Food Chemistry 158 (2014) 292–295
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Short communication
Solvent-free enzymatic synthesis of feruloylated structured lipids by the transesterification of ethyl ferulate with castor oil Shangde Sun ⇑, Sha Zhu, Yanlan Bi Lipid Technology and Engineering, School of Food Science and Engineering, Henan University of Technology, Lianhua Road, Zhengzhou 450001, Henan Province, PR China
a r t i c l e
i n f o
Article history: Received 10 August 2013 Received in revised form 21 January 2014 Accepted 25 February 2014 Available online 5 March 2014 Keywords: Solvent-free system Feruloylated structured lipids Enzymatic transesterification Ethyl ferulate Castor oil
a b s t r a c t A novel enzymatic route of feruloylated structured lipids synthesis by the transesterification of ethyl ferulate (EF) with castor oil, in solvent-free system, was investigated. The transesterification reactions were catalysed by Novozym 435, Lipozyme RMIM, and Lipozyme TLIM, among which Novozym 435 showed the best catalysis performance. Effects of feruloyl donors, reaction variables, and ethanol removal on the transesterification were also studied. High EF conversion (100%) was obtained under the following conditions: enzyme load 20% (w/w, relative to the weight of substrates), reaction temperature 90 °C, substrate molar ratio 1:1 (EF/castor oil), 72 h, vacuum pressure 10 mmHg, and 200 rpm. Under these conditions, the transesterification product consisted of 62.6% lipophilic feruloylated structured lipids and 37.3% hydrophilic feruloylated lipids. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Ferulic acid (FA) is a phenolic acid widely occurring in the plant kingdom, which can be used as a potential UV protective ingredient and antioxidant (Itagaki et al., 2009; Kanski, Aksenova, Stoyanova, & Butterfield, 2002; Saija et al., 2000; Warner & Laszlo, 2005). However, these applications of FA in food, cosmetics, and other fields are limited because of its poor solubility in hydrophilic and lipophilic media (Karboune, St-Louis, & Kermasha, 2008; Zheng et al., 2010). Therefore, modifications of FA, using hydrophilic and lipophilic moieties, have attracted much attention (Pinedo, Peñalver, Pérez-Victoria, Rondón, & Morales, 2007; Reddy, Ravinder, & Kanjilal, 2012; Sabally, Karboune, Yeboah, & Kermasha, 2005; Sun, Song, Bi, Yang, & Liu, 2012; Xin et al., 2009; Yang, Guo, & Xu, 2012; Yang, Mu, Chen, Xiu, & Yang, 2013). Owning to the oxidizability and heat sensitivity of FA, chemical modification of FA was limited. Therefore, enzymatic biosynthesis of FA lipids has been used as an attractive alternative to the conventional chemical processes (Choo & Birch, 2009; Compton, Laszlo, & Berhow, 2000; Sun et al., 2007). Castor oil mainly consists of the esters of 12-hydroxy-9-octadecenoic acid (ricinoleic acid), which make castor oil widely used in skin and personal-care products due to its excellent emolliency, lubricity, and noncomedogenicity (Ogunniyi, 2006; Mutlu & Meier, 2010). Feruloylated structured lipids, types of FA esters, can be
⇑ Corresponding author. Tel./fax: +86 371 67758022. E-mail address: [email protected] (S. Sun). http://dx.doi.org/10.1016/j.foodchem.2014.02.146 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
prepared by the esterification of castor oil with FA. The novel structured lipids may offer many combined beneficial properties of both the castor oil and the FA (Compton, Laszlo, & Isbell, 2004). However, there is only one report on the chemical esterification of castor oil with cinnamic acid (CA) and 4-methoxycinnamic acid (MCA) (Compton et al., 2004). In this report, 85% CA conversion and 50% MCA conversion were obtained using molecular sieve as a dehydrant at 200 °C for 24 h under a nitrogen atmosphere. However, no study focussed on the enzymatic transesterification of castor oil with ethyl ferulate (EF) was found. The aim of the current work was to investigate a novel enzymatic route of feruloylated structured lipids synthesis by the transesterification of castor oil with EF in solvent-free system (Scheme 1). Enzyme screening was also evaluated. Effects of feruloyl donors (FA and EF), reaction variables (enzyme load, reaction temperature, reaction time, and substrate ratio) and ethanol removal, on the transesterification were investigated. 2. Materials and methods 2.1. Materials Ferulic acid (FA) and ethyl ferulate (EF) were purchased from Suzhou Chang Tong Chemical Co., Ltd. (Suzhou, China). Castor oil was purchased from Shanghai Reagent Factory (Shanghai, China). Novozym 435, Lipozyme RMIM, and Lipozyme TLIM were from Novozymes A/S (Bagsvaerd, Denmark). Glacial acetic acid and Methanol were of HPLC grade. All other regents were of analytical grade.
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S. Sun et al. / Food Chemistry 158 (2014) 292–295
OH O O H C O C (CH2)7CH CHCH2CH(CH2)5CH3 2 C O CH H2C O C (CH2)7CH CHCH2CH(CH2)5CH3
OH H3C(CH2)5CHCH2CH
CH(CH2)7
HC CH COOCH2CH3
+ OH
OCH3
OH
O
Ethyl ferulate
Castor oil
Novozym 435 O
OCH3
H2C O C CH HC HO CH H2C OH
OH
O OH
+
H3C(CH2)5CHCH2CH
Glyceryl ferulate
+
OCH3
O
H2C O C CH HC OH CH(CH2)7 C O CH O H2C O C (CH2)7CH CHCH2CH(CH2)5CH3 Ferulated diricinoleic acyl structured lipid
O
OCH3
H2C O C CH HC HO CH O H2C O C CH HC
OH OCH3 OH
O
+
OH
OCH3
H2C O C CH HC OH HO CH CH2 O CH2 (CH2)7CH CHCH2CH(CH2)5CH3 O OH
Glyceryl diferulate
Ferulated moricinoleic acyl structured lipid
Scheme 1. Enzymatic synthesis of feruloylated structured lipids by the transesterification of castor oil with EF.
2.2. Enzymatic transesterification Transesterifications were performed in 25 mL round-bottom flasks containing EF and castor oil (1:1, mol/mol ratio), and lipase. The reaction mixtures were incubated at various temperatures using a water bath with a magnetic stirrer under 10 mmHg vacuum pressure. Samples (20 ll) were withdrawn by a micro pipettor at specified time intervals. 2.3. Analytical methods Reactants and products were analysed by HPLC (Waters 2695) with a C18 reverse phase column (5 lm, 250 4.6 mm) fitted with a dual absorbance detector (Waters 2487) at 325 nm. Elution was conducted with solvent A (methanol) and solvent B (water, containing 0.5% of acetic acid) at a flow rate of 1 mL/min. The elution sequence consisted consecutively of a linear gradient from 50% (v/v) A to 90% A (v/v) over 10 min, then to 100% A for 30 min, followed by 100% A for 20 min at 35 °C. Components in the sample were identified with regard to the relevant major ions detected by HPLC–ESI–MS according to the previous report (Sun et al., 2007, 2008).
Fig. 1. Effect of feruloyl donors (EF and FA) on the transesterification. Reaction conditions: castor oil/EF 1:3 (mol/mol), enzyme load 20% (relative to the total weight of substrates), 70 °C, vacuum pressure 10 mmHg, and 200 rpm.
EF was a better and preferred feruloyl donor than FA for the transesterification.
2.4. Statistical analyses All experiments were performed at least in triplicate. Results were expressed as averages ± S.E.M. For orthogonal array design analysis of experiments, a two-way analysis of variance (ANOVA) was used. Statistical significance was considered at p < 0.05. 3. Results and discussion 3.1. Effect of feruloyl donors Initially, attempts were made to prepare feruloylated structured lipids by esterification of castor oil with FA. Results showed that no reaction (FA conversion < 1%) was found (Fig. 1). However, using EF as the feruloyl donor, EF conversion was 53.5 ± 2.5% at 48 h, which was much higher than that (
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Short communication
Solvent-free enzymatic synthesis of feruloylated structured lipids by the transesterification of ethyl ferulate with castor oil Shangde Sun ⇑, Sha Zhu, Yanlan Bi Lipid Technology and Engineering, School of Food Science and Engineering, Henan University of Technology, Lianhua Road, Zhengzhou 450001, Henan Province, PR China
a r t i c l e
i n f o
Article history: Received 10 August 2013 Received in revised form 21 January 2014 Accepted 25 February 2014 Available online 5 March 2014 Keywords: Solvent-free system Feruloylated structured lipids Enzymatic transesterification Ethyl ferulate Castor oil
a b s t r a c t A novel enzymatic route of feruloylated structured lipids synthesis by the transesterification of ethyl ferulate (EF) with castor oil, in solvent-free system, was investigated. The transesterification reactions were catalysed by Novozym 435, Lipozyme RMIM, and Lipozyme TLIM, among which Novozym 435 showed the best catalysis performance. Effects of feruloyl donors, reaction variables, and ethanol removal on the transesterification were also studied. High EF conversion (100%) was obtained under the following conditions: enzyme load 20% (w/w, relative to the weight of substrates), reaction temperature 90 °C, substrate molar ratio 1:1 (EF/castor oil), 72 h, vacuum pressure 10 mmHg, and 200 rpm. Under these conditions, the transesterification product consisted of 62.6% lipophilic feruloylated structured lipids and 37.3% hydrophilic feruloylated lipids. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Ferulic acid (FA) is a phenolic acid widely occurring in the plant kingdom, which can be used as a potential UV protective ingredient and antioxidant (Itagaki et al., 2009; Kanski, Aksenova, Stoyanova, & Butterfield, 2002; Saija et al., 2000; Warner & Laszlo, 2005). However, these applications of FA in food, cosmetics, and other fields are limited because of its poor solubility in hydrophilic and lipophilic media (Karboune, St-Louis, & Kermasha, 2008; Zheng et al., 2010). Therefore, modifications of FA, using hydrophilic and lipophilic moieties, have attracted much attention (Pinedo, Peñalver, Pérez-Victoria, Rondón, & Morales, 2007; Reddy, Ravinder, & Kanjilal, 2012; Sabally, Karboune, Yeboah, & Kermasha, 2005; Sun, Song, Bi, Yang, & Liu, 2012; Xin et al., 2009; Yang, Guo, & Xu, 2012; Yang, Mu, Chen, Xiu, & Yang, 2013). Owning to the oxidizability and heat sensitivity of FA, chemical modification of FA was limited. Therefore, enzymatic biosynthesis of FA lipids has been used as an attractive alternative to the conventional chemical processes (Choo & Birch, 2009; Compton, Laszlo, & Berhow, 2000; Sun et al., 2007). Castor oil mainly consists of the esters of 12-hydroxy-9-octadecenoic acid (ricinoleic acid), which make castor oil widely used in skin and personal-care products due to its excellent emolliency, lubricity, and noncomedogenicity (Ogunniyi, 2006; Mutlu & Meier, 2010). Feruloylated structured lipids, types of FA esters, can be
⇑ Corresponding author. Tel./fax: +86 371 67758022. E-mail address: [email protected] (S. Sun). http://dx.doi.org/10.1016/j.foodchem.2014.02.146 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
prepared by the esterification of castor oil with FA. The novel structured lipids may offer many combined beneficial properties of both the castor oil and the FA (Compton, Laszlo, & Isbell, 2004). However, there is only one report on the chemical esterification of castor oil with cinnamic acid (CA) and 4-methoxycinnamic acid (MCA) (Compton et al., 2004). In this report, 85% CA conversion and 50% MCA conversion were obtained using molecular sieve as a dehydrant at 200 °C for 24 h under a nitrogen atmosphere. However, no study focussed on the enzymatic transesterification of castor oil with ethyl ferulate (EF) was found. The aim of the current work was to investigate a novel enzymatic route of feruloylated structured lipids synthesis by the transesterification of castor oil with EF in solvent-free system (Scheme 1). Enzyme screening was also evaluated. Effects of feruloyl donors (FA and EF), reaction variables (enzyme load, reaction temperature, reaction time, and substrate ratio) and ethanol removal, on the transesterification were investigated. 2. Materials and methods 2.1. Materials Ferulic acid (FA) and ethyl ferulate (EF) were purchased from Suzhou Chang Tong Chemical Co., Ltd. (Suzhou, China). Castor oil was purchased from Shanghai Reagent Factory (Shanghai, China). Novozym 435, Lipozyme RMIM, and Lipozyme TLIM were from Novozymes A/S (Bagsvaerd, Denmark). Glacial acetic acid and Methanol were of HPLC grade. All other regents were of analytical grade.
293
S. Sun et al. / Food Chemistry 158 (2014) 292–295
OH O O H C O C (CH2)7CH CHCH2CH(CH2)5CH3 2 C O CH H2C O C (CH2)7CH CHCH2CH(CH2)5CH3
OH H3C(CH2)5CHCH2CH
CH(CH2)7
HC CH COOCH2CH3
+ OH
OCH3
OH
O
Ethyl ferulate
Castor oil
Novozym 435 O
OCH3
H2C O C CH HC HO CH H2C OH
OH
O OH
+
H3C(CH2)5CHCH2CH
Glyceryl ferulate
+
OCH3
O
H2C O C CH HC OH CH(CH2)7 C O CH O H2C O C (CH2)7CH CHCH2CH(CH2)5CH3 Ferulated diricinoleic acyl structured lipid
O
OCH3
H2C O C CH HC HO CH O H2C O C CH HC
OH OCH3 OH
O
+
OH
OCH3
H2C O C CH HC OH HO CH CH2 O CH2 (CH2)7CH CHCH2CH(CH2)5CH3 O OH
Glyceryl diferulate
Ferulated moricinoleic acyl structured lipid
Scheme 1. Enzymatic synthesis of feruloylated structured lipids by the transesterification of castor oil with EF.
2.2. Enzymatic transesterification Transesterifications were performed in 25 mL round-bottom flasks containing EF and castor oil (1:1, mol/mol ratio), and lipase. The reaction mixtures were incubated at various temperatures using a water bath with a magnetic stirrer under 10 mmHg vacuum pressure. Samples (20 ll) were withdrawn by a micro pipettor at specified time intervals. 2.3. Analytical methods Reactants and products were analysed by HPLC (Waters 2695) with a C18 reverse phase column (5 lm, 250 4.6 mm) fitted with a dual absorbance detector (Waters 2487) at 325 nm. Elution was conducted with solvent A (methanol) and solvent B (water, containing 0.5% of acetic acid) at a flow rate of 1 mL/min. The elution sequence consisted consecutively of a linear gradient from 50% (v/v) A to 90% A (v/v) over 10 min, then to 100% A for 30 min, followed by 100% A for 20 min at 35 °C. Components in the sample were identified with regard to the relevant major ions detected by HPLC–ESI–MS according to the previous report (Sun et al., 2007, 2008).
Fig. 1. Effect of feruloyl donors (EF and FA) on the transesterification. Reaction conditions: castor oil/EF 1:3 (mol/mol), enzyme load 20% (relative to the total weight of substrates), 70 °C, vacuum pressure 10 mmHg, and 200 rpm.
EF was a better and preferred feruloyl donor than FA for the transesterification.
2.4. Statistical analyses All experiments were performed at least in triplicate. Results were expressed as averages ± S.E.M. For orthogonal array design analysis of experiments, a two-way analysis of variance (ANOVA) was used. Statistical significance was considered at p < 0.05. 3. Results and discussion 3.1. Effect of feruloyl donors Initially, attempts were made to prepare feruloylated structured lipids by esterification of castor oil with FA. Results showed that no reaction (FA conversion < 1%) was found (Fig. 1). However, using EF as the feruloyl donor, EF conversion was 53.5 ± 2.5% at 48 h, which was much higher than that (
