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Hydrophilization of bixin by lipase-catalyzed transesterification with sorbitol
Food Chemistry 268 (2018) 203–209
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Hydrophilization of bixin by lipase-catalyzed transesterification with sorbitol
T
Amita Jahangiria,2, Anders Hauer Møllera,3, Marianne Danielsena,4, Bjoern Madsenb, ⁎ Bjarne Joernsgaardb, Signe Vaerbakb, Patrick Adlercreutzc, Trine Kastrup Dalsgaarda, ,1 a
Department of Food Science, Aarhus University, DK-8830 Tjele, Denmark Chr. Hansen Natural Colors A/S, Hoejbakkegaard Alle, 30, 2630 Taastrup, Denmark c Department of Chemistry, Division of Biotechnology, Lund University, SE-221 00 Lund, Sweden b
A R T I C LE I N FO
A B S T R A C T
Keywords: Bixin Transesterification Lipase-catalysis Candida antarctica lipase B Lipase immobilization Water activity
Bixin is one of the most used yellow-orange food colorants in the food industry. The polyene chain of bixin makes it highly hydrophobic and less suitable for water-based food formulations. Lipase-catalyzed reactions of bixin with sorbitol were studied to synthesize a new derivative of bixin with potential hydrophilic properties. Interestingly, we show that the lipase-catalyzed reaction of bixin leads to a transesterification reaction and formation of a transesterified product, sorbitol ester of norbixin (SEN). The reaction efficiency was optimized with various immobilized lipases at different water activity levels in the organic solvent, 2-methyl-2-butanol. Among the examined lipases, immobilized Candida antarctica lipase B (Novozyme 435) provided the highest reaction yield at a water activity close to zero. Tetrahydrofuran (THF) was used as co-solvent to improve bixin solubility. The optimization of the reaction conditions with 20% THF lead to a total reaction yield of 50% of SEN.
1. Introduction The annatto colorant is one of the most widely used natural colorants in the food industry. The colorant is extracted from the seeds of the tropical shrub, Bixa Orellana. The major color components of annatto extract are bixin and norbixin (Scotter, Wilson, Appleton, & Castle, 1998). The bixin structure contains a long polyunsaturated hydrocarbon chain with one carboxylic acid and one methyl ester group in each end of the chain, which results in a high degree of lipophilicity. Norbixin is mainly produced by saponification of bixin in alkaline solution to form the diacid derivative of bixin. Norbixin has better hydrophilic properties, which makes it more applicable for particular food products such as cheddar cheese (Kang, Campbell, Bastian, & Drake, 2010). The food industry lacks stable natural hydrophilic colorants in the range of yellow–red, which can be used in low pH water-based food products such as beverages. As implied above, bixin is not soluble in aqueous food items. Furthermore, norbixin, which has been known as the water soluble annatto colorant is only soluble in alkaline solutions
⁎
Corresponding author. E-mail address: [email protected] (T.K. Dalsgaard). 1 orcid.org/0000-0002-5635-4102. 2 orcid.org/0000-0002-0103-112X. 3 orcid.org/0000-0002-9455-6039. 4 orcid.org/0000-0002-8850-5649. https://doi.org/10.1016/j.foodchem.2018.06.085 Received 23 March 2018; Received in revised form 17 June 2018; Accepted 18 June 2018 Available online 19 June 2018 0308-8146/ © 2018 Elsevier Ltd. All rights reserved.
and precipitates in acidic and neutral conditions (Breukers, Øpstad, Sliwka, & Partali, 2009). One way to increase the water solubility of bixin could be by appending a hydrophilic molecule to bixin to increase its polarity and thereby improve the aqueous solubility. There is a lack of studies investigating the reaction of bixin with hydrophilic compounds. To our knowledge, only one paper concerning the modification of the bixin scaffold by enzymatic reaction with L-ascorbic acid has been reported (Humeau, Rovel, & Girardin, 2000). Humeau et al. suggested that the reaction of bixin with ascorbic acid can occur via the ester bond to form a new bixin derivative. Besides, as ascorbic acid is an antioxidant, it might protect bixin from heat or light induced degradation. The enzymatic reaction was performed with immobilized lipase from Candida antarctica. According to Humeau et al. the conversion ratio of ascorbic acid to ascorbyl ester of norbixin was only 25% after 144 h reaction. It was assumed that the low reaction yield might be due to the low solubility of substances in the reaction solvent 2-methyl-2-butanol (2M2B). Solvents such as tert-butanol and diacetone alcohol were examined, but none of them improved the solubility of bixin (Humeau
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Ethanol (0.5 mL) was added to 0.5 g of solid support, polypropylene MP 1000 and mixed thoroughly. Lipase (0.5 g) was dissolved in 10 mL phosphate buffer (50 mM, pH 7.0) and mixed and centrifuged at 2700g for 5 min to remove undissolved particles. Subsequently, the supernatant was mixed with the prepared polypropylene support solution. The immobilization was carried out by shaking the solution using an orbital shaker over night at room temperature. The preparations were filtered and gently washed with phosphate buffer (50 mM, pH 7.0) and then dried in a desiccator under vacuum overnight. The immobilization yield was determined by measuring the enzymatic activity of the enzyme solutions before and after immobilization based on the hydrolysis of p-nitrophenyl butyrate (Gitlesen, Bauer, & Adlercreutz, 1997).
et al., 2000). Hydrophilic derivatives of bixin can be synthesized through esterification or transesterification reactions using biocatalysts. Lipases are widely used as the biocatalyst for esterification or transesterification reactions and the immobilized ones have been shown to exhibit a better catalytic activity compared to the non-immobilized lipases in nonaqueous media (Adlercreutz, 2017). The use of organic solvents in enzymatic reactions can favor an esterification or transesterification reaction on bixin rather than a hydrolysis, to prevent the formation of norbixin. Among the most commonly used non-immobilized lipases for esterification or transesterification reactions are those from the microorganisms R. arrhizus, C. rugosa and P. cepacia. Immobilized lipases, such as Novozyme 435 (C. antarctica lipase B, CALB), Lipozyme RM-IM (lipase from R. miehei) and Lipozyme TL-IM (lipase from T. lanuginosus) are also commercially available. Although a lower water content is favorable for the esterification or transesterification reactions, we need to take into account that the activity of lipases are highly dependent on their hydration level. A low water content has also been shown essential in lipase-catalyzed reactions in non-aqueous solutions and the water content of the reactions can significantly impact the catalytic activity of lipases (Adlercreutz, 2013; Klibanov and Zaks, 1987). Water is known to act as a molecular lubricant for lipase in the non-aqueous media, whereas if the water layer is stripped away, the active site of the lipase might show conformational changes and thus display lower catalytic activity (Halling, 2004). Lipases respond differently to the amount of water in organic media. Some retain good activity at low water activity, e.g. the lipase from R. arrhizus and CALB, while some require higher water activity level to show good catalytic activity, e.g. lipase from P. cepacia (Wehtje & Adlercreutz, 1997). It is difficult to measure the amount of water bound to the lipase, but the water activity level of the reactions can be controlled and adjusted by different saturated salt solutions (Greenspan, 1977). In the present study, we aim to modify the chemical structure of bixin by introducing sorbitol as a hydrophilic compound into the bixin scaffold, using a lipase-catalyzed reaction. Sorbitol has previously been used to synthesis a derivate of astaxanthin in order to improve the water solubility (Lockwood et al., 2005). Moreover, we will investigate whether different lipases will favor the esterification or the transesterification reaction. Furthermore, we will find the optimal water activity level for the individual lipases and reaction solvents, in order to optimize the reaction yield.
2.3. Equilibration of water activity The thermodynamic water activity level of all experiments were adjusted by equilibrating the substrates and solvents with aqueous saturated salt solutions in separated vessels over two days. Equilibration was obtained using MgCl2 and Mg(NO3)2 at room temperature to reach the water activity levels of 0.33 and 0.53, respectively (Greenspan, 1977). The molecular sieve was used to dry solvents to reach a water activity level of zero. The substrates were dried under vacuum. 2.4. Synthesis of sorbitol ester of norbixin Bixin (59 mg, 50 mM), sorbitol (27 mg, 50 mM) and immobilized lipase 12 mg (20% of the total weight of bixin) were weighed into a 4 mL septum capped vial. A mixture of 2M2B:THF (3 mL) was added in different ratios based on reaction conditions from 0 to 100% of THF. The reaction mixture was shaken using a MHR11-TH21 thermo shaker (HLC, Germany) at 58 °C and 750 rpm. After 24 h of reaction, the lipase was separated from the reaction mixture by centrifugation (centrifuge model 4417R, Hamburg, Germany) at 20,200g for 10 min. The supernatant was separated and stored at −20 °C until further analysis using ultra-High Performance Liquid Chromatography (uHPLC) with reverse phase separation. 2.5. Purification The SEN was isolated by glass column chromatography. The column was prepared with silica gel using a mixture of chloroform/methanol/ water/acetic acid 86:12:1:1 (v/v/v/v) as elution solvent. The fractions containing the product were combined, the solvent was evaporated using a vacuum centrifuge and the residue was analyzed and quantified by uHPLC.
2. Materials and methods 2.6. Thin layer chromatography analysis 2.1. Reagents Thin layer chromatography (TLC) analysis was used to monitor the reaction progress. The reaction mixtures were diluted in THF 1:50 (v/v) and analyzed on TLC plates (TLC Silica gel 60, 5 × 7.5 cm, Merck, Germany) with the elution system chloroform/methanol/water/acetic acid 86:12:1:1 (v/v/v/v). Under these conditions, the retention factors (Rf) were 0.19, 0.62 and 0.81for SEN, norbixin and bixin, respectively.
Bixin was provided by Chr. Hansen A/S (Taastrup, Denmark). Lipase acrylic resin from Candida antarctica, ≥5,000 U/g (Novozyme 435), Lipozyme RM-IM (lipase from R. miehei) and Lipozyme TL-IM (lipase from T. lanuginosus), were purchased from Sigma-Aldrich. Nonimmobilized lipases, Rhizopus arrhizus, Candida rugosa and Pseudomonas cepacia were purchased from Sigma-Aldrich. Porous polypropylene (Accurel MP 1000) was purchased from Membrana GmbH (Obernburg, Germany). P-nitrophenyl butyrate (p-NPB, 98% pure), D-Sorbitol (≥98% pure) and all other solvents were of analytical grade. Molecular sieve (3 Å, 2 mm bead, 10 mesh) were purchased from Merck (Darmstadt, Germany). Silica gel (60 Å, 70–230 mesh, 63–200 µm) were purchased from Sigma-Aldrich.
2.7. Quantification Quantification was performed on an uHPLC Shimadzu LC-2040C 3D system (Duisburg, Germany), equipped with a photodiode array detector (PDA). The analyte mixture was diluted 50 times in THF and 2 µL was injected onto a C18 column (2.6 µm, 100 mm, 4.6 mm, Kinetex), at 1.6 mL/min flow rate and a column temperature of 35 °C. The mobile phases consisted of solvent A with 2% aqueous acetic acid and solvent B with acetonitrile with 0.2% acetic acid. The step linear gradients were; 45% B at 0 min, 70% B at 4 min, 70% B at 9 min, 100% B at 10 min, 100% B at 14 min, 45% B at 15 min and 45% B at 20 min. Detection was
2.2. Lipase immobilization Lipases from R. arrhizus, C. rugosa and P. cepacia were immobilized separately on polypropylene with particle size smaller than 500 µm. 204
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though the evidence for the identification of the reaction product was limited (Humeau et al., 2000). Absorption spectra of SEN and bixin were compared. Both compounds showed the same absorption pattern indicating that appending sorbitol to bixin did not effect on the conjugated polyene chain and they appear the same color (data not shown). The result from the reverse phase separation indicated bixin and norbixin eluted at 6.70 and 4.47 min, respectively. Two separated peaks with the same molecular mass represented of SEN eluted with a major peak at 2.61 min and minor peak at 2.15 min. The major peak was approximately three times higher than the minor peak. Both peaks corresponding to two isomers of SEN were integrated and the sum was used for the quantification. Bixin is naturally found as 9′-cis-isomer (M. Scotter, 2009), through the heating process of the reaction the 9′,13′-dicis isomer of bixin can form. Therefore, we suggested that SEN has also two cis- and di-cis isomers. At 40 °C a yield of 8% of SEN was obtained and the yield of norbixin was 3% after 72 h reaction. This reaction yield was not satisfactory high and consequently the reaction conditions needed optimization. Therefore, the following parameters were optimized; the source of lipase, water activity, temperature and solvent composition.
carried out at 460 nm. Peak areas for bixin, norbixin and SEN were integrated. Data acquisition and processing were performed using the LabSolution software program (Shimadzu, Kyoto, Japan). 2.8. Identification LC-MS identification was carried out using an Agilent 6560 Ion Mobility LC Q-TOF (Santa Clara, CA, USA) equipped with LC (Agilent 1290 Infinity II) system using diode array detector (DAD) and Mass Hunter software (Agilent, Santa Clara, CA, USA). The used column was a C18 column (2.6 µm, 100 mm, 4.6 mm, Kinetex) and the mobile phase consisted of solvent A with 2% aqueous acetic acid and solvent B with acetonitrile with 0.2% acetic acid. The LC was run at isocratic conditions at 70% solvent B and 0.3 mL/min flow rate. The column temperature was maintained at 35 °C. The Q-TOF instrument was equipped with an Agilent jet stream electro spray ionization source. The fragmentor voltage was set at 400 V. The flow rate of drying-gas (N2) was 11 L/min with a temperature of 150 °C and the nebulizer pressure was 35 psi. The sheath gas was 11 L/min with a temperature of 350 °C. The nozzle voltage was 1500 V and the capillary voltage was set at 3500 V. Mass spectra were recorded over a mass-to-charge ratio (m/z) range of 100–1000 in negative ion detection mode, and the collision energy was adjusted to 20 eV.
3.2. Lipase screening One way to improve the reaction yield would be to find a lipase better suited for the desired transesterification reaction. Lipases originating from R. arrhizus, C. rugosa and P. cepacia were chosen for a screening study and their activity in the hydrolysis of p-nitrophenyl butyrate (Gitlesen et al.) was measured. C. rugose lipase had the highest catalytic activity, followed by the lipases from P. cepacia and R. arrhizus (Table 1). Immobilization of lipases can often improve their catalytic activity in organic solvents and therefore the investigated lipases were immobilized on a hydrophobic support material, microporous polypropylene (Accurel MP 1000), which previously has been shown to be an efficient support for lipase immobilization (Nordblad & Adlercreutz, 2013). An immobilization yield above 88% were resulted for all the lipases (Table 1). In addition, three commercially available immobilized lipases were included in the screening study; Lipozyme RMIM (originating from R. miehei), Lipozyme TL-IM (originating from T. lanuginosus) and Novozyme 435 (immobilized C. antarctica lipase B), the enzyme used in the preliminary experiments. Since lipases differ with respect to water activity dependence, all lipases were tested at three different water activity levels; 0, 0.33 and 0.53. The yield of SEN was analyzed after 24 h of reaction and Novozyme 435 was the only lipase catalyzing the transesterification of bixin and sorbitol, while the rest of the lipases did not catalyze the reaction under these conditions. There have been several studies on the conformational changes of the active site region of C. antarctica lipase during the catalytic action (Haeffner, Norin, & Hult, 1998; Orrenius et al., 2009; Raza, Fransson, & Hult, 2001). The C. antarctica lipase is known as a lipase which lacks the typical lid that is normally present in many lipases (Uppenberg, Hansen, Patkar, & Jones, 1994). In principle, the active site lid needs to be open to some extent during the catalytic event to transfer the substrates and the products in and out. Lipases with a lid normally works at an interface that will favor opening of the lid (Paul Woolley, 1994). Therefore, the properties of the solvent, as well as the substrates can change the active site conformation. Uppenberg et al. suggested that the active site region of C. antarctica lipase consists of four hydrophobic residues, which provide a channel for substrates to cross and a small helix appears to act as a lid (Uppenberg, et al., 1994). It was also reported that the catalytic behavior of C. antarctica is more similar to an esterase rather than a lipase with no interfacial activation (Martinelle, 1995). This property might be one of the possible reasons that C. antarctica lipase catalyzed the reaction between bixin and sorbitol, while the other lipases displayed no activity neither in transesterification nor
3. Results and discussion 3.1. Lipase-catalyzed reaction of bixin 3.1.1. Transesterification versus hydrolysis In the present study, we aimed to synthesize a novel ester from bixin with the linear sugar alcohol, sorbitol using an enzymatic reaction in organic medium. Finding an adequate organic solvent can be a challenge. In many non-polar solvents, bixin solubility is low. In contrast, bixin shows higher solubility in polar solvents, such as ethanol and THF, but polar solvents might cause lipase inactivation. Initially, 2M2B, a well-known solvent for pigment applications, was selected as the organic medium. The immobilized lipase B from C. antarctica, which has previously been shown efficient in transesterification reactions (Humeau et al., 2000) was used as the biocatalyst. The reaction was carried out under dehydrated conditions using a molecular sieve to dry out the solvent and thereby minimize the hydrolysis reaction. The reaction was conducted at 40 °C and the formation of the new compound was monitored by TLC. The reaction mixture was analyzed by LC Q-TOF to identify the new product. The mass of the newly formed product was determined to m/z 543.2596, compared to the theoretical mass of SEN, m/z 543.2594, corresponding to a ± 0.3 ppm error (Fig. 1a). The fragmentation pattern gave most intense peaks at m/z 335, 291, 379 and 181, which supported the identification of the suggested SEN (Fig. 1b). Three cleavage sites around the ester bond with the sorbitol moiety and one at the carboxylic acid end of bixin chain were observed. The sorbitol moiety was cleaved of, resulting in the specific fragment m/z 181 [C6H13O6]-. The m/z 335 [C23H27O2]− corresponded to α-cleavage of a carbonyl bond resulting in the conjugated unsaturated carbon chain and the esterified sorbitol moiety, corresponding to [C7H13O7]−. Subsequently, the conjugated unsaturated carbon chain lost the carboxylic acid group generating the fragment at m/z 291, [C22H27]−. The m/z 379 [C24H27O4]− derived from loss of a m/z 165-fragment [C6H13O5]−, corresponding to the sorbitol moiety with one oxygen molecule. The results from the LC-MS/MS suggested the transesterification reaction at the methyl ester group to be the favorable reaction (Fig. 2), rather than the esterification reaction at the carboxylic acid group. This is the first report on enzymatic transesterification of bixin with sorbitol. Previously, Novozyme 435 has been suggested to catalyze a similar transesterification reaction between bixin and ascorbic acid via the methyl carboxylic acid group and not the carboxylic acid group, even 205
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Fig. 1. (a) MS spectra of sorbitol ester of norbixin. Theoretical mass m/z 543.2594, detected mass m/z 543.2596 (0.3 ppm error). (b) MS-MS spectra of SEN. The reaction was carried out in 2-methyl-2-butanol at 40 °C, 12 mL reaction mixture using bixin (237 mg, 50 mM), sorbitol (109 mg, 50 mM) and Novozyme 435, 47 mg (20% of the total weight of bixin) in 50 mL glass tube with screw cap.
reaction rate by eliminating the inhibition effect of the water molecules (Nordblad & Adlercreutz, 2013). Although many lipases show better activity at high water activity levels, the C. antarctica lipase apparently has a different water activity profile and requires less water to show high catalytic activity.
in hydrolysis reaction. 3.3. Reaction optimization 3.3.1. Effect of water activity on Novozyme 435 The optimal water activity level was determined for Novozyme 435 (Fig. 3). The production of SEN was favored at low water activity (aw = 0), whereas the hydrolysis reaction was favored at higher water activities, aw = 0.33 and 0.53. Low water activity favored the transesterification reaction rather than hydrolysis. At higher water activity levels, Novozyme 435 showed lower catalytic activity towards both the transesterification and the hydrolysis reactions, indicating an overall reduction of the catalytic activity. These results correlate with literature confirming that the immobilized C. antarctica lipase displays higher activity in organic medium when the water activity is close to zero. For example, in the synthesis of ascorbyl palmitate in 2M2B, the highest initial reaction rate was obtained when the reaction mixture was preequilibrated at water activity level of 0.07 (Humeau, Girardin, Rovel, & Miclo, 1998a, 1998b). Nordblad et al. investigated the transesterification of ethyl acrylate by octanol using immobilized CALB on MP1000 and demonstrated that lower water activity (0.06) provides higher
3.3.2. Temperature study The effect of temperature on Novozyme 435 was studied at three different reaction temperatures, 40 °C, 50 °C and 58 °C and the reaction yield was monitored for 72 h (Fig. 4). The reaction yield increased gradually with increasing reaction time at 40 °C and 50 °C and reached 8% and 18% after 72 h reaction, respectively. The reaction yield at 58 °C increased rapidly and was doubled compared to 50 °C, after both 23 h and 46 h of reaction time. Thereafter, the reaction slowed down until 72 h of reaction, reaching a yield of 31%. From the results presented, it is obvious that higher temperatures provided higher reaction yields, which demonstrating that Novozyme 435 has a high thermostability. According to other studies, the C. antarctica lipase has shown thermal stability between 30 and 70 °C in alcohols, with especially good stability in long-chain alcohols (Ghaly, Dave, Brooks, & Budge, 2010; Gumel, Annuar, Heidelberg, & Chisti, 2011; Yoshida, Kimura, & Adachi, 206
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Fig. 2. Enzymatic reaction of bixin with sorbitol using immobilized C. antarctica lipase B (Novozyme 435) in solvent 2-methyl-2-butanol. (1) 9′-cis-Bixin, (2) Sorbitol, (3) 9′-cis-sorbitol ester of norbixin.
Table 1 Lipase immobilization results. Lipase source
Enzymatic activity1 [µmol/min.mg]
Immobilization yield %
R. arrhizus C. rugosa P. cepacia
0.040 2.971 0.350
88.7 93.3 92.9
1
Enzymatic activities were measured on free lipase.
2006). The high temperature tolerance of the lipase provides the possibility of increasing the reaction temperature and thus, improving the solubility of the substrates in the reaction media, which is important when the substrates otherwise have low solubility in organic media. Moreover, a higher reaction temperature can improve the mass transfer conditions by accelerating the diffusion of the substrates into the lipase active site and formation of acyl-lipase complex (Jiugao, Zhang, Zhao, & Ma, 2008). However, in our study the reaction temperature was kept at 58 °C for the rest of the experiments. Fig. 3. Selectivity between transesterification and hydrolysis reaction using immobilized C. antarctica lipase B (Novozyme 435) at three different water activity levels. The reaction was carried out in 2-methyl-2-butanol and 10% tetrahydrofuran for 24 h at 58 °C with bixin (59 mg, 50 mM), sorbitol (27 mg, 50 mM) and Novozyme 435, (12 mg, 20% of the total weight of bixin) in 4 mL septum capped vial.
3.3.3. Solvent effect The solubility of the substrates is another parameter that was considered. One of the main issues in the enzymatic reaction of bixin with sorbitol was to select a proper organic solvent. The solvent should show solubility for both substrates as well as retaining the lipase activity. Bixin and sorbitol show very low solubility in non-polar solvents and have better solubility in polar solvents such as THF, ethanol and methanol. However, these solvents might lead to lipase inactivation. Zhi and co-workers studied the solubility of sorbitol in different solvents at different temperature. Sorbitol solubility was high in many alcohols due
to the formation of hydrogen bonds and it was moderately soluble in THF, in which solubility increased with increasing temperature (Zhi, Hu, Yang, Kai, & Cao, 2013). Our previous experiments showed that the solubility of bixin and 207
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the active site of the C. antarctica lipase stays stable in non-polar solvents (log p > 4), while polar solvent (log p < 2) can interact with the active site and breaking the hydrogen bonds between the amino acid residues important for lipase activity (Cong Li, Zhang, & Feng, 2010). Moreover, Park et al. observed higher catalytic efficiency for the C. antarctica lipase in tert-butanol than methanol using molecular dynamics simulations (Park, Park, & Yoo, 2013). This indicates that 2M2B should be a better solvent than THF for sustaining lipase activity. On the other hand, adding THF improves substrate solubility, and the solvent mixture of 80% 2M2B and 20% THF turned out to be a good compromise, resulting in the highest yield. 4. Conclusion The hydrophobic characteristics of bixin limits its food application in water-based food products. In this paper, we introduced a simple method to catalyze the reaction between bixin and sorbitol, thereby synthesizing a new derivate of bixin, SEN, which is likely to have better hydrophilic properties. Transesterification of bixin was carried out using immobilized lipases from different sources at different water activity levels. Novozyme 435 was the only lipase, which efficiently catalyzed the transesterification of bixin with sorbitol at a water activity close to zero. A solvent mixture of 80% 2M2B and 20% THF provided the highest reaction yield of 50%. The sugar alcohol moiety on the bixin molecule is likely to change the chemical properties of bixin towards a hydrophilic colorant. Moreover, the modified colorant might show emulsifier properties, which even further increases its application in the food industry. Further studies are needed to determine the aqueous solubility, as well as stability, of the new sorbitol derivate of bixin.
Fig. 4. Temperature study of reaction between bixin with sorbitol using immobilized C. antarctica lipase B (Novozyme 435) at water activity level of zero performed at three different temperatures. The reactions were carried out in 2methyl-2-butanol, 12 mL reaction mixture using bixin (237 mg, 50 mM), sorbitol (109 mg, 50 mM) and Novozyme 435, (47 mg, 20% of the total weight of bixin) in 50 mL glass tube with screw cap.
Acknowledgements This study was part of the Annatto project funded by the Innovation Fund Denmark, grant no. 4097-00004B-2014 and Aarhus University. The authors would like to thank Dr. Bashar Amer for fruitful discussions. References Adlercreutz, P. (2013). Immobilisation and application of lipases in organic media. Chemical Society Reviews, 42(15), 6406–6436. Adlercreutz, P. (2017). Comparison of lipases and glycoside hydrolases as catalysts in synthesis reactions. Applied microbiology biotechnology, 101(2), 513–519. Breukers, S., Øpstad, C. L., Sliwka, H. R., & Partali, V. (2009). Hydrophilic carotenoids: Surface properties and aggregation behavior of the potassium salt of the highly unsaturated diacid norbixin. Helvetica Chimica Acta, 92(9), 1741–1747. Cong Li, T. T., Zhang, Haiyang, & Feng, Wei (2010). Analysis of the conformational stability and activity of Candida antarctica lipase B in organic solvents. Journal of Biological Chemistry, 285(37), 28434–28441. Ghaly, A. E., Dave, D., Brooks, M. S., & Budge, S. (2010). Production of biodiesel by enzymatic transesterification: Review. American Journal of Biochemistry and Biotechnology, 6(2), 54–76. Gitlesen, T., Bauer, M., & Adlercreutz, P. (1997). Adsorption of lipase on polypropylene powder. Biochimica Et Biophysica Acta, 1345(2), 188–196. Greenspan, L. (1977). Humidity fixed-points of binary saturated aqueous-solutions. Journal of Research of the National Bureau of Standards Section a-Physics and Chemistry, 81(1), 89–96. Gumel, A. M., Annuar, M. S. M., Heidelberg, T., & Chisti, Y. (2011). Lipase mediated synthesis of sugar fatty acid esters. Process Biochemistry, 46(11), 2079–2090. Haeffner, F., Norin, T., & Hult, K. (1998). Molecular modeling of the enantioselectivity in lipase-catalyzed transesterification reactions. Biophysical Journal, 74(3), 1251–1262. Halling, P. J. (2004). What can we learn by studying enzymes in non-aqueous media? Philosophical Transactions of the Royal Society B Biological Sciences, 359(1448), 1287–1296 discussion 1296-1287, 1323-1288. Humeau, C., Girardin, M., Rovel, B., & Miclo, A. (1998a). Effect of the thermodynamic water activity and the reaction medium hydrophobicity on the enzymatic synthesis of ascorbyl palmitate. Journal of Biotechnology, 63(1), 1–8. Humeau, C., Girardin, M., Rovel, B., & Miclo, A. (1998b). Enzymatic synthesis of fatty acid ascorbyl esters. Journal of Molecular Catalysis B-Enzymatic, 5(1–4), 19–23. Humeau, C., Rovel, B., & Girardin, M. (2000). Enzymatic esterification of bixin by Lascorbic acid. Biotechnology Letters, 22(2), 165–168. Jiugao, Yu, Zhang, J., Zhao, Ang, & Ma, Xiaofei (2008). Study of glucose ester synthesis by immobilized lipase from Candida sp. Catalysis Communications, 1369–1374. Kang, E. J., Campbell, R. E., Bastian, E., & Drake, M. A. (2010). Invited review: Annatto
Fig. 5. Reaction yield of sorbitol ester of norbixin using immobilized C. antarctica lipase B (Novozyme 435) in 2-methyl-2-butanol with different level of tetrahydrofuran at water activity level of zero, at 58 °C, with bixin (59 mg, 50 mM), sorbitol (27 mg, 50 mM) and Novozyme 435, (12 mg, 20% of the total weight of bixin) in 4 mL septum capped vial. The yield was quantified after 24 h. The reaction with 20% tetrahydrofuran was repeated and the data is presented as mean ± standard deviation.
sorbitol were not high in 2M2B, and therefore, in order to facilitate the solubility of bixin and sorbitol in the organic medium, different amounts of THF were added as a co-solvent. The reaction yield of SEN and the hydrolysis product, norbixin were quantified (Fig. 5). The results show that varying the solvent content had a large influence on the reaction yield. Addition of 20% of THF improved the yield resulting in an overall reaction yield of 50%, while adding even more THF decreased the yield of SEN. The formation of norbixin was less influenced by the THF content. Many studies have attempted to explain the effect of solvent polarity on the conformation and catalytic activity of the C. antarctica lipase. Hydrophobicity is commonly described by the log p parameter, which explains the tendency of the solvent to partition between phases with different polarities. 2M2B and THF are considered as polar solvents with log p values of 1.3 and 0.49, respectively. Cong et al. showed that 208
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Raza, S., Fransson, L., & Hult, K. (2001). Enantioselectivity in Candida antarctica lipase B: A molecular dynamics study. Protein Science, 10(2), 329–338. Scotter, M. (2009). The chemistry and analysis of annatto food colouring: A review. Food Additives and Contaminants Part a-Chemistry Analysis Control Exposure & Risk Assessment, 26(8), 1123–1145. Scotter, M. J., Wilson, L. A., Appleton, G. P., & Castle, L. (1998). Analysis of annatto (Bixa orellana) food coloring formulations. 1. Determination of coloring components and colored thermal degradation products by high-performance liquid chromatography with photodiode array detection. Journal of Agricultural and Food Chemistry, 46(3), 1031–1038. Uppenberg, J., Hansen, M. T., Patkar, S., & Jones, T. A. (1994). The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica. Structure, 2(4), 293–308. Wehtje, E., & Adlercreutz, P. (1997). Water activity and substrate concentration effects on lipase activity. Biotechnology and Bioengineering, 55(5), 798–806. Yoshida, Y., Kimura, Y., & Adachi, S. (2006). Thermal inactivation of immobilized lipase in 1-alcohols. Journal of Bioscience and Bioengineering, 102(1), 66–68. Zhi, W. B., Hu, Y. H., Yang, W. G., Kai, Y. M., & Cao, Z. (2013). Measurement and correlation of solubility of D-sorbitol in different solvents. Journal of Molecular Liquids, 187, 201–205.
usage and bleaching in dairy foods. Journal of Dairy Science, 93(9), 3891–3901. Klibanov, A., & Zaks, A. A. (1987). Enzymatic catalysis in nonaqueous solvents. Journal of Biological Chemistry, 263, 3194–3201. Lockwood, S. F, O'Malley, S., Watumull D. G., Hix L. M., Jackson, H., & Nadolski, G. (2005). Carotenoid ester analogs or derivatives for the inhibition and amelioration of ischemic reperfusion injury. US20050037995. Martinelle, Mats, Holmquist, Mats, & Hult, Karl (1995). On the interfacial activation of Candida antarctica lipase A and B as compared with Humicola lanuginosa lipase. Biochini. Biophys. Acta, 272–276. Nordblad, M., & Adlercreutz, P. (2013). Immobilisation procedure and reaction conditions for optimal performance of Candida antarcticalipase B in transesterification and hydrolysis. Biocatalysis and Biotransformation, 31(5), 237–245. Orrenius, C., Hbffner, F., Rotticci, D., öhrner, N., Norin, T., & Hult, K. (2009). Chiral recognition of alcohol enantiomers in acyl transfer reactions catalysed By Candida Antarctica lipase B. Biocatalysis and Biotransformation, 16(1), 1–15. Park, H. J., Park, K., & Yoo, Y. J. (2013). Understanding the effect of tert-butanol on Candida antarctica lipase B using molecular dynamics simulations. Molecular Simulation, 39(8), 653–659. Paul Woolley, S. B. P. (1994). Lipases: Their structure biochemistry and application. Cambridge University Press159–180.
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Hydrophilization of bixin by lipase-catalyzed transesterification with sorbitol
T
Amita Jahangiria,2, Anders Hauer Møllera,3, Marianne Danielsena,4, Bjoern Madsenb, ⁎ Bjarne Joernsgaardb, Signe Vaerbakb, Patrick Adlercreutzc, Trine Kastrup Dalsgaarda, ,1 a
Department of Food Science, Aarhus University, DK-8830 Tjele, Denmark Chr. Hansen Natural Colors A/S, Hoejbakkegaard Alle, 30, 2630 Taastrup, Denmark c Department of Chemistry, Division of Biotechnology, Lund University, SE-221 00 Lund, Sweden b
A R T I C LE I N FO
A B S T R A C T
Keywords: Bixin Transesterification Lipase-catalysis Candida antarctica lipase B Lipase immobilization Water activity
Bixin is one of the most used yellow-orange food colorants in the food industry. The polyene chain of bixin makes it highly hydrophobic and less suitable for water-based food formulations. Lipase-catalyzed reactions of bixin with sorbitol were studied to synthesize a new derivative of bixin with potential hydrophilic properties. Interestingly, we show that the lipase-catalyzed reaction of bixin leads to a transesterification reaction and formation of a transesterified product, sorbitol ester of norbixin (SEN). The reaction efficiency was optimized with various immobilized lipases at different water activity levels in the organic solvent, 2-methyl-2-butanol. Among the examined lipases, immobilized Candida antarctica lipase B (Novozyme 435) provided the highest reaction yield at a water activity close to zero. Tetrahydrofuran (THF) was used as co-solvent to improve bixin solubility. The optimization of the reaction conditions with 20% THF lead to a total reaction yield of 50% of SEN.
1. Introduction The annatto colorant is one of the most widely used natural colorants in the food industry. The colorant is extracted from the seeds of the tropical shrub, Bixa Orellana. The major color components of annatto extract are bixin and norbixin (Scotter, Wilson, Appleton, & Castle, 1998). The bixin structure contains a long polyunsaturated hydrocarbon chain with one carboxylic acid and one methyl ester group in each end of the chain, which results in a high degree of lipophilicity. Norbixin is mainly produced by saponification of bixin in alkaline solution to form the diacid derivative of bixin. Norbixin has better hydrophilic properties, which makes it more applicable for particular food products such as cheddar cheese (Kang, Campbell, Bastian, & Drake, 2010). The food industry lacks stable natural hydrophilic colorants in the range of yellow–red, which can be used in low pH water-based food products such as beverages. As implied above, bixin is not soluble in aqueous food items. Furthermore, norbixin, which has been known as the water soluble annatto colorant is only soluble in alkaline solutions
⁎
Corresponding author. E-mail address: [email protected] (T.K. Dalsgaard). 1 orcid.org/0000-0002-5635-4102. 2 orcid.org/0000-0002-0103-112X. 3 orcid.org/0000-0002-9455-6039. 4 orcid.org/0000-0002-8850-5649. https://doi.org/10.1016/j.foodchem.2018.06.085 Received 23 March 2018; Received in revised form 17 June 2018; Accepted 18 June 2018 Available online 19 June 2018 0308-8146/ © 2018 Elsevier Ltd. All rights reserved.
and precipitates in acidic and neutral conditions (Breukers, Øpstad, Sliwka, & Partali, 2009). One way to increase the water solubility of bixin could be by appending a hydrophilic molecule to bixin to increase its polarity and thereby improve the aqueous solubility. There is a lack of studies investigating the reaction of bixin with hydrophilic compounds. To our knowledge, only one paper concerning the modification of the bixin scaffold by enzymatic reaction with L-ascorbic acid has been reported (Humeau, Rovel, & Girardin, 2000). Humeau et al. suggested that the reaction of bixin with ascorbic acid can occur via the ester bond to form a new bixin derivative. Besides, as ascorbic acid is an antioxidant, it might protect bixin from heat or light induced degradation. The enzymatic reaction was performed with immobilized lipase from Candida antarctica. According to Humeau et al. the conversion ratio of ascorbic acid to ascorbyl ester of norbixin was only 25% after 144 h reaction. It was assumed that the low reaction yield might be due to the low solubility of substances in the reaction solvent 2-methyl-2-butanol (2M2B). Solvents such as tert-butanol and diacetone alcohol were examined, but none of them improved the solubility of bixin (Humeau
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Ethanol (0.5 mL) was added to 0.5 g of solid support, polypropylene MP 1000 and mixed thoroughly. Lipase (0.5 g) was dissolved in 10 mL phosphate buffer (50 mM, pH 7.0) and mixed and centrifuged at 2700g for 5 min to remove undissolved particles. Subsequently, the supernatant was mixed with the prepared polypropylene support solution. The immobilization was carried out by shaking the solution using an orbital shaker over night at room temperature. The preparations were filtered and gently washed with phosphate buffer (50 mM, pH 7.0) and then dried in a desiccator under vacuum overnight. The immobilization yield was determined by measuring the enzymatic activity of the enzyme solutions before and after immobilization based on the hydrolysis of p-nitrophenyl butyrate (Gitlesen, Bauer, & Adlercreutz, 1997).
et al., 2000). Hydrophilic derivatives of bixin can be synthesized through esterification or transesterification reactions using biocatalysts. Lipases are widely used as the biocatalyst for esterification or transesterification reactions and the immobilized ones have been shown to exhibit a better catalytic activity compared to the non-immobilized lipases in nonaqueous media (Adlercreutz, 2017). The use of organic solvents in enzymatic reactions can favor an esterification or transesterification reaction on bixin rather than a hydrolysis, to prevent the formation of norbixin. Among the most commonly used non-immobilized lipases for esterification or transesterification reactions are those from the microorganisms R. arrhizus, C. rugosa and P. cepacia. Immobilized lipases, such as Novozyme 435 (C. antarctica lipase B, CALB), Lipozyme RM-IM (lipase from R. miehei) and Lipozyme TL-IM (lipase from T. lanuginosus) are also commercially available. Although a lower water content is favorable for the esterification or transesterification reactions, we need to take into account that the activity of lipases are highly dependent on their hydration level. A low water content has also been shown essential in lipase-catalyzed reactions in non-aqueous solutions and the water content of the reactions can significantly impact the catalytic activity of lipases (Adlercreutz, 2013; Klibanov and Zaks, 1987). Water is known to act as a molecular lubricant for lipase in the non-aqueous media, whereas if the water layer is stripped away, the active site of the lipase might show conformational changes and thus display lower catalytic activity (Halling, 2004). Lipases respond differently to the amount of water in organic media. Some retain good activity at low water activity, e.g. the lipase from R. arrhizus and CALB, while some require higher water activity level to show good catalytic activity, e.g. lipase from P. cepacia (Wehtje & Adlercreutz, 1997). It is difficult to measure the amount of water bound to the lipase, but the water activity level of the reactions can be controlled and adjusted by different saturated salt solutions (Greenspan, 1977). In the present study, we aim to modify the chemical structure of bixin by introducing sorbitol as a hydrophilic compound into the bixin scaffold, using a lipase-catalyzed reaction. Sorbitol has previously been used to synthesis a derivate of astaxanthin in order to improve the water solubility (Lockwood et al., 2005). Moreover, we will investigate whether different lipases will favor the esterification or the transesterification reaction. Furthermore, we will find the optimal water activity level for the individual lipases and reaction solvents, in order to optimize the reaction yield.
2.3. Equilibration of water activity The thermodynamic water activity level of all experiments were adjusted by equilibrating the substrates and solvents with aqueous saturated salt solutions in separated vessels over two days. Equilibration was obtained using MgCl2 and Mg(NO3)2 at room temperature to reach the water activity levels of 0.33 and 0.53, respectively (Greenspan, 1977). The molecular sieve was used to dry solvents to reach a water activity level of zero. The substrates were dried under vacuum. 2.4. Synthesis of sorbitol ester of norbixin Bixin (59 mg, 50 mM), sorbitol (27 mg, 50 mM) and immobilized lipase 12 mg (20% of the total weight of bixin) were weighed into a 4 mL septum capped vial. A mixture of 2M2B:THF (3 mL) was added in different ratios based on reaction conditions from 0 to 100% of THF. The reaction mixture was shaken using a MHR11-TH21 thermo shaker (HLC, Germany) at 58 °C and 750 rpm. After 24 h of reaction, the lipase was separated from the reaction mixture by centrifugation (centrifuge model 4417R, Hamburg, Germany) at 20,200g for 10 min. The supernatant was separated and stored at −20 °C until further analysis using ultra-High Performance Liquid Chromatography (uHPLC) with reverse phase separation. 2.5. Purification The SEN was isolated by glass column chromatography. The column was prepared with silica gel using a mixture of chloroform/methanol/ water/acetic acid 86:12:1:1 (v/v/v/v) as elution solvent. The fractions containing the product were combined, the solvent was evaporated using a vacuum centrifuge and the residue was analyzed and quantified by uHPLC.
2. Materials and methods 2.6. Thin layer chromatography analysis 2.1. Reagents Thin layer chromatography (TLC) analysis was used to monitor the reaction progress. The reaction mixtures were diluted in THF 1:50 (v/v) and analyzed on TLC plates (TLC Silica gel 60, 5 × 7.5 cm, Merck, Germany) with the elution system chloroform/methanol/water/acetic acid 86:12:1:1 (v/v/v/v). Under these conditions, the retention factors (Rf) were 0.19, 0.62 and 0.81for SEN, norbixin and bixin, respectively.
Bixin was provided by Chr. Hansen A/S (Taastrup, Denmark). Lipase acrylic resin from Candida antarctica, ≥5,000 U/g (Novozyme 435), Lipozyme RM-IM (lipase from R. miehei) and Lipozyme TL-IM (lipase from T. lanuginosus), were purchased from Sigma-Aldrich. Nonimmobilized lipases, Rhizopus arrhizus, Candida rugosa and Pseudomonas cepacia were purchased from Sigma-Aldrich. Porous polypropylene (Accurel MP 1000) was purchased from Membrana GmbH (Obernburg, Germany). P-nitrophenyl butyrate (p-NPB, 98% pure), D-Sorbitol (≥98% pure) and all other solvents were of analytical grade. Molecular sieve (3 Å, 2 mm bead, 10 mesh) were purchased from Merck (Darmstadt, Germany). Silica gel (60 Å, 70–230 mesh, 63–200 µm) were purchased from Sigma-Aldrich.
2.7. Quantification Quantification was performed on an uHPLC Shimadzu LC-2040C 3D system (Duisburg, Germany), equipped with a photodiode array detector (PDA). The analyte mixture was diluted 50 times in THF and 2 µL was injected onto a C18 column (2.6 µm, 100 mm, 4.6 mm, Kinetex), at 1.6 mL/min flow rate and a column temperature of 35 °C. The mobile phases consisted of solvent A with 2% aqueous acetic acid and solvent B with acetonitrile with 0.2% acetic acid. The step linear gradients were; 45% B at 0 min, 70% B at 4 min, 70% B at 9 min, 100% B at 10 min, 100% B at 14 min, 45% B at 15 min and 45% B at 20 min. Detection was
2.2. Lipase immobilization Lipases from R. arrhizus, C. rugosa and P. cepacia were immobilized separately on polypropylene with particle size smaller than 500 µm. 204
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though the evidence for the identification of the reaction product was limited (Humeau et al., 2000). Absorption spectra of SEN and bixin were compared. Both compounds showed the same absorption pattern indicating that appending sorbitol to bixin did not effect on the conjugated polyene chain and they appear the same color (data not shown). The result from the reverse phase separation indicated bixin and norbixin eluted at 6.70 and 4.47 min, respectively. Two separated peaks with the same molecular mass represented of SEN eluted with a major peak at 2.61 min and minor peak at 2.15 min. The major peak was approximately three times higher than the minor peak. Both peaks corresponding to two isomers of SEN were integrated and the sum was used for the quantification. Bixin is naturally found as 9′-cis-isomer (M. Scotter, 2009), through the heating process of the reaction the 9′,13′-dicis isomer of bixin can form. Therefore, we suggested that SEN has also two cis- and di-cis isomers. At 40 °C a yield of 8% of SEN was obtained and the yield of norbixin was 3% after 72 h reaction. This reaction yield was not satisfactory high and consequently the reaction conditions needed optimization. Therefore, the following parameters were optimized; the source of lipase, water activity, temperature and solvent composition.
carried out at 460 nm. Peak areas for bixin, norbixin and SEN were integrated. Data acquisition and processing were performed using the LabSolution software program (Shimadzu, Kyoto, Japan). 2.8. Identification LC-MS identification was carried out using an Agilent 6560 Ion Mobility LC Q-TOF (Santa Clara, CA, USA) equipped with LC (Agilent 1290 Infinity II) system using diode array detector (DAD) and Mass Hunter software (Agilent, Santa Clara, CA, USA). The used column was a C18 column (2.6 µm, 100 mm, 4.6 mm, Kinetex) and the mobile phase consisted of solvent A with 2% aqueous acetic acid and solvent B with acetonitrile with 0.2% acetic acid. The LC was run at isocratic conditions at 70% solvent B and 0.3 mL/min flow rate. The column temperature was maintained at 35 °C. The Q-TOF instrument was equipped with an Agilent jet stream electro spray ionization source. The fragmentor voltage was set at 400 V. The flow rate of drying-gas (N2) was 11 L/min with a temperature of 150 °C and the nebulizer pressure was 35 psi. The sheath gas was 11 L/min with a temperature of 350 °C. The nozzle voltage was 1500 V and the capillary voltage was set at 3500 V. Mass spectra were recorded over a mass-to-charge ratio (m/z) range of 100–1000 in negative ion detection mode, and the collision energy was adjusted to 20 eV.
3.2. Lipase screening One way to improve the reaction yield would be to find a lipase better suited for the desired transesterification reaction. Lipases originating from R. arrhizus, C. rugosa and P. cepacia were chosen for a screening study and their activity in the hydrolysis of p-nitrophenyl butyrate (Gitlesen et al.) was measured. C. rugose lipase had the highest catalytic activity, followed by the lipases from P. cepacia and R. arrhizus (Table 1). Immobilization of lipases can often improve their catalytic activity in organic solvents and therefore the investigated lipases were immobilized on a hydrophobic support material, microporous polypropylene (Accurel MP 1000), which previously has been shown to be an efficient support for lipase immobilization (Nordblad & Adlercreutz, 2013). An immobilization yield above 88% were resulted for all the lipases (Table 1). In addition, three commercially available immobilized lipases were included in the screening study; Lipozyme RMIM (originating from R. miehei), Lipozyme TL-IM (originating from T. lanuginosus) and Novozyme 435 (immobilized C. antarctica lipase B), the enzyme used in the preliminary experiments. Since lipases differ with respect to water activity dependence, all lipases were tested at three different water activity levels; 0, 0.33 and 0.53. The yield of SEN was analyzed after 24 h of reaction and Novozyme 435 was the only lipase catalyzing the transesterification of bixin and sorbitol, while the rest of the lipases did not catalyze the reaction under these conditions. There have been several studies on the conformational changes of the active site region of C. antarctica lipase during the catalytic action (Haeffner, Norin, & Hult, 1998; Orrenius et al., 2009; Raza, Fransson, & Hult, 2001). The C. antarctica lipase is known as a lipase which lacks the typical lid that is normally present in many lipases (Uppenberg, Hansen, Patkar, & Jones, 1994). In principle, the active site lid needs to be open to some extent during the catalytic event to transfer the substrates and the products in and out. Lipases with a lid normally works at an interface that will favor opening of the lid (Paul Woolley, 1994). Therefore, the properties of the solvent, as well as the substrates can change the active site conformation. Uppenberg et al. suggested that the active site region of C. antarctica lipase consists of four hydrophobic residues, which provide a channel for substrates to cross and a small helix appears to act as a lid (Uppenberg, et al., 1994). It was also reported that the catalytic behavior of C. antarctica is more similar to an esterase rather than a lipase with no interfacial activation (Martinelle, 1995). This property might be one of the possible reasons that C. antarctica lipase catalyzed the reaction between bixin and sorbitol, while the other lipases displayed no activity neither in transesterification nor
3. Results and discussion 3.1. Lipase-catalyzed reaction of bixin 3.1.1. Transesterification versus hydrolysis In the present study, we aimed to synthesize a novel ester from bixin with the linear sugar alcohol, sorbitol using an enzymatic reaction in organic medium. Finding an adequate organic solvent can be a challenge. In many non-polar solvents, bixin solubility is low. In contrast, bixin shows higher solubility in polar solvents, such as ethanol and THF, but polar solvents might cause lipase inactivation. Initially, 2M2B, a well-known solvent for pigment applications, was selected as the organic medium. The immobilized lipase B from C. antarctica, which has previously been shown efficient in transesterification reactions (Humeau et al., 2000) was used as the biocatalyst. The reaction was carried out under dehydrated conditions using a molecular sieve to dry out the solvent and thereby minimize the hydrolysis reaction. The reaction was conducted at 40 °C and the formation of the new compound was monitored by TLC. The reaction mixture was analyzed by LC Q-TOF to identify the new product. The mass of the newly formed product was determined to m/z 543.2596, compared to the theoretical mass of SEN, m/z 543.2594, corresponding to a ± 0.3 ppm error (Fig. 1a). The fragmentation pattern gave most intense peaks at m/z 335, 291, 379 and 181, which supported the identification of the suggested SEN (Fig. 1b). Three cleavage sites around the ester bond with the sorbitol moiety and one at the carboxylic acid end of bixin chain were observed. The sorbitol moiety was cleaved of, resulting in the specific fragment m/z 181 [C6H13O6]-. The m/z 335 [C23H27O2]− corresponded to α-cleavage of a carbonyl bond resulting in the conjugated unsaturated carbon chain and the esterified sorbitol moiety, corresponding to [C7H13O7]−. Subsequently, the conjugated unsaturated carbon chain lost the carboxylic acid group generating the fragment at m/z 291, [C22H27]−. The m/z 379 [C24H27O4]− derived from loss of a m/z 165-fragment [C6H13O5]−, corresponding to the sorbitol moiety with one oxygen molecule. The results from the LC-MS/MS suggested the transesterification reaction at the methyl ester group to be the favorable reaction (Fig. 2), rather than the esterification reaction at the carboxylic acid group. This is the first report on enzymatic transesterification of bixin with sorbitol. Previously, Novozyme 435 has been suggested to catalyze a similar transesterification reaction between bixin and ascorbic acid via the methyl carboxylic acid group and not the carboxylic acid group, even 205
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Fig. 1. (a) MS spectra of sorbitol ester of norbixin. Theoretical mass m/z 543.2594, detected mass m/z 543.2596 (0.3 ppm error). (b) MS-MS spectra of SEN. The reaction was carried out in 2-methyl-2-butanol at 40 °C, 12 mL reaction mixture using bixin (237 mg, 50 mM), sorbitol (109 mg, 50 mM) and Novozyme 435, 47 mg (20% of the total weight of bixin) in 50 mL glass tube with screw cap.
reaction rate by eliminating the inhibition effect of the water molecules (Nordblad & Adlercreutz, 2013). Although many lipases show better activity at high water activity levels, the C. antarctica lipase apparently has a different water activity profile and requires less water to show high catalytic activity.
in hydrolysis reaction. 3.3. Reaction optimization 3.3.1. Effect of water activity on Novozyme 435 The optimal water activity level was determined for Novozyme 435 (Fig. 3). The production of SEN was favored at low water activity (aw = 0), whereas the hydrolysis reaction was favored at higher water activities, aw = 0.33 and 0.53. Low water activity favored the transesterification reaction rather than hydrolysis. At higher water activity levels, Novozyme 435 showed lower catalytic activity towards both the transesterification and the hydrolysis reactions, indicating an overall reduction of the catalytic activity. These results correlate with literature confirming that the immobilized C. antarctica lipase displays higher activity in organic medium when the water activity is close to zero. For example, in the synthesis of ascorbyl palmitate in 2M2B, the highest initial reaction rate was obtained when the reaction mixture was preequilibrated at water activity level of 0.07 (Humeau, Girardin, Rovel, & Miclo, 1998a, 1998b). Nordblad et al. investigated the transesterification of ethyl acrylate by octanol using immobilized CALB on MP1000 and demonstrated that lower water activity (0.06) provides higher
3.3.2. Temperature study The effect of temperature on Novozyme 435 was studied at three different reaction temperatures, 40 °C, 50 °C and 58 °C and the reaction yield was monitored for 72 h (Fig. 4). The reaction yield increased gradually with increasing reaction time at 40 °C and 50 °C and reached 8% and 18% after 72 h reaction, respectively. The reaction yield at 58 °C increased rapidly and was doubled compared to 50 °C, after both 23 h and 46 h of reaction time. Thereafter, the reaction slowed down until 72 h of reaction, reaching a yield of 31%. From the results presented, it is obvious that higher temperatures provided higher reaction yields, which demonstrating that Novozyme 435 has a high thermostability. According to other studies, the C. antarctica lipase has shown thermal stability between 30 and 70 °C in alcohols, with especially good stability in long-chain alcohols (Ghaly, Dave, Brooks, & Budge, 2010; Gumel, Annuar, Heidelberg, & Chisti, 2011; Yoshida, Kimura, & Adachi, 206
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Fig. 2. Enzymatic reaction of bixin with sorbitol using immobilized C. antarctica lipase B (Novozyme 435) in solvent 2-methyl-2-butanol. (1) 9′-cis-Bixin, (2) Sorbitol, (3) 9′-cis-sorbitol ester of norbixin.
Table 1 Lipase immobilization results. Lipase source
Enzymatic activity1 [µmol/min.mg]
Immobilization yield %
R. arrhizus C. rugosa P. cepacia
0.040 2.971 0.350
88.7 93.3 92.9
1
Enzymatic activities were measured on free lipase.
2006). The high temperature tolerance of the lipase provides the possibility of increasing the reaction temperature and thus, improving the solubility of the substrates in the reaction media, which is important when the substrates otherwise have low solubility in organic media. Moreover, a higher reaction temperature can improve the mass transfer conditions by accelerating the diffusion of the substrates into the lipase active site and formation of acyl-lipase complex (Jiugao, Zhang, Zhao, & Ma, 2008). However, in our study the reaction temperature was kept at 58 °C for the rest of the experiments. Fig. 3. Selectivity between transesterification and hydrolysis reaction using immobilized C. antarctica lipase B (Novozyme 435) at three different water activity levels. The reaction was carried out in 2-methyl-2-butanol and 10% tetrahydrofuran for 24 h at 58 °C with bixin (59 mg, 50 mM), sorbitol (27 mg, 50 mM) and Novozyme 435, (12 mg, 20% of the total weight of bixin) in 4 mL septum capped vial.
3.3.3. Solvent effect The solubility of the substrates is another parameter that was considered. One of the main issues in the enzymatic reaction of bixin with sorbitol was to select a proper organic solvent. The solvent should show solubility for both substrates as well as retaining the lipase activity. Bixin and sorbitol show very low solubility in non-polar solvents and have better solubility in polar solvents such as THF, ethanol and methanol. However, these solvents might lead to lipase inactivation. Zhi and co-workers studied the solubility of sorbitol in different solvents at different temperature. Sorbitol solubility was high in many alcohols due
to the formation of hydrogen bonds and it was moderately soluble in THF, in which solubility increased with increasing temperature (Zhi, Hu, Yang, Kai, & Cao, 2013). Our previous experiments showed that the solubility of bixin and 207
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the active site of the C. antarctica lipase stays stable in non-polar solvents (log p > 4), while polar solvent (log p < 2) can interact with the active site and breaking the hydrogen bonds between the amino acid residues important for lipase activity (Cong Li, Zhang, & Feng, 2010). Moreover, Park et al. observed higher catalytic efficiency for the C. antarctica lipase in tert-butanol than methanol using molecular dynamics simulations (Park, Park, & Yoo, 2013). This indicates that 2M2B should be a better solvent than THF for sustaining lipase activity. On the other hand, adding THF improves substrate solubility, and the solvent mixture of 80% 2M2B and 20% THF turned out to be a good compromise, resulting in the highest yield. 4. Conclusion The hydrophobic characteristics of bixin limits its food application in water-based food products. In this paper, we introduced a simple method to catalyze the reaction between bixin and sorbitol, thereby synthesizing a new derivate of bixin, SEN, which is likely to have better hydrophilic properties. Transesterification of bixin was carried out using immobilized lipases from different sources at different water activity levels. Novozyme 435 was the only lipase, which efficiently catalyzed the transesterification of bixin with sorbitol at a water activity close to zero. A solvent mixture of 80% 2M2B and 20% THF provided the highest reaction yield of 50%. The sugar alcohol moiety on the bixin molecule is likely to change the chemical properties of bixin towards a hydrophilic colorant. Moreover, the modified colorant might show emulsifier properties, which even further increases its application in the food industry. Further studies are needed to determine the aqueous solubility, as well as stability, of the new sorbitol derivate of bixin.
Fig. 4. Temperature study of reaction between bixin with sorbitol using immobilized C. antarctica lipase B (Novozyme 435) at water activity level of zero performed at three different temperatures. The reactions were carried out in 2methyl-2-butanol, 12 mL reaction mixture using bixin (237 mg, 50 mM), sorbitol (109 mg, 50 mM) and Novozyme 435, (47 mg, 20% of the total weight of bixin) in 50 mL glass tube with screw cap.
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Fig. 5. Reaction yield of sorbitol ester of norbixin using immobilized C. antarctica lipase B (Novozyme 435) in 2-methyl-2-butanol with different level of tetrahydrofuran at water activity level of zero, at 58 °C, with bixin (59 mg, 50 mM), sorbitol (27 mg, 50 mM) and Novozyme 435, (12 mg, 20% of the total weight of bixin) in 4 mL septum capped vial. The yield was quantified after 24 h. The reaction with 20% tetrahydrofuran was repeated and the data is presented as mean ± standard deviation.
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