.7 0.9 0.6 MAG 2.8 3.1 2.7 4.7 FFA 0.4 0.6 0.7 0.7 Glycerol 1.9 2.3 1.3 1.7 Water 0.4 0.2 0.3 0.3 Table 7. Activation energy, kJ/mol, for the different steps in some glycerolysis systems Step Ea,i (this work) Ea,i [17]* Ea,i [11]** 1 35.18 18.30 27.91 2 40.12 5.36·10-5 0.06 3 0.20 -- 47.08 4 3.52 1.097·10-4 2.68·10-12 5 20.64 8.397·10-4 208.17 6 -- 0.35 11.46 7 -- 15.55 62.83 8 4.10 -- 13.33 9 0.001 2.33 1.11·10-13 10 4.20 -- 69.46 11 0.009 45.46 71.48 20 12 0.08 15.77 81.77 (*) Solvent = tert-butanol (**) Surfactant system Table 8. Root mean square deviation (wt%) for glycerolysis products obtained with our model equations (1-6) TAG DAG MAG FFA Glycerol Water Reference 4.74 0.63 4.02 0.60 1.65 0.25 This work 4.19 2.73 3.58 1.04 -- -- [17] (--) data not reported Figure 1. Effect of catalyst loading on initial reaction rate of MAG formation (T = 323 K, MR = 3:1). y = 4.1704x - 0.0277 R² = 0.9878 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.00 0.05 0.10 0.15 0.20 0.25 1/ro (L·min·mmol-1) 1/m (L·genzyme-1) 21 Figure 2. Effect of particle size (○) dp < 400 µm; (◇) unsieved lipase; (△) dp > 400 µm on MAG formation reaction: T = 323 K, 5 wt % Lipozyme 435 loading, MR =3:1. Standard uncertainty u (mole fraction) = 0.02. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 100 200 300 400 MAG mole fraction, lipid base time, min a) (b) (a) 22 Figure 3. Time course of the glycerolysis reaction at different mole ratios (MR): (a) 9:1, (b) 5:1, (c) 3:1 (d) 1:1; 323 K, 10 wt % Lipozyme  435 loading; □ MAG ○ FFA △ TAG ◇ glycerol × DAG. Continuous lines are for the model in this work. Standard uncertainty u (mole fraction) = 0.02. 0 10 20 30 40 50 60 70 80 90 100 012345678910 MAG equilibrium yield, MAG composition Molar ratio (MR) glycerol:sardine oil (c) (d) 23 Figure 4. MAG equilibrium yield (□) as a function of initial mole ratio (MR) glycerol:oil. The continuous line is for Eq. 20. MAG composition as mole percentage on a solvent and glycerol free-basis (●) and on a solvent free-basis (○). Continuous lines are the equilibrium composition obtained with the model in this work. Figure 5. Binodal curve of the ternary system glycerol + fish oil + tert-butanol at 303.15 K (−) and 323.15 K (-- -). Initial composition of glycerolysis reaction in tert-butanol medium: ● this work, ○ Sunflower oil [8], ◇ Sunflower oil [20], Δ Tuna oil [37], + Camellia oil [19], □ Olive oil [17], × Olive oil [16]. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Weight fraction of tert-butanol Weight fraction of glycerol One phase-region Two phases-region 24 Figure 6. Initial reaction rate as a function of initial mole ratio glycerol:oil (323 K, 10 wt % Lipozyme 435 loading): □ MAG ○ FFA △ TAG ◇ glycerol × DAG. 0 1 2 3 4 5 6 7 8 9 10 0246810 ro, mmoles·min-1·L-1 Mole ratio (MR) glycerol:sardine oil (a) (b) 25 Figure 7. Time course for the glycerolysis reaction at different temperatures: ( a) 303 K, (b) 313 K, (c) 323 K, (d) 333 K; 10 wt % Lipozyme  435 loading, MR =3:1; □ MAG ○ FFA △ TAG ◇ glycerol × DAG. Continuous lines are for the model in this work. Standard uncertainty u (mole fraction) = 0.02. Kinetic study of lipase-catalyzed glycerolysis of sardine oil in a homogeneous media. Comparison of glycerolysis kinetic parameters. SOLAESA Ángela G., SANZ M. Teresa*, BELTRÁN Sagrario, MELGOSA Rodrigo University of Burgos, Spain This work presents a detailed kinetic study of enzymatic glycerolysis of sardine oil catalyzed by the commercial lipase Lipozyme 435. Glycerolysis is carried out in tert-butanol to create a homogeneous system avoiding mass transfer limitations. Kinetic study of lipase-catalyzed glycerolysis of sardine oil in a homogeneous media.Comparison of 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0100 200 300 400 500 Composition, mole fraction time, min MAG TAG DAG FFA GLY Time course for glycerolysis reaction at 3:1 mole ratio, 323 K and 10 wt % Lipozyme  435 loading Lipozyme  435 CH 2 –O –CO – R 1 CH –O –CO – R 2 CH 2 –O –CO – R 3 CH 2 –O –CO – R 1 CH –OH (R 2 ) CH 2 –OH Tert-butanol media (DAG FFA) 2 CH 2 –OH CH –OH CH 2 –OH MAG 3 GlycerolTAG (c) (d)