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D-isoascorbyl palmitate: Lipase-catalyzed synthesis, structural characterization and process optimization using response surface methodology

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Isoascorbic acid is a stereoisomer of L-ascorbic acid, and widely used as a food antioxidant. However, its highly hydrophilic behavior prevents its application in cosmetics or fats and oils-based foods. To overcome this problem, D-isoascorbyl palmitate was synthesized in the present study for improving the isoascorbic acid's oil solubility with an immobilized lipase in organic media. The structural information of synthesized product was clarified using LC-ESI-MS, FT-IR, 1H and 13C NMR analysis, and process parameters for high yield of D-isoascorbyl palmitate were optimized by using One--factor-at-a-time experiments and response surface methodology (RSM). The synthesized product had the purity of 95% and its structural characteristics were confirmed as isoascorbyl palmitate by LC-ESI-MS, FT-IR, 1H, and 13C NMR analysis. Results from "one--factor-at-a-time" experiments indicated that the enzyme load, reaction temperature and D-isoascorbic-to-palmitic acid molar ratio had a significant effect on the D-isoascorbyl palmitate conversion rate. 95.32% of conversion rate was obtained by using response surface methodology (RSM) under the the optimized condition: enzyme load of 20% (w/w), reaction temperature of 53[degree sign]C and D- isoascorbic-to-palmitic acid molar ratio of 1:4when the reaction parameters were set as: acetone 20 mL, 40 g/L of molecular sieves content, 200 rpm speed for 24-h reaction time. The findings of this study can become a reference for developing industrial processes for the preparation of isoascorbic acid ester, which might be used in food additives, cosmetic formulations and for the synthesis of other isoascorbic acid derivatives.
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RES E AR C H A R T I C L E Open Access
D-isoascorbyl palmitate: lipase-catalyzed
synthesis, structural characterization and process
optimization using response surface methodology
Wen-Jing Sun
1,2,3*
, Hong-Xia Zhao
1
, Feng-Jie Cui
1,2*
, Yun-Hong Li
1
, Si-Lian Yu
2,3
, Qiang Zhou
2,3
, Jing-Ya Qian
1
and Ying Dong
1
Abstract
Background: Isoascorbic acid is a stereoisomer of L-ascorbic acid, and widely used as a food antioxidant. However,
its highly hydrophilic behavior prevents its application in cosmetics or fats and oils-based foods. To overcome this
problem, D-isoascorbyl palmitate was synthesized in the present study for improving the isoascorbic acids oil
solubility with an immobilized lipase in organic media. The structural information of synthesized product was
clarified using LC-ESI-MS, FT-IR,
1
H and
13
C NMR analysis, and process parameters for high yield of D-isoascorbyl
palmitate were optimized by using Onefactor-at-a-time experiments and response surface methodology (RSM).
Results: The synthesized product had the purit y of 95% and its structural characteristics were confirmed as
isoascorbyl palmitate by LC-ESI-MS, FT-IR,
1
H, and
13
C NMR analysis. Results from onefactor-at-a-time experiments
indicated that the enzyme load, reaction temperature and D-isoascorbic-to-palmitic acid molar ratio had a
significant effect on the D-isoascorbyl palmitate conversion rate. 95.32% of conversion rate was obtained by using
response surface methodology (RSM) under the the optimized condition: enzyme load of 20% (w/w), reaction
temperature of 53°C and D- isoascorbic-to-palmitic acid molar ratio of 1:4 when the reaction parameters were set
as: acetone 20 mL, 40 g/L of molecular sieves content, 200 rpm speed for 24-h reaction time.
Conclusion: The findings of this study can become a reference for developing industrial processes for the
preparation of isoascorbic acid ester, which might be used in food additives, cosmetic formulations and for the
synthesis of other isoascorbic acid derivatives.
Keywords: Isoascorbyl palmitate, Enzymatic synthesis, Structural characteristic, Response surface methodology,
Optimization
Background
D- isoascorbic acid (synonyms: Erythorbic acid) is a stereo-
isomer of ascorbic acid (Vitamin C). It is a novel food anti-
oxidant and preservative with excellent safe performance
[1]. D- isoascorbic acid can prevent the food oxidation, in-
hibit the decrease of color, aroma and flavors, and block
the production of the carcinogen ammonium nitrite during
food manufacturing process. It had been classified as gen-
erally recognized as safe (GRAS) additives by US Food and
Drug Administration (FDA). Now it can be used in
processed foods in accordance with Good Manufacturing
Practice (GMP) [2]. D-isoascorbic acid is freely soluble in
water. However, its highly hydrophilic behavior similar
with ascorbic acid prevents its application in cosmetics or
fats and oils-based foods [3]. Esterification process of
converting ascorbic acid to its acid esters has been regarded
as an effective solution for overcoming such problems. Fur-
thermore, the esterified ascorbic acid products also have bi-
functional activity including its original antioxidant activity
and the bioactivity of the connected group. For example,
the biosynthesized ascorbyl benzoate owned the antioxi-
dant and antimicrobial/ antifungal activities from original
ascorbic acid and connected benzoic acid group [4]. And
* Correspondence: sunwenjing1919@163.com; fengjiecui@163.com
1
School of Food and Biological Engineering, Jiangsu University, Zhenjiang
212013, P.R. China
2
Jiangxi Provincial Engineering and Technology Center for Food Additives
Bio-production, Dexing 334221, P.R. China
Full list of author information is available at the end of the article
© 2013 Sun et al.; licensee Chemistry Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Sun et al. Chemistry Central Journal 2013, 7:114
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the fatty acid ester of ascorbic acid also has the antioxidant
and surfactant functions with its potential application in
high-fat food and cosmetics [5-7]. As for the isoascorbic
acid, an erythorbyl fatty acid ester of erythorbyl laurate had
been recently synthesized for improving the lipophilicity
[8]. However, other erythorbyl fatty acid esters are still
needed for enlarging its application fields, especially in oil
& fat foods.
Oil-soluble ascorbic acid derivatives can be prepared by
enzymatic or chemical synthesis [9-11]. For the chemical
esterification process, a strongly corrosive acid including
hydrogen fluoride or sulfuric acid is used as a catalyst,
which results in a series of disadvantages, for example, for-
mation of many side-products and high energy consump-
tion [12]. Enzymatic synthesis is preferred because of its
advantages-high catalytic efficiency, mild reaction condi-
tion, and inherent selectivity of the natural catalyst [12-15].
As for isoascorbic acid industry, development of its ester
products is attractive for enlarging the application fields of
oil foods, cosmetics and pharmaceuticals. Furthermore,
other erythorbyl fatty acid esters are still needed for in-
crease its application fields. Optimizating the reaction pa-
rameters for esterification reaction plays an important
role for maximum yield and economical production of
isoascorbyl palmitate. V arious statistical optimization tech-
niques such as response surface methodology (RSM) with
Central Composite Rotatable design (CCRD), Box-Behnken
or uniform design method had been applied for ascorbyl
palmitate sysnthesis [13], L-ascorbyl laurate [16], ascorbyl
oleate [17] and L-ascorbyl lactate [18]. However, there have
been no detailed reports on the effects of the reaction pa-
rameters on isoascorbic esters production till now.
The objectives of this study were to: (1) synthesize
an oil-soluble isoascorbic acid palmitate by
enzymatic method in an organic solvent system, (2)
clarify the structural information using LC-ESI-MS,
FT-IR,
1
H and
13
C NMR analysis, (3) evaluate the
key reaction parameter for D-isoascorbyl palmitate
process, and (4) optimize the reaction parameters
for maximum conversion rate of D-isoascorbyl
palmitate using response surface methodology.
Results and discussion
Identification of isoascorbic acid and its esters by LC-MS
Figure 1 was the schematic diagram of D-isoascorbyl
palmitate catalyzed by lipase in organic media. To deter-
mine the production yield of the lipase- catalysed esterifi-
cation between palmitate acid and isoascorbic acid, the
wave full scan was conducted at the diode array detector
from 180 nm to 1000 nm to select the optimal determining
wavelength for the samples. Results showed that the ab-
sorbance of isoascorbic acid and isoascorbyl palmitate had
the maximum level when the wavelength was set as 254
nm. Thus the HPLC analysis was conducted at the wave-
length of 254 nm. Figure 2 showed the HPLC chromato-
graph of the reaction solution samples with ultraviolet
detector. Isoascorbic acid and isoascorbyl palmitate peaks
separated well with the retention times of 1.44 and 7.36
min, respectively. The mass spectra of LC-MS indicated
thatthesamplehadthemass-to-chargeratiosofthemo-
lecular ion peak (M -H) of 413.35 and (2M - H) of 827.00,
whileD-isoascorbylpalmitateshouldhaveamass-to-charge
ratio of 414 (M), which proved that the synthesized sample
is D-isoascorbyl palmitate
O
O
HO
OH
HO
HO H
O
O
OH
OH
HO
H
HO
O
O
O
+
Erythorbic acid
Palmitic acid
Er
y
thorbl
yp
almitate
+
H2O
Immobilized lipase Novozym 435
Figure 1 The scheme of lipase-catalysed synthesis of D-isoascorbyl palmitate.
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Structural characteristic analysis of the synthesized
D-isoascorbyl palmitate
The FT-IR spectrum for the sample isoascorbyl palmitate
was presented in Figure 3. The band in the region of 3423
cm
-1
is due to the hydroxyl stretching vibration. The band
in the region of 2930 cm
-1
and 2850 cm
-1
are due to C-H
stretching vibration in CH
2
and 1711 cm
-1
is the absorption
of C=O stretching vibration. Absorption at 1659 cm
-1
was
typical for dual bond C = C in isoascorbic acid. The band
of 1470 cm
-1
was characteristic absorption of CH
3
.The
characteristic absorption at 1341 cm
-1
, 1225 cm
-1
, 1151 cm
-1
,
1110 cm
-1
and 1054 cm
-1
in the FT-IR spectrum was indi-
cative of C-O-C linkage in the isoascorbyl palmitate while
the absorption at 721 cm
-1
also indicated the presence of
linked palmitic acid.
1
H,
13
CNMRspectraweredeterminedasfollows
(Figure 4),
1
H NMR (400 MHz,DMSO-d6):δ (ppm):11.258
(s,1H,-OH), 8.467 (s,1H,-OH), 5.577(s,1H,-OH), 4.739 (d,1H,
-CH,J=1.6 Hz), 4.017(m,3H,-OCH2-OH), 2.279 (t,2H,-CH2CO,
J = 7 . 6 H z ) , 1. 5 0 4 (t , 2 H , -CH2- , J = 6. 8 Hz), 1.236 (m,2H,12-CH2-),
0.855 (t,3H,-CH3,J=7.2 Hz).
13
C NMR(400MHz ,DMSO-d6):δ (ppm):(173.21 (C-
1=O), 170.60 (C-1'=O), 152.96 (C-2), 118.74 (C-3), 76.62
(C-4), 68.07(C-5), 63.84(C-6), 34.11 (C-2'), 33.80 (C-3'), 31.77-
28.94 (C-4'-C12'), 24.95 (C-13'), 24.81 (C-14'), 22.57 (C-15'),
14.39 (C-16').
Figure 2 HPLC-PDA chromatogram for the components obtained from the immobilized lipase-catalysed esterification between
isoascorbic acid and palmitic acid.
Figure 3 FT-IR spectra of isoascorbyl palmitate synthesized by lipase.
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The
13
C NMR spectrum of isoascorbyl palmitate showed
the carbonyl group at C-1 and double bonds between C-2
and C-3 in isoascorbic moiety were intact which indicated
that the enzymatic reaction happened in other position.
The C-6 signal at 65.6 ppm in the synthesized isoascorbyl
ester had a down-field shift of 3.9 ppm in comparison with
that of isoascorbic acid (61.7 ppm). These results proved
thepresenceofanesterbondonC-6of the isoascorbyl
moiety and correspond with the pattern of chemical shift
reported by Park et al. [8] and Stamatis et al. [19].
Figure 4
1
H (a) and
13
C NMR (b) spectra of isoascorbyl palmitate synthesized by lipase in present study (400 MHz, DMSO-d6).
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One-factor-at-a-time experiments for isoascorbyl
palmitate synthesis process
Effect of lipase source on D-isoascorbyl palmitate synthesis
Lipases (E.C. 3.1.1.3) generally catalyze the hydrolysis of
oils and fats [20,21]. Under specific conditions , they also
catalyze the hydrolysis reactions in organic solvents by
direct esterification with free acid, transesterification,
acidolysis, alcoholysis and aminolysis [22,23]. The li-
pases sourc es ha d the difference in structure including
the lid region structure which affe cted the catalytic ac-
tivity, regioselectivity and stereoselectivity.
All the lipases used in the present study were listed in
Table 1 with their optimum catalytic activities given from
the providers. The screening experiments were conducted
under a preliminary set of reaction conditions that may
not have been the optimum set for all the lipases. In a typ-
ical reaction, 150 mg of immobilized derivative was added
to the mixture of D-isoascorbic acid: palmitic acid at 1:4
molar ratio using 2-methyl-2-butanol as solvent. Results
obtained showed that Novozym 435 had the highest cata-
lytic efficiency with the conversion rate of 41.3% (m/m),
which was in accordance with previous reported results
[24,25]. Using RMIM from Rhizomucor miehei,hada
lower performance of conversion (15.2%). However, other
lipases of LVK-H100 and LBK-B400 had no catalytic effect
on the D-isoascorbyl palmitate synthesis. Hence, Novozym
435 from Candida antarctica was screened as a catalyst
for the D-isoascorbyl palmitate lipase-catalyzed synthesis.
Effect of reaction medium source on D-isoascorbyl
palmitate synthesis
A nonaqueous solvent is essential for lipase synthesis of
fatty acid esters. A suitable solvent must be able to dissolve
sufficient amounts of both the substrates, i.e. D-isoascorbic
acid and palmitic acid. The hydrophobicity of the organic
solvent significantly influenced the catalytic power of en-
zyme by changing the three dimensional conformation of
protein, and therefore significantly alters conversion and
rate [26-28]. The log P value, defined as logarithm of the
partition coefficient of a given compound in the standard
two phase system of octano/water, ha s been the most
commonly used to express solvent e ffe ct on the a ctivity
and /or stability of enzymes. Differences in solvent log P
have been widely used to explain their effect on the
catalytic activity and enzymes spe cificity [29]. A series of
solvents, such as ethanol, acetone, chloroform, tert-amyl al-
cohol, n-hexanol and petroleum ether with the log P value
from 0.24 to 3.53 were used for D-isoascorbyl palmitate
synthesis. The conversion rates of D-isoascorbyl palmitate
were shown in Table 2. Among all the solvents, acetone
with the log P value of 0.23 gave the highest molar con-
version (57.8%). A slightly lower performance was achieved
in 2-methyl-2-butanol (log P = 1.31) (molar conversion =
49.6%). However, ethanol (log P = 0.24), chloroform (log
P = 2.0), and petroleum ether (log P = 2.62) had no
benefits for the proposed reaction. These obtained results
were somewhat inconsistent with general reports that sol-
vents with log P < 2 are less suitable for biocatalysis [30,31].
2-Methyl-2-butanol is a choice as the reaction solvent for
ascrobyl palm ester production with a high conversion from
70 to 75%. However, 2-Methyl-2-butanol has the higher
price and toxicity comparing with other solvents including
acetone [32]. In conclusion, acetone was selected as the re-
action medium for the D-isoascorbyl palmitate synthesis in
the following experiments.
Influence of enzyme load on D-isoascorbyl palmitate
synthesis
The immobilized lipase load volume directly influences
the rate and efficiency of the esterification reaction. In
the present study, lipase Novozym 435 load varying from
0 to 30% (weight % of substrates) was used (Figure 5).
From Figure 5, no D-isoascorbyl palmitate was synthe-
sized when the catalyst Novozym 435 was absent. The
convention rate increased from 28.79% to the maximum
level of 72.05% with the increase of Novozym 435 load
from 1% to 15% (w/w). However, further increase of en-
zyme (above 15%) load declined conversion ratio to
55.23%. This may be contributed to the high amount of
immobilized enzyme was added, especially in the solv ent
Table 1 Influence of the lipase source on the synthesis of D- isoascorbyl palmitate
Lipase Origin Immobilized
matrixe
Effective
temperature (°C)
Specific
activity
Water
content
Conversion
rate (%)
a
Novozyme 435 Candida antarctica Macroporous acrylic resin 40-60 10,000PLU/g
b
1-2% 41.30 ± 2.6
Lipozyme TLIM Thermomyces lanuginosus Silica granulation 55-70 250IUN/g
c
5% 4.30 ± 1.9
Lipozyme RMIM Rhizomucor miehei Anionic exchange resin 30-70 5-6BAUN/g
d
2-3% 15.20 ± 3.5
LVK-H100 Aspergillus nige 15-45 20,000U/g 0
LBK-B400 Aspergillus nige 25-65 30,000U/g 0
a: Reaction conditions: D-Isoascorbic 2.5 mmol palmitic acid 10 mmol (Molar ratio was 1:4), lipase load: 15% (weight % of substrates), temperature 50°C, tert-amyl
alcohol 20 mL, 50 g/L molecular sieve 4 Å and 200 rpm speed for 24 h.
b: PLU is based on a reaction between propyl alcohol and lauric acid.
c: Interesterification Unit ( IUN) is international unit, based on tributyrin assay.
d: Batch Acidolsis Units Novo (BAUN) is based on a reactionbetween high oleic sunflower oil and decanoic acid at 70- 80°C for 60 min.
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system, the viscosity of the reaction medium was in-
creased and then further led to the less effective transfer
of the substrates to the active sites of the excess enzyme
molecules inside the bulk of enzyme particles [33,34].
Similar results also were previous reported by Sun et al.
[35] by obtaining maximum transesterification yield of
coconut oil with fusel alcohols when the immobilized
lipase TLIM loading volume was 15% (w/w). Thus,
immobilized lipase load of 15% w/w) appeared to be the
optimal for D-isoascorbyl palmitate synthesis.
Effect of reaction time on D-isoascorbyl pal mitate synthesis
To check the highest efficiency of D-isoascorbyl palmi-
tate synthesis, the time course of the esterification of D-
isoascorbic and palmitic acid catalyzed by the Novozy m
435 was monitored. Results were shown in Figure 6. The
conversion rate increased rapidly to 80.09% during the
24-h reaction, and then possibly reached to the stable
level. For the palm-based ascorbyl est ers synthesis, the
rapid reaction time was 16-h [32]. Although, maximum
conversion ratio of 81% was finally achieved after 36-h
synthesis, increase in reaction time also led to a decrease
in the reactor working efficiency, which is not econom-
ical. For this study, 24-h reaction time was selected.
Effect of reaction temperature on D-isoascorbyl palmitate
synthesis
Reaction temperature had the direct influence of the sta-
bility and the activity of the lipase, the solubility of the
substrates, the rate of the reaction and the position of
the reaction equilibrium [36]. In order to understand the
influence of temperature on the D-isoascorbyl palmitate
synthesis, the reaction with 2.5 mmol of D-isoascorbic
acid and 10 mmol of palmititic acid (Molar ratio was
1:4) loading 15% of Novozym 435 was conducted at five
temperatures ranging from 30°C to 70°C (Figure 7). The
conversion was significantly af fected by the temperature
(P < 0.01). The maximum conversion rate of 82.05%
was obtained at 50°C after 24-h of reaction. The in-
crease of temperature to 60°C inhibited the enzyme ca -
talysis process with the conversion rate of 69.01%. From
the Figure 7, Novozym 435 had no catalytic activity when
the te m pe rat u r e was set as 70°C with no D-is o as c o r by l
palmitate production. This result was in consistence with
those previously reported that Novozym 435 to be active in
nonaqueous systems (organic solvents, solvent-free system,
supercritical fluid) at temperatures of 40-60°C [37]. There-
fore, 50°C appeared to be the optimal temperature for D-
isoascorbyl palmitate production by using Novozym 435 as
the catalyst.
Effect of substrate molar ratio on D-isoascorbyl palmitate
synthesis
The influence of six substrate molar ratios of D-isoascorbic
to palmitic acid, ranging from 1:1 to 1:10 (m/m), on D-
isoascorbyl palmitate production performance was investi-
gated. As shown in Figure 8, the conversion rate increased
Table 2 Influence of the organic solvent on the synthesis
of D- isoascorbyl palmitate
Solvent Log P Conversion rate (%)
a
Ethanol 0.24 0
Acetone 0.23 57.8 ± 1.8
2-Methyl-2-butanol 1.31 49.6 ± 2.3
Chloroform 2 0
Petroleum ether 2.62 0
N-hexane 3.53 25.28 ± 3.9
a: D-isoascorbic 2.5 mmol palmitic acid 10 mmol (Molar ratio was 1:4),
Novozym 435 load: 15% (weight % of substrates), temperature 50°C, 50 g/L
molecular sieve 4 Å and 200 rpm speed for 24 h.
0
20
40
60
80
100
0 5 10 15 20 25 30
Conversion rate(%)
Enz
y
me load (%)
Figure 5 Effect of enzyme load (weight % of substrates) on lipase-catalyzed synthesis of D-isoascorbyl palmitate. (Temperature: 50°C;
fermentation time: 24 h; molar ratio: 1:4; acetone 20 mL; 4 Å molecular sieves content: 50 g/L; speed: 200 rpm).
Sun et al. Chemistry Central Journal 2013, 7:114 Page 6 of 13
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substantially from 16.66 to 89.21% when substrate molar ra-
tio increased from 1:1 to 1:6 (m/m)(P<0.001). Further in-
creases in molar ratio (beyond 1:6) had no decreased effect
on the isoascorbyl palmitate production, which may contrib-
ute to the inhibitory effect of high acid concentration on en-
zyme activity [37]. The same effect was also observed in
another study in which oleyl oleate production was investi-
gated using Novozym 435 in a solvent- free system [37,38].
In the present study, substrate molar ratio of 1:6 was opti-
mal for isoascorbyl palmitate production with the highest
conversion rate of 84.21%, and used in the following tests.
Effect of molecular sieves content on D-isoascorbyl
palmitate synthesis
The ester formation process requires low water content.
Lipase catalysis needs a minimal amount of water to ensure
its optimal conformation and optimal activity. However,
excess of water also negatively decreases the enzyme activ-
ity from kinetic and thermodynamic points. Hence, during
the lipase-catalyzed synthesis process, removal of the water
by using pervaporation or microwave irradiation was un-
realistic. Addition of a desiccant such as molecular sieves is
an effective method due to the low cost and easy to be sep-
arated and regenerated [36]. For the D-isoascorbyl palmi-
tate synthesis reaction, molecular sieves had the use of
drying the reaction mixture and adsorbing the produced
water to shift the reaction equilibrium. To evaluate the ef-
fect of molecular sieves on the conversion performance of
isoascorbyl palmit ate, 4 Å molecular sieves volume varying
from 0 to 100 g/L were added. As shown in Figure 9, lowest
conversion rate of 8.64% was obtained without adding 4 Å
molecular sieves. A gradual increase up to maximum D-
isoascorbyl palmitate conversion rate of 81.31% was ob-
served with the increase of the molecular sieve content to
0
20
40
60
80
100
0 3 6 9 12 15 18 21 24 27 30 33 36
Conversion rate(%)
Reaction time (h)
Figure 6 Effect of time course on lipase catalyzed synthesis of D-isoascorbyl palmitate. (Enzyme load 15% (weight % of substrates);
temperature: 50°C; molar ratio: 1:4; acetone 20 mL; 4 Å molecular sieves content: 50 g/L; speed: 200 rpm).
Conversion rate(%)
0
10
20
30
40
50
60
70
80
90
100
30 40 50 60 70
Tem
p
erature(
)
Figure 7 Effect of temperature on lipase-catalyzed synthesis of D-isoascorbyl palmitate. (Enzyme load 15% (weight % of substrates); time:
24 h; molar ratio: 1:4; acetone 20 mL; 4 Å molecular sieves content: 50 g/L; speed: 200 rpm).
Sun et al. Chemistry Central Journal 2013, 7:114 Page 7 of 13
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40 g/L. Further increases in molecular sieves content (be-
yond 40 g/L) had the negative effect on the isoascorbyl
palmitate production. The conversion rate decreased to
65.25% when 4 Å molecular sieves content was 80 g/L.
Similar results were also obtained by He et al. [39] that the
higher molecular sieves concentration up to 80 g/L would
result in the lower conversion rate about of 40% by de-
creasing the activity of lipase. Based on these obtained re-
sults, 40 g/L of 4 Å molecular sieves content was used for
subsequent experiments in the synthesis of D-isoascorbyl
palmitate.
Response surface optimization
The key parameters including the enzyme load, reaction
temperature and molar ration, significantly influencing
on the conversion rate of D-isoascorbyl palmitate were
obtained based on the one-factor-at-a-time(OFAT )
experiments, which by changing one factor at a time, and
keeping other variables constant. Tables 3 and 4 gave the
factors, their values, and the experimental design, respect-
ively. Other reaction parameters are set as follows, 20 mL
of acetone 40 g/L of molecular sieves content , 200 rpm of
rotation speed for 24-h during the course of optimization
experiments. Table 3 showed that the considerable variation
in the conversion rate of D-isoascorbyl palmitate under dif-
ferent reaction composition. The isoascorbyl palmitate
conversion rate ranged from 37.07% to 93.28%, and the
run #10 and #1 had the minimum and maximum ratio
values, respectively.
Model fitting
Table 5 showed the analysis of variance (ANOVA) for
this experiment, and the coefficient of determination
(R
2
) was shown as 97.34%. This indicated that, the ac-
curacy and general ability of the polynomial model was
good, analysis of the response trends using the model
was considered to be reasonable. A precision ratio of
15.79 indicates an adequate signal. A ratio greater than 4
is desirable. The relatively low coefficient of variation
value (CV=6.15%) indicated the good precision and
0
10
20
30
40
50
60
70
80
90
100
1246810
Molar ratio(D-isoascorbic to
p
almitic acid)
Conversion rate(%)
Figure 8 Effect of molar ratio (D-isoascorbic to palmitic acid) on lipase-catalyzed synthesis of D-isoascorbyl palmitate. (Enzyme load
15% (weight % of substrates); temperature: 50°C; time: 24 h; acetone 20 mL; 4 Å molecular sieves content: 50 g/L; speed: 200 rpm).
0
10
20
30
40
50
60
70
80
90
100
020406080100
Conversion rate (%)
Molecular sieve content(
g
/L)
Figure 9 Effect of molecular sieves on lipase catalyzed
synthesis of D-isoascorbyl palmitate. (Enzyme load 15% (weight
% of substrates); time: 24 h; molar ratio: 1:6; acetone 20 mL;
temperature: 50°C; speed: 200 rpm)
Table 3 Variables and experimental design levels for
response surface
Independent variables Coded symbols Levels
10 1
Enzyme load(%, w/w) A(X
1
) 5 13 20
Temperature(°C) B(X
2
)405060
Molar ratio(D-isoascorbic: palmitic acid) C(X
3
)246
Sun et al. Chemistry Central Journal 2013, 7:114 Page 8 of 13
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reliability. The regression coefficients, along with the
corresponding P-values, for the model of the conversion
rate of isoascorbyl palmitate, were presented in Table 5.
The P-values are used as a tool to check the significance
of each coefficient, which also indicate the interaction
strength between each independent variable. The smaller
the P values, the bigger the significance of the corre-
sponding coefficient [40]. Table 5 showed that the quad-
ratic model was highly significant (p<0.01). Meanwhile
the lack-of-fit the P values of 0.0027 indicated that the
lack of fit was significan t. Enzyme load and molar ratio
of D-isoascorbic to palmitic acid had a highly linear ef-
fect at 1% le vel. Temperature was also significant at 5%
level. While the interaction effects of independent vari-
ables were found no sign ificant quadratic effect (p-value:
AB=0.2665, BC=0.4343).
Using the designed experimental data (Table 3), the
polynomial model for conversion rate (%) Y
conversion rate
was regressed by only considering the significant terms
and was shown as below:
Y
conversion rate
¼ 84:66 þ 16:90X
1
þ 5:05X
2
þ 8:16X
3
7:15X
1
X
3
1:94X
2
X
3
4:88X
1
2
10:79X
3
2
ð2Þ
Where Y is the response variable (isoascorbyl palmi-
tate conversion rate, %), and X
1
, X
2
and X
3
are enzyme
load, temperature and molar ratio of D-isoascorbic to
palmitic acid, respectively. Figure 10 shows the observed
and predicted conversion rate determined by the model
Eq. (2) which indicated an excellent agreement between
actual and predicted responses.
Mutual effect of parameters and attaining optimum
condition
The response surface and contour plots in Figure 11 show
the main, interaction, and quadratic effect of 2 independ-
ent variables on conversion rate. Figure 11(a, b) shows the
effect of enzyme load (X
1
)andtemperature(X
3
)onthe
conversion rate of isoascorbyl palmitate at the molar ratio
keptataconstantlevel.Itwasobviousthattheconversion
rate of isoascorbyl palmitate was sensitive even when en-
zyme load was subject to small alteration. An increase in
the conversion rate could be significantly improved with
the increase of enzyme load and temperate. Figure 11(c, d)
shows the effect of enzyme load (X
1
)andmolarratio(X
2
)
on the conversion rate of isoascorbyl palmitate at the
temperature kept at a constant level. From the figures, we
can see that a higher conversion rate will obtained using a
high molar ratio of D-isoascorbic to palmitic acid. Figure 11
(e, f) shows the interaction between molar ratio (X
2
)and
temperature (X
3
) on the conversion rate of isoascorbyl
palmitate at the enzyme load kept at a constant level. As it
is shown, the conversion rate increased slightly with in-
creasing temperature from 40°C to 60°C in the range of
molar ratio. The optimal conditions for D- isoascorbyl
palmitate synthesis was obtained by using the Point predic-
tion function of software Design-Expert 7.1.1 to calculate
Table 4 Experimental designs and the results of
Box-Behnken design for optimizing reaction conditions
for the production of D- isoascorbyl palmitate
Runs Coded levels Conversion rate (%)
A B C I II Average Predicted
1 1(20) 1(40) 0(4) 92.70 93.86 93.28 ± 0.82 90.33
2 0(13) 1(60) 1(6) 84.78 85.74 85.26 ± 0.68 80.98
3 1(5) 1(60) 0(4) 62.69 64.63 63.66 ± 1.37 66.61
4 1(5) 0(50) 1(2) 65.89 66.25 66.07 ± 0.25 67.40
5 0(13) 0(50) 0(4) 84.03 84.75 84.39 ± 0.51 84.66
6 1(20) 0(50) 1(2) 86.89 85.53 86.21 ± 0.96 84.88
7 1(20) 0(50) 1(6) 85.78 87.46 86.62 ± 1.19 86.91
8 0(13) 1(40) 1(2) 50.00 50.60 50.30 ± 0.42 54.58
9 0(13) 1(60) 1(2) 70.98 71.38 71.18 ± 0.28 68.53
10 1(5) 0(50) 1(2) 37.08 37.06 37.07 ± 0.01 36.78
11 0(13) 1(40) 1(6) 72.88 71.36 72.12 ± 1.07 74.78
12 1(5) 1(40) 0(4) 53.89 55.75 54.82 ± 1.32 50.83
13 1(20) 1(60) 0(4) 90.22 91.24 90.73 ± 0.72 94.72
14 0(13) 0(50) 0(4) 83.97 86.01 84.99 ± 1.44 84.66
15 0(13) 0(50) 0(4) 85.02 84.20 84.61 ± 0.58 84.66
Table 5 Results of ANOVA analysis of a full second-order
polynomial model for reaction conditions for the
production of D- isoascorbyl palmitate
Source Sum of
squares
df Coefficient
estimate
F-Value P-Value
Model 3798.88 9 422.10 20.35 0.0020**
A 2285.56 1 2285.56 110.17 0.0001**
B 203.11 1 203.11 9.79 0.0260*
C 533.17 1 533.17 25.70 0.0039**
AB 32.43 1 32.43 1.56 0.2665
AC 204.35 1 204.35 9.85 0.0257*
BC 14.98 1 14.98 0.72 0.4343
A
2
87.99 1 87.99 4.24 0.0945*
B
2
63.87 1 63.87 3.08 0.1397
C
2
429.81 1 429.81 20.72 0.0061**
Residual 103.73 5 20.75
Lack of fit 103.55 3 34.52 374.63 0.0027**
Pure error 0.18 2 0.092
Cor total 3902.61 14
R-squared = 0.9734 Adj-Squared = 0.9256 C.V.% = 6.15
** Significant at 1% level * Significant at 5% level Adeq Precision=15.9.
Sun et al. Chemistry Central Journal 2013, 7:114 Page 9 of 13
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maximum level of conversion rate. The maximum conver-
sion rate of D- isoascorbyl palmitate was 96.98% under the
reaction conditions as follows: enzyme load of 20% (w/w),
reaction temperature of 53°C and D- isoascorbic-to-pal-
mitic acid molar ratio of 1:4.
Validation of the model
The availability of the regression model (Eq. (2)) of the
conversion rate of isoascorbyl palmitate was tested using
the calculated optimal condition, viz. acetone 20 mL,
40 g/L of molecular sie ves content, 200 rpm speed, 20%
enzyme load, D- isoascorbic-to-palmitic aci d molar ratio
of 1:4, temperature of 53
o
for 24-h during the course of
optimization experiments. The mean value of the myce-
lial biomass was 95.32 ± 0.17% , which agreed with the
predicted value (96.98%) well that indicated the high val-
idity and adequacy of the model.
Experimental
Materials
D-isoascorbic acid (purity > 99%) was provided from
Parchn Sodium Isovitamin C Co., Ltd (Dexing , Jiangxi,
China). Palmitic acid (purity > 99.5%) was obtained
from Sinopharm Chemical Reagent Co., Ltd (Shanghai,
China). Novozym 435 wa s purchased from Novo
NordiskCo.,Ltd(Beijing,China).LipozymeTLIM,a
lipase from Thermomyce s l anug inosus immobilized on
silicagranulationandLipozymeRMIM,alipasefrom
Rhizomucor miehei, immobilized on an anionic ex-
change resin, also purcha sed from Novo Nordisk Co.,
Ltd (Beijing, China). Lipase LVK-H100 and LBK-B400,
were kindly gifted by Leveking bio-engineering Co.,
Ltd (Shenzhen, China). The properties of all lipa ses are
showninTable1.
2-Methyl-2-butanol, n-hexane, ethanol, chloroform, pet-
roleum ether, acetone and acetic ether were analytical re-
agent grade purchased from Sinopharm Chemical Reagent
Co., Ltd (Shanghai, China). HPLC-grade methanol was
purchased from Tedia, USA. All reagents were dehydrated
by molecular sieve 4 Å (Shanghai world molecular sieve
Co., Ltd., Shanghai, China) for at least 24 h and filtered
using a membrane filter (0.45 μm) prior to use as a reac-
tion medium.
Procedure for lipase-catalysed esterification
D-isoascorbic acid (2.5 mmol), palmitic acid (10 mmol)
and the immobilized lipase (150 mg, about 5% of the sub-
strates amount) were weighed into a 150 mL conical flask.
20 mL of 2-methyl-2-butanol and 1.0 g of molecular sieve
4 Å were then added. The stoppered flasks were shaken at
the speed of 200 rpm on a thermo-constant orbital shaker
at 50°C for 48 h. The sampled reaction mixture was filtered
through a membrane filter (0.45 μm), and 20 μL of each
aliquot were injected into the HPLC for further analyzing
concentrations of the substrate isoascorbic acid and the
produced D-isoascorbyl palmitate.
Purification of produced D- isoascorbyl palmitate
The purification process was conducted according to the
method described by Park et al. [8] and Bradoo et al.
[41] with a slight modification. Briefly, the reaction solu-
tion was filtered with a membrane filter (0.45 μm) to remove
the lipase and molecular sieve. The mixture solution of D-
isoascorbyl palmitate, isoascorbic acid and palmitic acid was
Figure 10 Plot of predicted and observed conversion rate (%) of D-isoascorbyl palmitate.
Sun et al. Chemistry Central Journal 2013, 7:114 Page 10 of 13
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Figure 11 Response surface and 3D contour plots indicating the effect of interaction between reaction parameters on D- isoascorbyl
palmitate conversion rate (a, b) interaction between enzyme load and temperature while holding molar ratio at 4 (c, d) interaction
between enzyme load and molar ratio while holding temperature of 50°C (e, f) interaction between temperature and molar ratio while
holding enzyme load at 13% (w/w).
Sun et al. Chemistry Central Journal 2013, 7:114 Page 11 of 13
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obtained by vacuum evaporating the 2-Methyl-2-butanol,
and resolved in ethyl acetate. The same quota of deonized
water was added for removing the residue isoascorbic acid,
and hexane was used to washing out the palmitic acid. The
insoluble D- isoascorbyl palmitate was then finally obtained
by vacuum drying for 2 h.
Structural analysis
Produced D-isoascorbyl palmitate and residual isoascorbic
acid was identified by mass spectrometry with a quadru-
pole ion trap Thermo Finnigan LXQ LC-ESI-MS (San
Jose, CA , USA) equipped with a degasser, LC-20AD binary
pumps, a model SIL-20AC autosampler, a model CTO-
20A thermostat, an electro-spray ionization (ESI) inter-
face, and a model CBM-20A system controller. FT-IR
spectra with Thermo-Nicolet Nexus 670 Fourier Trans-
form Infrared Spectrometer (San Jose, CA, USA),
1
Hand
13
C NMR spectra with a Bruker AVANCE NMR Spec-
trometer (Switzerland) at 400 MHz.
Products quantification
Produced D-isoascorbyl palmitate and residual isoascorbic
acid were quantitatively analyzed by using a Waters Alli-
ance LC-20AT (SHIMADZU, Japan) liquid chromatog-
raphy connected to a model 2996 (DAD) diode array
detector and controlled by LC Driver Ver.2.0 for Waters
Empower software. The column equipped in the HPLC
system was ZORBAX Eclipse XDB-C18 (150 mm×4.6 mm,
5 μm, Torrance, CA, USA). The mobile phase was
methanol/water (90:10, v/v) at 1.0 ml/min flow rate for
15 min. Samples of 20 μL were injected automatically. The
purity of sample was 95% with a sole peak in the HPLC
chromatograph, which could be used as a standard. Purified
D-isoascorbyl palmitate had the purity of 95% deter-
mining with HPLC (data not shown) as the standards
(0.2, 0.5, 1.0, 1.5, 2.0, and 2.5 g/L) were used to obtain
the D-isoa scorbyl palmitate calibration cur ve. The
conversion rate (%) wa s calculated by dividing the ini-
tial molar amount of D-isoascorbic acid by the pro-
duced molar amount of isoascorbyl palmitate.
Experimental design and evaluation
According to the results of onefactor-at-a-time experi-
ments, which vary only one factor or variable at a time
while keeping others fixed, a response surface method-
ology (RSM) was used to influence of enzyme load (w/w),
temperature and molar ration (D-isoascorbic : palmitic
acid) on the conversion rate of the D-isoascorbyl palmitate
by lipase-catalyzed synthesis. A three factors, three levels
Box-Behnken factorial design was used for fitting a second
order response surface, using the software Design Expert
7.1.1 (Stat-Ease, Minneapolis, MN, USA). All other factors,
for example reaction time, molecular sieves content were
maintained constant. A mathematical model, describing
the relationships between the process indices (the conver-
sion rate of D-isoascorbyl palmitate) and the medium
component contents in second order equation, was devel-
oped. The conversion rate of D-isoascorbyl palmitate was
multiply regressed with respect to the reaction parameters
by the least squares method as follow:
Y ¼ A
0
þ A
i
X
i
þ A
ii
X
2
i
þ A
ij
X
i
X
j
ð1Þ
Where Y is the predicted response variable (conversion
rate, %); A
o
,A
i
,A
ii
,A
ij
are constant regression coeffi-
cients of the model, and X
i
,X
j
(i=1, 3; j=1, 3, ij) repre-
sent the independent variables (reaction parameters) in
the form of coded values. The accuracy and general abil-
ity of the above polynomial model could be evaluated by
the coefficient of determination R
2
.
Conclusions
Isoascorbyl palmitate was successfully synthesized by
using lipase-catalysed esterification of isoascorbic acid
and palmitic acid under the mild reaction conditions. It
structure was characterized by LC-MS, FT-IR,
1
H, and
13
C NMR. The effect of various parameters on synthesis
of D-isoascorbyl palmitate, such as enzyme source, type
of organic, enzyme load, reaction time, temperature, mo-
lecular sieves content and D-isoascorbic-to-palmitic acid
molar ratio were discussed using onefactor-at-a-time
experiments and Response surface methodology. The
optimized condition was obtained a s follow: enzyme
load of 20% (w/w), reaction temperature of 53°C and
D-isoascorbic-to-palmitic acid molar ratio of 1:4. Under
these optimal conditions , 95.32% of conversion rate was
obtained which wa s in agreement with the predicted
value (96.9 8%). The results are of a reference for develop-
ing industrial processes for the preparation of isoascorbic
acid ester, which might be used in food additives, cosmetic
formulations and for the synthesis of other isoascorbic
acid derivatives.
Competing interests
The authors declare that they have no competing interests.
Authors
contributions
W-JS and F-JC conceived of the study, participated in its design and
coordination, and drafted the manuscript. H-XZ performed experiments and
analyzed results and helped to draft the manuscript. Y-HL helped to do
experiments. QZ, S-LY, J-YQ and YD performed partial experiments and
analyzed results. All authors read and approved the manuscript.
Acknowledgements
This work was supported by funding from the National High Technology
Research and Development Program (2012AA022103), China Postdoctoral
Science special Foundation (2013T60648), China Postdoctoral Science
Foundation (2012M511222), 2012 Excellent Key Young Teachers Project of
Jiangsu University, Graduate Research and Innovation Projects of Jiangsu
Province (CX10B_021X, CXLX12_0670), Advanced Programs of Jiangxi
Postdoctoral Science Foundation ([2012]195), the Research Foundation for
Advanced Talents of Jiangsu University and Science & Technology Platform
Construction Program of Jiangxi Province.
Sun et al. Chemistry Central Journal 2013, 7:114 Page 12 of 13
http://journal.chemistrycentral.com/content/7/1/114
Author details
1
School of Food and Biological Engineering, Jiangsu University, Zhenjiang
212013, P.R. China.
2
Jiangxi Provincial Engineering and Technology Center for
Food Additives Bio-production, Dexing 334221, P.R. China.
3
Parchn Sodium
Isovitamin C Co. Ltd, Dexing 334221, P.R. China.
Received: 9 May 2013 Accepted: 4 July 2013
Published: 8 July 2013
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doi:10.1186/1752-153X-7-114
Cite this article as: Sun et al.: D-isoascorbyl palmitate: lipase-catalyzed
synthesis, structural characterization and process optimization using
response surface methodology. Chemistry Central Journal 2013 7 :114.
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... However, immobilizing enzymes on hydrophilic supports may reduce the enzymatic activity and the solubility of hydrophobic substrates [27]. Four different commercial immobilized lipases on distinct supports were examined in this work: Novozym 435 (originated from Candida antarctica lipase B), Lipozyme RM IM (originated from Rhizomucor mehei), Lipozyme TL IM (originated from Thermomyces lanuginosus), and Lipase PS Amano IM (originated from Burkholderia cepacia) ( Table 2) [28][29][30][31][32]. The respective conversion yields of formic acid to phenethyl formate using these lipases were 47.83% (Novozym 435), 0.28% (Lipozyme RM IM), 0.34% (Lipozyme TL IM), and 0.60% (Lipase PS Amano IM) ( Table 2). ...
... However, with the quantity of enzyme above 30 mg, both the conversion and reaction rates decreased. Similarly, Sun et al. [28] observed an initial increase in conversion yield for the synthesis of erythorbyl palmitate using a Novozym 435 load from 1% to 15% (w/w), with a subsequent decrease in conversion when this loading was exceeded. This phenomenon is likely responsible for the observed decrease in the conversion of the reactants to phenethyl formate beyond the enzyme concentration of 15 g/L in our work. ...
... Therefore, the enzyme activity was not generally affected by the changes in temperature used in this work. This is in excellent agreement with the reported heat tolerance of Novozym 435 (20 to 110 • C) and studies, which have clearly shown good activity at 90 • C [28]. It is reported that the presence of a small volume of water is essential for lipase activity. ...
Article
Full-text available
Current methods for the production of esters, including chemical synthesis and extraction from natural sources, are hindered by low yields and environmental pollution. The enzymatic synthesis of these compounds could help overcome these problems. In this study, phenethyl formate, a commercially valuable formate ester, was synthesized using commercial immobilized lipases. The effects of specific enzymes, enzyme concentration, formic acid:phenethyl alcohol molar ratio, temperature, and solvent were studied in order to optimize the synthesis conditions, which were identified as 15 g/L of Novozym 435 enzyme, a 1:5 formic acid:phenethyl alcohol molar ratio, a 40 °C reaction temperature, and 1,2-dichloroethane as the solvent. Under these conditions, phenethyl formate was obtained in a conversion yield of 95.92%. In addition, when 1,2-dichloroethane was replaced with toluene as the solvent, the enzyme could be recycled for at least 20 reactions with a steady conversion yield above 92%, testifying to the economic aspects of the process. The enzymatic synthesis of phenethyl formate using the proposed method is more environmentally friendly than methods currently employed in academic and laboratory settings. Moreover, the method has the potential to enhance the value-added properties of formic acid owing to its downstream use in the production of commercially essential esters.
... However, when the enzyme is in excess, a problem arises in the diffusion of the substrate [25,26], leading to a reduction in the conversion of octyl formate. This occurs due to the limited internal and external mass transfer associated with high enzyme loading [4,7,26,27]. The non-contributing behavior of excessive immobilized enzyme towards higher molar conversion, and associated reduced product yield, was also reported in the production of amyl isobutyrate [28] and citronellyl acetate [15]. ...
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Octyl formate is an important substance used in the perfume industry in products such as cosmetics, perfumes, and flavoring. Octyl formate is mostly produced by chemical catalysts. However, using enzymes as catalysts has gathered increasing interest due to their environment-friendly proprieties. In the present study, we aimed to identify the optimal conditions for the synthesis of octyl formate through immobilized enzyme-mediated esterification. We investigated the effects of enzymatic reaction parameters including the type of immobilized enzyme, enzyme concentration, molar ratio of reactants, reaction temperature, and type of solvent using the optimization method of one factor at a time (OFAT). The maxium conversion achieved was 96.51% with Novozym 435 (15 g/L), a 1:7 formic acid to octanol ratio, a reaction temperature of 40 °C, and with 1,2-dichloroethane as solvent. Moreover, we demonstrated that the Novozym 435 can be reused under the optimal conditions without affecting the octyl formate yield, which could help reduce the economic burden associated with enzymatic synthesis.
... This could possibly be due to the decrease in the activity of the enzyme by excessive water removal or due to increase in the overall viscosity of the reaction medium. Similar results have been reported by other researchers [32]. Thus, 100 mg/mL was used as the optimum molecular sieve load for further optimization experiments. ...
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The main challenge associated with the upgrading of crude rice bran oil (RBO) rests with their high content of free fatty acids (FFA). In this study, we describe a green and sustainable process for the deacidification of high-acid RBO using D-isoascorbic acid as a novel acyl acceptor in combination with a newly prepared immobilized Aspergillus Niger lipase (ANL). The process contributed a high deacidification efficiency of 97.53% with a desirable D-isoascorbic acid ester (DIAE) content of 36.10%. The immobilized ANL could be used consecutively for at least 10 batches with only 7.86% of activity loss. Scale-up reaction was implemented to verify the amenability of the procedure, and a low acid value of 1.46 mg KOH/g conformed to the quality criteria of edible RBO was then obtained. Physicochemical indices analysis indicated that this procedure was amicable to bioactive phytochemicals in RBO. In addition, the Rancimat test suggested that this process greatly improved the shelf life of deacidified RBO due to the formation of DIAE. This is the first time that D-isoascorbic acid has been used in the refining of RBO. Overall, we report an economically and efficiently viable process for the upgrading of high-acid RBO.
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This paper describes the regioselective production of palm‐based sorbitol monoesters via esterification catalyzed by Lipozyme® TL IM (Thermomyces lanuginosus lipase adsorbed onto silica gel, Novozymes, Inc., Franklington, NC, USA). Effects of various reaction parameters including types of solvent, substrate molar ratio, molecular sieve and lipase concentration, temperature, reaction time, and fatty acid chain length were investigated. Approximately 76% conversion of sorbitol to sorbitol esters was achieved within 24 h under optimal conditions: sorbitol (0.4 M), fatty acid (0.8 M), 20 wt% Lipozyme® TL IM in 100 mL tert‐butanol at 55 °C for 24 h in the presence of 25 wt% 3 Å molecular sieve as water absorbent. The reactions were conducted in an orbital incubator shaker at a shaking rate of 200 rpm. Lipozyme® TL IM was highly regioselective, esterifying exclusively at sorbitol's primary hydroxyl groups, producing 1‐O‐ and 6‐O‐sorbitol monoesters. The biocatalyst also exhibited substrate selectivity toward shorter chain acyl donors, with caprylic acid exhibiting the highest conversion of sorbitol. In addition, Lipozyme® TL IM was reused up to four successive reaction cycles without significant loss of activity. The biocatalytic process reported in this paper is a one‐step process to produce biobased surfactants that does not involve the use of toxic or expensive solvents that are commonly employed for derivatization of sugars, or pre‐derivatization of the substrates molecules.
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This review is concerned with lipase catalyzed synthesis of sugar fatty acid esters in water immiscible organic solvents. Sugar esters are widely used nonionic and nontoxic biosurfactants. Certain sugar esters inhibit microbial growth and have other activities. Lipase mediated synthesis has important advantages over conventional chemical synthesis of sugar esters. Lipase catalyzed synthesis is typically carried out in organic solvents having a low water activity to drive the reaction towards synthesis instead of towards ester hydrolysis. The impact of the various reaction conditions on enzymatic synthesis of sugar esters in nonaqueous media is discussed. Considered in particular are the solvent effects; the effects of water activity; the influence of the nature and concentration of the reactants (sugars and fatty acids); the influence of temperature; and the effects associated with the specific nature of the lipase catalyst used.
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The synthesis of palm-based ascorbyl esters through transesterification of ascorbic acid and palm oil in tert-amyl alcohol catalyzed by immobilized lipase is described. Highest conversion (70–75%) was determined after 16h reaction at 40°C using lipase (Novozyme 435 from Candida antartica) with an ascorbic acid to palm oil mole ratio of 1:8. The purified product was further characterized by 13C NMR and GC–MS and the mixture of ascorbyl monoesters obtained were identified as ascorbyl monooleate (61%), ascorbyl monopalmitate (30%) and ascorbyl monostearate (9%). The antioxidant activity of palm-based ascorbyl esters was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) test. The results showed that pure palm-based ascorbyl esters have an antioxidant activity with an IC50 value of 0.1mg/mL.
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L-ascorbyl acetate was synthesized through lipase-catalyzed esterification using Lipozyme TLIM and Novozym 435. Four solvents, including methanol, ethanol, acetonitrile, and acetone were investigated for the reaction, and acetone and acetonitrile were found to be suitable reaction media. The influences of several parameters such as water activity (a w), substrate molar ratio, enzyme loading, and reaction temperature on esterification of L-ascorbic acid were systematically and quantitatively analyzed. Through optimizing the reaction, lipase-catalyzed esterification of L-ascorbic acid gave a maximum conversion of 99%. The results from using Lipozyme TLIM and Novozym 435 as biocatalysts both showed that a w was an important factor for the conversion of L-ascorbic acid. The effect of pH value on lipase-catalyzed L-ascorbic acid esterification in acetone was also investigated. Furthermore, results from a kinetic characterization of Lipozyme TLIM were compared with those for Novozym 435, and suggested that the maximum reaction rate for Lipozyme TLIM was greater than that for Novozym 435, while the enzyme affinity for substrate was greater for Novozym 436.
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The kinetics of l-ascorbyl oleate synthesis catalyzed by immobilized lipase from Candida antarctica in acetone was investigated. Significant inhibition of synthesis with an excess of ascorbic acid was observed. Experimental data were successfully fitted with a ping–pong bi–bi kinetic model with substrate inhibition, and related kinetic constants were determined. The kinetic study was performed at optimum experimental factors (temperature, initial water content, and enzyme concentration), which were determined using response surface methodology. Then, a model for predicting product–time progress curves was developed by expanding the obtained ping–pong model with terms describing ester hydrolysis. Kinetic constants of the reverse reaction were determined, and good congruence between the model and experimental data was achieved. Calculated kinetic constants revealed that lipase has the highest affinity for ascorbyl oleate, slightly lower activity with ascorbic acid, and the lowest activity with oleic acid. The obtained results are valuable for elucidating the reaction mechanism and represent an important contribution for reaction optimization and creating strategies to increase the productivity of vitamin C ester synthesis.
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The conversion of coconut oil and fusel alcohols, the sustainable and low-cost natural materials, to valuable products through Lipozyme TL IM-catalysed transesterification was investigated in a solvent-free system. Flavour esters, especially the octanoic acid esters (ethyl-, butyl-, isobutyl-, propyl- and (iso)amyl octanoate), were formed during the transesterification reactions. The reaction parameters were optimised as follows: molar ratio of 3.0:1 (alcohol to oil), enzyme loading of 15% wt/wt, reaction temperature of 23 °C, shaking speed of 130 rpm, and reaction time of 20 h. Further, the operational stability of Lipozyme TL IM was improved through washing with solvents, after which the enzyme could be continuously used for at least 100 h within 5 batches’ reactions without significant loss of activity. The results indicate that coconut oil and fusel alcohols can be effectively converted to valuable flavour esters through the Lipozyme TL IM-catalysed transesterification.
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In recent years, researchers have focused on finding green alternative media to organic solvents for enzyme-catalyzed reactions. Thereby, ionic liquids (IL) have emerged as fascinating media for enzymatic reactions. One drawback to the wider development of these solvents in biocatalysis is their cost and the difficulty of product recovery. Recently, a novel medium with similar properties to IL but with additional advantages regarding cost, environmental impact and synthesis has been created: Deep Eutectic Solvents (DESs). These DESs result from the association of an ammonium salt and a hydrogen-bond donor. This study aimed at analyzing the advantages and limitations of several DESs as ‘green solvents’ for biotransformation using immobilized Candida antarctica lipase B as catalyst. The transesterification of vinyl laurate was chosen as model reaction and the influence of substrate polarity was assessed using alcohols of various chain lengths. Results showed that grinding of immobilized lipase was essential parameters for good lipase activity and some DESs cannot be used as media for iCALB-catalyzed reaction, especially DESs based on dicarboxylic acids and ethylene glycol. Finally, the best DES’s specific activity - and stability up to five days incubation time - were analyzed and compared with conventional organic solvents. Experiments revealed that iCALB is less influenced by the chain length of alcohol in DES than organic solvents and it is preserves its activity with minimally destructive to protein structure.
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Ascorbyl benzoate was synthesized through lipase-catalyzed esterification in organic media, and its properties studied. A series of organic solvents with a logP from −1.30 to 2.50 were investigated, in which cylcohexanone (logP=0.96) was found to be the most suitable. The optimum reaction conditions in cylcohexanone were pH 6.0, aw 0.33, a substrate concentration form 0.06M to 0.1M, 65°C, and above 150rpm speeds of shaking. Experimental results also demonstrated that benzoic acid was not an ideal substrate of lipase, which led to low conversion rates, but its limitation could be overcome by excess l-ascorbic acid. Schaal oven test illustrated that the antioxidant activity of ascorbyl benzoate was comparable to that of ascorbyl palmitate, and minimal inhibitory concentration (MIC) data showed that its antimicrobial activity was weaker than that of benzoic acid.
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Ascorbyl Palmitate, Ascorbyl Dipalmitate, Ascorbyl Stearate, Erythorbic Acid, and Sodium Erythorbate are related ingredients that function as antioxidants in cosmetic formulations. Ascorbyl Palmitate, Ascorbyl Dipalmitate, and Ascorbyl Stearate are esters and diesters of ascorbic acid with long-chain fatty acids. Erythorbic Acid is a stereoisomer of ascorbic acid and Sodium Erythorbate is the sodium salt of Erythorbic Acid. Although all of these ingredients are used, uses of Ascorbyl Palmitate and Erythorbic Acid predominate, with combined uses in over a thousand cosmetic formulations at low concentrations. Ascorbyl Palmitate is used at concentrations between 0.01 and 0.2% , and Erythorbic Acid is used at concentrations of 0.5-1% . Ascorbyl Palmitate has vitamin C activity approximately equal to that of L-ascorbic acid, whereas Erythorbic Acid has only 5% activity. The esters are likely to penetrate the skin readily, but the acid and its salt are not likely to penetrate. These ingredients exhibit low acute oral toxicity in animals. In chronic feeding studies, decreased body weight gain, the formation of oxalate stones in the bladder, and hyperplasia were seen in rats fed high levels of Ascorbyl Palmitate. Ascorbyl Palmitate (10%) and Ascorbyl Dipalmitate (100%) were not irritating to the intact skin of albino rabbits. Ascorbic Acid (30 % ) itself caused barely perceptible erythema and Sodium Erythorbate powder caused no irritation to the intact and abraded skin of rabbits. In animal studies, Ascorbic acid was not a sensitizer, and Erythorbic Acid (10%) applied topically to porcine skin reduced ultraviolet B (UVB)-induced phototoxicity. In clinical studies, Ascorbyl Palmitate caused no dermal irritation or sensitization. These ingredients are minimally irritating to the eye. Sodium Erythorbate did not cause fetal or maternal toxicity or developmental toxicity in rats and mice fed high levels. Although these ingredients were generally negative in a wide range of genotoxicity tests, Erythorbic Acid and Sodium Erythorbate did produce isolated positive genotoxicity test results. As antioxidants, these ingredients have been studied in animals after initiation with various carcinogens. In some cases reductions in tumor incidence were seen, in others no effect was noted. In no case did treatment with these ingredients increase tumor incidence. The highest use concentrations of Erythorbic Acid and Sodium Erythorbate are in oxidative hair dyes, where they are completely consumed in the chemical reaction that takes place at mixing. The fatty acid esters of ascorbic acid are used at lower concentrations in leave-on formulations. In consideration of these uses and based on the available safety test data, Ascorbyl Palmitate, Ascorbyl Dipalmitate, Ascorbyl Stearate, Erythorbic Acid, and Sodium Erythorbate are safe for use as cosmetic ingredients in the present practices of use.
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Stanols are more effective and safer than sterols in lowering plasma total cholesterol, while stanol esters are advantageous since this compound has a better solubility than the free stanols, which contribute to the practical application in foods. The current work focuses on the synthesis of phytostanyl esters from plant stanols and fatty acid such as lauric acid, including screening of various source lipases and selecting solvents of different LogP values. Among lipases from different origins, the immobilized Novozym 435 lipase from microorganism was found to be the best biocatalyst, while plant lipase from Carica papaya and animal lipase from Porcine pancreas are quite ineffective as biocatalyst for the esterification of plant stanols with fatty acid. The highest phytostanyl laurate esterification degree of 79.3% was obtained under the selected conditions: 25μmol/mL plant stanols, 100μmol/mL lauric acid, 80mg/mL 3Å molecular sieves and 40mg/mL Novozym 435 at 150rpm and 55°C for 96h in 10mL of n-hexane. The chemical structure of sitostanyl laurate was confirmed by FT-IR, MS and NMR. The comparison of solubility of plant stanols and phytostanyl laurate in plant oil was done.
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The enzymatic synthesis of biodiesel from used palm oil and ethanol using immobilized lipases in a solvent-free system was attempted. Five immobilized lipases, Lipase AK from Pseudomonas fluorescens, Lipase PS from Pseudomonas cepacia, Lipase AY from Candida rugosa, Lipozyme TL IM from Thermomyces lanuginosa and Novozym 435 from Candida antarctica, were screened based on their catalytic activities on reactions involved in biodiesel synthesis. The combined use of Lipase AY and Lipase AK gave a higher yield of biodiesel than using Lipase AK alone. The optimal conditions for biodiesel synthesis using mixed lipases in a batch system were: 2% water content, 10% enzyme dosage and 3:1 molar ratio of ethanol to oil. The mixed lipases could be used in 15 replicates with retained relative activity higher than 50%. In a continuous system using mixed lipases packed in packed-bed reactor, >67% of biodiesel was achieved.