Synthesis and properties of ascorbyl esters catalyzed by lipozyme TL im using triglycerides as acyl donors

Article abstract of DOI:10.1007/s11746-010-1643-5

Esters of l-ascorbic acid with long-chain fatty acids (E-304) are employed as antioxidants in foods rich in lipids. Although their enzymatic synthesis offers some advantages compared with the current chemical processes, most of the reported methods employ the immobilized lipase from Candida antarctica as biocatalyst and free fatty acids or activated esters as acyl donors. In order to diminish the cost of the process, we have investigated the synthesis of ascorbyl oleate and ascorbyl palmitate esters with the immobilized Thermomyces lanuginosus lipase Lipozyme TL IM-which is significantly less expensive than Novozym 435-and triglycerides as source of fatty acids. Lipozyme TL IM gave rise to a lower yield of 6-O-ascorbyl oleate than Novozym 435 when using triolein (64 vs. 84%) and olive oil (27 vs. 33%) as acyl donors. Both 6-O-ascorbyl oleate and 6-O-ascorbyl palmitate displayed excellent surfactant and antioxidant properties. The Trolox Equivalent Antioxidant Capability values for the oleate and palmitate were 71 and 84%, respectively, of those obtained with l-ascorbic acid; however, both derivatives were able to stabilize soybean oil towards peroxide formation.

Full text of DOI:10.1007/s11746-010-1643-5

J Am Oil Chem Soc (2011) 88:57–64
DOI 10.1007/s11746-010-1643-5
ORIGINAL PAPER
Synthesis and Properties of Ascorbyl Esters Catalyzed
by Lipozyme TL IM using Triglycerides as Acyl Donors
D. Reyes-Duarte
F. Comelles J. L. Parra
A. Ballesteros F. J. Plou
•
N. Lopez-Cortes
•
P. Torres
A. V. Ugidos
•
•
•
S. Pe n˜ a
•
•
•
Received: 17 March 2010 / Revised: 5 May 2010 / Accepted: 7 July 2010 / Published online: 23 July 2010
Ó AOCS 2010
Abstract Esters of L-ascorbic acid with long-chain fatty
acids (E-304) are employed as antioxidants in foods rich in
lipids. Although their enzymatic synthesis offers some
advantages compared with the current chemical processes,
most of the reported methods employ the immobilized
lipase from Candida antarctica as biocatalyst and free fatty
acids or activated esters as acyl donors. In order to diminish
the cost of the process, we have investigated the synthesis of
ascorbyl oleate and ascorbyl palmitate esters with the
immobilized Thermomyces lanuginosus lipase Lipozyme
TL IM—which is significantly less expensive than
Novozym 435—and triglycerides as source of fatty acids.
Lipozyme TL IM gave rise to a lower yield of
with L-ascorbic acid; however, both derivatives were able to
stabilize soybean oil towards peroxide formation.
Keywords Vitamin C ꢀ Ascorbyl esters ꢀ
L-Ascorbyl oleate ꢀ Lipase ꢀ Thermomyces lanuginosus ꢀ
Enzymatic transesterification ꢀ Alkyl esters ꢀ Triglycerides ꢀ
Surface tension ꢀ CMC ꢀ Antioxidant activity
Introduction
The modification of natural antioxidants to improve their
chemical, oxidative and/or thermal stability, or to alter their
hydrophile–lipophile balance (HLB), yields a series of
‘‘semisynthetic’’ antioxidants with a great impact in
industry [1]. These derivatives are generally prepared under
harsh conditions using strongly corrosive acids such as
sulfuric acid or hydrogen fluoride [2, 3]. To overcome these
shortcomings, new approaches based on the use of biolog-
ical catalysts are being evaluated, which are characterized
by mild reaction conditions, low energy requirements and a
minimization of the isomerization and rearrangement side
reactions [1, 4]. In addition, biocatalysts are biodegradable
and display chemo-, regio- and/or stereospecificity resulting
in decreased by-product formation thus avoiding the need
for functional group protection and activation [5, 6].
6
-O-ascorbyl oleate than Novozym 435 when using triolein
64 vs. 84%) and olive oil (27 vs. 33%) as acyl donors. Both
-O-ascorbyl oleate and 6-O-ascorbyl palmitate displayed
(
6
excellent surfactant and antioxidant properties. The Trolox
Equivalent Antioxidant Capability values for the oleate and
palmitate were 71 and 84%, respectively, of those obtained
D. Reyes-Duarte ꢀ N. Lopez-Cortes ꢀ P. Torres ꢀ
A. Ballesteros ꢀ F. J. Plou (&)
Departamento de Biocat a´ lisis, Instituto de Cat a´ lisis
y Petroleoqu ´ı mica, CSIC, Marie Curie 2, 28049 Madrid, Spain
e-mail: fplou@icp.csic.es
D. Reyes-Duarte
Departamento de Procesos y Tecnolog ´ı a,
Universidad Aut o´ noma Metropolitana (UAM),
Unidad Cuajimalpa, 01120 M e´ xico, DF, M e´ xico
L-Ascorbic acid (vitamin C) is the major water-soluble
natural antioxidant. Acting as a free radical scavenger,
L-ascorbic acid and its derivatives react with oxygen, thus
removing it in a closed system. The combination of
L-ascorbic acid and primary antioxidants like a-tocopherol
renders a synergic effect that results in the ‘‘vitamin E
recycling system’’ [7]. This mixture of vitamins is usually
added to fatty products to retard the autooxidation of
unsaturated fatty acids. However, due to the low miscibility
F. Comelles ꢀ J. L. Parra
Departamento de Tensioactivos, Instituto de Qu ´ı mica Avanzada
de Catalu n˜ a, CSIC, 08034 Barcelona, Spain
S. Pe n˜ a ꢀ A. V. Ugidos
Biotecnolog ´ı as Aplicadas BTSA, Sierra de Grazalema 4,
2
8946 Fuenlabrada, Spain
1
23

5
8
J Am Oil Chem Soc (2011) 88:57–64
˚
solvent 2-methyl-2-butanol was dried before use over 3-A
of L-ascorbic acid with a-tocopherol or with any oil-based
formula, it is well established the use of ascorbyl fatty acid
esters instead of vitamin C [8].
molecular sieves (Sigma) at least for 24 h.
Ascorbyl palmitate and stearate, classified by European
Union with the code E-304 (fatty acid esters of ascorbic
acid) are produced by reacting ascorbic acid with sulfuric
acid followed by re-esterification with the corresponding
fatty acid, and subsequently purified by recrystallization
Determination of Lipase Activity
The hydrolytic activity was measured titrimetrically at pH
8.0 and 30 °C using a pH-stat (Mettler, Model DL 50). The
reaction mixture contained tripropionin (0.3 ml, final con-
centration 80 mM), acetonitrile (0.7 ml) and Tris–HCl
buffer (19 ml, 1 mM, pH 8.0) containing NaCl (0.1 M).
The immobilized biocatalyst was then added and the pH
automatically maintained at 8.0 using 0.1 N NaOH as
titrant. Experiments were done in triplicate. One enzyme
unit (U) was defined as that catalyzing the formation of
1 lmol of fatty acid per min.
[
9]. This chemical process has some disadvantages such as
the use of strong acids, the low yields due to non-regio-
selective reactions, the formation of by-products due to
instability of vitamin C and the need of tedious product
isolation [10, 11]. As an alternative, lipases, especially that
from Candida antarctica, have been successfully used to
catalyze the synthesis of ascorbyl esters in tertiary alcohols,
acetone and even in ionic liquids [12], employing as acyl
donors saturated and unsaturated free fatty acids, alkyl and
vinyl esters [13, 14]. The enzymatic process is specific
for the primary alcohol, yielding the corresponding
Enzymatic Synthesis of 6-O-Ascorbyl Esters
All reactions were performed in 30-ml amber glass sealed
vessels at 40 °C, with orbital shaking (250 rpm). Ascorbic
acid (0.5 mmol) and the corresponding acyl donor (fatty
acid, alkyl or vinyl ester, 1.5 mmol) were dissolved in 5 ml
of dried 2-methyl-2-butanol. The mixture was equilibrated
for 10 min, and the biocatalyst (125 mg) was added. Ali-
quots were removed at intervals, filtered using a 0.45 lm
6
-O-ascorbyl ester. Although ascorbyl palmitate is more
soluble in fats than ascorbic acid itself, its low solubility
and miscibility in edible fats and oils limits its use [9].
Ascorbyl esters of unsaturated fatty acids, e.g. oleate or
linoleate, exhibit improved miscibility with a-tocopherol,
oils or fatty products, and can be also employed as food
additives (E-304).
Ò
Durapore membrane coupled to an eppendorf tube and
In the present work, we have investigated the enzymatic
synthesis of L-ascorbic acid fatty acid esters, in particular
ascorbyl oleate and palmitate. In order to reduce the cost of
the process, we have analyzed: (1) the use of less expensive
biocatalysts than the immobilized lipase from C. antarc-
tica, in particular the silica-granulated lipase from Ther-
momyces lanuginosus—Lipozyme TL IM [15]; (2) the use
of triglycerides and oils as acyl donors as an alternative to
fatty acids or activated esters.
analyzed by HPLC. For the reactions involving the use of
triglycerides (tripalmitin, triolein) as acyl donors, the pro-
cedure was as described above but the amount of ascorbic
acid and triglyceride was varied in the range 0.5–1.5 mmol
(0.1–0.3 M). In the case of olive oil, the weight of oil
added was the same (88–265 mg/ml) as in the experiments
with triolein. Experiments were performed in duplicate.
Standard deviations were lower than 5%.
HPLC Analysis
Experimental Procedures
Reactions aliquots were analyzed by HPLC, using a 9012
pump (Varian) and a Nucleosil 100-C18 column (4.6 9
250 mm, An a´ lisis Vinicos, Tomelloso, Spain), maintained
at 45 °C. Integration was carried out using the Varian Star
4.0 software. Detection was performed using an evapora-
tive light-scattering detector DDL-31 (Eurosep) equili-
brated at 60 °C. Methanol:water 95:5 (v/v) containing
0.1% (v/v) acetic acid was used as mobile phase (flow rate
1.2 ml/min) for 6 min. Then, a gradient from this eluent to
pure methanol was performed in 1 min (min 6 to 7), after
which the flow rate was increased to 1.7 ml/min in 1 min
(min 7 to 8) and held for another minute (min 8 to 9).
A new gradient from methanol to methanol:acetone (50:50
v/v) was performed in 2 min (min 9 to 11) and held for
6 min (min 11 to 17) at 1.7 ml/min. After this, the gradient
returned to the initial conditions (min 17 to 25).
Chemicals and Enzymes
Immobilized lipases from Thermomyces lanuginosus
Lipozyme TL IM) and Candida antarctica (Novozym
35) were a kind gift from Novozymes A/S. Triolein,
(
4
methyl oleate, oleic acid, tripalmitin, ethyl palmitate, pal-
mitic acid, 2-methyl-2-butanol (2M2B, tert-amyl alcohol),
(
R)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic
0
acid [(R)-Trolox] and 2,2 -Azino-bis (3-ethylbenzthiazo-
line-6-sulfonic acid (ABTS) were from Sigma-Aldrich.
Vinyl palmitate was from TCI (Tokyo, Japan). L-ascorbic
acid (vitamin C) was supplied by Fluka. Extra-virgin olive
oil was from La Espa n˜ ola (Aceites del Sur S.A., Spain).
All other reagents were of the highest available purity. The
1
23

J Am Oil Chem Soc (2011) 88:57–64
59
Purification and Characterization
microplate reader (model Versamax, Molecular Devices)
and the decrease of absorbance (DA734) for each concen-
tration was calculated determining the area under the
curve. Experiments were performed at 30 °C in quadru-
plicate. The DA734nm versus concentration curve was
plotted for the different compounds and used to calculate
the equivalent Trolox concentration. The TEAC value was
determined as the ratio between the slopes of concentra-
tion-DA734 curves for the corresponding antioxidant and
Trolox.
of 6-O-Ascorbyl Oleate
6
-O-Ascorbyl oleate was synthesized as described in the
general procedure and purified by semipreparative HPLC.
The column was a Nucleosil-C18 (10 9 250 mm, 5 lm,
Analisis Vinicos, Spain), and the mobile phase was 95:5
(
v/v) methanol/H O at 4.8 ml/min. The fractions contain-
2
ing the product were pooled and the solvent evaporated.
NMR spectra were recorded on a Varian Unity spectrom-
1
13
eter ( H NMR, 500 MHz; C NMR, 125 MHz), with a
reverse probe and a gradient unit. The spectra were
obtained at 30 °C, for samples of around 5 mg in solutions
Evaluation of Oxidative Stability Under Accelerated
Conditions
of 0.6 ml of deuterated methanol (CD OD). Proton
3
chemical shifts refer to the methanol multiplet (3.30 ppm).
Carbon chemical shifts refer also to the methanol multiplet
Oil stability was determined at 110 °C and 20 ml air/h
using a Rancimat apparatus (Metrohm). The ascorbyl
derivatives (50 ppm) were added to soybean oil. The range
of conductivity was 200 lS. The soybean oil with no added
antioxidant was used as control.
(
49.0 ppm). Mass spectrometry was determined with a
MALDI-TOF system Reflex III (Bruker-Franzen). For the
experiments the matrix employed was a saturated solution
of 2,5-hydroxybenzoic acid.
Surfactant Properties
Results and Discussion
Determination of surface tension in water of ascorbyl esters
was performed at 20 °C according to the Wilhelmy plate
method, with a Kr u¨ ss tensiometer (Processor tensiometer
K-12, Hamburg, Germany), which determines directly the
real tension values at the equilibrium, using a series of
aqueous solutions of ascorbyl esters at various concentra-
tions. The equilibrium time of the surface before the sur-
face tension measurements was at least 1 h. The critical
micelle concentration (CMC) was determined graphically
from the abrupt change in the slope of the plot representing
the surface tension versus logarithm of surfactant concen-
tration (expressed in mM).
The enzymatic synthesis of ascorbyl esters has been widely
investigated using saturated and unsaturated acyl donors. It
is noteworthy that most of the studies have been carried out
with the lipase B from Candida antarctica immobilized in
different supports (lipases Novozym 435, Chirazyme L-2,
etc.). The high cost of these biocatalysts, approx. $900/kg
[16], makes it difficult their application to food processes.
The lipases from B. stearothermophilus [17] and Candida
sp. [14] also catalyze the esterification of L-ascorbic acid
with fatty acids; however, these lipases are not commer-
cially available in immobilized form at large scale.
Most of the processes developed up to now are based on
esterification strategies with different fatty acids, whereas
transesterification with alkyl and vinyl esters have been
also successfully employed. We have found only one report
using triglycerides (in particular, palm oil) as acyl donor
[18]. An interesting two-step route for the synthesis of
ascorbyl esters from oils has been also described, i.e. their
transformation into biodiesel followed by the transesteri-
fication of the corresponding methyl esters with ascorbic
acid [19].
TEAC Assay
To measure the antioxidant activity of the new compounds
we used the Trolox Equivalent Antioxidant Capability
(
TEAC) assay with some modifications to adapt to 96-well
plates. This assay is based on the ability of antioxidants in
reducing ABTS radical. Briefly, ABTS (7 mM final con-
centration) was added to an aqueous solution of 2.45 mM
potassium persulfate and kept in the dark at room tem-
perature for 15 h to obtain the ABTS radical, which was
In order to develop an efficient and affordable strategy
to synthesize ascorbyl fatty acid esters, we have assessed
the use of less expensive biocatalysts than those based on
lipase B from C. antarctica. In particular, we have focused
on the silica-granulated lipase from Thermomyces lanugi-
nosus, Lipozyme TL IM [20, 21], whose price is 8 and 10
times lower than other immobilized lipases such as Lipo-
zyme RM IM and Novozym 435, respectively [22]. The
hydrolytic activity of Lipozyme TL IM (2,490 U/g),
ꢀ
?
stable for 2 days. The ABTS solution was diluted with
ethanol to get an absorbance of 0.70 (± 0.02) at 734 nm and
equilibrated at room temperature. In each well, 20 ll of a
solution of Trolox (standard) or of the antioxidants
(
0.5–25 lM) in ethanol were added to 230 ll of adjusted
ꢀ
?
ꢀ?
ABTS solution. The decrease of absorbance of ABTS
solution was monitored at 734 nm during 6 min using a
1
23

6
0
J Am Oil Chem Soc (2011) 88:57–64
measured in the hydrolysis of tripropionin, is slightly
higher than that of Novozym 435 (1,725 U/g). On the other
hand, we have explored the use of triglycerides and oils as
acyl donors, which could represent another improvement to
the cost of the process.
donors. It is well reported that the transesterification of
hydroxylated compounds with vinyl esters is about 20–100
times faster than with alkyl esters [24]. In addition, the
vinyl alcohol formed during the process tautomerizes to the
low-boiling-point acetaldehyde, shifting the equilibrium
towards the ester formation thus increasing yield [23]. In
fact, the monoester yield passed from 20% with palmitic
acid and ethyl palmitate to 100% employing the vinyl ester
Synthesis of Ascorbyl Esters Using Fatty Acids,
Alkyl and Vinyl Esters
(Table 1).
The low solubility of ascorbic acid (hydrophilic) in non-
polar solvents is the major hurdle for its acylation with
fatty acids. Different solvents such as 2-methyl-2-butanol
We also studied the transesterification with methyl
oleate. The yield was significantly higher than the obtained
with ethyl palmitate (50 vs. 20%, see Table 1). We con-
firmed by mass spectrometry and NMR (data not shown)
that the major products obtained with Lipozyme TL IM
were the monoesters at hydroxyl 6-OH, as occurs with the
lipase from C. antarctica.
(
tert-amyl alcohol), t-butanol and acetone have been
selected for the enzymatic synthesis of ascorbyl esters of
fatty acids [8]. We used tert-amyl alcohol as reaction
medium since ascorbic acid is sufficiently soluble in this
solvent and most of the lipases are stable and active [23].
Compared with other polyhydroxylated compounds such as
carbohydrates, the lower hydrophilicity of ascorbic acid
made not necessary to add cosolvents like dimethyl sulf-
oxide [20].
Potential Use of Natural Oils as Substrates
for the Synthesis of Ascorbyl Esters
Vinyl esters give rise to the fastest reactions and highest
yields. However, from the economical point of view, vinyl
esters of long-chain fatty acids are still too expensive
[25, 26]. Oils, which are mainly composed of triglycerides,
offer an alternative route for the synthesis of ascorbyl
esters. Acylation of ascorbic acid directly using oils usually
yields a mixture of ascorbyl fatty esters with acyl chains of
different length depending on the oil composition [8].
Burham et al. [18] reported the synthesis of mixed ascorbyl
esters using palm oil and Novozym 435.
In order to explore the applicability of Lipozyme TL IM
in these processes, the synthesis of ascorbyl palmitate was
attempted by esterification with palmitic acid and by
transesterification with different palmitic acid donors
(
ethyl, vinyl) (Fig. 1). A 3/1 M ratio acyl donor/ascorbic
acid was selected. It is important to consider that the
hydrolysis of the alkyl or vinyl ester to fatty acid catalyzed
by the lipase is an undesirable but unavoidable reaction
(
water can also act as nucleophile) yielding free fatty acid.
As shown in Fig. 1, the activated vinyl ester gave a
In a first attempt, we assayed tripalmitin and triolein as
acyl donors (in a 100 mM equimolar ratio with L-ascorbic
acid, which is equivalent to threefold excess of fatty acid
with respect to vitamin C) using Lipozyme TL IM (Fig. 2).
As shown, reaction is slightly faster with triolein, probably
due to its higher miscibility with the substrates, yielding
approx. 64% of 6-O-ascorbyl oleate in 140 h. Figure 3
illustrates a typical chromatogram obtained with triolein, in
which the formation of mono- and di-glycerides as by-
products is clearly observed. As the formed mono- and di-
acylglycerols could also serve as emulsifiers [27], it would
not be necessary to remove them from the final product. As
illustrated in Fig. 2, Novozym 435 produced a higher yield
of 6-O-ascorbyl oleate than Lipozyme TL IM (84 vs. 64%).
Different concentrations of L-ascorbic acid and triolein
were tested (Table 1). Using 300 mM triolein and 100 mM
L-ascorbic acid (which represents a 9/1 M ratio of oleic
acid to L-ascorbic acid), the yield was not improved sig-
nificantly. Interestingly, when assaying 300 mM triolein
and 300 mM L-ascorbic acid the yield obtained with
Lipozyme TL IM was 70.5%, which is equivalent to a
theoretical concentration of 6-O-ascorbyl oleate of nearly
97 g/l.
faster reaction and a higher yield than the rest of acyl
Fig. 1 Time course of enzymatic synthesis of L-ascorbyl palmitate in
2
-methyl-2-butanol catalyzed by Lipozyme TL IM using different
acyl donors. Experimental conditions: 100 mM L-ascorbic acid,
00 mM acyl donor, 25 mg/ml biocatalyst, 40 °C
3
1
23

J Am Oil Chem Soc (2011) 88:57–64
61
Table 1 Effect of the nature of
Ascorbic acid (mM)
Acyl donor
Acyl donor (mM)
Biocatalyst
Yield (%)
acyl donor, molar ratio donor/
acceptor and biocatalyst on the
yield of fatty acid ascorbyl
esters
1
1
1
1
1
1
1
1
1
3
00
00
00
00
00
00
00
00
00
00
Ethyl palmitate
Palmitic acid
Vinyl palmitate
Methyl oleate
Tripalmitin
Triolein
300
300
300
300
100
100
100
300
300
300
Lipozyme TL IM
Lipozyme TL IM
Lipozyme TL IM
Lipozyme TL IM
Lipozyme TL IM
Lipozyme TL IM
Novozym 435
20
20
100
50
50
64
Triolein
84
Triolein
Lipozyme TL IM
Novozym 435
71
Triolein
82
a
Triolein
Lipozyme TL IM
Lipozyme TL IM
Novozym 435
70.5
27
The amount of olive oil was
a
the same as the employed in the
experiments with 100 mM
triolein, on a weight basis
100
00
Olive oil
88 mg/ml
a
1
Olive oil
88 mg/ml
33
Fig. 3 HPLC chromatogram showing the different products formed
in the synthesis of 6-O-ascorbyl oleate from L-ascorbic acid and
triolein. Analytical conditions described in the ‘‘Experimental’’
Section
Fig. 2 Kinetics of acylation of L-ascorbyl acid in 2-methyl-2-butanol
using triolein and tripalmitin catalyzed by Lipozyme TL IM and
Novozym 435. Experimental conditions: 100 mM L-ascorbic acid,
1
00 mM triglyceride, 25 mg/ml biocatalyst, 40 °C
not very significant, with Novozym 435 giving a higher
yield in 168 h (33 vs. 27%).
Olive oil is the main source of fat in the Mediterranean
diet and is associated to a low incidence of cardiovascular
disease [28]. The composition of the virgin olive oil
depends on the variety and the grade of the olive. The
saponifiable fraction (96–98%) is constituted mainly by
triglycerides formed of different fatty acids with oleic acid
as the major component (60–80%), and variable amounts of
palmitic, palmitoleic, stearic and linoleic acids. We tested
virgin olive oil as acyl donor for L-ascorbic acid acylation.
We observed in the HPLC chromatograms (data not shown)
the presence of other monoesters apart from the monool-
eate, as expected considering the typical composition of the
oil. We determined the formation of 6-O-ascorbyl oleate
with both Lipozyme TL IM and Novozym 435 lipases
CMC and Surface Tension of Ascorbyl Esters
For a surfactant to act as an emulsifier, it must show good
surface activity and generate a low interfacial tension in the
particular system in which it is to be used. This means that
it must have a tendency to migrate to the interface, rather
than to remain dissolved in either one of the bulk phases
[29]. Few data are available characterizing the surfactant
properties of ascorbyl esters [30]. Figure 5 illustrates the
variation of surface tension as a function of concentration
of the ascorbyl esters synthesized in this work.
The synthesized 6-O-ascorbyl derivatives displayed
excellent surface-active properties. The minimum surfac-
tant concentration needed to form micelles, the called
(
Fig. 4). The differences between the two biocatalysts were
1
23

6
2
J Am Oil Chem Soc (2011) 88:57–64
Fig. 4 Comparative kinetics of acylation of L-ascorbyl acid using
olive oil in 2-methyl-2-butanol catalyzed by Lipozyme TL IM and
Novozym 435. Experimental conditions: 100 mM L-ascorbic acid,
1
00 mM olive oil, 25 mg/ml biocatalyst, 40 °C
critical micelle concentration (CMC), was determined
graphically from the inflexion appearing in the plots of
surface tension versus logarithm of concentration. When
considering 6-O-ascorbyl oleate, the characteristic inflec-
tion point at the critical micelle concentration was slightly
distorted by the minimum appearing in the graph. This is
usually related to the presence of byproducts, even in small
traces. However, the estimated CMC value (0.1–0.3 mM)
was in the same range as that reported previously for the
same compound at 25 °C (0.22 mM) [30]. Although the
CMC of 6-O-ascorbyl palmitate (0.1 mM) was closely
similar to that of the oleyl derivative, the palmitoyl ester
solubility in water is much lower than for the unsaturated
ester, corroborating that a C16 saturated alkyl chain is
more insoluble that a C18 unsaturated one. On the other
hand, the surface tension at CMC of the palmitoyl ester
was 32 mN/m, higher than the obtained with oleic acid
ester (26–27 mN/m), which indicates the higher effec-
tiveness of the oleyl derivative.
Fig. 5 Variation of surface tension as a function of concentration for
-O-ascorbyl oleate (a) and 6-O-ascorbyl palmitate (b)
6
applications as antioxidants in lipophilic food products, it is
interesting to determine their antioxidant activity. In this
work, the antioxidant activity of the synthesized derivatives
was analyzed using the TEAC assay. The results of the
experiments are represented in Fig. 6. We observed that the
addition of an acyl chain in the position 6-OH of L-ascorbic
acid caused a loss of antioxidant activity. The TEAC values,
calculated from the slopes in Fig. 6, are presented in Table 2.
6-O-ascorbyl palmitate and 6-O-ascorbyl oleate showed 84
and 71% efficiency compared with L-ascorbic acid.
The amphiphilic character of fatty acid ascorbyl esters
may serve to solubilize and stabilize drug molecules. Thus,
the hydrophobic drug stays in the inner hydrophobic core
of the micelle, whereas the antioxidant nature of the
hydrophilic shell protects the drug against oxidation. In this
context, Palma et al. [31] reported the stabilization and
solubilization of several lipophilic molecules in micelles of
6
-O-ascorbyl octanoate.
Antioxidant Activity
It seems that the selection of the assay to measure
antioxidant activity may render different conclusions. In
this context, Lo Nostro et al. [32, 33] reported that the
addition of an acyl chain to the L-ascorbic acid in the
6-position, retained or even enhanced its physiological and
In physiological systems, fatty acid ascorbyl esters are
hydrolyzed to vitamin C and fatty acids—without forming
any hazardous components [8]. However, for their
1
23

J Am Oil Chem Soc (2011) 88:57–64
63
a good miscibility with oils, an excellent surfactive behavior
and maintains a substantial level of antioxidant activity,
which facilitates its use in foods and also as a micellar
vehicle to solubilize and stabilize hydrophobic drugs and
other substances.
Acknowledgments We thank Prof. Manuel Bernab e´ (Instituto de
Qu ´ı mica Org a´ nica, CSIC, Madrid, Spain) for help with NMR anal-
ysis. We thank Ramiro Mart ´ı nez (Novozymes A/S, Madrid, Spain) for
supply of lipase samples and for suggestions. We are grateful to
Comunidad de Madrid for a research contract to P. Torres and to
CONACYT for grant 020256 to D. Reyes. This research was sup-
ported by the Spanish CSIC (Project 200680F0132) and by the Latin-
American Science & Technology Development Programme (CYTED
Project 108RT0346).
ꢀ?
Fig. 6 Effect of antioxidant concentration on the decrease of ABTS
absorbance for Trolox, L-ascorbic acid and its fatty acid esters
References
1
. Torres P, Kunamneni A, Ballesteros A, Plou FJ (2008) Enzymatic
modification for ascorbic acid and alpha-tocopherol to enhance
their stability in food and nutritional applications. Open Food Sci
J 2:1–9
Table 2 Antioxidant properties of fatty acid ascorbyl esters
Sample
TEAC
Hours
Rancimat
Stability increase,
Rancimat (%)
2
. Stamatis H, Sereti V, Kolisis FN (2001) Enzymatic synthesis of
hydrophilic and hydrophobic derivatives of natural phenolic acids
in organic media. J Mol Catal B Enzym 11:323–328
. Bonrath W, Netscher T (2005) Catalytic processes in vitamins
synthesis and production. Appl Catal A Gen 280:55–73
. Torres P, Reyes-Duarte D, Lopez-Cortes N, Ferrer M, Ballesteros
A, Plou FJ (2008) Acetylation of vitamin E by Candida antarc-
tica lipase B immobilized on different carriers. Process Biochem
Control
1.06
0.89
0.75
6.83
8.27
8.22
–
3
4
6
6
-O-Ascorbyl palmitate
-O-Ascorbyl oleate
21.1
20.4
antioxidant activity measured by DPPH (a,a-diphenyl-b-
picrylhydrazyl) method. Song et al. [33] demonstrated that
4
3:145–153
5. Alcalde M, Ferrer M, Plou FJ (2007) Environmental biocatalysis:
from remediation with enzymes to novel green processes. Bio-
catal Biotransform 25:113
. Wohlgemuth R (2007) Modular and scalable biocatalytic tools for
practical safety, health and environmental improvements in the
production of speciality chemicals. Biocatal Biotransform
6
-O-ascorbyl oleate had a better protective effect on human
umbilical cord vein endothelial cells than other ascorbyl
derivatives.
6
The accelerated test Rancimat was applied to evaluate
the oxidative stability of soybean oil in presence of the
fatty acid ascorbyl esters (Table 2). The results indicated
that both ascorbyl oleate and palmitate produce stabiliza-
tion of nearly 20% towards peroxide formation. In this
context, Viklund et al. [34] compared the efficiency of
ascorbyl oleate and palmitate to retard peroxide develop-
ment in rapeseed oil; they concluded that the oleate
derivative was more effective than the palmitate in a
2
5:178–185
7
. Kochhar SP, Rossell JB (1990) Detection, estimation and eval-
uation of antioxidants in food systems. In: Hudson BJF (ed) Food
antioxidants. Elsevier, Essex, pp 19–64
8. Karmee SK (2009) Biocatalytic synthesis of ascorbyl esters and
their biotechnological applications. Appl Microbiol Biotechnol
8
1:1013–1022
9
. Valentin HE, Qi QG (2005) Biotechnological production and
application of vitamin E: current state and prospects. Appl
Microbiol Biotechnol 68:436–444
1
1
1
0. Humeau C, Girardin M, Coulon D, Miclo A (1995) Synthesis of
-O-palmitoyl L-ascorbic-acid catalyzed by Candida antartica
lipase. Biotechnol Lett 17:1091–1094
1. Yan YC, Bornscheuer UT, Schmid RD (1999) Lipase-catalyzed
synthesis of vitamin C fatty acid esters. Biotechnol Lett
1
8-week period.
6
Conclusion
2
1:1051–1054
2. Park S, Viklund F, Hult K, Kazlauskas RJ (2003) Ionic liquids
create new opportunities for nonaqueous biocatalysis with polar
substrates: acylation of glucose and ascorbic acid. In: Rogers RD,
Seddon KR (eds) Ionic liquids as green solvents: progress and
prospects. ACS Symposium Series, American Chemical Society,
vol 856, pp 225–238
We have demonstrated the applicability of immobilized
Thermomyces lanuginosus lipase (Lipozyme TL IM) to
synthesize fatty acid ascorbyl esters using triglycerides and
natural oils as acyl donors. Although yields are slightly
lower than those obtained with immobilized Candida
antarctica lipase preparations, its tenfold lower price may
compensate the reduced efficiency. Ascorbyl oleate presents
1
3. Jun HK, Bae KM, Kim YH (1998) Identification of L-ascorbic
acid 2-O-alpha-glucoside, a stable form of ascorbic acid, in
kimchi. J Microbiol Biotechnol 8:710–713
1
23

6
1
4
J Am Oil Chem Soc (2011) 88:57–64
4. Song QX, Wei DZ (2002) Study of vitamin C ester synthesis by
immobilized lipase from Candida sp. J Mol Catal B Enzym
enol esters as acylating reagents—preparative enantioselective
and regioselective syntheses of alcohols, glycerol derivatives,
sugars, and organometallics. J Am Chem Soc 110:7200–7205
25. Cao LQ, Bornscheuer UT, Schmid RD (1999) Lipase-catalyzed
solid-phase synthesis of sugar esters. Influence of immobilization
on productivity and stability of the enzyme. J Mol Catal B Enzym
6:279–285
1
8:261–266
1
5. Ferrer M, Plou FJ, Fuentes G, Cruces MA, Andersen L, Kirk O,
Christensen M, Ballesteros A (2002) Effect of the immobilization
method of lipase from Thermomyces lanuginosus on sucrose
acylation. Biocatal Biotransform 20:63–71
1
1
1
1
2
6. Borole AP, Davison BH (2007) Techno-economic analysis of
biocatalytic processes for production of alkene epoxides. Appl
Biochem Biotechnol 137:437–449
7. Bradoo S, Saxena RK, Gupta R (1999) High yields of ascorbyl
palmitate by thermostable lipase-mediated esterification. J Am
Oil Chem Soc 76:1291–1295
8. Burham H, Gafoor RA, Rasheed A, Noor NM, Badruddin S,
Sidek H (2009) Enzymatic synthesis of palm-based ascorbyl
esters. J Mol Catal B Enzym 58:153–157
9. Hsieh HJ, Chen JW, Giridhar R, Wu WT (2005) Synthesis of
mixed esters of ascorbic acid using methyl esters of palm and
soybean oils. Prep Biochem Biotech 35:113–118
0. Ferrer M, Soliveri J, Plou FJ, Lopez-Cortes N, Reyes-Duarte D,
Christensen M, Copa-Patino JL, Ballesteros A (2005) Synthesis
of sugar esters in solvent mixtures by lipases from Thermomyces
lanuginosus and Candida antarctica B, and their antimicrobial
properties. Enzyme Microb Technol 36:391–398
26. Reyes-Duarte D, Lopez-Cortes N, Ferrer M, Plou FJ, Ballesteros
A (2005) Parameters affecting productivity in the lipase-catalysed
synthesis of sucrose palmitate. Biocatal Biotransform 23:19–27
27. Plou FJ, Barandiaran M, Calvo MV, Ballesteros A, Pastor E
(1996) High-yield production of mono- and di-oleoylglycerol by
lipase-catalyzed hydrolysis of triolein. Enzyme Microb Technol
18:66–71
28. Brenes M, Garcia A, Dobarganes MC, Velasco J, Romero C
(2002) Influence of thermal treatments simulating cooking pro-
cesses on the polyphenol content in virgin olive oil. J Agric Food
Chem 50:5962–5967
29. Ferrer M, Comelles F, Plou FJ, Cruces MA, Fuentes G, Parra JL,
Ballesteros A (2002) Comparative surface activities of di- and
trisaccharide fatty acid esters. Langmuir 18:667–673
30. Adamczak M, Bornscheuer UT (2009) Improving ascorbyl oleate
synthesis catalyzed by Candida antarctica lipase B in ionic liq-
uids and water activity control by salt hydrates. Process Biochem
44:257–261
31. Palma S, Manzo RH, Allemandi D, Fratoni L, Lo Nostro P (2002)
Solubilization of hydrophobic drugs in octanoyl-6-O-ascorbic
acid micellar dispersions. J Pharm Sci 91:1810–1816
32. LoNostro P, Capuzzi G, Pinelli P, Mulinacci N, Romani A,
Vincieri FF (2000) Self-assembling and antioxidant activity of
some vitamin C derivatives. Colloid Surf A Physicochem Eng
Asp 167:83–93
2
1. Reyes-Duarte D, Polaina J, Lopez-Cortes N, Alcalde M, Plou FJ,
Elborough K, Ballesteros A, Timmis KN, Golyshin P, Ferrer M
(2005) Conversion of a carboxylesterase into a triacylglycerol
lipase by a random mutation. Angew Chem Int Edit 44:7553–7557
2. Awang R, Basri M, Ahmad S, Salleh AB (2005) Thermomyces
lanuginosus lipase-catalyzed esterification of 9,10-dihydroxyste-
aric acid and monohydric alcohol. J Oleo Sci 54:305–309
3. Plou FJ, Cruces MA, Ferrer M, Fuentes G, Pastor E, Bernabe M,
Christensen M, Comelles F, Parra JL, Ballesteros A (2002)
Enzymatic acylation of di- and trisaccharides with fatty acids:
choosing the appropriate enzyme, support and solvent. J Bio-
technol 96:55–66
2
2
33. Song QX, Wei DZ, Zhou WY, Xu WQ, Yang SL (2004) Enzy-
matic synthesis and antioxidant properties of L-ascorbyl oleate
and L-ascorbyl linoleate. Biotechnol Lett 26:1777–1780
34. Viklund F, Alander J, Hult K (2003) Antioxidative properties and
enzymatic synthesis of ascorbyl FA esters. J Am Oil Chem Soc
80:795–799
2
4. Wang YF, Lalonde JJ, Momongan M, Bergbreiter DE, Wong CH
(1988) Lipase-catalyzed irreversible transesterifications using
1
23