Food Chemistry 158 (2014) 292295Contents lists available at ScienceDirect

Food Chemistry

journal homepage: www.elsevier .com/locate / foodchemShort communicationSolvent-free enzymatic synthesis of feruloylated structured lipidsby the transesterification of ethyl ferulate with castor oilhttp://dx.doi.org/10.1016/j.foodchem.2014.02.1460308-8146/ 2014 Elsevier Ltd. All rights reserved.

Corresponding author. Tel./fax: +86 371 67758022.E-mail address: sunshangde@hotmail.com (S. Sun).Shangde Sun , Sha Zhu, Yanlan BiLipid 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 oArticle history:Received 10 August 2013Received in revised form 21 January 2014Accepted 25 February 2014Available online 5 March 2014

Keywords:Solvent-free systemFeruloylated structured lipidsEnzymatic transesterificationEthyl ferulateCastor oila b s t r a c t

A novel enzymatic route of feruloylated structured lipids synthesis by the transesterification of ethyl fer-ulate (EF) with castor oil, in solvent-free system, was investigated. The transesterification reactions werecatalysed by Novozym 435, Lipozyme RMIM, and Lipozyme TLIM, among which Novozym 435 showedthe best catalysis performance. Effects of feruloyl donors, reaction variables, and ethanol removal onthe transesterification were also studied. High EF conversion (100%) was obtained under the followingconditions: enzyme load 20% (w/w, relative to the weight of substrates), reaction temperature 90 C, sub-strate molar ratio 1:1 (EF/castor oil), 72 h, vacuum pressure 10 mmHg, and 200 rpm. Under these condi-tions, the transesterification product consisted of 62.6% lipophilic feruloylated structured lipids and 37.3%hydrophilic feruloylated lipids.

2014 Elsevier Ltd. All rights reserved.1. Introduction prepared by the esterification of castor oil with FA. The novel struc-Ferulic acid (FA) is a phenolic acid widely occurring in the plantkingdom, which can be used as a potential UV protective ingredi-ent 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, andother fields are limited because of its poor solubility in hydrophilicand lipophilic media (Karboune, St-Louis, & Kermasha, 2008;Zheng et al., 2010). Therefore, modifications of FA, using hydro-philic 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 theoxidizability and heat sensitivity of FA, chemical modification ofFA was limited. Therefore, enzymatic biosynthesis of FA lipidshas been used as an attractive alternative to the conventionalchemical processes (Choo & Birch, 2009; Compton, Laszlo, &Berhow, 2000; Sun et al., 2007).

Castor oil mainly consists of the esters of 12-hydroxy-9-octa-decenoic acid (ricinoleic acid), which make castor oil widely usedin 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 betured lipids may offer many combined beneficial properties of boththe castor oil and the FA (Compton, Laszlo, & Isbell, 2004).

However, there is only one report on the chemical esterificationof castor oil with cinnamic acid (CA) and 4-methoxycinnamic acid(MCA) (Compton et al., 2004). In this report, 85% CA conversion and50% MCA conversion were obtained using molecular sieve as adehydrant at 200 C for 24 h under a nitrogen atmosphere. How-ever, no study focussed on the enzymatic transesterification of cas-tor oil with ethyl ferulate (EF) was found.

The aim of the current work was to investigate a novel enzy-matic route of feruloylated structured lipids synthesis by thetransesterification of castor oil with EF in solvent-free system(Scheme 1). Enzyme screening was also evaluated. Effects of feru-loyl donors (FA and EF), reaction variables (enzyme load, reactiontemperature, reaction time, and substrate ratio) and ethanolremoval, on the transesterification were investigated.

2. Materials and methods

2.1. Materials

Ferulic acid (FA) and ethyl ferulate (EF) were purchased fromSuzhou Chang Tong Chemical Co., Ltd. (Suzhou, China). Castor oilwas purchased from Shanghai Reagent Factory (Shanghai, China).Novozym 435, Lipozyme RMIM, and Lipozyme TLIM were fromNovozymes A/S (Bagsvaerd, Denmark). Glacial acetic acid andMethanol were of HPLC grade. All other regents were of analyticalgrade.

H2CCH

H2C

O CO

O C

(CH2)7CHH3C(CH2)5CHCH2CH C

CHCH2CH(CH2)5CH3

OH

CH

OH

(CH2)7CH CHCH2CH(CH2)5CH3OH

(CH2)7

Novozym 435

OHOCH3

HC CH COOCH2CH3+

OO

O

+

++

H2CCH

H2C

O C

OH

O

HCCHHO

H2CCH

H2C

O CO

O CH3C(CH2)5CHCH2CH CCH

OH

(CH2)7CH CHCH2CH(CH2)5CH3OH

(CH2)7

OO

O

H2CCHCH2

O CHO

OHCCH

O CH2 (CH2)7CH CHCH2CH(CH2)5CH3OHO

Castor oil Ethyl ferulate

Glyceryl ferulate

Glyceryl diferulate

Ferulated diricinoleic acyl structured lipid

Ferulated moricinoleic acyl structured lipid

OHOCH3

H2CCH

H2C

O C

O

O

HCCHHO

OHOCH3

C

O

HCCH OHOCH3

OHOCH3

HCCH OHOCH3

Scheme 1. Enzymatic synthesis of feruloylated structured lipids by the transesterification of castor oil with EF.

Fig. 1. Effect of feruloyl donors (EF and FA) on the transesterification. Reactionconditions: castor oil/EF 1:3 (mol/mol), enzyme load 20% (relative to the totalweight of substrates), 70 C, vacuum pressure 10 mmHg, and 200 rpm.

S. Sun et al. / Food Chemistry 158 (2014) 292295 2932.2. Enzymatic transesterification

Transesterifications were performed in 25 mL round-bottomflasks containing EF and castor oil (1:1, mol/mol ratio), and lipase.The reaction mixtures were incubated at various temperaturesusing a water bath with a magnetic stirrer under 10 mmHg vac-uum pressure. Samples (20 ll) were withdrawn by a micro pipet-tor 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 witha dual absorbance detector (Waters 2487) at 325 nm. Elution wasconducted with solvent A (methanol) and solvent B (water,containing 0.5% of acetic acid) at a flow rate of 1 mL/min. Theelution sequence consisted consecutively of a linear gradient from50% (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 samplewere identified with regard to the relevant major ions detected byHPLCESIMS according to the previous report (Sun et al., 2007,2008).

2.4. Statistical analyses

All experiments were performed at least in triplicate. Resultswere expressed as averages S.E.M. For orthogonal array designanalysis 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 structuredlipids by esterification of castor oil with FA. Results showed that noreaction (FA conversion < 1%) was found (Fig. 1). However, using EFas the feruloyl donor, EF conversion was 53.5 2.5% at 48 h, whichwas much higher than that (

Fig. 2. Effects of different lipases on the transesterification. Reaction conditions:castor oil/EF 1:3 (mol/mol), enzyme load 20% (relative to the total weight ofsubstrates), 70 C, vacuum pressure 10 mmHg, and 200 rpm.

Fig. 4. Effect of enzyme load on the transesterification. Reaction conditions: castoroil/EF 1:3 (mol/mol), 70 C, vacuum pressure 10 mmHg, and 200 rpm.

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

294 S. Sun et al. / Food Chemistry 158 (2014) 292295first 10 h, initial EF conversion rate of atmospheric pressure wassimilar to that of the vacuum system (Fig. 3). After 10 h, EF conver-sion of the vacuum system was smoothly increased up to 100% at72 h. However, EF conversions of the atmospheric pressure systemalmost maintained the same levels (68.6 2.0%). These resultswere attributed to the efficient removal of by-product ethanolunder the vacuum system.

3.4. Effect of reaction variables

Initial EF conversion rate increased linearly with the increasingof enzyme load from 5% to 20%; then the initial reaction ratesmoothly increased when the enzyme load was above 20%(Fig. 4), which can be attributed to the decrease of catalyst effi-ciency and diffusional limitations of the reaction system at higherenzyme loads. And in the product, with the increase of enzymeload, more lipophilic feruloylated structured lipids were formed,such as, 34.2% lipophilic feruloylated structured lipids yield of60% enzyme load and 16.3% of 5% enzyme load at 72 h.

When the temperature increased from 60 to 90 C, EF conver-sion increased dramatically (Fig. 5). In the transesterification prod-uct, the lipophilic feruloylated structured lipids smoothlyincreased to 58.7% at 90 C. This phenomenon can be attributedto reduction of the viscosity of the reaction system and fastertransfer 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. WithFig. 3. Effect of ethanol removal on the transesterification. Reaction conditions:enzyme load 20% (relative to the total weight of substrates), 90 C, 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 Cfor 72 h, which can be attributed to the enhancement (by hightemperature) of the hydrolysis of EF.

Varying the molar ratio of castor oil to EF from 1:1 to 1:5resulted in a concomitant decrease of EF conversion. Furthermore,the maximum EF conversion (100%) was obtained with the sub-strate ratio 1:1 at 72 h (Fig. 6). The optimum transesterificationconditions were as follows: enzyme load 20%, reaction tempera-ture 90 C, and substrate ratio 1:1 (castor oil/EF, mol/mol). Underthese conditions, the maximum EF conversion was 100% at72 h, which is higher than that (85% CA conversion and 50% MCAconversion) of a previous report (Compton et al., 2004). And inthe transesterification product, the lipophilic and hydrophilicferuloylated structured lipids were 62.6% and 37.3%, respectively.

The stirring speeds between 80 and 800 rpm had minor effectson the transesterification (data not shown), which suggests thatthe external transfer limitation can be neglected and an internalmass transfer limitation may account for the low initial reactionrates. These results can also be confirmed by the effect of enzymeload. At the tested stirring speed (200 rpm), initial EF conversionrate increased linearly with the increasing of enzyme load from5% to 20%, which also indicates that the external transfer limitationcould be eliminated (Fig. 4). A similar effect of mass transferlimitation on the enzymatic reaction was also found in theprevious reports (Guo & Sun, 2007; Sabeder, Habulin, & Knez,2006; Sun, Shan, Jin, Liu, & Wang, 2007).

Fig. 6. Effect of substrate ratio on the transesterification. Reaction conditions:enzyme load 20% (relative to the total weight of substrates), 90 C, vacuum pressure10 mmHg, and 200 rpm.

S. Sun et al. / Food Chemistry 158 (2014) 292295 2954. Conclusion

In this study, a novel enzymatic route for feruloyl structuredlipids synthesis by the transesterification of EF with castor oil ina solvent-free system was successfully achieved. Novozym 435showed the best catalysis performance, which made the workmore suitable for feruloyl structured lipids preparation, due tothe oxidizability and heat sensitivity of ethyl ferulate. EF was thebest feruloyl donor for the transesterification. The optimumtransesterification reaction conditions were as follows: enzymeload 20%, reaction temperature 90 C and substrate ratio 1:1 (cas-tor oil/EF, mol/mol). Under these optimised conditions, the maxi-mum EF conversion (100%) was obtained at 200 rpm for 72 hand, in the transesterification product, the lipophilic and hydro-philic feruloylated structured lipids were 62.6% and 37.3%,respectively.

Acknowledgements

The authors gratefully acknowledge financial support fromNational Natural Science Foundation of China (31101301), FundingScheme for Young Teachers in Colleges and Universities in HenanProvince (2012GGJS-83), and Plan for Scientific Innovation Talentof Henan University of Technology (11CXRC03).

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