Lipase immobilized on ionic liquid-functionalized magnetic silica composites as a magnetic biocatalyst for production of trans-free plastic fats

Food Chemistry 257 (2018) 15–22

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Lipase immobilized on ionic liquid-functionalized magnetic silica composites as a magnetic biocatalyst for production of trans-free plastic fats

T

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Wenlei Xie , Xuezhen Zang School of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou 450001, PR China

A R T I C L E I N F O

A B S T R A C T

Keywords: Magnetic nanoparticle Immobilized lipase Interesterification Vegetable oil Ionic liquid Plastic fat

The main objective of this study is to develop an efficient and environmentally gentle process for production of trans-free plastic fats. To acheive this, the core–shell structured magnetic composites were prepared, and then imidazole-based ionic liquids (ILs) were covalently grafted on the magnetic composites. Thereafter, Candida rugosa lipase was immobilized on the magnetic IL-functionalized composites. The immobilized lipase could be facilely separated using an external magnetic filed. With the magnetic biocatalyst, enzymatic interesterifications of solid palm stearin and liquid rice bran oil blends were performed at 45 °C. It was shown that the total fatty acid (FA) compositions of the binary blends were almost unchanged after the interesterifications, whereas the FA positional distribution and triacylglycerol species were significantly varied. As compared with the physical blends, the interesterified products had a lower slip melting point, and the interesterification could result in an obvious change in the microstructure of the final products.

1. Introduction Most native vegetable oils have their inherent disadvantages as usually employed in their original forms for the edible food industry, such as poor plasticity, tractility and shortening property (Lee, Akoh, Himmelsbach, & Lee, 2008). During recent years, the consumers preference is towards products containing healthier fat and less fat contents. Therefore, for the purpose of improving the functional characteristics of vegetable oils, the modification method can be used most often to produce new and more valuable oil products with specific properties by varying the compositions of fatty acids (FAs) in native oils, so as to better meet the demand of edible food industry (Xu, 2000). For instance, plastic fats, such as margarine and shortening, are usually produced by the partial hydrogenation of vegetable oils. However, trans fats are commonly generated during the partial hydrogenation processes, which have been known to increase the risk factor of coronary heart disease (CHD). Thus, many European countries have banned the use of trans fat in food formulations. As recommended by the Food and Agriculture Organization (FAO) and World Health Organization (WHO), trans fat content of food should not exceed 4% (Dhaka, Gulia, Ahlawat, & Khatkar, 2011). In this connection, considerable efforts have already been made to develop suitable alternative approaches to produce trans-free plastic base stocks for various food applications to replace the traditional hydrogenation process (Costales-Rodríguez, Gibon, Verhé, & De Greyt, 2009). The alternative method as often used

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Corresponding author. E-mail address: [email protected] (W. Xie).

https://doi.org/10.1016/j.foodchem.2018.03.010 Received 8 October 2017; Received in revised form 1 March 2018; Accepted 3 March 2018 0308-8146/ © 2018 Elsevier Ltd. All rights reserved.

to produce zero-trans plastic fat is based on the lipase-catalyzed interesterification between solid fats and liquid oils. Basically, the interesterification can lead to the change of the distribution of FAs on the glycerol backbone of triacylglycerol (TAG) molecules with the formation of new TAG species, which has essentially no possibility to form trans FA residues in the end-products (Paula, Nunes, de Castro, & Santos, 2015). Accordingly, the interesterified product can be tailored to meet specific nutritional and functionality requirements by substantially varying the FA composition and altering the FA positional distribution. Oftentimes, the interesterification is performed by either enzymatically or chemically catalyzed procedures, with a concurrent variation in the textural and functional characteristics of original oils (Costales-Rodríguez et al., 2009). The most commonly used catalysts are metallic sodium, sodium hydroxide and sodium alkoxide, which are efficient, cheap and easy to control in the interesterification processes (Fauzi, Rashid, & Omar, 2013). Unfortunately, these homogeneous catalysts usually suffer from many drawbacks including the need of cleaning process to remove the residual catalyst after the reaction, thereby resulting in undesirable wastewater in the downstream purification processes (Casas, Pérez, & Ramos, 2017). To triumph such weaknesses, enzymatic interesterification has been therefore investigated deeply. In comparison with the chemical catalysts, the major merits of enzymes lie in their selectivity, milder reaction condition, and ease of product recovery (Speranza, Ribeiro, & Macedo, 2015).

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2012; Rosenholm, Zhang, Sun, & Gu, 2011; Xie & Wan, 2018; Yang, Li, & Ma, 2012). Accordingly, the integration of mesoporous silica into magnetic nanoparticle seems to be promising and attractive carries for the immobilization of lipase. In fact, as far as we know there are only a few published literatures on the magnetic immobilized lipase for the organic transformations (Baharfar & Mohajer, 2016; Netto et al., 2013; Shaw, Chen, Ou, & Ho, 2006). As a part of our ongoing project designing efficient and environmentally gentle processes for the modification of vegetable oils (Xie and Zang, 2017), in this contribution, we focused on the synthesis of IL-functionalized core–shell magnetic silica composites used as magnetic carriers for the immobilization of lipase, in which the imidazole-based ILs containing silane coupling groups were covalently grafted on the magnetic silica composites. Thereafter, Candida rugosa lipase was then loaded onto the IL-functionalized magnetic composites. The magnetic silica composite and immobilized lipase were characterized in detail by Fourier transform infrared (FT-IR) spectra, transmission electron microscopy (TEM), vibrating-sample magnetometer (VSM), X-ray powder diffraction (XRD), and N2 adsorption–desorption techniques. By using this biocatalyst, the binary blends of palm stearin (PS) and rice bran oil (RBO) were subjected to the interesterification reactions, and the physicochemical properties of the physical blends and interesterified products were comparatively investigated in terms of FA compositions, TAG profiles, slip melting points (SMPs), and fat crystal microsctures. Furthermore, the reusability of the biocatalyst was also tested in the current research. The novelty of the present study is associated with the preparation of immobilized lipase with magnetic core–shell structure and its application in the heterogeneous interesterification of PS and RBO. When taking the separation process into consideration, the magnetic feature makes the biocatalyst to be magnetically recyclable with minimal loss, which is a facile and efficient approach as compared with tedious method like filtration or centrifugation.

However, the high price of commercial lipases, together with their lack of long-term operational stabilities, has been considered as the main hindrances to the industrial applications for the production of commodity fats in food industry. Nevertheless, such hitches can be minimized by using immobilization techniques, since the immobilized lipase has advantages over free lipase in terms of feasible continuous operations, easy removal from the reaction mixture, simple product purification, and adaptability to industrial biocatalytic processes, hence offering more efficient and cleaner catalytic processes (Jeyarani & Reddy, 2010; Pacheco, Palla, Crapiste, & Carrín, 2013). Over the past decades, the immobilized lipase has been widely utilized for the modification of vegetable oils (Nunes, Pires-Cabral, & Ferreira-Dias, 2011; Xie & Zang, 2016). Specifically, several enzyme immobilization strategies have been explored, such as physical adsorption, covalent binding, physical entrapment and hydrophobic ion pair affinity ligation (Shuai, Das, Naghdi, Brar, & Verma, 2017). Among these methods, the adsorption approach is, by far, the most economical and environmental method in terms of its simplicity of design and low cost, simultaneously maintaining the intact 3D structure of enzymes (Zhai et al., 2010). However, the physical interaction between lipase and porous support is generally not strong enough, thus leading to the leakage of lipase during the biocatalytic processes. In order to address the leaching issues, surface modification of the porous support can be employed to adjust the interactions with the lipase and subsequently improve the stability of the immobilized lipase (Abdullah, Sulaiman, & Kamaruddin, 2009; Zou et al., 2014). It is shown that ionic liquids (ILs) are suitable media for enzymatic catalysis and various lipases were reported to possess excellent activities and extraordinary stabilities in ILs especially as N-methylimidazole cation is existed in the ILs (Adak, Datta, Bhattacharya, & Banerjee, 2015; Jia, Hu, Liu, Jiang, & Huang, 2013). In this regard, the surface modification of the porous support with ILs would enhance the electrostatic interaction, hydrophobic interaction, and hydrogen bond between the porous support and the lipase to efficiently prevent the leakage of lipase from the support (Qin, Zou, Lv, Jin, & Wang, 2016). From this point of view, the IL-modified support is of great potential for the immobilization of lipase since this surface modification can make beneficial improvements in the surface properties for the lipase activity and stability. More recently, nano-sized porous materials have been exploited as solid matrices in the immobilization of lipase, because the large surface area can greatly improve the lipase loading and the catalytic efficiency of the immobilized lipase (Shuai et al., 2017). However, the drawbacks originated from the nano-biocatalysis are the tedious recycling of the immobilized lipase with nanometer size from the liquid phase and their inevitable loss in the separation process by using filtration or centrifugation, thus resulting in the increased burden of solid–liquid separation for the nanoparticles in particular for the interesterification system of vegetable oils that have a higher viscosity as compared with the conventional reaction system (Netto, Toma, & Andrade, 2013; Xie & Huang, 2018). Therefore, it is of considerable interest to develop an attractive alternative approach to conventional filtration or centrifugation for the biocatalyst recovery. Among nanostructured materials, magnetic nanoparticles have been identified as a potent catalyst support in catalysis, since they can be readily separated from the reaction mixture especially with minimal loss of the catalyst by applying an external magnetic field (Netto et al., 2013; Xie, Han, & Tai, 2017). However, the unavoidable problems associated with magnetic nanoparticles are their instability over longer periods of time, and in most cases the tendency to aggregate into large clusters due to their magnetic dipole–dipole attractions and their large surface area to volume ratio (Baharfar & Mohajer, 2016). As such, the protection strategies are required to chemically stabilize the naked magnetic nanoparticles. It has been reported that a suitable passive material such as polymer, carbon, and silica can be coated on the magnetic nanoparticles to effectively suppress their aggregations and improve their chemical stabilities (Ranjbakhsh, Bordbar, & Abbasi,

2. Materials and methods 2.1. Materials Candida rugosa lipase, tetraethyl orthosilicate (TEOS, ≥98%), cetyltrimethylammonium bromide (CTAB, ≥99%), and 3-chloropropyltriethoxysiane (CPTES, ≥98%) were procured from Sigma-Aldrich. Commercial rice bran oil (RBO) was purchased from a local grocery store (Zhengzhou, China) having the FA composition: 41.2% oleic acid, 33.4% linoleic acid, 1.5% linolenic acid, 21.2% palmitic acid, 1.8% stearic acid, and 0.5% myristic acid. Palm stearin (PS) was provided by Wilmar Biotechnology Research and Development Center (Shanghai, China), and according to GC analysis the FA composition was as follows: 83.9% palmitic acid, 8.9% oleic acid, 4.4% stearic acid, 1.4% linoleic acid, and 1.3% linolenic acid. All other chemicals were analytical or chromatographical grades and used as supplied without further treatment. 2.2. Preparation of IL-functionalized core–shell magnetic composites Firstly, the Fe3O4 magnetite was synthesized by a chemical coprecipitation method according to the previously reported procedure (Shaw et al., 2006). Typically, FeSO4·7H2O (3.9 g) and FeCl3·6H2O (8.1 g) were dissolved in 150 mL deionized water till the resulting solution became yellowish-orange in color. Thereafter, a certain amount of ammonia solution (25%) was added into the above solution with stirring at room temperature to maintain the pH between 10 and 11. The black suspension, which was produced instantly, was then allowed to continuously stir for 1 h under nitrogen atmosphere. The formed magnetite precipitates were separated by an external permanent magnet, washed repeatedly with deionized water, and finally dried at 60 °C under vacuum for 24 h. 16

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Fig. 1. Synthesis of the magnetic immobilized lipase.

To improve the separation performance, the coating of a silica layer on the surface of Fe3O4 nanoparticles was achieved via the stöber method (Ji et al., 2010). Initially, 0.5 g of Fe3O4 nanoparticles was dispersed in a solution of ethanol (60 mL) and deionized water (120 mL). Subsequently, 1.2 mL of ammonia aqueous solution was added into the solution and allowed to stir for 1 h at room temperature. Thereafter, to the resultant solution 10 mL of CTAB solution (100 mmol/L) was added dropwise to the above solution. After stirring for 1 h, 1.05 g of TEOS was introduced to the solution and continued to stir for 24 h at room temperature. The solid products were isolated by applying an external magnetic filed, washed several times with ethanol and water, and dried under reduced pressure at 60 °C for 24 h. The core–shell magnetic silica composites were obtained by calcining the solids at 550 °C for 6 h. For the preparation of IL-functionalized magnetic silica composites (Fig. 1), 1-methylimidazole (20 mmol) and CPTES (20 mmol) were charged into a 250 mL three-necked flask, and then stirred for 26 h at 95 °C under nitrogen atmosphere. Afterwards, the core–shell magnetic silica composite (1.0 g) was dispersed in 50 mL of dry toluene by ultrasonication for 20 min, and the prepared IL containing silane coupling groups was subsequently added into the reaction mixture, followed by stirring at 90 °C for 24 h. After cooling to room temperature, the solid product was isolated by magnetic separation, washed with methanol and ether for several times, following by drying at 60 °C under reduced pressure.

processed by the Brunauer-Emmett-Teller (BET) method for surface areas (SBET) and by the Barrett-Joyner-Halenda (BJH) method for pore size distribution and pore volume. FT-IR spectra were tested on a Shimadzu IR-Prestige-21 spectrometer by using KBr pellet techniques with a spectral resolution of 4 cm−1. The magnetic behavior was determined on a vibrating sample magnetometer (LakeShore model 7304) at room temperature.

2.3. Lipase immobilization

2.6. Analytic method

Lipase was immobilized on the IL-functionalized magnetic silica composites by the procedure illustrated in Fig. 1. In a typical procedure, 2.0 g of free Candida rugosa lipase was mixed with 1.0 g of IL-functionalized magnetic silica composite in phosphate buffer solution (30 mL, 0.1 mol/L phosphate buffer, pH 7.0), and the resultant solution was allowed to incubate in a shaking water bath at 25 °C for 12 h until an adsorption equilibrium was achieved. After that, the immobilized lipase was obtained by magnetic separation, washed thoroughly with phosphate buffer solution (0.1 mol/L phosphate buffer, pH 7.0) to remove nonspecific lipases, and finally freeze-dried overnight at 4 °C for future use.

The FA profiles of the final products were determined by a gas chromatography (GC) based on the AOCS Official Method (AOCS, 2009). After complete methylation of FA residues into fatty acid methyl esters (FAMEs), the FAMEs to be analyzed were injected into an 6890 N gas chromatograph (Agilent Technologies, Santa Clara, CA. U.S.A.), fitted with a flame-ionization detector (FID) and a fused silica capillary column (SP-2380, 60 m × 0.25 mm) coated with 0.25 μm of BPX-70 (SGE, Australia). The injection volume was 20 µL and the elution flow rate was 1.2 mL/min. The temperatures of injector and detector were held at 260 °C and 300 °C, respectively. The oven temperature was initially held at 110 °C for 4 min, followed by temperature programming to 240 °C and maintained at 240 °C for 15 min. FAMEs were tentatively identified by comparison of their retention time of the GC peaks with those of the authentic standards, and the percentage of FAs was quantified as the ratio of partial area to total peaks area according to the AOCS Official method Ce 1–62 and Ce 2–66 (AOCS, 2009). The analyses were carried out in triplicate and the reported values are the average of three measurements. Analysis of TAG species was performed in a reversed phase highperformance liquid chromatography (HPLC). Each sample was dissolved in chloroform (10 mg/mL), and 20 µL aliquots were injected into

2.5. Enzymatic interesterification reaction For the batch enzymatic interesterification, PS was initially mixed with RBO at different substrate ratios, and then the binary blends were melted and homogenized at 70 °C for 40 min. After lowering the temperature to 45 °C, the immobilized lipase (10 wt%) was added to the reaction mixture to start the interesterification reaction. The enzymatic interesterification was carried out in a shaking incubator at 45 °C for approximately 48 h on a reciprocal shaker. At the end of the interesterification reaction, the immobilized lipase was separated magnetically from the reaction mixture. The interesterified product was then used for subsequent analyses. The reusability of the immobilized lipase was assayed by repeating the batch runs under the same interesterification condition. The immobilized lipase was recovered by using an external magnetic filed, washed with phosphate buffer (0.1 mol/L phosphate buffer, pH 7.0), and finally freeze-dried for use in the next interesterification reaction.

2.4. Characterizations Transmission electron microscopy (TEM) analysis was conducted on a JSM-6390LV transmission electronic microscopy using a 200 kV accelerating voltage. Powder X-ray diffraction (XRD) patterns were acquired on a Rigaku D/max-3B X-ray diffractometer (Tokyo, Japan) employing Cu Kα radiation (γ = 0.1542 nm). Pore size and surface area were measured by N2 adsorption–desorption analysis at −196 °C using a Quantachrome NOVA 1000e instrument. The obtained data were 17

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encapsulation of silica onto Fe3O4 is particularly promising because of the innovative functional properties that can not be obtained from the parent material alone. In this present case, the silanol groups on the silica layer can react with the imidazole-based ILs containing silane coupling groups to form the IL-functionalized magnetic silica composites. From this view of point, this IL-functionalized silica composites can act not only as a promising carrier for the lipase immobilization through the efficient interaction of IL microenvironment on the surface with the lipase, but also as magnetically separable materials for the facile separation of the biocatalyst. Thereafter, Candida rugosa lipase was readily immobilized on the IL-functionalized magnetic silica composites by physical adsorptions due to their chemically modifiable surface. By using this immobilization approach, it can be expected that the immobilized lipase possesses a rigid conformation structure and an excellent catalytic stability. For the immobilized lipase, there are multipoint interactions, such as electrostatic interaction, hydrophobic interaction, and hydrogen bond, between the IL groups and the lipase. Attractively, these interactions could prevent the conformational transition of the lipase during the enzymatic interesterification processes. The ILs possess an imidazole ring with positive charge, which could change the nature of support surface and enhance the interactions between the magnetic support and the lipase, thus improving the catalytic stability of the immobilized lipase. Even though the preparation of the lipase composites seems to be more complicated and higher cost as compared with chemical catalysts for the production of plastic fats, the immobilized lipase is of interest because of its magnetic properties, rendering the biocatalyst to be separated easily by using an external magnetic filed especially in the high-viscosity interesterification mixture. Apart from a better recovery of the biocatalyst as a technical advantage, the often-encountered aggregation issues for conventional magnetic nanocatalysts could be also addressed by such core–shell structured magnetic composites. In an attempt to demonstrate the successful immobilization of lipase on the magnetic support, FT-IR spectra of magnetic silica composites, IL-functionalized magnetic silica composites, and lipase- immobilized composites are shown in Fig. 2. For the magnetic silica composite, the IR band located at 3432 cm−1 could be ascribed to surface OeH stretching vibration, and the absorption peak at 564 cm−1 corresponded the FeeO vibration of the magnetite core (Shaw et al.,2006). Besides, the IR peak at 1649 cm−1 was attributed to the bending vibration of water present in the magnetic composites (Xie & Wang, 2014). As it can be seen from Fig. 2a, after the silica shell was coated on the Fe3O4 magnetite, the formed magnetic silica composites showed three obvious IR peaks at 1088 cm−1, 810 cm−1, and 465 cm−1, which were clearly responsible for the anti-symmetric, symmetric SieO stretching and deformation mode of mesoporous framework SieOeSi in the silica. Moreover, the IR absorption peak at 970 cm−1 for the magnetic silica composite was mainly due to the bending vibration of Si-OH groups in the silica shell (Cıtak, Erdem, Erdem, & Oksüzoğlu, 2011; Khorshidi & Shariati, 2014). Notably, these IR absorption bands associated with the silica shell and magnetite core were absolutely maintained after IL modification (Fig. 2b). For the IL-functionalized magnetic silica composite, the IR peaks at 1564 cm−1 and 1461 cm−1 were resulted from the stretching vibration of imidazole groups, thus confirming that the imidazole-based ILs were tethered on the magnetic silica composites (Wu et al., 2014; Zou et al., 2014). Furthermore, as compared with the IL-functionalized magnetic support, the immobilized lipase showed additional IR bands situated at 1658 cm−1 and 1539 cm−1, which were reasonably assignable to amide I and II, respectively, as an evidence for the efficient lipase loading on the core–shell magnetic support (Shaw et al., 2006; Xie & Zang, 2016). Based on the obtained IR results, it is reasonable, therefore, to suggest that the lipase has been successfully immobilized on the magnetic silica composites. The morphological character of the samples is shown in Fig. 3. As observed, the magnetic silica composites possessed regular spherical

Fig. 2. FT-IR spectra of samples: (a) magnetic silica composite; (b) IL-functionalized magnetic composite; and (c) immobilized lipase.

the HPLC system consisting of a Waters Model 600 (Milford, PA, USA) together with an Alltech 500 evaporative light-scattering detector (ELSD). The chromatographic separation of TAG species was carried out by a commercially packed Genesis C18 column (150 mm × 4.6 mm) with a mobile phase consisting of dichloromethane and acetonitrile. Individual TAG peak was identified tentatively by comparing the retention times with those of the standards and also by equivalent carbon number (ECN). The ECN of each TAG was calculated according to the AOCS Official Method Ce 5b-89 (AOCS, 2009). The TAG profiles were determined by internal normalization of chromatographic peak area in terms of the relative percentage (AOCS, 2009). The FA composition at the sn-2 position was evaluated by pancreatic lipase method (AOCS, 2009). The pancreatic lipase can be applied to selectively hydrolyze the TAGs to produce 2-monoacylglycerol (2MAG). After the lipase hydrolysis, each sample was separated on TLC plates with diethyl ether/hexane/acetic acid (50:50:1, by volume). The TLC bands corresponding to 2-MAG were carefully scraped from the TLC plates, and were then extracted with hexane. The sn-2 positional analysis of FA residues was conducted by GC techniques after methylation of 2-MAG as mentioned previously. The iodine value (IV) was assessed from the FA compositions as described in the AOCS Official Method Cd 1c-85 (AOCS, 2009). The slip melting point (SMP) was determined using the capillary tube method according to the AOCS Official Method Cc 3–25 (AOCS, 2009). The microstructure observation of the interesterified products was carried out using a polarized light microscope (PLM, XP-203). Samples were melted absolutely at 80 °C, and then 10 mg of melted sample was placed on a glass microscope slide. Thereafter, the sample was covered with a glass slip to give a homogeneous distribution. After cooling to 25 °C, the crystal morphology was taken at room temperature.

3. Results and discussion 3.1. Characteristics of the immobilized lipase At the present study, the core–shell structured magnetic composites were prepared and subsequently used for the immobilization of lipase. Initially, the chemical co-precipitation of Fe2+ and Fe3+ ions in basic solution afforded the magnetite nanoparticles by using sol–gel procedure. For the prevention of magnetic aggregation of Fe3O4 nanoparticles, a silica layer was coated on the magnetite particles by the hydrolysis and condensation of TEOS in a basic condition. Of interest, 18

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Fig. 3. TEM images of samples: (a, b) magnetic silica composite; and (c, d) immobilized lipase.

there was no obvious difference in the XRD patterns among the magnetic silica composite, IL-functionalized magnetic support and the immobilized lipase. This observation offers an evidence that the IL grafting and lipase immobilization did not vary the structure of the magnetic silica composite. No any XRD peak attributed to the silica shell was observed, which was assumed to be due to its amorphous behavior. Accordingly, the immobilized lipase could provide magnetic properties of the lipase composite materials, allowing it to be separated easily from the reaction mixture. The N2 adsorption–desorption isotherms obtained for the magnetic silica composite and immobilized lipase are presented in Fig. S2, Supplementary material. The two N2 adsorption–desorption isotherms could be classified as a type IV isotherm according to IUPAC classifications, which were characteristic of ordered mesoporous materials (Hassan et al., 2014; Rosenholm et al., 2011). Such a result demonstrated that the mesoporous structure remained intact after the IL surface modification and immobilization of lipase. The magnetic silica composite had a high surface area of 205 m2/g, pore volume of 0.13 cm3/g and mean pore size of 3.6 nm. With the introduction of ILs and loading of lipase, the surface area, pore volume and mean pore size for the immobilized lipase were changed to 62.8 m2/g, 0.06 cm3/g and 8.1 nm, respectively. This phenomenon reported here was mostly due to the blockage of the pores of the magnetic silica composites with the ILs

morphology with an average diameter of about 200 nm. After coating of silica on the Fe3O4 magnetite, the formed magnetic silica composite showed core–shell structures with Fe3O4 as the core and silica as the shell, and the mean size of the silica shell was approximately 40 nm (Fig. 3b). As can be seen from Fig. 3c, upon the grafting of ILs and immobilization of lipase, the surface of the magnetic silica composites became slightly rough. These results implied that the Fe3O4 magnetite was indeed coated with the silica shell. The core–shell magnetic silica composites not only have magnetic behaviors, but also possess abundant surface silanols for the binding of ILs and subsequently immobilization of lipase. In addition, as also seen from Fig. 3, the morphology and size for the immobilized lipase were quite similar to those of the magnetic silica support, thus evidencing that the morphology of the core–shell structured magnetic support basically remained almost unchanged after the IL grafting and lipase loading. The magnetic silica composite, IL-functionalized magnetic silica support and immobilized lipase were also characterized by XRD techniques, and the obtained XRD patterns are given in Fig. S1, Supplementary material. The Fe3O4 and magnetic silica composite exhibited the same diffraction peaks situated at 2θ of 30.1°, 35.5°, 43.1°, 53.4°, 57.0°, and 62.6°, which were indexed to the typical reflections of (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1), and (4 4 0) lattice planes of Fe3O4, respectively (Shaw et al., 2006; Xie & Wang, 2014). Moreover, 19

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applications. Practically, the magnetic biocatalyst responded rapidly to a magnetic filed and left a clear supernatant (inserted picture in Fig. 4), which allows facile biocatalyst separation and reduction of toxic waste. 3.2. Immobilized lipase-catalyzed interesterification of palm stearin and rice bran oil Palm stearin (PS), as a natural solid fat obtained by dry fractionation of palm oil, is an excellent substitute for animal fat for the production of plastic fat due to its high solid fat content (Adhikari et al., 2010). The enzymatic interesterification of PS with other liquid oils such as RBO, can be employed to improve its melting and crystallization characteristics (Oliveira, Rodrigues, Bezerra, & Silva, 2017). The major FAs of RBO were linoleic acid (33.4%), oleic acid (41.2%), palmitic acid (21.2%), linolenic acid (1.5%), stearic acid (1.8%) and myristic acid (0.5%), with the IV of 104.2 (Mayamol, Balachandran, Samuel, Sundaresan, & Arumughan, 2009). Unsaturated FAs in RBO amounted to be about 76.1% of the total FAs, and thus RBO is absolutely in liquid state at room temperature. These physicochemical attributes cannot confer any special properties for the utilization in the edible food, for example, margarines, spreads and shortenings. By contrast, PS was mainly composed of palmitric acid (83.9%), oleic acid (8.9%) and stearic acid (4.4%), with the IV of 19.3. Therefore, PS is indeed in solid state at room temperatures due to its higher content of saturated FAs which, in turn, can provide strength and textural structure to food products. Almost no interesterified product was detected as the magnetic silica support was employed as a catalyst for the interesterification of RBO with PS. However, after the loading of lipase, the immobilized lipase showed high catalytic activities towards the interesterification reaction. The interesterification activity of the immobilized lipase was assayed at different reaction condition settings. The FA compositions of the binary blends before and after the interesterification remained almost unchanged as expected, since the interesterification just altered the distribution of FAs on the TAG molecular (data not shown here). Correspondingly, the IV of the blends also kept nearly invariable after the enzymatic reaction (Table 1), validating that the interesterification did not change the unsaturation level of the blends. Even though the FA profiles of the blends were not varied after the reaction, the rearrangement of FAs within and between TAGs occurred, and concomitantly the physicochemical properties of the final products were significantly modified by the interesterification. The FA profile at the sn-2 position for various blends before and after the interesterification is listed in Table 1. For the RBO, 51.4% of linoleic acid, 38.9% of oleic acid, 5.6% of palmitic acid, and 3.1% of linolenic acid were primarily situated at the sn-2 position of TAG molecular. Meanwhile, the sn-2 position of PS significantly contained 83.4% of palmitic acid and 9.2% of oleic acid, with a small amount of stearic acid (4.1%), and linoleic acid (1.9%). More importantly, as seen in Table 1, the FA composition at the sn-2 position was greatly varied

Fig. 4. Room temperature magnetization curves for samples: (a) Fe3O4; (b) magnetic silica composite; (c) IL-functionalized magnetic composite; and (d) immobilized lipase.

and lipase. It is worth mentioning that, even though such a change in the textural parameters to some extent, the BET surface area for the target immobilized lipase still maintained at a high level, which was favorable for the biocatalytic processes. The magnetic hysteresis measurement was done in an applied magnetic field at room temperature. As observed from Fig. 4, the magnetization curves obtained for all samples displayed an S-shape over the applied magnetic filed. Moreover, hysteresis was absent with no remanence or coercivity at room temperature, suggesting that the samples were typical superparamagnetic materials (Shaw et al., 2006; Xie & Wang, 2014). The saturation magnetization value of the pristine Fe3O4 was determined to be 47.7 emu/g, and that of the magnetic silica composite was reduced to 28.4 emu/g. Presumably, the decreased saturation magnetization for the magnetic silica composite could be originated from the coating of Fe3O4 magnetite with nonmagnetic silica shell, thereof leading to the reduced weight ratio of magnetic components in the composites as compared with the bare Fe3O4. After incorporating the silica into Fe3O4 magnetite, the obtained magnetic silica composites still remained a high value of saturation magnetization. Moreover, the saturation magnetization was reduced to 24.3 emu/g when the magnetic silica composite was modified with the ILs. As for the immobilized lipase, the saturation magnetization was further decreased to 16.7 emu/g. Even so, such saturation magnetization is still high enough to ensure the easy recovery of the immobilized lipase from the reaction mixture using an external magnetic field. At the same time, it could be re-dispersed into the reaction medium by shaking without any aggregation once the external magnet was removed. Hence, the immobilized lipase prepared in the present study possesses a strong magnetic responsiveness, which is very useful in terms of the industrial

Table 1 Sn-2 positional fatty acid profiles (%) and iodine values of palm stearin, rice bran oil and their blends in different ratios before and after interesterification reaction. PS/RBO ratio

Myristic 14:0

Palmitic 16:0

Stearic 18:0

Rice bran oil 20:80 (before) 20:80 (after) 30:70 (before) 30:70 (after) 40:60 (before) 40:60 (after) 50:50 (before) 50:50 (after) Palm stearin

0.1 0.3 0.4 0.4 0.6 0.4 0.7 0.7 1.0 0.9

5.6 ± 0.2 20.3 ± 0.8 35.4 ± 1.1 24.8 ± 0.9 37.8 ± 1.0 29.1 ± 0.8 46.3 ± 1.2 33.1 ± 1.0 51.2 ± 1.2 83.4 ± 1.6

0.5 1.3 3.2 1.7 4.2 2.2 3.4 2.3 4.5 4.1

± ± ± ± ± ± ± ± ± ±

0.0 0.0 0.1 0.1 0.2 0.2 0.0 0.1 0.2 0.2

± ± ± ± ± ± ± ± ± ±

0.1 0.0 0.2 0.1 0.4 0.2 0.2 0.0 0.7 0.2

PS-palm stearin; RBO-rice bran oil; IV-iodine value; ND-not detected.

20

Oleic 18:1

Linoleic 18:2

Linolenic 18:3

IV

38.9 ± 0.5 34.3 ± 0.4 31.1 ± 0.5 33.5 ± 0.5 29.7 ± 0.3 32.5 ± 0.4 24.8 ± 0.3 31.5 ± 0.5 22.7 ± 0.4 9.2 ± 0.6

51.4 ± 0.8 41.5 ± 1.0 28.5 ± 0.8 37.2 ± 0.9 26.3 ± 1.1 33.6 ± 1.0 23.4 ± 0.9 30.3 ± 0.8 19.3 ± 0.5 1.9 ± 0.1

3.1 2.1 1.1 2.1 1.2 1.9 1.0 1.8 0.9 ND

104.2 88.6 89.2 67.8 68.2 62.3 61.8 57.3 57.1 19.3

± ± ± ± ± ± ± ± ±

0.4 0.3 0.4 0.5 0.3 0.3 0.2 0.3 0.1

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Table 2 Triacylglycerol composition (%) of rice bran oil and palm stearin, their blends in different ratios before and after interesterification reaction. ECN

40 42 42 44 44 46 46 46 48 48 48 48 50 50

TAG

LLLn LLL OLnL OLL PLL OLO PLO PPL OOO POO PPO PPP StPO StOO

Rice bran oil

0.2 ± 0.1 8.1 ± 0.3 0.8 ± 0.1 16.7 ± 0.5 17.3 ± 0.4 15.1 ± 0.4 19.3 ± 0.6 4.0 ± 0.2 8.3 ± 0.2 6.8 ± 0.2 1.5 ± 0.1 0.6 ± 0.1 0.7 ± 0.1 0.2 ± 0.0

20:80

30:70

40:60

50:50

Palm stearin

Before

After

Before

After

Before

After

Before

After

0.1 ± 0.0 6.0 ± 0.2 0.3 ± 0.0 11.6 ± 0.4 12.9 ± 0.3 10.2 ± 0.3 13.9 ± 0.3 3.3 ± 0.2 5.9 ± 0.2 5.7 ± 0.1 3.4 ± 0.1 24.1 ± 0.9 0.7 ± 0.1 1.6 ± 0.2

ND 4.1 ± 0.2 0.8 ± 0.1 10.2 ± 0.3 14.7 ± 0.4 11.1 ± 0.3 18.6 ± 0.6 8.6 ± 0.3 1.7 ± 0.1 8.2 ± 0.2 6.2 ± 0.2 12.6 ± 0.8 1.6 ± 0.1 1.2 ± 0.1

ND 4.0 ± 0.2 0.2 ± 0.0 9.0 ± 0.2 9.1 ± 0.2 7.8 ± 0.2 10.6 ± 0.2 2.6 ± 0.1 4.3 ± 0.2 4.8 ± 0.2 4.3 ± 0.2 39.3 ± 1.1 1.1 ± 0.1 2.4 ± 0.2

ND 1.5 ± 0.1 0.6 ± 0.1 7.8 ± 0.2 11.7 ± 0.3 9.4 ± 0.4 16.2 ± 0.3 8.1 ± 0.2 1.8 ± 0.1 6.8 ± 0.3 7.8 ± 0.3 24.1 ± 1.2 1.5 ± 0.2 2.3 ± 0.2

ND 3.6 ± 0.1 0.2 ± 0.0 6.5 ± 0.2 7.5 ± 0.2 6.1 ± 0.2 7.1 ± 0.2 2.5 ± 0.1 3.4 ± 0.2 4.0 ± 0.2 5.6 ± 0.2 48.7 ± 1.3 1.3 ± 0.1 3.2 ± 0.2

ND 1.2 ± 0.1 0.4 ± 0.0 5.2 ± 0.2 9.1 ± 0.3 8.4 ± 0.3 14.7 ± 0.3 7.3 ± 0.3 1.6 ± 0.1 6.5 ± 0.3 10.5 ± 0.4 30.6 ± 0.9 1.4 ± 0.2 2.8 ± 0.1

ND 2.8 ± 0.2 0.3 ± 0.0 6.2 ± 0.3 6.3 ± 0.2 4.9 ± 0.2 5.6 ± 0.2 2.5 ± 0.1 3.1 ± 0.2 3.9 ± 0.1 6.9 ± 0.3 52.5 ± 1.5 1.5 ± 0.1 3.0 ± 0.2

ND 1.2 ± 0.1 0.3 ± 0.1 5.0 ± 0.3 8.7 ± 0.3 6.7 ± 0.3 11.4 ± 0.4 7.1 ± 0.3 1.4 ± 0.1 6.3 ± 0.3 11.3 ± 0.4 35.2 ± 1.1 1.7 ± 0.2 3.1 ± 0.2

ND ND ND ND ND ND ND ND 0.2 ± 0.0 2.7 ± 0.2 8.7 ± 0.3 83.9 ± 2.1 2.5 ± 0.1 1.4 ± 0.1

ECN-equivalent carbon number; TAG-triacylglycerol; PS-palm stearin; RBO-rice bran oil; L-linoleic acid; Ln-linolenic acid; O-oleic acid; P-palmitic acid; St-stearic acid; ND-not detected;

properties. The slip melting point (SMP) was measured by the open capillary tube method at the present study, and the results are shown in Table 1S, Supplementary material. The physical blends had SMPs ranging from 46.1 °C to 51.6 °C, and the SMP obtained here was enhanced with increasing the proportion of PS in the blends due to the increased saturated FAs. Interestingly, the interesterified products had a tendency to display lower SMPs for all products when compared with their corresponding physical blends, and the decrease in the SMPs could be explained due to the decrease in the high melting TAG species originated from the rearrangement of FAs among the TAGs. Also, Mayamol et al. have reported that the enzymatic interesterification of high melting point fat with liquid oil could result in a modified product with lower melting point (Mayamol et al., 2009). Furthermore, the interesterification caused a narrower melting scope for all blends evaluated, and the melting scope was varied according to the different ratio of PS and RBO. In general, fat bases as used for production of margarine and shortening should melt absolutely at body temperature to eliminate waxy mouthfeel of the final products. Hence, by changing the ratio of PS and RBO, the tailored-fats with desired melting characteristics can be produced by the lipase-catalyzed interesterification to better meet consumer preferences. The crystal morphology of the samples obtained by polarized light microscopy is presented in Fig. 3S, Supplementary material. As can be seen, PS showed densely-packed crystals of large rod-like spheruliticshaped microstructures, whereas RBO was absolutely in liquid state and no crystal was found at room temperature. In the case of the physical blends of RBO and PS, due to the dilution reason, loosely-packed and low-density aggregates of spherulitic crystals were observed. After the enzymatic interesterification, the interesterified products exhibited smaller aggregates of fat crystals with uniform and small clusters. Generally, larger size fat crystals can result in a sandy mouthful feel of the finished food product, and small compact crystals are desired for use in margarine fats and commercial shortenings since they can stabilize air bubbles to final food products, giving a fine and smooth texture.

after the enzymatic reaction for all blends, because of the rearrangement of FAs on the TAGs. For instance for the 40:60 blend of PS and RBO, it was observed that myristic acid, stearic acid, palmitic acid, oleic acid, linoleic acid, and linolenic acid at the sn-2 position were substantially changed from 0.4%, 2.2%, 29.1%, 32.5%, 33.6% and 1.9% to 0.7%, 3.4%, 46.3%, 24.8%, 23.4%, and 1.0%, respectively, following the interesterification reaction. By this observation, we can conclude that the immobilized lipase is capable of catalyzing the interesterification of PS and RBO. The TAG profile is the ultimate determinant in understanding the functional and textural characteristics of the end products. The TAG compositions of PS, RBO and their blends before and after the enzymatic modification are summarized in Table 2. As observed, the most abundant TAG species present in PS were shown to be PPP (83.9%), PPO (8.7%), POO (2.7%), and StPO (2.5%) (Oliveira et al., 2017). On the other hand, RBO contained higher levels of unsaturated TAG species, including PLO (19.3%), PLL (17.3%), OLL (16.7%), OLO (15.1%), OOO (8.3%), LLL (8.1%) and POO (6.8%) (Mayamol et al., 2009). Undoubtedly, after the enzymatic interesterification, the substantial changes of the TAG species in the interesterified products were observed as compared with the physical blends. As seen from Table 2, for different blends of PS and RBO, different TAG profiles were achieved relying on the different substrate ratios, and the desirable FA compositions could be obtained, which has a health benefit effect. For the 40:60 blend of RBO and PS, the final interesterified product showed an increased content of TAG species, such as PLL, OLO, PLO, PPL, POO, and PPO from 7.5%, 6.1%, 7.1%, 2.5%, 4.0%, and 5.6% to 9.1%, 8.4%, 14.7%, 7.3%, 6.5%, and 10.5%, respectively; on the contrary, the amount of other TAG species was concurrently decreased, such as LLL, OLL, OOO, and PPP from 3.6%, 6.5%, 3.4%, and 48.7% to 1.2%, 5.2%, 1.6%, and 30.6%, respectively. Moreover, according to the method as described by De Clercq et al., the degree of interesterification (DI) could be estimated from the TAG composition in the interesterified product (De Clercq, Danthine, Nguyen, Gibon, & Dewettinck, 2012). For the 20:80, 30:70, 40:60, and 50:50 blends of RBO and PS, the DI for the enzymatic interesterification reaction was calculated to be 92.6%, 94.5%, 97.3% and 95.8%, respectively. Besides, the TAG compositions of the enzymatically interesterified products had minor differences from those of randomly interesterified products (chemical interesterification process). Accordingly, it could be assumed that the almost randomization rearrangement of FAs was achieved at the end of the enzymatic reaction. Similar results were also reported in the other lipase-catalyzed interesterification reactions (De Clercq et al.,2012). By drawing on the results, it is reasonable to speculate that the interesterification is practically occurred over the immobilized lipase to produce the trans-free modified fat with desirable physicochemical

3.3. Reusability of the immobilized lipase The reusability of the immobilized lipase is of critical important from the view of practical applications. However, because of the very complex compositions in the interesterified products, the accurate determination of the interesterification degree seems to be difficult. Since the lowered SMPs were generally observed for all the tested blends after the lipase-catalyzed interesterification as reported above, the decrease extent of the SMPs after the reaction is probably considered as a 21

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qualitative parameter of the progress for the enzymatic interesterification. In this work, the reusability of the biocatalyst was tested in repetitive cycles under same reaction conditions. For the 40:60 blend of PS and RBO, when the immobilized lipase was utilized for 1, 2, 3, 4 cycles, the decrease extents of the SMPs after the interesterifications were determined to be 8.2 °C, 7.5 °C, 7.1 °C, and 6.5 °C. Moreover, according to the DI method as mentioned above (De Clercq et al., 2012), the corresponding DI for the interesterification reaction was 97.3%, 90.5%, 84.6%, and 74.8% as the immobilized lipase was employed for 1, 2, 3, 4 cycles. By drawing on these results, the magnetic biocatalyst could be reused for at least two times without significant loss of its activity, and the slight decrease of the activity is probably due to the protein denaturation during the interesterification processes.

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4. Conclusions In the current study, the lipase from Candida rugosa was successfully immobilized onto the IL-functionalized magnetic silica composites through electrostatic, dipole–dipole, and hydrogen binding interactions, and was then employed as a magnetically separable biocatalyst for the interesterification of PS and RBO in a shaking water bath. The characterization results showed that the core-shell structure of magnetic silica composites had no obvious change after the IL grafting and lipase immobilization. The immobilized lipase exhibited a strong magnetic responsiveness and had catalytic activities towards the interesterification of PS and RBO for the production of trans-free plastic fats with desirable physicochemical properties. The separation of the immobilized lipase can be facilely achieved by an external magnetic filed, without significant reduction of activity as it is reused for several times, which would have potential in green and clean production process. Acknowledgement This work was supported by the National Natural Science Foundation of China (Grant No. 21476062, 21776062). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2018.03.010. References Abdullah, A. Z., Sulaiman, N. S., & Kamaruddin, A. H. (2009). Biocatalytic esterification of citronellol with lauric acid by immobilized lipase on aminopropyl-grafted mesoporous SBA-15. Biochem. Eng. J. 44, 263–270. Adak, S., Datta, S., Bhattacharya, S., & Banerjee, R. (2015). Imidazolium based ionic liquid type surfactant improves activity and thermal stability of lipase of Rhizopus oryzae. J. Mol. Catal. B: Enzym. 119, 12–17. Adhikari, P., Zhu, X. M., Gautam, A., Shin, J. A., Hu, J. N., Lee, J. H., ... Lee, K. T. (2010). Scaled up production of zero-trans margarine fat using pine nut oil and palm stearin. Food Chem. 119, 1332–1338. AOCS (2009). AOCS Official and Recommended Practices (fifth ed.). Champain, IL, USA: AOCS Press. Baharfar, R., & Mohajer, S. (2016). Synthesis and characterization of immobilized lipase on Fe3O4, nanoparticles as nano biocatalyst for the synthesis of benzothiazepine and spirobenzothiazine chroman derivatives. Catal. Lett. 146, 1729–1742. Casas, A., Pérez, Á., & Ramos, M. J. (2017). Catalyst removal after the chemical interesterification of sunflower oil with methyl acetate. Org. Process Res. Dev. 21, 1253–1258. Cıtak, A., Erdem, B., Erdem, S., & Oksüzoğlu, R. M. (2011). Synthesis, characterization and catalytic behavior of functionalized mesoporous SBA-15 with various organosilanes. J. Colloid Interface Sci. 369, 160–163. Costales-Rodríguez, R., Gibon, V., Verhé, R., & De Greyt, W. (2009). Chemical and enzymatic interesterification of a blend of palm stearin: soybean oil for low transmargarine formulation. J. Am. Oil Chem. Soc. 86, 681–697. De Clercq, N., Danthine, S., Nguyen, M. T., Gibon, V., & Dewettinck, K. (2012). Enzymatic interesterification of palm oil and fractions: monitoring the degree of interesterification using different methods. J. Am. Oil Chem. Soc. 89, 219–229. Dhaka, V., Gulia, N., Ahlawat, K. S., & Khatkar, B. S. (2011). Trans fats-sources, health risks and alternative approach-a review. J. Food Sci. Technol. 48, 534–541.

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