Enzymatic Synthesis of Ascorbyl Palmitate in Organic Solvents: Process Optimization and Kinetic Evaluation
ABSTRACT This work is focused on the optimization of reaction parameters for the synthesis of ascorbyl palmitate catalyzed by Candida antarctica lipase in different organic solvents. The sequential strategy of experimental designs proved to be useful in maximizing the
conditions for product conversion in tert-butanol system using Novozym 435 as catalyst. The optimum production were achieved
at ascorbic acid to palmitic acid mole ratio of 1:9, stirring rate of 150rpm, 70°C, enzyme concentration of 5wt.% at 17h
of reaction, resulting in an ascorbyl palmitate conversion of about 67%. Reaction kinetics for ascorbyl palmitate production
showed that very satisfactory reaction conversions (∼56%) could be achieved in short reaction times (6h). The kinetic empirical
model proposed showed ability to satisfactory represents and predict the experimental data.
KeywordsBiocatalysis–Neural network–Kinetics–Lipase–Ascorbyl palmitate–Experimental design
- SourceAvailable from: Lindomar Lerin[Show abstract] [Hide abstract]
ABSTRACT: The main focus of this work was to investigate the residual esterification activity and the product conversion after 10 successive cycles of utilization of a commercial lipase in three systems: esterification of 2-ethyl hexanol and palmitic acid in a solvent-free system; esterification of ascorbic acid and palmitic acid in tert-butanol; and transesterification of glycerol and methyl benzoate in 2-propanol. These systems were chosen based on previous results by our research group in terms of product conversion. Before scale-up, there is a need for evaluating several cycles of utilization of the biocatalyst. The esterification of 2-ethyl hexanol showed that after 10 cycles the enzyme retained 90% of its activity. The system consisting of ascorbic acid, palmitic acid, Novozym 435 and tert-butanol showed that a reduction in enzyme activity was accompanied by a reduction in reaction conversion; the same behavior was not observed for the third system.Brazilian Journal of Chemical Engineering 06/2011; 28(2):181-188. · 0.91 Impact Factor
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ABSTRACT: In the present study, a dimethyl sulfoxide (DMSO)–acetone mixed solvent was used as a reaction medium to study lipase-catalyzed esterification of l-ascorbic acid with lauric acid as the acyl donor. Compared with the solvent of acetone, the mixed solvent not only could greatly improve the solubility of l-ascorbic acid but also could observably increase the enzymatic rate. Within the investigated range of substrate concentrations, a lauric acid/ascorbic acid mole ratio of 3:1 was found to be optimum. Moreover, the optimum production was achieved in the reaction medium dewatered by molecular sieves and at a lipase loading of 40 mg/mL. As a result, enzymatic synthesis of ascorbyl laurate gave a maximum conversion of 94% in the DMSO–acetone mixed solvent using lauric acid as the acyl donor.Industrial & Engineering Chemistry Research. 08/2013; 52(34):11875–11879.
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ABSTRACT: Kinetics and thermodynamics of lipase-catalyzed esterification of L-ascorbic acid in acetone was investigated by using vinyl acetate as acyl donor. The results showed that L-ascorbic acid could generate inhibition effect on lipase activity. A suitable model, Ping-Pong Bi-Bi mechanism having substrate inhibition, was thus introduced to describe the enzymatic kinetics. Furthermore, the kinetic and thermodynamic parameters were calculated from a series of experimental data according to the kinetic model. The inhibition constant of L-ascorbic acid was also obtained, which seemed to imply that enhancing reaction temperature could depress the substrate inhibition. Besides, the activation energy of the first-step and the second-step reaction were estimated to be 37.31 and 4.94kJ/mol, respectively, demonstrating that the first-step reaction was the rate-limiting reaction and could be easily improved by enhancing temperature.Journal of Biotechnology 11/2013; · 3.18 Impact Factor
Enzymatic Synthesis of Ascorbyl Palmitate in Organic
Solvents: Process Optimization and Kinetic Evaluation
Lindomar A. Lerin & Aline Richetti & Rogério Dallago & Helen Treichel &
Marcio A. Mazutti & J. Vladimir Oliveira & Octávio A. C. Antunes &
Enrique G. Oestreicher & Débora de Oliveira
Received: 24 February 2010 /Accepted: 21 June 2010 /Published online: 7 July 2010
# Springer Science+Business Media, LLC 2010
Abstract This work is focused on the optimization of
reaction parameters for the synthesis of ascorbyl palmitate
catalyzed by Candida antarctica lipase in different organic
solvents. The sequential strategy of experimental designs
proved to be useful in maximizing the conditions for
product conversion in tert-butanol system using Novozym
435 as catalyst. The optimum production were achieved at
ascorbic acid to palmitic acid mole ratio of 1:9, stirring rate
of 150 rpm, 70 °C, enzyme concentration of 5 wt.% at 17 h
of reaction, resulting in an ascorbyl palmitate conversion of
about 67%. Reaction kinetics for ascorbyl palmitate
production showed that very satisfactory reaction conver-
sions (∼56%) could be achieved in short reaction times
(6 h). The kinetic empirical model proposed showed ability
to satisfactory represents and predict the experimental data.
Keywords Biocatalysis.Neural network.Kinetics.
Lipase.Ascorbyl palmitate.Experimental design
Antioxidants have been added to food for decades to
control the oxidation process, being widely used nowadays
for better food preservation. Development of new antiox-
idants, with better capacity and less toxicity, is desirable for
prevention and/or treatment of a number of diseases and for
enhancing the shelf-life of several food products (de Pinedo
et al. 2007; Khiari et al. 2009; Jing et al. 2009).
L-Ascorbic acid, a natural hydrophilic antioxidant, has
been used but its application is limited in hydrophobic
foods and cosmetics (Liu et al. 1996). Due to the steady
growing demand for natural materials, the synthesis of
esters of ascorbic acid by lipase-catalyzed reactions has
become of a current commercial interest. An optimized
enzymatic synthesis of L-ascorbyl esters with improved
yield at reduced cost would be more appealing to the
consumer and of benefit to the manufacturers (Chang et al.
The importance of enzymatic synthesis catalyzed by
lipases to produce L-ascorbyl esters via esterification in
water-miscible organic solvents has been emphasized in
several works (Humeau et al. 1998; Yan et al. 1999;
Treichel et al. 2010). Palmitic, caprilic, capric, lauric, and
stearic acids have been widely used as acyl donors. Among
the unsaturated acids we can mention oleic, linolenic,
arachidonic, and docosahexaenoic acids (Karmee 2009).
However, no systematic study involving process conversion
optimization as well as the understanding of the kinetics of
this kind of reaction, of primary importance to determine
the optimal operational conditions, is presented in the
Based on the abovementioned information, the present
work is focused on the optimizing the reaction conditions
for ascorbyl palmitate (AP) synthesis by a sequential
strategy of experimental planning. Then, the kinetic
experimental data and modeling for this reactional system
was also investigated in the optimized experimental con-
ditions obtained in the experimental design. To the best of
L. A. Lerin:O. A. C. Antunes:E. G. Oestreicher
Instituto de Química-IQ/UFRJ,
CT, Bloco A,
Rio de Janeiro 21945-900 RJ, Brazil
A. Richetti:R. Dallago:H. Treichel (*):M. A. Mazutti:
J. V. Oliveira:D. de Oliveira
Universidade Regional Integrada do Alto Uruguai e das Missões,
URI - Campus de Erechim,
Av. Sete de Setembro, 1621,
99700-000 Erechim, RS, Brazil
Food Bioprocess Technol (2012) 5:1068–1076
our knowledge, the systematic study for maximizing the
product synthesis as well as experimental data and kinetic
modelingconcerningtheenzymatic esterification ofascorbic
acid has not been reported in any published literature yet.
Material and Methods
The substrates used in the esterification reactions were
commercial palmitic acid (Vetec, 98% purity) and L-(+)-
ascorbic acid (Vetec, 99% purity). Novozym 435, a lipase
from Candida antarctica, and Lipozyme RM IM, a lipase
from Rhizomucor miehei, purchased from Novozymes S/A
(Araucária-PR-Brazil), were used as catalysts. Novozym
435, immobilized on a macroporous anionic resin, 1.4 wt.%
water (Karl Fischer titration method, DL 50, Mettler-
Toledo) presented an initial enzyme activity of around
60 U/g of support, determined as the initial rates in
esterification reactions between lauric acid and propanol
at a mole ratio of 3:1, 60 °C and enzyme content of 5 wt.%
in relation to the substrates (Oliveira et al. 2006).
Acetone, tert-butanol, acetic acid, ethanol, n-propanol, 2-
propanol, and methanol of HPLC grade were obtained from
Vetec (SP, Brazil) and the standard of 6-Ο-palmitoyl-L-
ascorbic acid was obtained from Sigma–Aldrich (Fluka,
Tert-butanol, ethanol, n-propanol, 2-propanol, and petro-
leum ether, and two commercial immobilized lipases
(Novozym 435 and Lipozyme IM) were evaluated as
organic solvents and catalysts, respectively.
In all preliminary experiments, the reaction mixture was
stirred at 150 rpm, 50 °C, ascorbic to palmitic acid mole
ratio of 1:5, 20 mL of solvent, 5 wt.% of enzyme (based on
substrates), and 24 h of reaction, an experimental condition
defined in a previous work presented in the literature
(Humeau et al. 1995). Experiments were carried out in
conical flasks (125 mL) in orbital shaker (Excella E25,
New Brunswick Scientific, NJ). Samples were then filtered,
washed by solvent, and evaporated in rota-evaporator
(Fisatom-Model 803). After selecting the organic solvent
and enzyme, in terms of experimental conversion (%), a
kinetic run (36 h of reaction) was performed to define the
reaction time to be used in the experimental design.
Sequential Strategy of Experimental Designs
To determine the best reaction conditions, the first
experimental design included the evaluation of the effect
of substrates mole ratio (1:1 to 1:5), enzyme concentration
(5 to 15 wt.%), solvent volume (10 to 30 mL), and
temperature (40–70 °C), in terms of ascorbyl palmitate
production. For this experimental planning, the levels of
variables were defined from preliminary experiments, and
the agitation and reaction time were kept constant at
150 rpm and 17 h (time fixed after a previously kinetic
evaluation), respectively. Three replicates at the central
point of each planning were carried out to determine the
After analyzing the results of the first experimental
design, a central composite rotatable design 22was carried
out by adjusting the substrates mole ratio (1:3 to 1:9) and
temperature (50 to 70 °C) (Rodrigues and Iemma 2006;
Kinetic Study of Enzymatic Esterification
Taking into account the results obtained in the experimental
designs, reaction kinetic experiments were performed by
adopting ascorbic acid to palmitic acid mole ratios of 1:1,
1:3, 1:5, and 1:7, enzyme concentration of 1, 10, 15, and
20 wt.% (based on the total amount of substrates—ascorbic
acid and palmitic acid), temperature of 60 and 70 °C, and
solvent volume of 20, 30, 40, and 50 mL. Samples were
taken from the bulk reaction system at 15, 30 min, and 1, 2,
6, 12, 18, and 24 h.
Hybrid Model Formulation
This study was concerned with the development of a hybrid
model to predict the AP production. The model consisted of
a mass balance for the ascorbyl palmitate production in the
batch reactor with no density and volume changes accord-
ing to the following equation:
The main difficulty in the above model was to determine
the reaction rate (v), since there were no data referring the
kinetics of substrates employed. An alternative custom
effective was to use a feed-forward neural network (ANN)
to predict the reaction rate in Eq. 1. The ANN was
employed with the following topology: the inputs for
ANN were reaction time, temperature, initial enzyme
concentration, palmitic acid, and ascorbic acid concentra-
tions; only one hidden layer was used, and the number of
nodes in this layer was determined by trial and error; the
transfer function employed was the hyperbolic tangent in
the output and hidden layer; the output layer composed of
one node related to the reaction rate of ascorbyl palmitate
production (Mazutti et al. 2009).
Food Bioprocess Technol (2012) 5:1068–1076 1069
The above inputs were combined to minimize the
objective function F defined according to Eq. 2.
Where NPE was the number of experimental points
where the F was calculated, [AP]jexpand [AP]jcalcwere the
experimental and calculated ascorbyl palmitate concentra-
During the training procedure, the weights and the bias
were optimized using the simulated annealing algorithm
combined with Nelder and Mead (Press et al. 1992). The
parameters of the simulated annealing algorithm as the
initial artificial annealing temperature (TA) and cooling rate
(!) were used as 10.0 and 0.98, respectively.
(Fig. 3a, c, e, and f) and two additional experimental ones
were used for the validation step (Fig. 3b and d). The hybrid
model was developed by the Process Simulation Group of
DEA/URI and implemented in FORTRAN 90 language.
Quantitative analyses of the products were conducted using
an HPLC system from Agilent Series, equipped with a
refractive index. The following instrumentation and con-
ditions were used: Zorbax C18column (4.6 m×250 mm,
5 μm), flow rate of 1.0 mL/min, column temperature of
35 °C; the mobile phase, acetone/methanol/H20 with 0.5%
of acid acetic (75:25:5, v/v/v). The mobile phase was used
as a sample dissolving solvent, and the injection volume
was 20 μL. Quantification was carried out using authentic
standards of ascorbyl palmitate (6-O-palmitoyl-L-ascorbic
acid). Calibration curves were built with the product
concentrations of 240; 480; 960; 1,920; 2,880; 3,840; and
4,800 ppm. Reaction conversion was calculated based on
the content of ascorbyl palmitate in the analyzed sample
and the reaction stoichiometry.
The statistical analysis related to the estimated effects of
each variable and process optimization was performed
using the global error and the relative standard deviation
between the experimental and predicted data. It may be
important to mention that the kinetic results subsequently
presented in this work are in fact mean values of triplicate
runs, which resulted in an overall absolute deviation of
reaction conversion of around 5%. The quality of the hybrid
model formulation was verified using the correlation
coefficient (r). All analysis was performed using the
software Statistica version 6.0 (Statsoft Inc, USA).
Results and Discussion
The solubility of substrates in organic solvents can
directly affect the synthesis of the palmitoyl ester of
ascorbic acid. In this sense, different solvents were tested
in this work; ethanol (log P (partition coefficient)=−0.24),
2-propanol (log P=0.05), n-propanol (log P=0.25), tert-
butanol (log P=0.80), and petroleum ether (log P=3.20).
Results obtained in this step permitted us to identify tert-
butanol as the suitable solvent for this work, since no
conversion was obtained when other solvents were used.
Wescott and Klibanov (1993) showed that the hydro-
phobicity of the organic solvent greatly influenced the
enzyme activity and the substrate specificity. In our case, L-
ascorbic acid is hydrophilic and dissolves easily in polar
solvents, but the palmitic acid and ascorbyl palmitate are
hydrophobic and preferred by nonpolar solvents. Therefore,
organic solvents with an appropriate polarity are significant
for the substrate and product solubility and mass transfer in
reactional system (Lv et al. 2007). Different organic
solvents have different ability to distort the essential water
layer around the immobilized lipase. Log P (polarity
constant, the partition coefficient of the solvent between
in octanol and water) is widely used to represent the
characteristics of the organic phase and to predict enzymat-
ic activity (Laane et al. 1987; Cui et al. 1997).
It is generally reported that solvents with log P<2 are
less suitable for biocatalytic purpose (Chen 1996). Howev-
er, in the present work, tert-butanol (log P=0.80) seemed to
be the most appropriate organic solvent for this reaction,
leading to conversions of 36.34% and 16.64%, respectively,
for Novozym 435 and Lipozyme IM. The reaction product
was not inspected in other tested solvents. This result can
be related to the fact that the solvents with high polarity
may strip water from enzyme molecules easily, and the
enzyme cannot get enough water for keeping its active
As an example, Lv et al. (2007), evaluating the effect of
the organic solvent on ascorbyl benzoate production,
observed that cycle-hexanone (log P=0.96) and acetone
(log P=−0.23) led to higher conversions, indicating that the
interaction between solvent and enzyme should be consid-
ered. Song and Wei (2002) also tested different solvents for
oleoyl ester of ascorbic acid production and observed that
only t-amyl alcohol (log P=1.31) conducted to production
When testing Novozym 435 and Lipozyme IM as
potential catalysts for the specific reaction of interest in
this work, in a general sense, we could observe that the use
of Novozym 435 (36.34%) led to higher conversions
compared to Lipozyme IM (16.64%). This enzyme has a
1070Food Bioprocess Technol (2012) 5:1068–1076
broad substrate specificity to promote a reaction between a
wide range of primary and secondary alcohols and
carboxylic acids (Novo Nordisk 1992). Therefore, Novo-
zym 435 was used in the subsequent steps of this work.
Using the same procedure cited above, a last preliminary
study was carried out with the main purpose of defining the
reaction time to be used in the experimental design step.
Results obtained in this experimental condition showed that
conversions of 42.87% and 45.57% were obtained after 17
and 30 h of reaction, respectively. Based on this result, in
the next step of this work, the reaction time was kept
constant at 17 h.
Hsieh et al. (2006), evaluating the effect of reaction time
on ascorbyl palmitate synthesis by surfactant-coated lipase,
observed higher conversion (47%) after 24 h of reaction,
using an ascorbic acid to palmitic acid mole ratio of 1:5 and
50 °C. In the evaluation of palm-based ascorbyl esters
synthesis, other authors obtained a maximum production
after 16 h of reaction and the rate decreased gradually after
18 h, which was attributed to enzyme inhibition by the
excess of free fatty acids (Burham et al. 2009).
Sequential Strategy of Experimental Designs
From the results obtained previously, a 24−1experimental
design with triplicate of the central point was carried out to
evaluate the effect of temperature, enzyme concentration,
volume of solvent, and substrates mole ratio on reaction
conversion. Stirring rate and reaction time were kept
constant (150 rpm and 17 h, respectively) in all experi-
ments. Table 1 presents the matrix of the experimental
design with the real and coded values and the responses in
terms of ascorbyl palmitate conversion. From this table, one
can verify that higher conversions were obtained in experi-
ments 2 (54.41%) and 8 (38.91%), corresponding to higher
temperatures (70 °C) and substrates mole ratio (1:5).
Data presented in Table 1 were statistically treated, and
the main variables effects are presented in Fig. 1, where we
can observe that substrate mole ratio and temperature
presented a positive significant (p<0.05) effect on reaction
conversion. The volume of solvent and enzyme concentra-
tion did not present significant effect on ascorbyl palmitate
Based on the results of the first experimental design, a
full 22was carried out, keeping the enzyme concentration
constant (5 wt.%), the solvent volume (10 mL), the stirring
rate (150 rpm), and the reaction time (17 h), varying the
temperature (50, 60, and 70 °C) and the substrates mole
ratio (1:3, 1:6, and 1:9). Table 2 presents the matrix of the
second experimental design with the responses as ascorbyl
palmitate production. Highest conversions (67.34%) were
achieved in experiment 4, related to temperatures of 70 °C
and substrates mole ratio of 1:9, showing the positive effect
of these variables on product conversion.
Results obtained in the second experimental design were
statistically analyzed, and this permitted the development of
an empirical coded model for ascorbyl palmitate conversion
as a function of substrates mole ratio and temperature. The
resulting empirical model was validated by ANOVA. The
R2, the F test for regression (calculated value about three
times the listed one) proved that the model (Eq. 3) was
capable of well representing the experimental data of
ascorbyl palmitate conversion in the range of factors
investigated and allowed the construction of response
surface presented in Fig. 2. An analysis of this figure
permitted us to conclude that higher conversions are
obtained at the higher levels of temperature and mole ratio
within the studied range. This implies a satisfactory
Table 1 Matrix of the first experimental design (coded and real values) with responses in terms of ascorbyl palmitate conversion
Trial Temperature (°C)Enzyme concentration (wt.%) Solvent volume (mL)Mole ratio (MR)a
Stirring rate and reaction time were kept constant at 150 rpm and 17 h, respectively
aAscorbic acid/palmitic acid
Food Bioprocess Technol (2012) 5:1068–10761071
representation of the process by the empirical model, as
illustrated by the predicted conversion (5th column of
Table 2) and relative error deviation (6th column of
Conversion ð%Þ ¼ 51:46 þ 6:93 ? T þ 12:04 ? MR
? 2:30 ? T ? MR
Where T is temperature and MR is ascorbic acid to
palmitic acid mole ratio.
As can be observed from Eq. 3, the variables temperature
and mole ratio presented a positive significant effect (p<
0.05) and the interaction between them a negative effect on
ascorbyl palmitate conversion. Higher values of temperature
and an excess of palmitic acid seem to promote a good
Comparing the results obtained here with the literature,
one can cite the work of Humeau et al. (1995). The authors
tested an immobilized lipase from C. antarctica (Novozym
435) to synthesize 6-O-palmitoyl L-ascorbic acid in 2-
methyl-2-butanol and obtained an ester yield of 68% in 8 h
at 55 °C and 1:5 substrate mole ratio.
Viklund et al. (2003) reported the enzymatic synthesis of
ascorbyl palmitate using 10 mL of t-amyl alcohol at 60 °C
and 50 mg of Novozym 435 and 400 mg of molecular
sieves, varying the substrates mole ratio (1:1, 1:1.5, and
1:2) for 50 h, obtaining yields of 71%, 80%, and 86% for
each mole ratio, respectively.
Table 2 Matrix of the second experimental design (coded and real values) with responses in terms of ascorbyl palmitate conversion
TrialTemperature (°C)Mole ratio (MR)a
Experimental conversion (%)Predicted conversion (%)RED (%) RED ¼
??? ? 100
The fixed parameters were the enzyme concentration (5 wt.%), the solvent volume (10 mL), the stirring rate (150 rpm), and the reaction time
RED relative error deviation
aAscorbic acid:palmitic acid
(2) Enzyme concentration (wt%)
(3) Solvent volume (mL)
(4) Molar ratio
Effect Estimate (Absolute Value)
Fig. 1 Pareto chart of the
effects of all independent
studied variables on the ascorbyl
palmitate production (p<0.05).
Experimental data and
conditions shown in Table 2
1072Food Bioprocess Technol (2012) 5:1068–1076
Humeau et al. (1998) obtained 15 g/L (56%) of product
in 8 h using 1:9 mole ratio of ascorbic acid to palmitic acid,
Novozym 435 in 20 mL of t-amyl alcohol, 55 °C, and
400 rpm. Song et al. (2004) evaluated the production of
ascorbyl oleate and ascorbyl linoleate by enzymatic
esterification using Novozym 435 and obtained yields of
38% and 44% in 12 h of reaction, respectively.
Novozym 435 was found to be selective towards long
chain fatty acids (FA). In addition, C18 unsaturated FA
gave better yield as compared to C18 saturated ones
(Bradoo et al. 1999).
Earlier reports of enzymatic synthesis of ascorbyl
palmitate in t-amyl alcohol or t-butanol reported yields of
only 6% (Humeau et al. 1998) or 14% (Song et al. 2006)
using a 1:1 mole ratio of ascorbic acid and acyl donor.
Bradoo et al. (1999) reported a 50% yield in hexane when
using equimolar amounts of ascorbic and palmitic acids
catalyzed by Bacillus stearothermophilus lipase without
Hsieh et al. (2006) carried out the production of ascorbyl
palmitate by surfactant-coated lipase and free lipase and
observed that higher conversions were achieved (47%)
using molecular sieves in 24 h, 50 °C, and mole ratio of
1:6. The use of free lipase, on the other hand, leads to
conversions of only 6%.
Burham et al. (2009) investigated the synthesis of palm-
based ascorbyl esters and concluded that the reaction was
strongly influenced by type of immobilized lipase used,
percentage of organic solvent, substrates mole ratio,
reaction time, enzyme, and molecular sieve loading.
Conversions of about 70% were obtained after 16 h of
reaction and 40 °C.
Kinetic Study of Enzymatic Production of Ascorbyl
The effects of ascorbic acid to palmitic acid mole ratio,
temperature, enzyme concentration, and solvent volume
were investigated on the kinetics of ascorbyl palmitate
Effect of Acid to Alcohol Mole Ratio
To evaluate the effect of ascorbic to palmitic acid mole ratio
on ascorbyl palmitate conversion, temperature was fixed at
70 °C, enzyme concentration at 5 wt.%, 10 mL of solvent,
and 150 rpm, making possible to build the experimental
curves of conversion versus reaction time, as presented in
Fig. 3a and b. These subpanel figures also present the
empirical kinetic model fitting obtained using the proposed
hybrid neural network approach.
From these subpanel figures, one can verify that an
enhancement on mole ratio led to higher conversions.
Using mole ratio of 1:7, higher conversions were obtained
(58.74% and 60.03%, respectively, for reaction times of 12
and 17 h). These results are in agreement with that obtained
in the experimental design that pointed out the mole ratio of
1:9 as being the most appropriate for this reaction system.
Similar results were observed by Burham et al. (2009),
where higher conversions (about 70%) in palm-based
ascorbyl esters were obtained at mole ratio of 1:8.
However, for mole ratio value of palm oil higher than 8,
reduction in production of palm based ascorbyl esters was
Chang et al. (2009) studied the enzymatic esterification
of ascorbyl laurate and observed that an enhancement on
enzyme concentration and substrates mole ratio conducted
to conversion of about 80% (180 rpm, 45 °C, 3 mL of
acetonitrile, 6 h).
Lv et al. (2007) obtained the maximum conversion in
ascorbyl benzoate (79.48%) at a mole ratio of 10:1, and
higher mole ratios were not tested due to the limitation on
solubility. This result indicated that an excess of L-ascorbic
acid has a stronger promotion effect on the esterification
than an excess of benzoic acid.
Hsieh et al. (2006) evaluated the effect of the substrates
mole ratio for the synthesis of ascorbyl palmitate by
surfactant-coated lipase and observed higher conversions
(47%) at mole ratios of 1:6, 24 h, and 50 °C.
The substrates mole ratio is usually one of the most
important parameters in enzymatic esterification reactions.
Since the reaction is reversible, an enhancement on the
concentration of one substrate (particularly, the palmitic
acid) can displace the chemical equilibrium, resulting in
higher conversions. On the other side, high palmitic acid
concentrations may reduce the reaction rate due to the
Fig. 2 Response surface for ascorbyl palmitate production as a
function of temperature and ascorbic acid to palmitic acid mole ratio.
Experimental data and conditions shown in Table 2
Food Bioprocess Technol (2012) 5:1068–10761073
inhibition effect (Burham et al. 2009; Chang et al. 2009; Lv
et al. 2007; Hsieh et al. 2006).
Effect of Enzyme Concentration
The effect of enzyme concentration on ascorbyl palmitate
conversion was evaluated at 70 °C keeping the ascorbic to
palmitic acid mole ratio constant at 1:9, 10 mL of solvent,
and 150 rpm, varying the enzyme concentration of 1, 10,
15, and 20 wt.% (based on the substrates amount).
Figure 3c shows the experimental data and kinetic
modeling results obtained in this step. When using 10, 15,
and 20 wt.% of enzyme, it can be observed that high initial
reaction rates were obtained, leading to high conversions in
Fig. 3 Kinetics of ascorbyl
palmitate production at a ascor-
bic acid to palmitic acid mole
ratio of 1:5 and 1:7, b ascorbic
acid to palmitic acid mole ratio
of 1:1 and 1:3 (*validation run),
c enzyme concentration of 15
and 20 wt.%, d enzyme
concentration of 1 and 10 wt.%
(*validation run), e temperature
of 60 and 70 °C, f solvent
volume of 20 and 50 mL. The
reaction was carried out at
70 °C, ascorbic acid to palmitic
acid mole ratio of 1:9, 10 mL of
solvent, and 150 rpm, except for
when the kinetic study of the
referred variable was performed
1074Food Bioprocess Technol (2012) 5:1068–1076
short reaction times. The use of 10, 15, and 20 wt.% of
Novozym 435 does not present significant difference on
ascorbyl palmitate production. On the other hand, at the
experimental condition with 1 wt.% enzyme concentration,
low initial reaction rate was observed, reaching 34.94% of
conversion after 6 h of reaction.
Comparing Fig. 3c and d one can observe that best
conversions were reached with 15 wt.% of enzyme and fast
reaction rates were obtained, resulting in high conversions
with relatively short reaction times.
A possible explanation might be related to the fact that
an excess of enzyme in the reactional medium could not
contribute to the conversion enhancement, since high
enzyme concentration may form aggregates, thus not
making the enzyme active site available to the substrates.
The enzyme molecules on external surface of such particles
are exposed to high substrate concentrations, but the mass
transport could drastically limit the substrate concentration
inside the particles. Lower activities of the biocatalyst
reduce the efficiency of the enzyme, not enhancing the
reaction conversion (Karra-Châabouni et al. 2006).
Burham et al. (2009) evaluated the enzymatic synthesis of
palm-based ascorbyl esters reaching a production of 45 g/L
at 12 wt.% of enzyme, the reaction mixture composed of
1.5 mmol ascorbic acid, 12 mmol palm oil, 40 mg molecular
sieve, 0.5 mL tert-amyl alcohol, at 40 °C for 18 h.
Effect of Temperature
To evaluate the effect of temperature (60 and 70 °C) on
ascorbyl palmitate conversion, the mole ratio of ascorbic to
palmitic acid was kept fixed at 1:9, enzyme concentration at
5 wt.%, 10 mL of solvent, and 150 rpm, making possible to
follow the course of the reaction conversion, as presented in
Fig. 3e. This figure also shows the good results obtained
with the proposed empirical kinetic model.
From this figure, it can be observed that at 70 °C, in 18 h of
At temperatures of 60 and 70 °C similar behaviors were
observed.An experimentcarried outat 80°C (data notshown)
permitted us to verify that high initial rates were observed, but
after a certain period of time, the conversion was diminished,
probably related to the lost of enzymatic activity and
degradation of the ascorbyl palmitate by high temperature.
Lv et al. (2007) carried out a study of the role of the
temperature on ascorbyl benzoate production and observed
that the conversion enhanced with the temperature until
70 °C but decreased in higher temperatures, results similar
to that obtained in the present work.
Temperature presents two important roles in this kind of
reactional system. Firstly, an increase in temperature can
reduce mixture viscosity, enhance mutual solubility, and
improve diffusion process of substrates, thus reducing mass
transfer limitations and favoring interactions between
enzyme particles and substrates. Furthermore, enzymes
generally have an optimal working temperature value, and
in the case of Novozym 435, it is situated in the range of 40
to 70 °C (Kristensen et al. 2005).
Song and Wei (2002) evaluating the effect of the
temperature on the biosynthesis of the vitamin C ester
verified that higher conversions (18.5 g/L) were obtained at
temperatures of 55 °C.
Finally, it may be relevant to mention that measurements
of enzyme activity before (fresh) and after (used) reaction
experiments (data not shown) revealed no important changes
in residual lipase activity at temperatures of 60 and 75 °C.
Effect of Solvent Volume
The effect of solvent volume on ascorbyl palmitate
conversion was evaluated at 70 °C keeping the ascorbic
acid to palmitic acid mole ratio constant at 1:9 and
150 rpm, varying the solvent volume of 20, 30, 40, and
50 mL. The kinetic experimental and model curves
obtained at this step are presented in Fig. 3f.
We can observe from this figure that higher initial rate
(12.13 min−1) was obtained when using 20 mL of solvent.
The use of 30, 40 (data not shown), and 50 mL of solvent
led to lower conversions, probably due to the lost on
enzymatic activity provoked by an excess of solvent.
Figure 3 shows that the hybrid model proposed in this
work was able to satisfactory describe the kinetics of the
ascorbic acid esterification.
The optimum production were achieved at ascorbic acid to
palmitic acid mole ratio of 1:9, stirring rate of 150 rpm, 70 °C,
an ascorbyl palmitate conversion of about 67%. Reaction
conversions ofabout56%couldbeachievedin6hof reaction.
The empirical kinetic model proposed showed ability to
satisfactory represents and predict the experimental data.
financial support and scholarships.
The authors thank CNPq and CAPES for the
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