Synthesis of Structured Lipids by Enzymatic ... - ACS Publications

Article pubs.acs.org/IECR

Synthesis of Structured Lipids by Enzymatic Interesterification of Milkfat and Soybean Oil in a Basket-Type Stirred Tank Reactor Ariela V. Paula,† Gisele F. M. Nunes,‡ Heizir F. de Castro,§ and Júlio C. Santos*,∥ †

Faculty of Pharmaceutical Sciences, State University Julio of Mesquita Filho, UNESP, Araraquara, São Paulo, Brazil Campus I, Departamento de Química, Centro Federal de Educaçaõ Tecnológica de Minas Gerais (CEFET/MG), Belo Horizonte, Minas Gerais, Brazil § Engineering School of Lorena, Department of Chemical Engineering and ∥Engineering School of Lorena, Department of Biotechnology, University of São Paulo, Lorena, São Paulo, Brazil ‡

ABSTRACT: Lipase from Rhizopus oryzae immobilized on polysiloxane−poly(vinyl alcohol) (SiO2−PVA) was used to study the interesterification reaction of the milkfat with soybean oil in a stirred tank reactor (STR) containing baskets for the immobilized enzyme retention in two different configurations: central or lateral. The progress of the reaction was followed by determining free fatty acids, composition in triacylglycerols (TAGs), and consistency. The central basket was chosen for assessing the biocatalyst operational stability by running 10 consecutive batch assays lasting 6 h each. Non-notable deactivation of the biocatalyst was observed during the total operation time (60 h). The potential of the evaluated system to make the milkfat-based fat more spreadable under cooling temperature and with lower saturated fatty acids content was demonstrated in this study. Both evaluated basket reactor designs have shown potential to be used in interesterification reactions of industrial interest.

■

INTRODUCTION The functional and physical properties, metabolic fate, and putative health benefits of various fats and oils are different because of their composition in fatty acid (FA) residues and position on the glycerol backbone of triacylglycerols (TAGs).1,2 However, novel TAGs can be obtained by incorporating new FAs or changing their positions in the molecule.2 These modified fats, known as structured lipids (SLs) or “taylormade fats”, can be produced either chemically or enzymatically1,2 and display important medical and functional properties for food applications.1,3 The enzymatic synthesis of structured lipids is catalyzed by lipases (E.C.3.1.1.3; triacylglycerol acylhydrolases). The interest in the lipase-catalyzed production of structured lipids (SL) having specific functional properties has greatly increased due to the benefits of the enzymatic route relative to chemical processes, such as milder conditions used, allowing reduced losses of oil or fats, fewer unit operations, lower requirements of investment capital, and decreased degradation of antioxidants such as tocopherols.4 The high cost of the enzymes can be minimized using immobilization techniques, which result in more stable catalysts and favor their reuse or application in consecutive batch or continuous runs.5 Among commercial fats, special emphasis is given to milkfat, an important lipid source for human nutrition, with a flavor and mouthfeel superior than those from any other edible fat.6 Its chemical structure is complex and includes more than 100 000 different TAGs with about 400 different fatty acids, from short to long chains.7 Particularly important is its content of conjugated linoleic acid and butyric acid that have been identified as beneficial to health, namely, in cancer prevention.8 On the other hand, the high content of saturated fatty acids in milkfat has been associated with cardiovascular diseases.9 © XXXX American Chemical Society

In the past, the consumption of butter was largely replaced by margarines produced by partial hydrogenation of vegetable oils, leading to the formation of high contents of trans isomers, whose consumption is even worse for health than that of saturated fatty acids.10,11 Currently, margarines and spreads are produced by an interesterification process, without trans isomer generation. Another possible alternative, allowing us to take the benefits of milkfat and at the same time lower the total content of saturated fatty acids, would be its interesterification with vegetable oils rich in unsaturated fatty acids.12 Interesterification reactions modify the physicochemical properties of fats and oils by rearranging the positional distribution of fatty acids located at the glycerol backbone of TAG molecules.13 This alternative can be also to extend the possibilities of application of milkfat, since physical properties, such as the texture of the obtained products, can be tuned to enhance spreadability under refrigerated conditions.14 In order to take advantage of the use of the lipase-catalyzed interesterification process and compensate for its cost, a correct choice of the bioreactor is fundamentally required. A common configuration that has been reported for milkfat interesterification is the stirred tank reactor, STR.15,16 This configuration generally consists of a reactor vessel containing a stirrer that improves the mixing efficiency. STR has a number of advantageous features, namely, easy temperature control and suitability for multiphasic systems.17 Moreover, it offers a closely controllable environment that is useful for slow reactions, where the composition and conditions (e.g., temperature, pH, coenzyme concentrations) varied throughout Received: August 11, 2014 Revised: November 24, 2014 Accepted: January 22, 2015

A

DOI: 10.1021/ie503189e Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Article

Industrial & Engineering Chemistry Research

Figure 1. Schematic representation of the lateral (a) and central (b) basket configurations, flat blade stirrer (c), and propeller with inclined blades (d) used to mechanically stir the medium. All measurements are shown in millimeters.

the reaction15,18 may be accurately monitored. Its main limitation, however, is the high shear stress imposed to the immobilized enzyme by the mechanical agitation.18,19 This effect can be reduced by isolating the immobilized enzyme in a basket adapted within the STR or even adapting basket stirrer blades or baffles in the system.20,21 Examples from the literature indicate that few studies have been dedicated to explore this reactor configuration.

Fernandes22 and Sheelu et al.,23 e.g., described the use of a basket accomplished in the mechanical blades of the reactor. The present work includes another approach, assessing baskets adapted into the reactor vessel; particularly, the reaction was carried out in a STR with a new design, containing a central basket which was proposed and compared with the performance attained in a lateral basket configuration. The enzymatic interesterification of milkfat with soybean oil catalyzed by lipase B


Article


h at 45 °C. The baskets were inserted into the reactor vessel, and two configurations were evaluated: lateral basket (Figure 1a) and central basket (Figure 1b). The baskets were made of stainless steel with screens of 200 mesh for the immobilized enzyme retention. The medium was mechanically stirred by using flat blade stirrer (Figure 1c) with an agitation of 500 rpm for the lateral basket or propeller with inclined blades (Figure 1d) with an agitation of 700 rpm for the central basket. The stirrer positions were adjusted considering the distance from the impeller to the vessel base in 20 and 50 mm, respectively, for the lateral and central basket configurations. R. oryzae lipase immobilized in SiO2−PVA was used as biocatalyst (500 units of activity per gram of reaction medium). The progress of the reaction was followed by determining free fatty acids, composition in triacylglycerols (TAGs), and consistency. Batch Operational Stability Tests. The operational stability of the immobilized enzyme was assayed in the interesterification reaction of milkfat with soybean oil in successive batches (45 °C/6 h) using the reactor coupled with a basket in the central configuration. At the end of each batch the interesterified product was removed from the reactor and the basket containing the immobilized lipase was washed three times with hexane in order to remove any substrate or product eventually retained in the matrix or basket. After solvent evaporation, the basket was introduced into fresh medium containing milkfat and soybean oil (mass proportion 65:35) and a new reaction performed.

from Rhizopus oryzae was used as a reaction model to evaluate the reactor configuration. This enzyme was purchased in its free form and immobilized in our lab on polysiloxane−poly(vinyl alcohol) (SiO2−PVA) prepared by a sol−gel technique, as previously reported.24,25 Soybean oil was used for interesterification with milkfat due to its high content of unsaturated fatty acids, mainly linoleic and linolenic acids.7

■

MATERIALS AND METHODS Materials. Milkfat was obtained from commercial unsalted butter, purchased in a local market, which was melted at 50−65 °C, in a microwave oven, followed by centrifugation at 3500 rpm for 10 min to separate the aqueous phase. Its fatty acid composition was as follows (w/w): 2.9% butyric acid, 2.2% caproic acid, 1.3% caprylic acid, 3.0% capric acid, 3.5% lauric acid, 11.3% myristic acid, 1.0% pentadecanoic acid, 33.0% palmitic acid, 2.0% palmitoleic acid, 0.6% margaric acid, 22.9% oleic acid, 10.6% stearic acid, 2.2% trans-elaidic acid, 2.0% linoleic acid, and 0.2% linolenic acid with average molecular weights of the TAGs of 785.14 g/mol g mol−1.26 Refined, bleached, and deodorized soybean oil was purchased from a local market. Commercial virgin olive oil (0.3% acidity), purchased in a local market, was used to determine the hydrolytic activity of the biocatalysts. A deep blue liposoluble dye (organic synthetic pigment) was obtained from Glitter Ind. Com. Imp. Exp. Ltd. (Carapicuiba, SP, Brazil) and used in the tests of homogenization of the medium. A commercial food-grade lipase from R. oryzae (L036P, Biocatalysts, Cardiff, England) in powder form was used without further purification. Tetraethoxysilane (TEOS) and poly(vinyl alcohol) (PVA, MW 88 000) were acquired from Aldrich Chemical Co. (Milwaukee, WI). Hydrochloric acid (minimum 36%), ethanol (minimum 99%), and polyethylene glycol (PEG 1500 g/mol) were supplied by Synth (São Paulo, Brazil). All solvents and reagents for analyses were chromatographic or analytical grade. Support Synthesis and Lipase Immobilization. A polysiloxane−poly(vinyl alcohol) hybrid support (SiO2−PVA) was prepared by the hydrolysis and polycondensation of tetraethoxysilane according to the methodology previously described.30 The activation of SiO2−PVA particles was carried out with sodium metaperiodate (0.5 M) according to the methodology described by Paula et al.27 The resulting SiO2− PVA particles were used to immobilize R. oryzae lipase by covalent binding.12,25 The immobilized derivative displayed an average hydrolytic activity of 3900 U·g−1, measured using emulsified olive oil as substrate, as described by Soares et al.28 The moisture content was 10% to reduce byproduct formation. Homogenization Tests. Before interesterification reactions, a simple test was carried out by dropping a liposoluble deep blue dye and visualizing the time needed to its dispersion in the reaction medium. In this way, the deep blue liposoluble dye was added to the reactor (about 4 drops), containing 200 g of medium composed by a mixture of milkfat and soybean oil (mass proportion 65:35). In each of the reactors, different stirring speeds were evaluated targeting to reach the shortest mixture time for the dye to blend completely. Interesterification Reactions in Basket-Type Stirred Tank Reactor. The interesterification reactions were carried out in a jacketed cylindrical glass reactor (internal diameter of 70 mm) filled with 200 g of reaction medium composed by a mixture of milkfat and soybean oil (mass proportion 65:35). The process was performed under inert atmosphere (N2) for 12

■

ANALYTICAL METHODS Free Fatty Acid Content. The AOCS method Ca 5a-4029 was used for determination of total free fatty acids (FFA), expressed in terms of the percentage of free oleic acid. Triacylglycerols (TAGs) Composition. TAGs were analyzed by gas chromatography using a Varian CG 3800 model (Varian, Inc. Corporate Headquarters, Palo Alto, CA) equipped with a flame ionization detector and 3% OV1 SilptWBM 100/120 mesh in Silco Var packed column (Restek, Frankel Commerce of Analytic Instruments Ltd., SP, Brazil). Nitrogen was used as the carrier gas with a flow rate of 40 mL.min−1. The detector and injector temperatures were 350 and 370 °C, respectively. The column temperature was first set at 80 °C for 1 min and then programmed to increase at 50 °C min−1 to 210 °C, which was kept constant for 1 min. Finally, the column temperature was programmed to increase at 6 °C min−1 to 340 °C and kept constant for 2 min. The samples were prepared according to Nunes et al.25 The injection volume of this solution was 1 μL. The chromatograms were processed using the software Galaxie Chromatography Data System version 1.9. For determination of TAGs calibration curve, butterfat standard from the Community Bureau of Reference Materials30 was used. The groups of TAGs were identified by carbon number (CN), considering the total number of acyl-C atoms within the triglyceride. The interesterification degree (ID) was defined according to eq 1 ID(%) =

∑ (TAG I)t − ∑ (TAG I)0 ·100 ∑ (TAGD)0

(1)

where TAGI is the concentration (mM) of the triacylglycerols whose concentration increased during the reaction and TAGD is the concentration (mM) of the triacylglycerols whose concentration decreased during the reaction. The subscripts t C


Article

Industrial & Engineering Chemistry Research and 0 represent the concentrations of TAG at a given time t and at the initial reaction time, respectively. Consistency. The consistency of the raw materials and IE products were determined using a texture analyzer (model QTS-25 Brookfield, Middleboro, MA) controlled by the Texture Pro software v. 2.1. Samples were heated in a microwave oven (55−62 °C) for complete melting of the crystals and conditioned in cubic silicone molds (edge of 25 mm) for 48 h at 10 °C. The probe TA15 was used, corresponding to an acrylic cone with angle of 45°. Tests were carried out under the following conditions: total of cycles 1, distance = 10 mm, speed = 120 mm·min1, time = 5 s; determination of the force in compression (gf), in duplicate. Measurements were used to calculate the “yield value”, defined according to eq 231 C=K

W p1.6

reactor configurations, and the progress of the synthesis was monitored by determining the content of free fatty acids (%), composition of triacylglycerols (TAGs), and consistency of the interesterified products (Figures 2 and 3).

(2)

where C is the yield value (gf/cm2), K is a constant depending on cone angle (4700 for 45°), W is the maximum compression force (gf) for 5 s, and p is the penetration depth (in 0.1 mm).

■

Figure 2. Interesterification degree () and free fatty acid content (---) obtained in the reactions carried out in the basket-type stirred tank reactor.

RESULTS AND DISCUSSION Tests of Homogenization of the Medium. The damage caused to the enzyme particles after using stirred-tank reactors would also affect the productivity, final product quality, and downstream processing.32 To overcome such limitations and prevent possible contact of the immobilized biocatalyst with mechanical stirrers, the reactor was adapted with baskets inside. Baskets in two different configurations were used: lateral and central (Figures 1a and 1b). Since insertion of baskets in the reactor vessel could impair the efficiency of the stirrers to homogenize the reaction mixture, tests were carried out to verify the mixing efficiency of the two different stirrers: flat blade stirrer (Figure 1c) and propeller with inclined blades (Figure 1d). According to the literature, the force applied to the fluid by the stirrer produces movement, and this promotes circulation, distribution, and interpenetration and, therefore, mixing of the components.33 The mixing efficiency will be influenced by the flow rate and velocity of circulation, which in turn are dependent on the energy expended per unit time in the blender. Thus, mixing, circulation, and agitation rate are closely linked.33 Circulation patterns caused by the stirrer are of two types: radial or axial. In the first case the circulation is chambered, and in the second, the liquid follows a longer and noncompartmentalized circuit.33 The homogenization tests were performed by dropping liposoluble dye inside the reactor vessel and visualized its dispersion. Such procedure helped to adjust the kind, position, and impeller velocity for each proposed basket configuration. The results allowed setting up the best stirrer for each basket: for the lateral basket, the medium was mechanically stirred with a flat blade stirrer (Figure 1c; radial flow direction), while using the central basket configuration, a propeller with inclined blades was used (Figure 1d; axial flow direction). The agitation speeds of 500 and 700 rpm, respectively, were used for the flat blade stirrer (lateral basket) and propeller with inclined blades (central basket). Interesterification Reaction Performance in STR Coupled with Basket (Central and Lateral). The interesterification reactions were performed for 12 h in both

Figure 3. Composition of the medium as a function of time in interesterification reactions of milkfat with soybean oil: TAGs composition in the systems using a lateral basket (a) and central basket (b). D


Article


functional aspect for plastic fats, which are mixtures of solid fat crystals and liquid oil. The ratio between the two phases and the crystalline character of the solid phase determine sample consistency and firmness.24,35 Moreover, consistency is a critical factor in determining the functionality and consumer acceptance for table spreads.25,36 The consistency value for pure milkfat was 6074.00 ± 406.80 gf/cm2 (Table 1). The addition of soybean oil to this raw material allowed the reduction of the consistency to 1228.84 ± 78.24 gf/cm2 (NIE blend, Table 1). The consistency values for the IE products were 68.43% and 58.18% lower than the values measured for NIE blends for the system with the lateral and central basket, respectively (Table 1). Both products showed consistency values within the ideal range (200−800 gf/cm2), with satisfactory properties of spreadability according to Haighton.31 These results suggest that both basket configurations have similar performance. Biocatalyst Operational Stability. A fundamental parameter, when working with processes that involve immobilized enzymes, is its operational stability that depends on several parameters such as the biocatalyst itself, water content, and presence of oxidation products, related to the degree of refining of these fats.37 In the present work, the biocatalyst operational stability was evaluated in 10 consecutive batch runs (1 batch = 6 h) using the reactor coupled with the central basket which provides easier handling for biocatalyst recovered. In this set of experiments free fatty acid contents, TAGs composition of the IE products, ID, and consistency values were quantified along with the consecutive interesterification reactions. Results displayed in Figure 4 indicated that the free fatty acids levels were similar in all batches, and the maximum value

The results displayed in Figure 2 indicated that the free fatty acids content increased up to the end of the reactions for the two basket configurations. The maximum values were achieved in 12 h with the lateral basket (5.92 ± 0.54%) and central basket (4.61 ± 0.42%). Taking into account that only slight differences have been observed, it appears that the basket configurations do not have an influence on the free fatty acid formation (hydrolysis reactions). The occurrence of this side reaction may be associated with the water content in the immobilized lipase, which was set up to 10% as recommended in a previous work.12 This level of water was found to be suitable for the biocatalyst to maintain its physical and chemical interactions giving adequate configuration to the enzyme to express its catalytic activity.34 Lowering the water content of the biocatalyst may also decrease the extension of the hydrolysis reaction and consequently formation of free fatty acids at the expense of reducing the lipase activity. The TAGs composition of the interesterified products (IE) was determined at different reaction times using the reactor operated in batch mode adapted with central and lateral baskets, and results were compared with the triglycerides profile in the noninteresterified (NIE) blend (Figure 3). Using the lateral basket (Figure 3a), the concentration of TAG carbon species 24−30 increased until 6 h, decreasing after this time. On the other hand, the concentration of TAG carbon species 36−50 decreased until 3 h, increasing until the end reaction. A similar behavior was verified in the reaction carried out (Figure 3b) using the central basket. In this case, the concentration of TAG carbon species 24−26 increased and the concentration of TAG carbon species 36−50 decreased until 6 h, increasing after this time. Minor changes have been observed in the concentrations of TAGs 28−30 until the end of the reaction. For two basket configurations, TAG carbon species 54 decreased after 4 h reaction. Similar TAG profiles were also found by Paula et al.12 on assays carried out in STR without a basket. The authors observed that in interesterification reactions of milkfat and soybean oil (mass proportion 65:35), the concentration of TAG C34−42 and C54 decreased while the concentration of TAG 46−52 increased.12 From the TAGs concentrations, the interesterification degree (ID, %) values were calculated and are plotted in Figure 2, which compares the performance of both baskets configurations. The reaction carried out in the lateral basket reactor was faster up to 3 h of interesterification. Then the maximum ID (8.14 ± 0.63%) was obtained in 3 h, but at 12 h this value has been decreased (6.11 ± 0.64%). For the central basket configuration, a maximum ID value (9.19 ± 0.60%) was achieved in 4 h of process. After this time, the ID decreased until 7.31 ± 0.99% in 12 h. The IE products, obtained in 12 h of reaction, were also evaluated in relation to their consistency, and the results are displayed in Table 1. The consistency is an important

Figure 4. Interesterification degree (-■-) and free fatty acid content (-□-) obtained at different times (2, 4, and 6 h) for each repeated batch: evaluation of the R. oryzae lipase operational stability.

(3.16 ± 0.51%) was attained at the first batch, which is in agreement with the previous result obtained in single-batch run (3.58 ± 0.23%, Figure 2). In subsequent batches the free fatty acid contents were lower than 3.00%. This may also suggest that the dehydration technique applied to the biocatalyst (washing with hexane) could restore its initial water level and, therefore, its original activity. The TAGs composition of the IE products was determined at different reaction times (2, 4, and 6 h), and a similar profile was found for all batch runs as observed in Figure 5, which

Table 1. Consistency Values for Noninteresterified (NIE) Blend and Interesterified Products Obtained by Use of Central or Lateral Basket Reactor Configuration

pure milkfat NIE blend central basket lateral basket

consistency value (gf/cm2)

consistency reduction (%)

± ± ± ±

58.18 68.43

6074.00 1228.84 513.82 387.89

406.80 78.24 52.12 105.83

E


Article


minimum consistency value was obtained (524.00 ± 50.75 gf/ cm2). Also, according to previously related studies, in this batch the maximum free fatty acid content (3.16 ± 0.51%) was obtained. The presence of free fatty acids indicates the occurrence of hydrolysis, and a small product amount resulting from partial TAG hydrolysis, as mono- and diglycerides, can act as an emulsifier and change the material texture.24,38 Loss of mass of the biocatalyst was not verified during the consecutive batch runs. In general, low variations in ID and consistency values were observed (Figure 6), which indicates that the biocatalyst maintained its activity over 60 h during 10 batch runs. Such results are favorable compared with those reported in the literature. Osorio et al.,37 for example, evaluated the operational stability of the lipase from Candida parapsilosis, immobilized in Accurel MP 1000, in the interesterification reactions of blends containing polyunsaturated fatty acids. In that work, the reactions were carried out in batch reactors, and the authors reported a biocatalyst half-life of 10 h. Then, the high operational stability showed by the immobilized derivative may be suitable for future industrial application. This may be attributed to the immobilizing procedure applied to the lipase that is a method developed by our research group.24,25 The carrier silica−poly(vinyl alcohol) (SiO2−PVA) was prepared by the sol−gel technique using a simple but effective procedure which gave an immobilized lipase preparation with good activity and stability that has been successfully used in different processes.24,25,39,40

Figure 5. Composition of the medium as a function of time in the interesterification reactions of milkfat with soybean oil in repeated batch runs to assess the biocatalyst operational stability using the central basket configuration.

displays the average concentrations of TAGs. The results also showed similarity with those previously obtained using this basket configuration (Figure 3b): the concentration of TAG carbon species 24−30 was increased, while the concentration of TAG carbon species 36−52 was decreased or almost did not change during the 6 h of reaction. Moreover, the concentration of TAG 54 was decreased in comparison with the NIE blend. From the TAGs concentrations, the interesterification degree (ID, %) at different times (2, 4, and 6 h) for each batch run was calculated as shown in Figure 4. This allows comparing the performance of the immobilized enzyme. In general, the maximum ID values were obtained in 4 h of process, and after this time a decrease in these values was verified. The highest ID value (8.56 ± 1.37%) was achieved in batch 5 at 4 h of reaction. The IE products, obtained in 6 h of reaction, were also evaluated in relation to their consistency. The results are displayed in Figure 6, which allow comparing the physical

■

CONCLUSIONS The use of the R. oryzae lipase decreased the consistency value of the IE product in relation to NIE blend, which demonstrated the potential of this immobilized derivative to yield milkfatbased fat more spreadable under cooling temperature and with lower saturated fatty acids contents. Both evaluated basket reactor designs have shown potential to be used in interesterification reactions of industrial interest. In addition, the potential of the reactor coupled with the central basket was demonstrated by maintaining both the performance of the batch reactions relatively stable during the repeated tests and the biocatalyst activity over 60 h during 10 batch runs.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support from Fundaçaõ de Amparo à Pesquisa do Estado de São Paulo (FAPESP), CNPq, and CAPES, Brazil.

■

Figure 6. Consistency (bars) of IE products (6 h) interesterification degree (line) as a function of reaction batch.

REFERENCES

(1) Ferreira-Dias, S.; Sandoval, G.; Plou, F.; Valero, F. The potential use of lipases in the production of fatty acid derivatives for the food and nutraceutical industries. Electron. J. Biotechnol. 2013, 16, 1. (2) Khodadadi, M.; Azis, S.; St-Louis, R.; Kermasha, S. Lipasecatalyzed synthesis and characterization of flaxseedoil-based structured lipids. J. Funct. Foods 2013, 5, 424. (3) Gupta, R.; Rathi, P.; Bradoo, S. Lipase mediated upgradation of dietary fats and oils. Crit. Rev. Food Sci. 2003, 43, 635. (4) Soares, F. A. S. M.; Osório, N. M.; Silva, R. C.; Gioielli, L. A.; Ferreira-Dias, S. Batch and continuous lipase-catalyzed interester-

properties and ID values in this reaction time. For all batches, the consistency of the IE products was lower than the initial NIE blend (1228.84 ± 78.24 gf/cm2, Table 1), except for batch 8 where samples showed consistency values outside the ideal range (200−800 gf/cm2), according to Haighton.31 The average value for the consistency in 6 h of process, from batch 2 to batch 10, was 738.19 ± 43.43 gf/cm2. In batch 1, the F


Article

Industrial & Engineering Chemistry Research ification of blends containing olive oil for trans-free margarines. Eur. J. Lipid Sci. Technol. 2013, 115, 413. (5) Nunes, G. S.; Marty, J. L. Immobilization of enzymes and cells; Humana Press: Totowa, 2006. (6) Aguedo, M.; Hanon, E.; Danthine, S.; Paquot, M.; Lognay, G.; Thomas, A.; Vandenbol, M.; Thonart, P.; Wathelet, J. P.; Blecker, C. Enrichment of anhydrous milk fat in polyunsaturated fatty acid residues from linseed and rapeseed oils through enzymatic interesterification. J. Agric. Food Chem. 2008, 56, 1757. (7) Firestone, D. Physical and Chemical Characteristics of Oils, Fats, and Waxes; AOCS Press: Urbana, IL, 2006. (8) Ledoux, M.; Chardign, J.-M.; Darbois, M.; Soustre, Y.; Sébédio, J.-L.; Laloux, L. Fatty acid composition of French butters, with special emphasis on conjugated linoleic acid (CLA) isomers. J. Food Compos. Anal. 2005, 18, 409. (9) Shin, J. A.; Akoh, C. C.; Lee, K. T. Production and physicochemical properties of functional - butterfat through enzymatic interesterification in a continuous reactor. J. Agric. Food Chem. 2009, 57, 888. (10) Akoh, C. C. Healthful Lipids; AOSC Press: Champaign, IL, 2005. (11) Wassell, P.; Bonwick, G.; Smith, C. J.; Almiron-Roig, E.; Young, N. W. G. Towards a multidisciplinary approach to structuring in reduced saturated fat-based systems-a review. Int. J. Food Sci. Technol. 2010, 45, 642. (12) Paula, A. V.; Nunes, G. F. M.; Freitas, L.; De Castro, H. F.; Santos, J. C. Interesterification of milkfat and soybean oil blends catalyzed by immobilized Rhizopus oryzae lipase. J. Mol. Catal. B: Enzym. 2010a, 65, 117. (13) Shin, J.; Akho, C. C.; Lee, K. Enzymatic interesterification of anhydrous butterfat with flaxseed oil and palm stearin to produce lowtrans spreadable fat. Food Chem. 2010, 120, 1. (14) Wrigth, A. J.; Scanlon, R. W.; Hartel, R. W.; Margoni, A. G. Rheological properties of milkfat and butter. J. Food Sci. 2001, 66, 1056. (15) Teixeira, J. A.; Fonseca, M. M.; Vicente, A. Reactores Biológicos: Fundamentos e Aplicaçoẽ s; Lidel: Lisboa, 2007. (16) Nunes, G. F. M.; Paula, A. V.; De Castro, H. F.; Santos, J. C. ́ da gordura do leite. Quim. Nova 2010, 33, Modificaçaõ bioquimica 431. (17) Hill, C. H.; Root, T. W. Introduction to Chemical Engineering Kinetics and Reactor Design; Wiley: New York, 2014. (18) Willis, W. M.; Marangoni, A. G. Food lipids: chemistry, nutrition, and biotechnology; CRC Press: Boca Raton, FL, 2008. (19) Watanabe, T.; Sugiura, M.; Sato, M.; Yamada, N.; Nakanishi, K. Diacylglycerol production in a packed bed bioreactor. Process Biochem. 2004, 40, 637. (20) Carvalho, P. D.; Campos, P. R. B.; Noffs, M. D.; Oliveira, J. G.; Shimizu, M. T.; Silva, D. M. Application of microbial lipases to concentrate polyunsaturated fatty acids. Quim. Nova 2003, 26, 75. (21) Fernandes, P.; Ferreira, B. S.; Cabral, J. M. S. Engenharia enzimática; Lidel: Lisboa, 2003. (22) Fernandes, P. Enzymes in Food Processing: A Condensed Overview on Strategies for Better Biocatalysts. Enzyme Res. 2010, 1. (23) Sheelu, G.; Kavitha, G.; Fadnavis, N. W. Efficient immobilization of Lecitase in gelatin hydrogel and degumming of rice bran oil using a spinning basket reactor. J. Am. Oil Chem. Soc. 2008, 85, 739. (24) Paula, A. V.; Nunes, G. F. M.; Santos, J. C.; De Castro, H. F. Interesterification of milkfat with soybean oil catalysed by Rhizopus oryzae lipase immobilised on SiO2-PVA on packed bed reactor. Int. J. Food Sci. Technol. 2011, 46, 2124. (25) Nunes, G. F. M.; Paula, A. V.; De Castro, H. F.; Santos, J. C. Optimization of the enzymatic interesterification of milk fat and canola oil blends using immobilized Rhizopus oryzae lipase by response surface methodology. Food Technol. Biotechnol. 2011, 49 (3), 385−390. (26) Paula, A. V. 2012. Reestruturaçaõ da gordura de leite por interesterificaçaõ enzimática empregando lipase imobilizada: otimizaçaõ das condições reacionais e operacionais. Tese (Doutorado em

Ciências); Escola de Engenharia de Lorena, Universidade de São Paulo, Lorena, SP, 2011; p 212.. (27) Paula, A. V.; Nunes, G. F. M.; Silva, J. L.; De Castro, H. F.; Santos, J. C. Screening of food grade lipases to be used in esterification and interesterification reactions of industrial interest. Appl. Biochem. Biotechnol. 2010b, 160, 1146. (28) Soares, C. M. F.; Castro, H. F.; Moraes, F. F.; Zanin, G. M. Characterization and utilization of Candida rugosa lipase immobilized on controlled pore silica. Appl. Biochem. Biotechnol. 1999, 77−9, 745. (29) American Oil Chemists’ Society. Official Methods and Recommended Practices of the AOCS; AOCS Press: Champaign, IL, 2004. (30) Precht, D.; Molkentin, J. The certification of the triglyceride contents of an anhydrous butter fat reference material with additional value for free cholesterol CRM 519; Comission of the European Communities: Luxembourg, 1997. (31) Haighton, A. J. The measurement of the hardness of margarine and fats with cone penetrometers. J. Am. Chem. Soc. 1959, 36, 345. (32) Akho, C. C; Xu, X. Lipid Biotechnology; CRC Press: Boca Raton, FL, 2002. (33) Alves, S. S.; Teixeira, J. A.; Vasconcelos, J. M. T.; Reis, N. Reactores Biológicos: Fundamentos e Aplicaçoẽ s; Lidel: Lisboa, 2007. (34) Bodalo, A.; Bastida, J.; Maximo, M. F.; Montiel, M. C.; Murcia, M. D.; Ortega, S. Influence of the operating conditions on lipase catalysed synthesis of ricinoleic acid estolides in solvent-free systems. Biochem. Eng. J. 2009, 44, 214. (35) Ribeiro, A.P. B.; Basso, R. C.; Grimaldi, R.; Gioielli, L. A.; Gonçalves, L. A. G. Instrumental methods for the evaluation of interesterified fats. Food Anal. Methods 2009, 2, 282. (36) Silva, V. C.; Contesini, F. J.; Carvalho, P. O. Enantioselective behavior of lipases from Aspergillus niger immobilized in different supports. J. Ind. Microbiol. Biot. 2009, 36, 949. (37) Osório, N. M.; Dubreucq, E.; Da Fonseca, M. M. R.; FerreiraDias, S. Operational stability of immobilised lipase/ acyltransferase during interesterification of fat blends. Eur. J. Lipid Sci. Technol. 2009, 111, 358. (38) Kaewthong, W.; Sirisansaneeyakul, S.; Prasertsan, P.; HKittikun, A. Continuous production of monoacylglycerols by glycerolysis of palm olein with immobilized lipase. Process Biochem. 2005, 40, 1525. (39) Freitas, L.; Da Ros, P. C. M.; Santos, J. C.; De Castro, H. F. An integrated approach to produce biodiesel and monoglycerides by enzymatic interestification of babassu oil (Orbinya sp). Process Biochem. 2009, 44, 1068. (40) Da Ros, P. C. M.; Silva, G. A. M.; Mendes, A. A.; Santos, J. C.; De Castro, H. F. Evaluation of the catalytic properties of Burkholderia cepacia lipase immobilized on non-commercial matrices to be used in biodiesel synthesis from different feedstocks. Bioresour. Technol. 2010, 101, 5508.

G


Synthesis of Structured Lipids by Enzymatic ... - ACS Publications

Recommend Documents