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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
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