Food Chemistry 158 (2014) 292295

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Short communication

Solvent-free enzymatic synthesis of feruloylated structured lipids

by the transesterication 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 transesterication
Ethyl ferulate
Castor oil

a b s t r a c t
A novel enzymatic route of feruloylated structured lipids synthesis by the transesterication of ethyl ferulate (EF) with castor oil, in solvent-free system, was investigated. The transesterication 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 transesterication 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 transesterication product consisted of 62.6% lipophilic feruloylated structured lipids and 37.3%
hydrophilic feruloylated lipids.
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, & Buttereld, 2002; Saija et al., 2000; Warner & Laszlo,
2005). However, these applications of FA in food, cosmetics, and
other elds are limited because of its poor solubility in hydrophilic
and lipophilic media (Karboune, St-Louis, & Kermasha, 2008;
Zheng et al., 2010). Therefore, modications of FA, using hydrophilic and lipophilic moieties, have attracted much attention
(Pinedo, Pealver, Prez-Victoria, Rondn, & 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 modication 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: sunshangde@hotmail.com (S. Sun).
http://dx.doi.org/10.1016/j.foodchem.2014.02.146
0308-8146/ 2014 Elsevier Ltd. All rights reserved.

prepared by the esterication of castor oil with FA. The novel structured lipids may offer many combined benecial properties of both
the castor oil and the FA (Compton, Laszlo, & Isbell, 2004).
However, there is only one report on the chemical esterication
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 transesterication 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
transesterication 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 transesterication 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) 292295

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

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

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

OCH3

H2C O C CH HC
HO CH O
H2C O C CH HC

OH
OCH3
OH

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 transesterication of castor oil with EF.

2.2. Enzymatic transesterication

Transesterications were performed in 25 mL round-bottom
asks 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 specied 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) tted 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 ow 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 identied with regard to the relevant major ions detected by
HPLCESIMS according to the previous report (Sun et al., 2007,
2008).

Fig. 1. Effect of feruloyl donors (EF and FA) on the transesterication. 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

transesterication.

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 signicance 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 esterication 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 (<2% at 48 h) of FA. These results can be
explained by the fact that a lower melting point (64 C) of EF than
that (174 C) of FA and good solubility of EF with castor oil. Thus,

3.2. Screening of lipases

For the tested lipases, Novozym 435 showed higher catalyst
performance than did Lipozyme RMIM and Lipozyme TLIM
(Fig. 2). At 48 h, using Novozym 435 as biocatalyst, EF conversion
(53.5 2.5%) was 3.8 times that (14.1 2.0%) of Lipozyme RMIM
and 17.8 times that (3.0%) of Lipozyme TLIM. And in the transesterication product, the molar ratio of lipophilic and hydrophilic
feruloylated structured lipids was 1:1. These results showed that
Novozym 435 exhibited high transesterication activity toward
EF and castor oil.
3.3. Effect of ethanol removal
In order to investigate the effect of by-product (ethanol)
removal on the transesterication, reactions were conducted under
atmospheric pressure and vacuum pressure, respectively. In the

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S. Sun et al. / Food Chemistry 158 (2014) 292295

Fig. 2. Effects of different lipases on the transesterication. 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.

Fig. 4. Effect of enzyme load on the transesterication. Reaction conditions: castor

oil/EF 1:3 (mol/mol), 70 C, vacuum pressure 10 mmHg, and 200 rpm.

rst 10 h, initial EF conversion rate of atmospheric pressure was

similar to that of the vacuum system (Fig. 3). After 10 h, EF conversion of the vacuum system was smoothly increased up to 100% at
72 h. However, EF conversions of the atmospheric pressure system
almost maintained the same levels (68.6 2.0%). These results
were attributed to the efcient removal of by-product ethanol
under the vacuum system.
3.4. Effect of reaction variables
Initial EF conversion rate increased linearly with the increasing
of enzyme load from 5% to 20%; then the initial reaction rate
smoothly increased when the enzyme load was above 20%
(Fig. 4), which can be attributed to the decrease of catalyst efciency and diffusional limitations of the reaction system at higher
enzyme loads. And in the product, with the increase of enzyme
load, more lipophilic feruloylated structured lipids were formed,
such as, 34.2% lipophilic feruloylated structured lipids yield of
60% enzyme load and 16.3% of 5% enzyme load at 72 h.
When the temperature increased from 60 to 90 C, EF conversion increased dramatically (Fig. 5). In the transesterication product, the lipophilic feruloylated structured lipids smoothly
increased to 58.7% at 90 C. This phenomenon can be attributed
to reduction of the viscosity of the reaction system and faster
transfer rate of feruloyl to castor oil at high temperature. However,
too high a temperature will lead to higher lipase deactivation rates,
which resulted in the decrease of EF conversion at 100 C. With

Fig. 3. Effect of ethanol removal on the transesterication. Reaction conditions:

enzyme load 20% (relative to the total weight of substrates), 90 C, and 200 rpm.

Fig. 5. Effect of reaction temperature on the transesterication. Reaction conditions: castor oil/EF 1:3 (mol/mol), enzyme load 20% (relative to the total weight of
substrates), vacuum pressure 10 mmHg, and 200 rpm.

increase of reaction temperature, more by-product ferulic acid

(FA) was found, and the maximum FA yield was 32.4% at 90 C
for 72 h, which can be attributed to the enhancement (by high
temperature) of the hydrolysis of EF.
Varying the molar ratio of castor oil to EF from 1:1 to 1:5
resulted in a concomitant decrease of EF conversion. Furthermore,
the maximum EF conversion (100%) was obtained with the substrate ratio 1:1 at 72 h (Fig. 6). The optimum transesterication
conditions were as follows: enzyme load 20%, reaction temperature 90 C, and substrate ratio 1:1 (castor oil/EF, mol/mol). Under
these conditions, the maximum EF conversion was 100% at
72 h, which is higher than that (85% CA conversion and 50% MCA
conversion) of a previous report (Compton et al., 2004). And in
the transesterication product, the lipophilic and hydrophilic
feruloylated structured lipids were 62.6% and 37.3%, respectively.
The stirring speeds between 80 and 800 rpm had minor effects
on the transesterication (data not shown), which suggests that
the external transfer limitation can be neglected and an internal
mass transfer limitation may account for the low initial reaction
rates. These results can also be conrmed by the effect of enzyme
load. At the tested stirring speed (200 rpm), initial EF conversion
rate increased linearly with the increasing of enzyme load from
5% to 20%, which also indicates that the external transfer limitation
could be eliminated (Fig. 4). A similar effect of mass transfer
limitation on the enzymatic reaction was also found in the
previous reports (Guo & Sun, 2007; Sabeder, Habulin, & Knez,
2006; Sun, Shan, Jin, Liu, & Wang, 2007).

S. Sun et al. / Food Chemistry 158 (2014) 292295

Fig. 6. Effect of substrate ratio on the transesterication. Reaction conditions:

enzyme load 20% (relative to the total weight of substrates), 90 C, vacuum pressure
10 mmHg, and 200 rpm.

4. Conclusion
In this study, a novel enzymatic route for feruloyl structured
lipids synthesis by the transesterication of EF with castor oil in
a solvent-free system was successfully achieved. Novozym 435
showed the best catalysis performance, which made the work
more suitable for feruloyl structured lipids preparation, due to
the oxidizability and heat sensitivity of ethyl ferulate. EF was the
best feruloyl donor for the transesterication. The optimum
transesterication reaction conditions were as follows: enzyme
load 20%, reaction temperature 90 C and substrate ratio 1:1 (castor oil/EF, mol/mol). Under these optimised conditions, the maximum EF conversion (100%) was obtained at 200 rpm for 72 h
and, in the transesterication product, the lipophilic and hydrophilic feruloylated structured lipids were 62.6% and 37.3%,
respectively.
Acknowledgements
The authors gratefully acknowledge nancial support from
National Natural Science Foundation of China (31101301), Funding
Scheme for Young Teachers in Colleges and Universities in Henan
Province (2012GGJS-83), and Plan for Scientic Innovation Talent
of Henan University of Technology (11CXRC03).
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