Feruloylated Products from Coconut Oil and Shea Butter

Article abstract of DOI:10.1007/s11746-017-2953-7

The synthesis of feruloylated coconut oil and feruloylated shea butter were demonstrated in 0.5-L scale, shaken, batch reactions. Ethyl ferulate and the vegetable oil/fat were combined in a 1.0:1.3?mol ratio in the presence of Candida antartica lipase B immobilized on an acrylic resin (Novozym 435) at 60?°C. The transesterification of ethyl ferulate with coconut oil and shea butter reached equilibrium conversions, after 22?days, of 63 and 70%, respectively, with the shea butter transesterifications producing a white precipitate not observed in the coconut oil transesterifications. The faster transesterification rates, equilibrium conversions and white precipitate were shown to result from di- and monoacylglycerols (DAG and MAG) present in the shea butter. The transesterification of ethyl ferulate and coconut oil was also tested in a continuous, enzymatic, packed-bed bioreactor using Novozym 435 at 60?°C to produce feruloylated coconut oil at rates of 0.5–0.9?kg/day over 4.5?months. The feruloylated coconut oil acylglycerol species were identified by LC–MS analysis of transesterification reactions of ethyl ferulate with medium chain triacylglycerol (TAG) standards, C8–C14. The feruloylated vegetable oils possessed an ultra violet (UV) absorbing λ_max 328?nm, making them good UVAII absorbers, as defined by the U.S. Food and Drug Administration. The feruloylated coconut oil possessed a 17.5% higher absorption capacity than feruloylated shea butter on a per weight basis. All the feruloylated vegetable oils possessed rapid antioxidant capacity (50% reduction of initial radical concentration <5?min) at the concentrations tested, 0.5–2.5?mM. Feruloylated coconut oil possessed chemical and physical characteristics that suggested it would be fungible for feruloylated soybean oil in current retail formulations.

Full text of DOI:10.1007/s11746-017-2953-7

J Am Oil Chem Soc
DOI 10.1007/s11746-017-2953-7
ORIGINAL PAPER
Feruloylated Products from Coconut Oil and Shea Butter
David L. Compton1 · John R. Goodell2 · Mark A. Berhow3 · James A. Kenar3 ·
Steven C. Cermak4 · Kervin O. Evans1
Received: 21 October 2016 / Revised: 12 January 2017 / Accepted: 12 January 2017
© AOCS (outside the USA) 2017
Abstract The synthesis of feruloylated coconut oil and
feruloylated shea butter were demonstrated in 0.5-L scale,
shaken, batch reactions. Ethyl ferulate and the vegetable
oil/fat were combined in a 1.0:1.3 mol ratio in the pres-
ence of Candida antartica lipase B immobilized on an
acrylic resin (Novozym 435) at 60 °C. The transesteri-
fication of ethyl ferulate with coconut oil and shea butter
reached equilibrium conversions, after 22 days, of 63 and
70%, respectively, with the shea butter transesterifications
producing a white precipitate not observed in the coconut
oil transesterifications. The faster transesterification rates,
equilibrium conversions and white precipitate were shown
to result from di- and monoacylglycerols (DAG and MAG)
present in the shea butter. The transesterification of ethyl
ferulate and coconut oil was also tested in a continuous,
enzymatic, packed-bed bioreactor using Novozym 435 at
60 °C to produce feruloylated coconut oil at rates of 0.5–
0.9 kg/day over 4.5 months. The feruloylated coconut oil
acylglycerol species were identified by LC–MS analysis of
transesterification reactions of ethyl ferulate with medium
chain triacylglycerol (TAG) standards, C8–C14. The feru-
loylated vegetable oils possessed an ultra violet (UV)
absorbing λmax 328 nm, making them good UVAII absorb-
ers, as defined by the U.S. Food and Drug Administration.
The feruloylated coconut oil possessed a 17.5% higher
absorption capacity than feruloylated shea butter on a per
weight basis. All the feruloylated vegetable oils possessed
rapid antioxidant capacity (50% reduction of initial radical
concentration <5 min) at the concentrations tested, 0.5–
2.5 mM. Feruloylated coconut oil possessed chemical and
physical characteristics that suggested it would be fungible
for feruloylated soybean oil in current retail formulations.
Mention of trade names or commercial products in this
publication is solely for the purpose of providing specific
information and does not imply recommendation or endorsement
by the United States Department of Agriculture (USDA). USDA
is an equal opportunity provider and employer.
Keywords Biotechnology · Biocatalysis · Biobased
products · Fats and oils
* David L. Compton
david.compton@ars.usda.gov
1
Renewable Product Technology Research Unit, United States
Department of Agriculture, Agricultural Research Service,
National Center for Agricultural Utilization Research,
1815 N. University St., Peoria, IL 61604, USA
Introduction
2
Coconut oil is a vegetable oil obtained from the seed ker-
nel of the coconut palm consisting of mostly saturated,
short to medium chain (C6–C14) fatty acids. Coconut oil
is widely consumed in tropical populations and has gar-
nered increased global popularity as a functional oil, and
its health, metabolic, and antimicrobial properties have
been recently reviewed [1, 2]. Freshly pressed coconut oil
is a mixture of TAG (~85%), DAG (2–7%), MAG (0–3%),
and FFA (0–1%) [3, 4]. Consisting mostly of lauric acid
iActive Naturals/Biotechnology Research and Development
Corporation, 801 W Main St., Peoria, IL 61606, USA
3
Functional Foods Research Unit, United States Department
of Agriculture, Agricultural Research Service, National
Center for Agricultural Utilization Research, 1815 N.
University St., Peoria, IL 61604, USA
4
Bio-Oils Research Unit, United States Department
of Agriculture, Agricultural Research Service, National
Center for Agricultural Utilization Research, 1815 N.
University St., Peoria, IL 61604, USA
1 3

J Am Oil Chem Soc
(C12:0, 45–53%) and myristic acid (C14:0, 17–23%) with
other saturated, shorter chain fatty acids, refined coconut
oil, sans DAG and MAG, is a white solid at room tempera-
ture (mp 24 °C). The saturated fatty acids limit oxidation
and rancidity making coconut oil an ideal candidate for
lipid-based products. Coconut oil has recently gained much
interest as a natural ingredient in the cosmetic and personal
care industry and is used in products ranging from facial
cleansers to deodorants to insect repellants [2, 5, 6].
enzymatic, packed-bed bioreactor over a 4.5 months pro-
duction run. The advancement from small scale, biocata-
lytic transesterifications of ethyl ferulate using alterna-
tive vegetable oils to a pilot-scale, continuous, bioreactor
production is crucial for the commercial adoption of these
feruloylated lipids by the personal care and cosmetic
industries.
Shea butter is the fat extracted from the fruit kernel of
the African shea tree that is prevalent throughout the savan-
nahs of West and Central Africa. The ecological diversity of
the expansive geographical growing region of the shea tree
results in exported oils (butter) with a wide range of fatty
acid compositions [7]. Shea butter consists mainly of oleic
acid (C18:1, 37–62%) and stearic acid (C18:0, 25–50%)
with some palmitic acid (C16:0, 2–10%) and other minor
fatty acid components. This diversity results in shea butters
with a wide range of chemicophysical properties and minor
bioactive components (e.g. triterpene alcohols, phenols,
sterols) [8]. Africa exports over 350,000 tons/year of dry
kernel (115,000 tons shea butter/year) to the United States
and Europe for mostly food and specialty fat uses [7, 9].
More recently, the solid (mp 32–45 °C), off-white, malle-
able shea kernel extract has been increasingly used as an
exotic fat and oil ingredient in the cosmetic, health, and
personal care industries [8, 10, 11].
The biocatalytic modification of fats and oils to produce
bio-based active ingredients with enhanced functionaliza-
tion for the cosmetic and personal care industries has stead-
ily increased over the past decade [12–15]. Of particular
interest is the modification of vegetable oils via biocatalytic
transesterification with phenylpropanoid esters (e.g. feru-
lic, caffeic, sinapic) [16–18]. This enzymatic, solventless
process uses the vegetable oil as the reaction medium and
has been studied extensively using soybean oil, evolving
from a laboratory-scale, stirred, batch process into a con-
tinuous, enzymatic, packed-bed bioreactor process [19–21].
There has been limited examination of the transesterifica-
tion using other vegetable oils, all at the laboratory scale
[22, 23]; however, none have examined vegetable oils that
are solid at room temperature and none have demonstrated
their use in a continuous, enzymatic, packed-bed bioreactor
process.
Experimental Procedures
Materials
Coconut oil used for the batch syntheses was purchased
from Better Body Foods and Nutrition (Lindon, UT, USA)
from a local grocery. Coconut oil used for the continu-
ous, enzymatic, packed-bed synthesis was purchased from
Columbus Vegetable Oils (Des Plains, IL, USA). Shea but-
ter (100% Natural African—Ultra Refined) was purchased
from Green Island Traders (Chagrin Falls, OH, USA).
Non-GMO soybean oil was generously donated by Colum-
bus Vegetable Oils. Ethyl ferulate (4-hydroxy-3-methox-
ycinnamic acid ethyl ester) was purchased from Capot
Chemical Co., Ltd. (Hangzhou, Zhejiang, China). Novo-
zym 435 (Candida antarctica lipase B immobilized on
acrylic beads) was purchased from Brenntag Great Lakes,
LLC. (Wauwatosa, WI, USA). All solvents and other rea-
gents were purchased from Sigma-Aldrich (St. Louis, MO,
USA).
Transesterification Reactions
Batch
Batch reactions on a 500-g scale were conducted with
the following general procedure. Coconut oil, shea but-
ter, or soybean oil (400–500 g depending on the vegetable
oil, 0.57 mol) and ethyl ferulate (100 g, 0.45 mol) were
weighed into a 1-L Schlenk Flask and stirred under vacuum
(11.6 Pa) at 60 °C for 18 h. The dried solution was trans-
ferred to a 2-L, baffled Erlenmeyer shake flask and Novo-
zym 435 (75 g) was added. The shake flask was stoppered
and flushed with nitrogen for 15 min. The flask was shaken
in an Innova 4080 Incubator Shaker (Eppendorf Inc., for-
merly New Brunswick Scientific, Hauppauge, NY, USA) at
60 °C and 115 rpm for 21 days. The oil product was col-
lected by vacuum filtration through a double layer of What-
man 54 filter paper (Fisher Scientific, Pittsburgh, PA, USA)
using a Büchner funnel (135 × 60 mm) and a 2-L collec-
tion flask. The Novozym 435 was recovered and used for
subsequent reactions for a minimum of three replicates.
Transesterifications of glyceryl trioctanoate, glyc-
eryl tridecanoate, glyceryl tridodecanoate, glyceryl
We examined shaken, batch transesterification reactions
of ethyl ferulate with the solid vegetable oils, extra virgin
coconut oil and ultra refined shea butter and compared their
performance to soybean oil. The feruloylated coconut oil
and feruloylated shea butter were examined for antioxidant
and ultraviolet (UV) absorption efficacy and some of their
physical properties determined. Based on these findings,
the transesterification of ethyl ferulate and extra virgin
coconut oil was attempted in a 1/4-tonne/year continuous,
1 3

J Am Oil Chem Soc
tritetradecanoate, and purified shea butter with ethyl feru-
late were conducted using the same general procedure as
described above but on a 1/10-scale.
light scattering detection (ELSD) with nitrogen as the car-
rier gas. Parameters were set to 4 min peak width, 0.1 V
collection threshold, 40 °C spray temperature, 70 °C drift
temperature, and UV detection at 340 nm. Eluent peaks
were collected in 480-mL French bottles. Raw shea but-
ter (5.0 g) was dissolved in acetone (5.0 mL) with heating
to 60 °C for 10 min. The solution was loaded by syringe
onto a dry 220 g RediSep Rf High Performance Gold silica
column with 1 column volume (CV) equaling 135 mL at
83 mL/min flow rate. The RediSep silica column was aspi-
rated to dryness for 1 h. The sample loaded RediSep silica
column was developed with a hexane/ethyl acetate gradient
at 83 mL/min: 0% ethyl acetate for 1 CV, increased to 2%
ethyl acetate over 4 CV, held at 2% ethyl acetate for 2.7
CV, increased to 10% ethyl acetate over 4.5 CV, increased
to 100% ethyl acetate over 0 CV, held at 100% ethyl acetate
for 3 CV, decreased to 0% ethyl acetate over 0 CV, held
at 0% ethyl acetate for 2 CV. The column was air purged
for 2 min and then aspirated to dryness overnight for the
next sample loading. The purified shea butter (containing
only TAG) eluted during the 2% ethyl acetate to 10% ethyl
acetate gradient. Solvent was removed from eluent under
vacuum to produce a white, waxy solid. The procedure was
repeated five more times. Yield: 21.4 g (71.2 wt% relative
to the raw shea butter).
Continuous, Enzymatic, Packed‑Bed Bioreactor
The four-column bioreactor used for the continuous enzy-
matic esterification of coconut oil and ethyl ferulate was
1/4-scale of the bioreactor design reported for the produc-
tion of feruloylated soybean oil [21] with the following
modifications. The bioreactor used four 304-stainless steel
columns (6.35 cm I.D. × 81.28 cm, 2.57 L). A bleed valve
was fitted between column A and column B for sampling.
The feed pump was a PULSAtron E Plus pump (model
#LPK2SA-PTC4, Pulsafeeder, Punta Gorda, FL, USA)
set at 5% stroke length, 7.5% stroke rate (~0.60 mL/min)
and regulated by a GE (Fairfield, CT, USA) basic 125 V,
15 Amp, 30 min interval timer. Each column was plumbed
with a PULSAtron E Plus pump (P2, model #LE13SA-
PTC2) set at 24% stroke length, 40% stroke rate (~4.0 mL/
min) for a bottom-up flow.
Batches (20 kg) of the coconut oil/ethyl ferulate sub-
strate were prepared following the same basic procedure as
previously described for soybean oil [21] except at a 4:1
coconut oil:ethyl ferulate mass ratio (1.3:1.0 mol:mol). The
heated (65–75 °C) coconut oil/ethyl ferulate substrate was
filtered through a cooking oil filter (Chard, Two Rivers, WI,
USA, Model OPF-10) before being dried for 3–4 h at 60 °C
under partial vacuum with a nitrogen sparge and then trans-
ferred to the bioreactor feed tank as previously described
[21].
Each column was charged in the bioreactor heating
cabinet (60 °C) with a slurry of Novozym 435 (500 g) and
coconut oil (2.0 L) preheated to 60 °C. The excess coco-
nut oil was gravity drained to pack the column and form
the enzyme bed. The column ends were capped with glass
wool and filter screen gaskets and the system plumbed as
previously described [21]. The coconut oil was displaced
with coconut oil/ethyl ferulate substrate at a feed rate of
~0.65 mL/min. The bioreactor was then run in stop mode,
feed pump off and recirculation pumps at ~4 mL/min, for
22 days to equilibrate the Novozym 435. The bioreactor
was then switched to flow mode, feed pump at ~0.6 mL/
min and recirculation pumps at ~4 mL/min, for continuous
production of feruloylated coconut oil at 0.75–1.0 kg/day.
New, dried batches of coconut oil/ethyl ferulate substrate
(20 kg) were added to the feed tank as needed.
Analytical Methods
HPLC
HPLC analysis for feruloyl soy glycerol species’ quanti-
fication was based on previously published methods [24].
Analyses were performed on two systems. HPLC1 was a
Thermo Separation Product (Thermo Fischer Scientific
Inc., Waltham, MA, USA) HPLC system consisting of a
Spectra System AS3000 autosampler, a Spectra System
P4000 pump, a Spectra System UV6000LP detector, an
Alltech (Deerfield, IL, USA) 500 Evaporative Light-Scat-
tering Detector (ELSD), and a Thermo Separation Prod-
ucts SCM 1000 Membrane Degasser. HPLC2 was an Agi-
lent (Santa Clara, CA, USA) 1100 system consisting of a
G1322A Degasser, G1311A QuatPump, G1313A Autosa-
mpler, G1316A Column Heater, and G1315A Diode Array
Detector. Solvents were filtered using a Whatman 0.45-μm
nylon membrane (Sigma-Aldrich) before use, and samples
were developed on a Luna Phenyl-Hexyl column (5 μm,
250 × 4.6 mm, Phenomenex, Torrance, CA, USA).
Soybean oil and shea butter products were developed
with a gradient of solvent A: water (268 mL), methanol
(70 mL), 1-butanol (11 mL), and glacial acetic acid (1 mL),
solvent B: water (93 mL), methanol (245 mL), 1-butanol
(33 mL), and glacial acetic acid (1 mL), and solvent C:
methanol. The column method at 1.0 mL/min was a 5 min
Flash Chromatography Purification of Shea Butter
Raw shea butter was purified using a Teledyne Isco, Inc.
(Lincoln, NE, USA) CombiFlash Rf 200i flash chroma-
tography system. Products were monitored by evaporative
1 3

J Am Oil Chem Soc
isocratic flow of 3:1 A:B, a 2 min linear gradient to 100%
B, a 5 min isocratic flow of 100% B, a 2 min linear gradient
to 100% C, a 13 min isocratic flow of 100% C, followed by
a 3 min linear gradient to 3:1 A:B. The UV (325 nm, 7 nm
bandpass) and diode array (325 nm) detector response was
calibrated with ethyl ferulate and ferulic acid. Injection vol-
umes were 10 μL.
Coconut oil species were determined using a modified
gradient at 1.0 mL/min with a 5-min isocratic flow of 3:1
A:B, a 2-min linear gradient to 100% B, a 5-min isocratic
flow of 100% B, a 2-min linear gradient to 1:9 B:C, a
13-min isocratic flow of 1:9 B:C, followed by a 3-min lin-
ear gradient to 3:1 A:B. The UV (325 nm, 7 nm bandpass)
and diode array (325 nm) detector response was calibrated
with ethyl ferulate and ferulic acid. Injection volumes were
10 μL.
GC
GC analysis was conducted on a Hewlett-Packard 6890N
Series gas chromatograph (Palo Alto, CA, USA), equipped
with a flame ionization detector, autosampler/injector, and
a SP 2380 30 m × 0.32 mm i.d. (Supelco, Bellefonte, PA,
USA) column. SP 2380 analysis was adapted from that
of Isbell et al. [25] with a 1.4 mL/min column flow with
a helium head pressure of 138 kPa; split ratio 10:1; pro-
grammed ramp 120–135 °C at 10 °C/min, 135–175 °C
at 3 °C/min, and 175–265 °C at 10 °C/min; injector and
detector temperatures set at 250 °C. Saturated and unsatu-
rated C8–C30 fatty acid methyl ester standards were used
for assignment of fatty acid methyl esters obtained from
soybean oil, coconut oil, and shea butter.
Methyl esters were prepared by reacting vegetable oils
(10 mg) with 0.5 M KOH/methanol (0.5 mL) in a sealed
vial for 1 h at 100 °C in a heating block. The sample was
cooled to room temperature and 1.5 mL of 1 M H2SO4/
methanol was added, the vial resealed, and the vial heated
to 100 °C for 15 min. The mixture was transferred to a 2
dram vial (7.5 mL) and 1 mL of water added. The solution
was extracted with 1 mL of hexane. The hexane extract
was dried over sodium sulfate and injected into the GC for
FAME analysis.
LC–ESI–MS
Samples were run on a Thermo Electron LTQ Orbitrap Dis-
covery Mass Spectrometer (Thermo Fisher Scientific Inc.)—
a linear ion trap (LTQ XL) MS, coupled to a high precision
electrostatic ion trap (Orbitrap) MS with a high energy col-
lision (HCD) cell—with an Ion Max electrospray ionization
(ESI) source, and a Thermo Scientific (Thermo Fischer Sci-
entific Inc.) ACCELA series HPLC system (ACCELA 1250
UHPLC pump, ACCELA1 HTC cool stack autoinjector, and
an ACCELA 80 Hz PDA detector) all running under Thermo
Scientific Xcalibur 2.1.0.1140 LC–MS software. The MS
was typically calibrated at least monthly with a standard cali-
bration mixture recommended by Thermo Scientific and the
signal detection optimized by running the autotune software
feature as needed. The MS was run with the ESI probe in the
negative mode. The source inlet temperature was 300 °C, the
sheath gas rate was set at 50 arbitrary units, the auxiliary gas
rate was set at 5 arbitrary units and the sweep gas rate was
set at 2 arbitrary units. The maximal mass resolution was set
at 30,000, the spray voltage was set at 3.0 kV, and the tube
lens was set at −100 V. Other parameters were determined
and set by the calibration and tuning process. The column
was a Luna Phenyl-Hexyl column (3 µm, 3 × 150 mm,
100 Å, Phenomenex).
NMR Spectroscopy
1H- and 13C-NMR spectra were obtained on a Bruker
Avance 500 spectrometer (500 MHz H/125.77 MHz 13C)
1
using a 5 mm BBI probe. All samples were dissolved in
CDCl3 and all spectra were acquired at 27 °C. Chemical
shifts are reported as ppm from tetramethylsilane calcu-
lated from the lock signal (ΞD = 15.350609%).
Karl Fischer Titration
Water concentration was determined using a Mettler Toledo
DL 32 Karl Fischer Coulometer (Columbus, OH, USA),
following AOCS Method Ca 2e-84 [26] with 70:30 (v/v)
Hydranal AG-H/chloroform as the analyte.
For LC separation analysis, the initial solvent condi-
tions were 25% methanol in water with 0.25% formic
acid at a flow rate of 0.25 mL/min. After injection (1 µL
or less) the column was developed with a linear gradient
to 100% methanol with 0.25% formic acid in 5 min then
held at 100% methanol with 0.25% formic acid for 26 min.
The column effluent was monitored at 325 nm in the PDA
detector. The software package was set to collect mass data
between 100 and 2000 amu. Generally, the most significant
sample ions generated under these conditions are [M−H]−
and [M+HCOO]−.
Total Acid Value
Total acids were determined using a Metrohm Ltd. (Her-
isau, Switzerland) 751 GPD Titrino equipped with a
Metrohm pH electrode, following the AOCS Method Cd
3d-63 [27] with ethanol substituted for methanol.
Free Radical Scavenging
Free radical scavenging capacity was determined using
a DPPH assay [28] with modifications [29] where DPPH
1 3

J Am Oil Chem Soc
R
spontaneously forms a free radical in solution. Feruloylated
soy glycerides were mixed with 0.2 mM DPPH in ethanol.
The examined concentration range of antioxidant (feru-
loylated soy glyceride) was 0.5–2.5 mM. Reduction of the
DPPH radical was followed at 517 nm for 30 min at 2 min
intervals at room temperature (22–24°C). Experiments
were performed in triplicate. The DPPH radical remaining
(%) over time was calculated as the ratio of sample absorb-
ance to a control DPPH sample at the same time, multiplied
by 100.
R
R
CH2CH3
Fr
+
β
α
α
ethyl ferulate (EF)
vegetable oil (R = fatty acid)
60 °C Novozym 435
Fr
OH
OH
Fr
Fr
H
Fr
ferulic acid
(FA)
OH
diferulyol glycerol
ferulyol glycerol
(FG)
(F2G)
Fr
Fr
Fr
R
Fr
R
R
Lovibond Color
R
OH
diferuloyl monoacylglycerol
ferulyol monoacylglycerol
feruloyl diacylglycerol
(FMAG)
(FDAG)
(F2MAG)
O
Lovibond color was measured on an AOCS Tintometer
(model ca. 1950s, Lovibond Tintometer LTD., Salisbury,
England) following the AOCS Method Cc 13e-92 [30]. The
AOCS Color Comparison Tube (Kimax #45290, 15.4 × 2.0
i.d. cm, Capitol Scientific, Austin, TX, USA) was filled to
the 5 ¼″ line (13.335 cm) with sample and the color com-
pared to Lovibond colored glass filters.
O
O
feruloyl moiety (Fr)
HO
Fig. 1 Structures of feruloylated species obtained from the solvent-
less transesterification of vegetable oils with ethyl ferulate. The α and
β glycerol hydrogens of the vegetable oil are labeled for reference of
1H-NMR frequency assignments in Fig. 4
Differential Scanning Calorimetry
DSC data was obtained using a TA instruments (New Cas-
tle, DE) model 2920 V4.3A calibrated with an indium
standard (156.618 °C, enthalpy of melting = 28.86 J/g).
Data sampling and temperature controls were controlled by
the “TA Instrument Control” software program. Approxi-
mately 5.0–10.0 ( 0.1) mg of melted samples were
weighed into an aluminum DSC pans and hermetically
sealed. The sealed DSC samples were referenced against
an identical empty aluminum pan and run using modula-
tion ( 0.40 °C every 40 s) at a linear heating rate (β) of
5.0 °C/min from −55.0 to 90.0 °C. To eliminate tempera-
ture history effects and evenly distribute the samples in the
pans, the samples were first cycled across the temperature
range. The samples were subsequently cooled to −55 °C at
5.0 °C/min, held isothermally for 1 min, and then reheated
across the melting region to obtain the melting profile.
TA Universal Analysis 2000 software, version V4.3A (TA
Instruments, DE) was used to obtain heat flow (W/g) versus
temperature (°C) curves from each experiment. These DSC
thermograms were subsequently analyzed to determine
peaks, onset, and total enthalpies.
with aromatic moieties (e.g., caffeoyl, feruloyl, sinapoyl)
possessing UV absorbing and antioxidant functionalities.
The transesterification results in multiple molecular species
depicted in Fig. 1. The most studied of these reactions is the
transesterification of ethyl ferulate with soybean oil using
soybean oil as the solvent [21, 31]. Ethyl ferulate melts and
is miscible with soybean oil at the reaction temperature
of 60 °C. These reactions typically require 14–22 days to
reach an equilibrium of ~65% conversion of ethyl ferulate
into the molecular species in Fig. 1, which have been iden-
tified and quantified by HPLC–MS [24]. The feruloylated
acylglycerols, unreacted ethyl ferulate, unreacted soybean
oil, and fatty acid ethyl ester byproducts remain a homo-
geneous solution at room temperature, except when F2G
forms >2% of the total feruloylated species and precipitates
from the solution [32].
The transesterification of ethyl ferulate has been con-
ducted with other vegetable oils (e.g., canola, flaxseed,
olive) [18, 31] but not with vegetable oils that are solid at
room temperature. Coconut oil and shea butter are room
temperature vegetable fats that have gained prominence as
ingredients in the personal care industry. The fatty acid pro-
files of coconut oil, shea butter, and non-genetically modi-
fied organism (non-GMO) soybean oil were determined
(Table 1). Coconut oil contained mostly saturated short and
medium chain fatty acids compared to the shea butter and
soybean oil. Shea butter and soybean oil contained mostly
C18 fatty acids but soybean oil is more highly unsaturated.
Feruloylated soybean oil is produced commercially in a
1-metric tonne/year continuous, enzymatic, packed-bed
Results and Discussion
Feruloylated Coconut Oil and Shea Butter
The transesterification of vegetable oils with phenylpropa-
noic acids and their esters catalyzed by lipases results in
the exchange of fatty acid groups from the triacylglycerol
1 3

J Am Oil Chem Soc
Table 1 Fatty acid profile
of vegetable oils used for the
transesterification reactions
Fatty acid
Coconut oil (%)
Shea butter (%)
Raw
Soybean oil
(%)
Purified
C6:0
0.77 0.10
9.02 0.54
6.31 0.09
49.10 0.26
19.01 0.27
7.50 0.09
2.90 0.05
4.57 0.07
0.74 0.01
–
–
–
–
C8:0
–
–
–
C10:0
C12:0
C14:0
C16:0
C18:0
C18:1
C18:2
C18:3
C20:0
C22:0
C24:0
Other
Mol. wt.
–
–
–
0.21 0.01
0.14 0.00
5.43 0.01
28.41 0.02
60.80 0.10
1.83 0.01
0.36 0.00
1.12 0.01
0.14 0.00
0.09 0.00
1.47 0.10
882.94
0.21 0.03
0.15 0.02
5.69 0.04
28.36 0.22
62.24 0.27
1.73 0.03
0.32 0.03
1.09 0.03
0.14 0.02
0.08 0.01
–
–
–
10.87 0.06
3.83 0.01
21.58 0.07
53.53 0.15
7.96 0.02
0.32 0.01
0.38 0.01
–
–
–
–
–
1.71 0.02
873.38
670.39
882.74
Results are presented as means SD (n = 5 trials)
Raw shea butter as obtained from the supplier and after purification by flash chromatography
Mol. wt. was the average molecular weight (g/mol) of the vegetable oil determined by the relative quantity
of fatty acids
80
60
40
20
0
bioreactor [21]. It was not certain that lipase-catalyzed
transesterification of ethyl ferulate with coconut oil and
shea butter would proceed similarly to that of soybean oil;
ethyl ferulate being miscible in the vegetable oil, the reac-
tion mixture remaining a solution over the course of the
reaction, and the product being a liquid. The transesteri-
fications were first examined in 0.5-L scale shaken, batch
reactions.
Ethyl ferulate and coconut oil or shea butter were com-
bined at a 1.0:1.3 mol ratio. The substrates were heated to
60 °C and dried under vacuum to <35 ppm water. Ethyl
ferulate formed miscible solutions with both vegetable oils
but the solutions quickly solidified when allowed to cool to
room temperature. The substrate mixtures were, therefore,
kept at 60 °C for all manipulations. Novozym 435 was used
at a 0.75 wt% relative to ethyl ferulate to catalyze the reac-
tions. The time course of the conversion of ethyl ferulate
to the feruloylated vegetable oils are shown in Fig. 2, the
transesterification with non-GMO soybean oil was done
as a comparative control. The transesterifications of each
vegetable oil were performed in quadruplicate and in series
using the same enzyme load; the enzyme was collected
from a reaction by filtration and used in the subsequent
reaction. The data from the first reaction in the series was
discarded due to the adsorption of the ethyl ferulate by the
acrylic resin of the Novozym 435 beads, which resulted in
inaccurate conversion yields [20].
0
5
10
15
20
25
Time (d)
Fig. 2 Conversion of EF to feruloylated lipids via lipase-catalyzed
transesterifications with coconut oil (circles), shea butter (squares),
purified shea butter (diamonds), and non-GMO soybean oil (trian‑
gles) in shaken batch reactions. Data points are reported as the mean
with error bars denoting SD from the mean, n = 3 trials. For illustra-
tive purposes, SigmaPlot V11.2.0.5 exponential rise to maximum best
fit lines (f = y0 + a × (1 − exp(−b × x)) + c × (1 − exp(−d × x)))
are provided, coconut oil (solid), shea butter (dot), purified shea but-
ter (dash), and non-GMO soybean oil (dash-dot)
conversion after 22 days. The solution remained clear with
no visible precipitate forming over the course of the reac-
tion. The molecular species of feruloylated soybean oil
have been identified by LC–MS [24]. The HPLC chroma-
togram of feruloylated coconut oil (Fig. 3) is comparatively
The transesterification of ethyl ferulate and coconut oil
at 60 °C with gentle shaking reached equilibrium at 63%
1 3

J Am Oil Chem Soc
Table 2 Major products obtained from the transesterification of tria-
cylglycerol standards with ethyl ferulate
EF
TAG
Feruloylated species
FG FA F2G
FMAG
FMAG
F2MAG
FDAG
1
2
Rt (min)
C8
14.49
16.87
17.93
18.61
394.20
422.23
450.26
478.29
393.18
421.22
449.27
477.28
1.01
15.50
17.42
18.22
18.83
394.20
422.23
450.26
478.29
393.19
421.22
449.27
477.29
5.58
17.95
18.60
19.94
21.70
C10
C12
C14
C8
FMAG
F2MAG
FDAG
19.26
24.08
19.97
27.49
Calculated mass
Major ion (m/z)
Peak area (%)
570.25
598.28
626.31
654.79
569.24
597.27
625.30
653.33
12.30
520.30
576.37
632.43
688.49
519.30
575.36
631.42
687.48
55.33
C10
C12
C14
C8
3
0
5
10
15
20
25
30
Time (min)
C10
C12
C14
C8
Fig. 3 HPLC chromatograms (UV detector, 325 nm) of the transes-
terification reactions of coconut oil (1), tridodecanoate (2), and tritet-
radecanoate (3) with EF. FG, FA, F2G, and EF were determined vs
standards. FMAG, F2MAG, and FDG were identified by LC–MS (see
Table 2). See Fig. 1 for definitions of abbreviations
C10
C12
C14
1.36
5.98
14.35
44.40
1.10
5.40
15.13
47.15
0.99
5.52
15.59
44.46
Feruloylated species are defined in Fig. 1
more convoluted due to the greater diversity and relative
abundance of different fatty acids; soybean oil, >85% C18
vs coconut oil, 9% C8, 6% C10, 49% C12, 19% C14, 8%
C16 (Table 1). EF, FA, FG, and F2G (Fig. 1) were iden-
tified vs standards in the feruloylated coconut oil HPLC
chromatograms. The parent ion of each HPLC peak of the
feruloylated coconut oil samples could not definitively be
identify because of the coelution of molecular species. To
unambiguously determine retention times of the major fer-
uloylated coconut oil molecular species the transesterifica-
tion of ethyl ferulate was conducted with TAG standards,
trioctanoate, tridecanoate, tridodecanoate, and tritetrade-
canoate, using the same shaken, batch reaction parameters
at 1/10-scale.
The neat (without solvent) transesterifications of ethyl
ferulate with the TAG standards proceeded similarly to
those with coconut oil at 60 °C in shaken, batch reactions.
Transesterifications with trihexadecanoate (mp 66–67 °C)
could not be done neat under the examined reaction condi-
tions and were not attempted. The HPLC chromatograms
of the feruloylated tridodecanoate and tritetradecanoate
reactions are shown in Fig. 3. Each chromatogram shows
three distinct peaks >15.0 min Rt (retention time) that
were identified by LC–MS. The feruloyl monoacylglycer-
ols (FMAG, see Fig. 1) eluted before the diferuloyl mono-
acylglycerol (F2MAG) species. The feruloyl diacylglycerol
(FDAG) species eluted last. The Rt of the feruloylated spe-
cies and their major ions determined by LC–MS are listed
in Table 2. The FMAG peaks had a small preceding peak
with the same molecular weight, which was attributed to a
minor 1,2-FMAG isomer [24]. Fatty acid chain length had
Rt (min) were the retention times determined by HPLC diode array
detector (325 nm), see Fig. 3 for representative HPLC chromatograms
Major ions detected by LC–MS in negative ion mode, all determined
to be [M−H]−
Peak area (%) were relative to the total peak area of all feruloylated
species, including FA, F2G and EF, obtained from individual reac-
tions of each TAG with EF
the greatest influence on the Rt of the FMAG species, ∆Rt
3.4 min for C14 vs C12 (Fig. 3; Table 2). The influence of
fatty acid chain length on ∆Rt decreased with the decreas-
ing chain length. The difference in chain length also caused
longer fatty acid chain FMAG species to elute at the same
Rt as shorter fatty acid chain F2MAG species (Table 2).
For example, the C12 FMAG species eluted at the same
Rt 17.9 min as the C8 F2MAG species, and similarly, the
C14 FMAG species coelutes with the C10 F2MAG spe-
cies, Rt 18.6 min. The ratio, based on their relative abun-
dance by HPLC, of the feruloylated lipid species formed
during the transesterifications were similar for all four TAG
standards (Table 2). The most abundant were the triacylg-
lycerol products FMAG (44–55%) and F2MAG (12–16%).
The diacylglycerol species, FMAG, was the least preva-
lent (6–7%). The feruloyl, non-fatty acid products, F2G
and FA, were each less than 1% of the total feruloylated
species for both the coconut oil and TAG standard trans-
esterifications. The relative abundance of feruloylated lipid
species from the transesterification of the TAG standards,
FDAG ≫ F2MAG > FMAG, was different from those
obtained from the transesterification of ethyl ferulate with
1 3

J Am Oil Chem Soc
triolein, FMAG > FDAG > F2MAG [24]. This was possibly
due to the influence of higher water content in the triolein
reactions, 250 ppm [20], relative to the coconut transesteri-
fications, <35 ppm. The higher water content of the triolein
transesterifications also resulted in higher quantities of FA
and F2G, 2 and 6%, respectively. The transesterification of
octyl dihydrocaffeate with trioctanoate in a 1:1 mol:mol
ratio catalyzed by Novozym 435 in stirred batch reac-
tions also produced FMAG > FDAG > F2MAG [22]. The
authors did not report the water content of their phenolic
ester-trioctanoate substrate, and their experimental did not
indicate that precaution was taken to dry the substrate. This
suggested that chain length of the TAG, C8 of triocatante
vs C18 of triolein, was not responsible for the difference in
abundance of the feruloylated acylglycerol species formed
in the transesterifications of ethyl ferulate with the C8–C12
TAG standards compared to triolein. Because coconut oil
does not simply consist of a mixture of TAG standards but
instead consists of several, mixed fatty acid chain length
TAG, the number of feruloylated coconut oil species that
could potentially coelute made definitive peak identifica-
tion by LC–MS parent ion implausible. In fact, the HPLC
peaks of the feruloylated coconut oil species were so con-
voluted that the FMAG, F2MAG, and FDAG could not be
assigned general Rt ranges as was done for feruloylated
soybean oil [24].
While the transesterification of ethyl ferulate with coco-
nut oil performed similarly to that with soybean oil in the
shaken, batch reactions, the transesterification of ethyl
ferulate with shea butter produced unexpected results.
With very similar fatty acids profiles (Table 2), shea but-
ter (>95% C18: 0–3) and non-GMO soybean oil (>85%
C18: 0–3) were expected to produce very similar prod-
ucts. The transesterification with shea butter did result in
the same three-region feruloylated lipid profile as deter-
mined by HPLC Rt [24]. Under the very dry transesteri-
fication conditions (<35 ppm water) examined herein, the
relative abundance of the feruloylated lipid species of shea
butter was the same as those obtained from the transes-
terifications with the non-GMO soybean oil and C8–C12
TAG standards, FDAG > F2MAG > FMAG. After 24 h,
however, the shea butter transesterifications produced a
white precipitate not observed in the coconut oil, C8–C12
TAG standards, or soybean oil transesterifications. Also,
the ethyl ferulate transesterifications with the shea butter
reached equilibrium sooner than those with the other veg-
etable oils and reached a slightly higher conversion, 72%
(Fig. 2). The white precipitate was isolated and identified
by HPLC and 1H NMR as F2G, which was produced in the
shea butter transesterifications at 6–8%. This was a much
higher concentration of F2G than was formed in transester-
ifications with coconut oil (<1%), C8–C12 TAG standards
(<1%), and non-GMO soybean oil (<1%). Since the water
content was controlled for all of the transesterifications,
hydrolysis of the F2MAG was not suspected. There is prec-
edent that quantities of MAG and DAG leads to increased
quantities of F2G in similar transesterifications, and if
F2G is present in concentrations >1% it can precipitate
1
from the feruloylated vegetable oil product [32–34]. H-
NMR spectroscopy of the shea butter identified 16 mol%
of DAG was present in the shea butter as obtained from
the supplier (raw) (Fig. 4). Comparatively, coconut oil and
non-GMO soybean oil did not contain DAG as determined
by the absence of 1,2-DAG and 1,3-DAG alpha- and beta-
hydrogens [35].
To confirm that the DAG contaminants in the shea but-
ter were the cause of high concentrations of F2G and the
accelerated rate to equilibrium, transesterifications of ethyl
ferulate were conducted with shea butter TAG purified by
flash chromatography. Raw shea butter was developed with
a hexane/ethyl acetate gradient on silica gel to produce
three fractions (Fig. 5). The fractions were identified by 1H
NMR (Fig. 4) and found to be aromatic/phenolic contami-
nants containing some aliphatic material (aromatics), puri-
fied shea butter (TAG), and hydrolyzed shea butter (DAG
and MAG). The fatty acid profile of the purified shea butter
was very similar to the raw shea butter (Table 1), consist-
ing of >95% C18: 0–3. Shaken, batch transesterifications
of ethyl ferulate with purified shea butter (5-g scale) did
not produce a white precipitate and resulted in <1% F2G at
equilibrium. The rate of transesterification and the percent
conversion of the purified shea butter transesterifications
were more comparable to those of non-GMO soybean oil
than those of the raw shea butter. While it was possible to
obtain gram quantities of purified shea butter for analyti-
cal purposes it would have been cost prohibitive to purify
quantities (100 kg) needed to examine the transesterifica-
tion of ethyl ferulate and shea butter in a 1/4-tonne/year,
continuous, enzymatic, back-bed bioreactor.
Continuous, Enzymatic, Packed‑Bed Bioreactor
Production of Feruloylated Coconut Oil
Pilot scale (1/4-tonne/year) production of feruloylated
coconut oil was performed in a four-column, continuous,
enzymatic, packed-bed bioreactor [21]. The bioreactor
design ensured that the pumps, columns, valves, and feed
tank were maintained at 60 °C. The feruloylated coconut
oil product collection tank was exterior to the bioreactor
and kept at ambient temperature (22–24 °C). As observed
for the shaken, batch reactions, the harvested feruloylated
coconut oil remained liquid for up to 14 days before
solidifying; the feruloylated coconut oil product was eas-
ily melted and formed a homogenous oil upon reheating to
60 °C for manipulation and packaging. The melting point
of the feruloylated coconut oil is discussed below.
1 3

J Am Oil Chem Soc
1,2-DAGβ
TAGβ
1,3(2)-DAGα 1,3-DAGβ
TAGα
*
Shea butter (raw)
1
2
3
4
Shea butter (DAG & MAG)
Shea butter (aromatics)
Shea butter (TAG)
Coconut oil
Soybean oil
5
6
Fig. 4 1H-NMR (500 MHZ, *CDCl3) spectra of soybean oil (6),
coconut oil (5), and shea butter (1) as obtained from the suppliers.
Spectra 2–4 correspond to materials obtained from the flash chroma-
tography purification of raw shea butter (see Fig. 5) resulting in pure
shea butter TAG (4) and isolated aromatic (3) and DAG and MAG (2)
contaminants
Fig. 5 Flash chromatography purification of shea butter. Far left
axis (ELS) and curved trace correspond to the ELSD signal, the UV
absorbance (254 nm) signal was zero, and the right axis and darker
linear trace correspond to the percent ethyl acetate (v:v, Percent B) in
hexane during elution. Numbers above the trace denote correspond-
ing eluent collection bottles, and the bar below the trace denotes the
peak corresponding to pure shea butter TAG. See Fig. 4 for 1H-NMR
spectra of compounds isolated from collected peaks
The bioreactor conversion of ethyl ferulate and coconut
oil was modified from the previously described procedure
for the conversion ethyl ferulate and soybean oil to form
feruloylated soy glycerides [21]. After the columns were
packed with the Novozym 435-coconut oil slurry and
the coconut oil displaced with coconut oil:ethyl ferulate
1 3

J Am Oil Chem Soc
Table 3 Continuous, enzymatic, packed-bed, bioreactor operating
modes for the 4.5 months production of feruloylated coconut oil
substrate (1.3:1.0 mol:mol, 4:1 wt:wt) the bioreactor was
run in stop mode (feed pump off and recirculation pumps
on, see Table 3) for 22 days to allow the conversion of
ethyl ferulate to feruloylated coconut oil to reach equilib-
rium before product collection was started. This avoided
~20 kg of feruloylated coconut oil waste that would have
been unusable due to low conversion percentages, waste
that was produced with the analogous soybean oil biore-
actor runs [21]. After 22 days the bioreactor was switched
to flow mode (feed pump on and recirculation pumps on)
and feruloylated coconut oil continuously collected over
a 4.5 months period. Figure 6 shows the quantity of feru-
loylated coconut oil accumulated and the percent conver-
sion of ethyl ferulate to feruloylated coconut oil over the
4.5 months bioreactor run using a single charge of Novo-
zym 435. Over the first 30 days of the bioreactor run in flow
mode, the effective substrate feed rate of the feruloylated
Mode
Stop/flow times
Collection rate Collections
(h/h) (Feed pump
off)
(g/min)
(n)
A
B
C
Stop
24/0 12–12 am, pm 0.000 0.000
0/24 0.633 0.006
0
9
3
Flow
–
Stop-
flow
6/18 8–11 am, pm 0.468 0.010
D
E
Stop-
flow
8/16 8–10 am, pm 0.431 0.003
2–4 am, pm
3
9
Stop-
flow
12/12 8–11 am, pm 0.365 0.018
2–5 am, pm
Letters correspond to the lettered boxes in Fig. 6
Stop mode = feed pump off, column recirculation pumps on and flow
mode = feed pump on, column recirculation pumps on
Collection rates of the feruloylated coconut oil product are reported
coconut oil was 0.633
0.006 g/min based on the mass
as the means SD of n collections
Fig. 6 Conversion of ethyl
100
100
A
B
C
D
E
ferulate to feruloylated coconut
oil via lipase-catalyzed trans-
esterification in a continuous,
packed-bed bioreactor. Top
left axis feruloylated coconut
oil product collected from the
bioreactor over the course of the
continuous run (closed circles).
Top right axis conversion of
ethyl ferulate to feruloylated
coconut oil (closed squares).
Bottom left axis relative quantity
of the sum of FMAG, F2MAG,
and FDAG (open circles), EF
(open squares), FA (open up
triangles), and F2G (open down
triangles). Bottom right axis
total ferulate concentration rela-
tive to initial EF concentration
in the EF-coconut oil substrate
(diamonds). Boxes: bioreac-
tor operating modes, A–E, are
detailed in Table 3
80
60
40
20
80
60
40
20
0
0
100
100
80
60
40
20
0
80
60
40
20
0
0
20
40
60
80
100
120
140
Time (d)
1 3

J Am Oil Chem Soc
Table 4 Physical properties of the vegetable oils and the feruloylated vegetable oils
Sample
Total ferulates (mM)
Total acid (mg/g)
Tonset (°C)
Tpeak (°C)
Total ∆Hmelt (J/g)
Lovibond color (Y, R)
SBO
–
0.1415 0.0046 k
0.1702 0.0110 k
0.0452 0.0004 l
0.3965 0.0158 j
4.9914 0.0374 a
1.5831 0.0298 f
0.9592 0.0235 i
4.7419 0.0888 b
2.2629 0.0422 b
1.7998 0.0111 e
1.9642 0.0411 d
1.4243 0.0213 g
1.2025 0.0216 h
–
–
–
2.0, 0.0
–
SHBR
–
−26.5 (−2.5)
−33.6 (20.7)
7.8
−1.28 (24.9)
−0.62 (33.5)
22.1
65.1
91.9
103.9
–
SHBTAG
CNO
–
–
–
2.0, 0.0
8.0, 1.0
–
FSBO
866 45 c
831 38 c
821 38 c
1113 54 a
1021 6 b
1006 2 b
1022 9 b
1025 17 a,b
1018 18 b
–
–
FSHBR
−33.4 (28.6)
−46.2 (23.7)
−36.1 (−3.1)
−50.2 (−3.0)
−48.5 (−2.0)
−48.7 (−3.4)
−49.7 (−1.3)
−38.5 (−2.2)
4.82 (36.4)
12.6 (24.9)
−18.4 (16.5)
−18.4 (15.8)
−19.2 (16.8)
−19.2 (17.0)
−18.4 (17.4)
−19.1 (16.4)
49.9
69.8
61.7
69.9
74.4
72.3
81.5
80.4
FSHBTAG
FCNOSB
FCNOCBP1
FCNOCBP22
FCNOCBP42
FCNOCBP64
FCNOCBP86
–
2.0, 0.5
2.0, 0.0
1.0, 0.0
1.0, 0.0
0.5, 0.0
1.0, 0.0
Total ferulates and total acid (mg KOH/g of sample) are reported as the mean SD of n = 3 trials. Samples within a column not sharing the
same letter are significantly different based on Differences of Least Squares Means with a Bonferroni adjustment at p ≤ 0.05
Temperature onset and peak, numbers in parentheses correspond to additional melting peaks, as determined by DSC
Lovibond color is reported as yellow and red
SBO soybean oil, SHBR raw shea butter, SHBTAG shea butter purified by flash chromatography, CNO coconut oil, FSBO feruloylated soybean
oil, FSHBR feruloylated raw shea butter, FSHBTAG feruloylated purified shea butter, FCNOSB feruloylated coconut oil from shaken batch reac-
tions, FCNOCFB1‑86 feruloylated coconut oil from continuous bioreactor production with number indicating total accumulated mass (kg) col-
lected when sampled
collected every 4 days (n = 9 collections). The conversion
to feruloylated coconut oil during the 22 days of stop mode
reached an equilibrium of 65% (samples obtained from the
sampling valve between column A and column B during
stop mode operation), very similar to the equilibrium con-
version obtained from the shaken, batch reactions of ethyl
ferulate and coconut oil, 63% (Fig. 2). When switched to
flow mode at a feed rate of 0.633 0.006 g/min, however,
the conversion unexpectedly dropped to 54% over the next
8 days and remained at that conversion equilibrium for the
next 24 days. The total ferulate percentage relative to the
ferulate concentration of the ethyl ferulate-coconut oil sub-
strate was ~100% for the duration of the 4.5 months pro-
duction run (Fig. 6), indicating that ethyl ferulate and feru-
lated coconut oil species were not absorbed by the enzyme
substrate [20]. The enzyme substrate did absorb water as
evidenced by the very low concentration, <1%, of hydroly-
sis products, FA and F2G, detected by HPLC (Fig. 6) and
very low total acid number of the feruloylated coconut
oil product, which decrease from 0.18 to 0.12% over the
4.5 months run (Table 4), obtained from ethyl ferulate-
coconut oil substrate that varied in water content from 250
to 1400 ppm. The absorption of water by Novozym 435 in
other packed-bed bioreactor applications resulting in lim-
ited hydrolysis product formation and low total acid num-
bers has been shown before [21, 36].
Feruloyl soy glycerides are produced and marketed at
60–65% conversion of ethyl ferulate to feruloyl soy glyc-
erides, so it was desirable to attempt to increase the con-
version of ethyl ferulate to feruloylated coconut oil. It was
previously shown that slowing the feed rate of the substrate
to the bioreactor (i.e., increasing the substrate residence
time) increased the conversion equilibrium [21]; therefore,
the effective feed rate of the ethyl ferulate:coconut oil sub-
strate to the bioreactor was decreased. Already operating
at the lowest settings of the feed pump (0.633 0.006 g/
min) the bioreactor was run in a stop-flow mode where
the feed pump power was cycled on for 9 h and off for
3 h (8–11 am and pm), resulting in an effective feed rate
of 0.468 0.010 g/min based on the mass collected every
5 days (n = 3 collections), which was 3/4 of the original
feed rate (Table 3). The conversion of ethyl ferulate to fer-
uloylated coconut oil trended up to 57% for 10 days and
remained at 57% for the next 5 days. The stop-flow rate
was adjusted to further decrease the substrate feed rate to
0.431
0.003 g/min for the next 3 weeks. The conver-
sion rate temporarily increased to 60% but the returned to
57% after 18 days. The substrate feed rate was adjusted to
0.365 0.018 g/min for the final 63 days of the production
run. The substrate conversion again temporarily reached
60% conversion for 2 weeks, but slowly declined to 55%
conversion after seven additional weeks of continuous
1 3

J Am Oil Chem Soc
operation. The final equilibrium rate of conversion of ethyl
ferulate to feruloylated coconut oil in the continuous, enzy-
matic, packed-bed bioreactor, 55%, was ~15% lower than
the equilibrium conversion of ethyl ferulate to feruloylated
soybean oil obtained in a similar, x4-scale bioreactor [21].
The results of the shaken, batch transesterifications of ethyl
ferulate with soybean oil vs coconut oil (discussed earlier),
showed that the enzymatic feruloylation of coconut oil was
slower to reach equilibrium than that of soybean oil and
that the final equilibrium conversion was slightly lower
(Fig. 2). This suggested that the enzymatic transesterifi-
cation of ethyl ferulate with the medium chain fatty acid
vegetable oil resulted in lower kinetic rates of conversion
and conversion equilibrium and that the optimal reaction
parameters for the continuous, enzymatic, packed-bed bio-
reactor transesterification of ethyl ferulate with soybean oil
were not applicable to the transesterification coconut oil in
a similar bioreactor at ¼-scale.
Several studies have been conducted using response
surface methodology to determine the optimal vegetable
oil:ethyl ferulate ratio for lipase-catalyzed transesterifica-
tions to form feruloylated lipids [31, 33, 34, 37]. These
academic studies conclude optimal vegetable oil:ethyl
ferulate ratios of 4:1–10:1 (mol:mol) depending on reac-
tion temperature (>70 °C) and enzyme load (20–40 wt%
relative to ethyl ferulate). While interesting academic exer-
cises, the parameters determined from these small-scale,
stirred, batch reactions provide little practical insight for
large-scale, continuous, packed-bed, enzymatic syntheses
of feruloylated vegetable oils for use by the personal care
and cosmeceutical industries. To limit production costs by
avoiding post production refinement of the feruloylated
vegetable oils it was necessary to limit the mass of residual
vegetable oil in the product since it lowers the concentra-
tion of the feruloylated species (i.e. efficacy) in the reaction
product. The use of a 1.3:1.0 (mol:mol) ratio of vegetable
oil:ethyl ferulate at 60 °C in the bioreactor produced a feru-
loylated vegetable oil product, requiring no post produc-
tion refinement, with acceptable ethyl ferulate conversion
(>55%) and useful UV-absorbing and antioxidant efficacy.
1.5
1.0
0.5
0.0
UVB
UVA II
UVA I
300
320
340
360
380
400
Wavelength (nm)
Fig. 7 Ultraviolet (UV) absorption spectra of feruloylated coco-
nut oil (solid line), feruloylated shea butter (dashed line), and feru-
loylated soybean oil (dashed-dotted line) at 100 mg/L in ethanol.
The U.S. Food and Drug Administration categorizes UV irradiance
as UVC (<290 nm), UVB (290–320 nm), UVA II (320–340 nm), and
UVA I (340–400 nm) [39]
cinnamate (λmax 308) UVB absorber [32, 38], and 29 nm
less than avobenzone, a popular commercial dibenzometh-
ane (λmax 357) UVA I absorber [32, 39]. The feruloylated
vegetable oils’ λmax make them excellent UVA II and good
UVB absorbers (Fig. 7) and their addition to commercial
formulations would augment the commercial active ingre-
dients’ broad spectrum coverage. From our experience, skin
care formulators design formulations recipes on an ingredi-
ent weight percent basis and have a convoluted understand-
ing of formulating on a mole basis. Thus, including feru-
loylated vegetable oils with molecular weights of >800 g/
mol requires more material than octinoxate or avobenzone,
which have relatively lower molecular weights <315 g/mol,
to obtain the same efficacy, which requires equal moles
of chromophore. Feruloylated soy glycerides are used in
commercial formulations, but it would be advantageous if
a lower molecular weight triacylglyceride was used, pro-
ducing a feruloylated vegetable oil with improved efficacy
on a weight basis. Comparing the three feruloylated veg-
etable oils from the shaken, batch transesterifications by
integrating the area of the UV-absorption spectra repre-
sented in Fig. 7, FCnO = 108.8 0.2, FSbO = 97.6 0.6,
FShB = 92.6 0.4 (n = 3), showed that feruloylated coco-
nut oil had an 11.5% higher absorption capacity relative
to feruloylated soybean oil and 17.5% higher absorption
capacity than feruloylated shea butter. This was expected
based on the relative molecular weights of the three veg-
etable oils (Table 1), which resulted in a higher molar
concentration of ferulate chromophore in the feruloylated
coconut oil, ~1000 mM of total ferulates compared to the
feruloylated soybean oil and shea butter, ~830 mM of total
ferulates (Table 4).
Ultraviolet Absorbance and Antioxidant Properties
of Feruloylated Vegetable Oils
Commercial, UV-absorbing active ingredients function by
absorbing UV radiation and dissipating the energy through-
out their conjugated molecular structure. Specific chemi-
cal structures possess different UV-absorbing maxima,
thus skin care formulations require multiple active ingre-
dients to provide broad spectrum UV protection [38, 39].
The feruloylated vegetable oils possessed a UV-absorbing
λmax 328 nm (Fig. 7). This maxima was 20 nm greater than
octinoxate, a popular commercial methoxy-substituted
1 3

J Am Oil Chem Soc
concentrations, 0.50, 1.00, 1.75, and 2.50 mM, over the
course of 30 min. The controls showed that none of the
vegetable oils as obtained from the suppliers exhibited any
DPPH radical scavenging capacity; even the raw shea but-
ter that contained aromatic/phenolic contaminants did not
show any DPPH radical scavenging capacity under the
conditions tested. It was also confirmed that the purified
shea butter TAG did not possess DPPH radical scavenging
capacity. All three feruloylated vegetable oils were consid-
ered rapid free radical scavengers at the three highest con-
centrations tested. A rapid scavenger is defined as one that
reduced 50% of initial DPPH radical concentration in less
than 5 min [28]. Only the feruloylated raw shea butter was
considered a rapid free radical scavenger at the lowest con-
centration tested. Overall, there was minimal difference in
scavenging capacity of the three feruloylated vegetable oils
at equivalent total ferulate concentrations, confirming that
fatty acid chain length has no affect on scavenging capacity.
Soybean oil is a liquid at room temperature and feru-
loylated soybean oil is also a liquid at room temperature
and has been reported to remain so after 5 years of storage
[21]. The supplier reported the coconut oil and shea but-
ter, solids at room temperature, having melting points of
24.4 and 32–45 °C, respectively. The feruloylated coconut
oil from the shaken, batch reactions and from the continu-
ous, packed-bed bioreactor production had very similar
thermograms as measured by DSC (Table 4). The feru-
loylated coconut oil conveniently remained a liquid at room
temperature (23 °C) for several days before slowly cloud-
ing and eventually gelling. The feruloylated coconut oil
never completely solidified after several weeks of storage.
The gelled feruloylated coconut oil was easily liquefied
by microwaving 500 mL on high (1000 W) for 30 s. The
feruloylated shea butter synthesized from both raw shea
butter and shea butter TAG isolated by flash chromatogra-
phy solidified quickly upon sitting after filtration from the
shaken, batch reactions. Feruloylated coconut oil was much
lighter in color than feruloylated soybean oil. Lovibond
color measurements showed that the feruloylated coconut
oil possessed a much lower yellow tint and a slightly lower
red tint (Table 4). Lighter color ingredients are often pre-
ferred when attempting to produce white colored creams
and lotions. These observations suggested that feruloylated
coconut oil would be fungible with feruloylated soybean oil
in current retail formulation protocols while feruloylated
shea butter would require modification of current formula-
tion methods.
100
80
(A)
(B)
(C)
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
0
5
10
15
20
25
30
Time (min)
Fig. 8 Free radical, DPPH*, quenching kinetics of shea butter (a),
coconut oil (b), and soybean oil (c) as obtained from the suppliers
(closed circles), shea butter purified by flash chromatography (closed
squares), and feruloylated shea butter, feruloylated coconut oil, and
feruloylated soybean oil at 0.50 mM (open circles), 1.00 mM (open
triangles), 1.75 mM (open squares), and 2.50 mM (open diamonds).
Rapid antioxidants, as defined by Huang et al. [28], quench 50% of
the DPPH* within 5 min (area within the dashed box). Data points
are the means of n = 3 trials with error bars denoting SD from the
mean
The free radical scavenging capacity of the three feru-
loylated vegetable oils from the shaken, batch transes-
terification were determined by their ability to reduce the
DPPH radical in ethanol solutions [28]. Figure 8 shows
the percentage DPPH radical remaining in the presence of
the three feruloylated vegetable oils at four total ferulate
Conclusions
The enzymatic transesterification of coconut oil and shea
butter with ethyl ferulate were conducted in shaken, batch
1 3

J Am Oil Chem Soc
by ORAU under DOE contract number DE-AC05-06OR23100. All
opinions expressed in this paper are the authors’ and do not neces-
sarily reflect the policies and views of USDA, ARS, DOE, or ORAU/
ORISE. The authors would like to thank Elizabeth Lafond, Leslie
Smith, Amber Durham and Jeanette Little for their excellent techni-
cal assistance and Debra E. Palmquist, Agricultural Research Ser-
vice Midwest Area Statistician, for the statistical analysis of the data
reported in Table 4.
reactions and their kinetic and equilibrium conversions
compared with the well studied soybean oil transesteri-
fication. The ethyl ferulate transesterification with coco-
nut oil, with a very dissimilar fatty acid profile to soybean
oil, was slower to reach equilibrium, but reached the same
equilibrium conversion as soybean oil. The transesterifica-
tion of coconut oil produced a feruloylated triacylglycerol
product with a higher concentration of total ferulates. The
major feruloylated coconut oil species were identified by
LC–MS using transesterification reactions of ethyl ferulate
with medium chain TAG standards. The transesterification
of ethyl ferulate with shea butter, with a very similar fatty
acid profile to soybean oil, unexpectedly reach kinetic equi-
librium faster with a higher conversion of the ethyl feru-
late to feruloylated acylglycerol product than soybean oil.
The feruloylated shea butter reactions using raw shea butter
produced a white precipitate, identified as F2G, that was not
present in the soybean oil, coconut, and purified shea butter
transesterifications. It was shown that the unusual kinetic
and equilibrium conversion rates and white precipitate were
due to DAG and MAG contaminants in the raw shea butter.
The objective of the shaken, batch reactions was to
determine if coconut oil and shea butter were suitable veg-
etable oils/fats to use in the continuous, enzymatic, packed-
bed bioreactor process used to manufacture feruloylated
soy glycerides. The shea butter, as obtained from the sup-
plier containing DAG and MAG, produced the precipitate
F2G that could potentially accumulate and obstruct flow in
the bioreactor. Purifying quantities of shea butter required
for a bioreactor production run was cost prohibitive; there-
fore, the pilot-scale transesterification with shea butter was
not attempted. The enzymatic transesterification of ethyl
ferulate with coconut oil was tested in a 1/4-tonne/year
bioreactor using the same reaction parameters used for the
previously studied pilot-scale transesterification of ethyl
ferulate with soybean oil. While the conversion equilibrium
to feruloylated coconut initially reached similar equilib-
rium conversions as soybean oil, the equilibrium conver-
sion to feruloylated coconut oil declined by 15% over the
4.5 months production run. The feruloylated coconut oil
had the advantage of being 17% higher in total ferulate
concentration than feruloylated soybean oil or shea butter
and was a much lighter color than feruloylated soybean oil,
both advantageous characteristics preferred by skin care
formulators.
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