Antioxidative properties and enzymatic synthesis of ascorbyl FA esters

Article abstract of DOI:10.1007/s11746-003-0774-1

Efficient synthesis of unsaturated FA esters of ascorbic acid is possible with only a small excess of one of the reactants in t-amyl alcohol using Candida antarctica lipase as biocatalyst. Using free acids, we obtained yields that were comparable to yields reached using vinyl-activated acyl donors (71, 80, and 86% yields of esters with FA excesses of 1:1, 1:1.5, and 1:2, respectively). As very low water activity is needed to achieve sufficiently high yields of product, molecular sieves were used to improve the ascorbyl ester yields. Ascorbyl oleate is more amorphous and has a much lower m.p. and lower enthalpy of fusion than ascorbyl palmitate. This leads to a higher solubility of ascorbyl oleate in oil, resulting in an increased antioxidant effect compared to that of the palmitate. In an accelerated storage test using deodorized rapeseed oil, samples incubated with ascorbyl palmitate showed noticeable oxidation after 1 wk of storage, whereas samples incubated with ascorbyl oleate displayed negligible oxidation for 9 and 4 wk at 30 and 40°C, respectively.

Full text of DOI:10.1007/s11746-003-0774-1

Antioxidative Properties and Enzymatic
Synthesis of Ascorbyl FA Esters
Fredrik Viklunda, Jari Alanderb, and Karl Hulta,*
aRoyal Institute of Technology, AlbaNova University Center, Department of Biotechnology, SE-106 91 Stockholm, Sweden,
and bKarlshamns AB, Edible Oils R&D, SE-374 82 Karlshamn, Sweden
ABSTRACT: Efficient synthesis of unsaturated FA esters of
HO
ascorbic acid is possible with only a small excess of one of the
HO
O
O
reactants in t-amyl alcohol using Candida antarctica lipase as
biocatalyst. Using free acids, we obtained yields that were com-
parable to yields reached using vinyl-activated acyl donors (71,
80, and 86% yields of esters with FA excesses of 1:1, 1:1.5, and
1:2, respectively). As very low water activity is needed to
achieve sufficiently high yields of product, molecular sieves
were used to improve the ascorbyl ester yields. Ascorbyl oleate
is more amorphous and has a much lower m.p. and lower en-
thalpy of fusion than ascorbyl palmitate. This leads to a higher
solubility of ascorbyl oleate in oil, resulting in an increased an-
tioxidant effect compared to that of the palmitate. In an acceler-
ated storage test using deodorized rapeseed oil, samples incu-
bated with ascorbyl palmitate showed noticeable oxidation after
1 wk of storage, whereas samples incubated with ascorbyl
oleate displayed negligible oxidation for 9 and 4 wk at 30 and
40°C, respectively.
O
+
H
OH
HO
OH
oleic acid
ascorbic acid
Candida antarctica lipase B
t-amyl alcohol
O
O
HO
O
O
+
H2O
H
6-ascorbyl oleate
HO
OH
SCHEME 1
the reactants differ in polarity, the number of suitable solvents
available is limited. We used t-amyl alcohol because it dis-
solves the reactants at high concentrations and does not deac-
tivate the enzyme.
Paper no. J10512 in JAOCS 80, 795–799 (August 2003).
KEY WORDS: Antioxidant, ascorbyl oleate, ascorbyl palmi-
tate, esterification, lipase.
In 1971, Arakawa et al. (3) reported on the first enzymatic
synthesis of ascorbyl laurate in aqueous solution. In 1990,
Enomoto et al. (4) filed a patent on the esterification in or-
ganic solvents, and esterfications in t-amyl alcohol were re-
ported by Humeau et al. (5) in 1995. Since then, not only sat-
urated FA have been used, but also various unsaturated FA
(6–8) as well as of phenyl butyric acid (9) and methyl lactate
(10). These syntheses have typically reached yields below
50% unless activated acyl donors or a large excess of the FA
was used.
In this study we show that enzymatic catalysis can be used
for the synthesis of ascorbyl oleate. We also discuss some re-
quirements for a synthetically favorable reaction and some
potential pitfalls in this and similar syntheses when using ter-
tiary alcohols as solvent. We further assess some physical
properties of ascorbyl oleate and show that it has a favorable
antioxidative effect on rapeseed oil compared with ascorbyl
palmitate.
Ascorbyl palmitate is a fat-soluble antioxidant commonly
used in foods to prevent oxidation of sensitive fats and oils.
Ascorbyl palmitate has a disadvantage, however, in that it has
relatively low solubility in oils. The solubility can be im-
proved by the addition of lecithin, although this causes the
product to become hygroscopic. Another way to improve the
solubility of an ascorbyl FA ester is to modify its structure to
make it less crystalline. This can be done, for example, by in-
troducing a double bond in the FA. Oleic acid is unsaturated,
readily available, inexpensive, and approved for use in foods.
This makes it an attractive candidate to replace the palmitic
acid in an ascorbyl ester.
The chemical synthesis of saturated ascorbyl FA esters is
straightforward (1). A similar procedure for the synthesis of
ascorbyl oleate has been described in the literature (2) but has
proven hard to repeat. The difficulties are due to the double
bond of oleic acid, which is too sensitive for the reaction con-
ditions needed for the chemically catalyzed esterification. En-
zymatic catalysis, however, can be performed at lower reac-
tion temperatures and under milder conditions, enabling the
enzymatic synthesis of ascorbyl oleate (Scheme 1). Because
MATERIALS AND METHODS
Enzymatic synthesis. Ascorbic acid (99%), palmitic acid
(99%), and oleic acid (99%) were purchased from Sigma-
Aldrich (Stockholm, Sweden). Novozym 435, immobilized
Candida antarctica lipase B, was a gift from Novozymes A/S
*To whom correspondence should be addressed.
E-mail: kalle@biotech.kth.se.
Copyright © 2003 by AOCS Press
795
JAOCS, Vol. 80, no. 8 (2003)

796
F. VIKLUND ET AL.
(Bagsvaerd, Denmark). Syntheses of ascorbyl palmitate were minum pans and heated from 35 to 150°C at 5°C/min under
performed enzymatically with 100 mg (0.57 mmol) ascorbic nitrogen purging.
acid and palmitic acid in molar ratios of 1:1, 1:1.5, and 1:2
X-ray powder diffraction also was carried out to determine
(ascorbic acid/palmitic acid) in 10.0 mL t-amyl alcohol at ascorbyl ester crystallinity. A Siemens D5000 operating with
60°C. The reactions were started by the addition of 50 mg of a Cu anode at 40 mA and 40 kV at ambient temperature was
Novozym 435. Water activity of the reaction medium was used for the studies. Diffractograms were taken in the long-
kept constant with the addition of three portions of 3-Å molec- spacing (0.7–10° 2-θ) and the short-spacing regions (17–27°
ular sieves (400 mg), which were exchanged during the reac- 2-θ).
tion. Samples were taken throughout the reaction and did not
exceed 1% of the total volume.
RESULTS AND DISCUSSION
Ascorbyl oleate was synthesized by mixing 4.5 g oleic acid
(16 mmol) and 5.53 g (32 mmol) ascorbic acid in 184 mL of Control of water activity. The approaches used in the litera-
t-amyl alcohol together with 2.5 g molecular sieves and 600 ture for the enzymatic synthesis of ascorbyl esters can be di-
mg Novozym 435 at 65°C.
vided into two groups. The first approach is to remove the
HPLC analysis. Analysis was done with RP-HPLC using water or alcohol formed from the reaction with either molec-
a Supelco Discovery C18 column (15 cm × 4.6 mm, 5 µm; ular sieves or reduced pressure to shift the equilibrium, or to
Sigma-Aldrich), 1 mL/min of a 96:4 mixture of methanol/water use a vinyl ester as the acyl donor. The second approach uses
fortified with 0.5% acetic acid. Detection of ascorbic acid none of these measures to remove the co-product formed
and ascorbyl esters was done spectrophotometrically at 254 from the reaction mixture. Despite the well-known fact that
nm. FA were determined with a Sedex 45 ELSD (Sedere, Al- the yield of an equilibrium reaction benefits from having one
fortville, France) at 30°C and 2 bar air pressure using a cali- of the products removed from the mixture, relatively few pub-
bration curve obtained separately. The product conversion lished experiments use water- or methanol-removal strategies
was calculated as Cester/(Cascorbic acid + Cester) for each sam- such as molecular sieves or reduced pressure. The use of a
ple. Retention times were approximately 1.9, 3.3, and 4.5 vinyl ester as acyl donor, however, is commonly practiced.
min for the ascorbic acid, ascorbyl ester, and FA, respec-
tively.
The techniques used to control water activity that were
evaluated in this work included vapor phase equilibration
Purification. The ascorbyl oleate product was purified by with a salt solution (12), water diffusion through silicon tub-
washing with hexane (160 + 30 + 30 mL) to remove unre- ing with a circulating saturated salt solution (13), and molec-
acted oleic acid. Excess ascorbic acid was removed by dis- ular sieves. Vapor phase equilibration was considered unsuit-
solving the product in diethyl ether followed by filtration.
able because the t-amyl alcohol vapor reacted with the salts
NMR. Ascorbyl oleate was analyzed with a NMR spec- tested (LiCl and MgCl2), which resulted in irreversible trans-
1
trometer (Bruker, Rheinstetten, Germany): H NMR (500 port of t-amyl alcohol from the reaction vessel to the salt so-
MHz, CDCl3): δ 5.35 (m, 2H), 4.8 (s, 1H), 4.45 (s, 1H), 4.25 lution reservoir. This resulted in a change in reaction volume
(d, 2H), 2.35 (m, 2H), 2.05 (m, 4H), 1.65 (m, 2H); 1.3 (br, and loss of water activity control. Immersing silicon tubing,
20H), 0.9 (t, 3H).
through which saturated salt solutions circulated into the re-
Antioxidant effect. The antioxidant effect of the synthe- action mixture as a means of controlling the water activity,
sized ascorbyl oleate was compared with that of ascorbyl also was tried. The t-amyl alcohol, however, diffused through
palmitate (Danisco Ingredients, Brabrand, Denmark) in ac- the tubing and reacted with the salt solution, forming com-
celerated storage tests at 30 and 40°C. The antioxidants were plexes that precipitated and blocked the tubing and that also
added to fresh, deodorized low-erucic acid rapeseed oil (Karls- resulted in solvent loss from the reaction mixture and loss of
hamns AB, Sweden) at a concentration of 600 mg/kg. The FA water activity control. This problem is representative for a
composition of the oil was 58.5% oleic acid (18:1), 21.5% solvent that diffuses through silicon tubing and precipitates
linoleic acid (18:2), 10.5% linolenic acid (18:3), 4.5% the salt. In this respect, silicon tubing-mediated equilibration
palmitic acid (16:0), and 1.5% stearic acid (18:0). The tocoph- is perhaps better suited for water-insoluble solvents.
erols, analyzed by HPLC, comprised 200 ppm α-, 320 ppm
Because the common technique for controlling water ac-
γ-, and 7 ppm δ-tocopherols. Duplicate samples of 50 g were tivity with saturated salt solutions was not suitable for this re-
placed in dark glass jars covered by a loose aluminum foil. action system, molecular sieves were used to remove the
The jars were kept in a dark heating cabinet held at the de- water formed in the reaction. Molecular sieves have been
sired storage temperature. Jars were taken out at predeter- shown to dry t-butyl alcohol to a water content of 13 ppm but
mined time intervals and analyzed for PV using a standard only at a slow rate, thought to be dependent on its relatively
titrimetric method (11). The base oil with no added antioxi- high viscosity (14). To ensure a water activity as low and con-
dant was used as control.
Melting point and crystallinity. The melting properties of changed as the reaction proceeded and as water accumulated.
commercial ascorbyl palmitate and synthesized ascorbyl Reaction progress. We studied the esterification of ascor-
stant as possible throughout the reaction, the sieves were ex-
oleate were determined by DSC (Mettler TA8000). The ascor- bic acid with a FA in t-amyl alcohol on a small scale using
byl esters (6–8 mg) were weighed into standard 40-µL alu- palmitic acid as the acyl donor. The syntheses were carried
JAOCS, Vol. 80, no. 8 (2003)

PROPERTIES AND SYNTHESIS OF ASCORBYL ESTERS
797
out at three different FA concentrations at low water activity.
In this study, we used ascorbic acid/palmitic acid molar ra-
Ascorbic acid and palmitic acid were mixed in t-amyl alcohol tios of 1:1, 1:1.5, and 1:2 and dried the reaction system with
together with the molecular sieves. The reaction was started molecular sieves to obtain yields of 71, 80, and 86%, respec-
with the addition of Novozym 435 (immobilized lipase B tively. This can be compared to the results of Sakashita et al.
from C. antarctica). After about 50 h, the reactions had (16), who reported an 88% yield of ascorbyl stearate with
reached a steady state. The product yield, monitored with 2000 ppm water in t-butanol, and Yan et al. (17), who re-
time by HPLC, is shown in Figure 1.
ported an 84% yield using a 1.5-fold molar excess of vinyl
Earlier reports of enzymatic synthesis of ascorbyl palmi- stearate in dried t-butanol (Table 1).
tate in t-amyl alcohol or t-butanol reported only 6 (6) or 14%
From this it can be concluded that our reaction system,
yields (7) using a 1:1 ratio of ascorbic acid and acyl donor which uses free acid and careful drying of the reaction
(Table 1). Bradoo et al. (15) reported a 50% yield in hexane medium, seems to be as efficient in driving the reaction to-
when using equimolar amounts of ascorbic and palmitic acids ward completion as one using an activated acyl donor. Pre-
catalyzed by Bacillus stearothermophilus lipase without re- sumably, as the ascorbyl ester is formed it can be hydrolyzed
moving co-product. By increasing the palmitic acid mole con- in the presence of water.
centration to 2.5 times that of the ascorbic acid and adding
When using activated acyl donors such as vinyl palmitate,
molecular sieves, they obtained a 97% yield of ascorbyl ester. ascorbyl esters will form at a high rate and reach high yields, but
the ester product will hydrolyze if the medium is not carefully
dried, which results in the same final composition for vinyl-acti-
100
90
80
70
60
50
40
30
20
10
0
Ratio = 1:2
Ratio = 1:1.5
Ratio = 1:1
vated acyl donors as for the free acids (Viklund, F., and K. Hult,
unpublished results). Thus, it is very important to control the
water activity of the reaction to reach high ester yields.
Because the ascorbic acid and ascorbyl esters are sensitive
to oxidation, it also is important to check for oxidation of the
products as the reaction progresses to obtain a high yield. Re-
actant or product oxidation would be seen as a decrease in
yield with time, which is not the case (Fig. 1).
Ascorbyl oleate synthesis. Ascorbyl oleate was synthe-
sized in a scaled-up reaction with careful removal of the water
from the reaction with molecular sieves (Table 1). A twofold
excess of ascorbic acid was used to maximize the utilization
of the high-purity oleic acid. The reaction was stopped after
100 h. HPLC analysis indicated a yield of 87% based on oleic
acid, which agrees very well with the 86% achieved for the
palmitate ester when palmitic acid was used in excess. The
product was purified by washing with hexane to remove the
excess oleic acid, followed by dissolution in diethyl ether and
filtration to remove unreacted ascorbic acid. The purity was
Yield
86%
80%
71%
Exchange of molecular sieves
0
10
20
30
Time (h)
40
50
FIG. 1. Yield of ascorbyl palmitate in the lipase-catalyzed esterification of
ascorbic acid and palmitic acid as a function of time at three molar ratios
of ascorbic acid to palmitic acid in t-amyl alcohol and in the presence of
molecular sieves. Duplicates are shown for the reaction at ratio 1:1.
TABLE 1
Comparison of Lipase-Catalyzed Syntheses of Ascorbyl FA Esters
Acyl donor
Solvent
Catalyst
Novozym 435b
Temp. (°C)
55
Ratioa Yield (%)
Drying
Reference
6
Methyl palmitate
t-Amyl alcohol
1:1
1:5
6
47
14
26
32
80c
50
97
88
84
71
80
86
87
—
—
—
—
Methyl palmitate
t-Amyl alcohol
Novozym 435
50
1:1
1:3
7
1:5
1:5
1:1
—
Methyl palmitate
Palmitic acid
t-Amyl alcohol
Hexane
Novozym 435
Lipase from Bacillus stearothermophilus 50
70
Reflux distillation
—
18
15
1:2.5
1.5
Molecular sieves
2000 ppm water
Molecular sieves
Molecular sieves
Molecular sieves
Molecular sieves
Molecular sieves
Vinyl stearate
Vinyl palmitate
Palmitic acid
t-Butanol
t-Butanol
t-Amyl alcohol
Amano PS
40
40
60
16
17
This study
Chirazyme L-2b
Novozym 435
1.5
1:1
1:1.5
1:2
Oleic acid
t-Amyl alcohol
Novozym 435
65
2:1
This study
aRatio of ascorbic acid to acyl donor.
bNovozym 435 (Novozymes A/S, Bagsvaerd, Denmark) and Chirazyme L-2 are different preparations of immobilized lipase B from Candida antarctica.
cIsolated yield.
JAOCS, Vol. 80, no. 8 (2003)

798
F. VIKLUND ET AL.
>97% (w/w), and the isolated yield of ascorbyl oleate was
29% (2.1 g) based on oleic acid. To improve the workup pro-
cedure, preparative silica gel chromatography was tested;
however, silica gel chromatography was found to be unsuit-
able because the ascorbyl oleate oxidized and discolored on
the column.
The purified ascorbyl oleate was a white, waxy solid.
Structural verification of ascorbyl oleate was done with 1H
NMR. No oxidation of the oleoyl group was detected, and the
product was exclusively the 6-O-monoester. The reaction
rates (data not shown) and yields of ascorbyl palmitate and
oleate are similar, but the chemical sensitivity and the physi-
cal properties of ascorbyl oleate demand other synthetic
routes and workup procedures.
225
200
175
150
125
100
75
T = 40°C
Control
Ascorbyl palmitate
Ascorbyl oleate
50
25
0
0
1
2
3
4
5
6
7
8
9
10
Melting properties and crystallinity. Ascorbyl palmitate
melted cleanly in one single melting peak at 114–120°C, with
an enthalpy of fusion of 129 J/g. It crystallized in a double-
chain-packing crystal form (d = 45 Å) with short-spacing
diffraction peaks at 4.2, 4.3, 4.0, 3.9, and 3.8 Å. In contrast,
ascorbyl oleate exhibited only weak crystallinity, which was
characterized by two weak peaks at 4.5 and 4.2 Å and double-
chain packing (d = 35 Å). The amorphous structure of the
ascorbyl oleate also was evident in the DSC thermograms,
which had a broad melting peak at 83–84°C and an enthalpy of
fusion of only 20–30 J/g. The low enthalpy of fusion and the
observed diffraction pattern indicate that ascorbyl oleate solidi-
fies primarily in a liquid crystalline structure with nonordered
fatty acyl chains and a structured polar head group.
Time (wk)
FIG. 3. Comparison of antioxidant effects for ascorbyl palmitate and
ascorbyl oleate (600 mg/kg) in deodorized rapeseed oil at 40°C.
The remaining tocopherol content in the oil samples was
analyzed after 10 wk at 40°C. The base oil at 40°C had no de-
tectable tocopherol levels, whereas the sample with ascorbyl
palmitate contained 9–10 ppm (3% remaining) γ-tocopherol.
The oil protected by ascorbyl oleate had 184 ppm (57% re-
maining) γ-tocopherol and 6 ppm (86% remaining) δ-tocoph-
erol left after 10 wk at 40°C.
The results show that when used as an antioxidant in oil,
ascorbyl oleate is more effective than ascorbyl palmitate in
inhibiting oil oxidation.
Antioxidant effect. The PV, as a function of storage time
for the control and the two antioxidants at 30 and 40°C, are
shown in Figures 2 and 3, respectively. Ascorbyl oleate per-
formed significantly better than ascorbyl palmitate and the
control at both temperatures. For ascorbyl palmitate there was
a small but significant increase in the PV at short storage
times at both temperatures, whereas ascorbyl oleate had a
clear induction period of 4 wk at 40°C and 9 wk at 30°C be-
fore peroxide development accelerated.
ACKNOWLEDGMENTS
This work was supported by the Competence Centre for Surfactants
Based on Natural Products (SNAP).
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