Optimization of lipase-catalyzed synthesis of acetylated tyrosol by response surface methodology

Article abstract of DOI:10.1021/jf071685q

The ability of a noncommercial immobilized lipase from Staphylococcus xylosus (SXL_i) to catalyze the transesterification of tyrosol and ethyl acetate was investigated. Response surface methodology was used to evaluate the effects of the tempera

Full text of DOI:10.1021/jf071685q

10298 J. Agric. Food Chem. 2007, 55, 10298–10305
Optimization of Lipase-Catalyzed Synthesis of
Acetylated Tyrosol by Response Surface
Methodology
IMEN AISSA,†,‡ MOHAMED BOUAZIZ,‡ HANEN GHAMGUI,† AMEL KAMOUN,§
NABIL MILED,† SAMI SAYADI,‡ AND YOUSSEF GARGOURI*,†
Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS route de Soukra,
3038 Sfax-Tunisia, Laboratoire des Bioprocédés, Centre de Biotechnologie de Sfax, BP « K »,
3038, Sfax, Tunisie, and Laboratoire de Chimie Industrielle II, ENIS route de Soukra, 3038
Sfax-Tunisia
The ability of a noncommercial immobilized lipase from Staphylococcus xylosus (SXLi) to catalyze
the transesterification of tyrosol and ethyl acetate was investigated. Response surface methodology
was used to evaluate the effects of the temperature (40–60 °C), the enzyme amount (50–500 UI),
and the ethyl acetate/hexane volume ratio (0.2–1) on the tyrosol acetylation conversion yield. Two
independent replicates were carried out under the optimal conditions predicted by the model (reaction
temperature 54 °C, enzyme amount 500 UI, and volume ratio ethyl acetate/ hexane 0.2). The maximum
conversion yield reached 95.36 ( 3.6%, which agreed with the expected value (96.8 ( 3.7%). The
ester obtained was characterized by spectroscopic methods. Chemical acetylation of tyrosol was
performed, and the products were separated using HPLC. Among the eluted products from HPLC,
mono- and diacetylated derivatives were identified by positive mass spectrometry. Tyrosol and its
monoacetylated derivative exert similar antiradicalar activity on 2,2-diphenyl-1-picrylhydrazyle.
KEYWORDS: Acetylation; antioxidant; immobilized Staphylococcus xylosus lipase; response surface
methodology
INTRODUCTION
determined that tyrosol penetrates and accumulates in macro-
phages and improves the intracellular antioxidant defense
systems (12). A recent increase in serious research on the
commercial application of radical scavengers as beneficial
antiagingandphotoprotectioningredientsincosmeticproducts(13,14),
and the demand for nontoxic antioxidants that are active in
hydrophilic and lipophilic systems, led to the additional focus
on new natural antioxidants that can be used in oil-based
formulas and emulsions. The acetylation of biophenols increases
their hydrophobicity and, therefore, lipid solubility and may
modify their bioavailability (15), antioxidant effect in food
emulsions (16), stability, and color stability (17). This acetylation
can be explored by reaction with acid chlorides or acid
anhydrides, but these routes do not meet the requirements
necessary for food applications. We can overcome the disad-
vantages by the use of enzymes in nonaqueous media. Guyot
et al. (18) have reported for the first time the enzymatic
esterification of phenolic acid and alcohols with lipase from
Candida antartica B. Then, Buisman et al. (19) studied the
esterification of cinnamic acid and some benzoic acid derivatives
with fatty alcohols varying between 4 and 12 carbon atoms
(C4–C12). Recently, the hydroxytyrosol and the homovanillic
alcohol were subjected to chemoselective lipase-catalyzed
acylations, affording with good yield of 10 derivatives bearing
C2, C3, C4, C10, and C18 acyl chains at C-1. The hydroxytyrosol
Polyphenolic compounds produced by plants are of consider-
able research interest, both as functional food ingredients and
as nutraceuticals, because of their antioxidant properties (1) and
other beneficial biological activities (2). Extra-virgin olive oil
is the principal fat component of the Mediterranean diet, and
its chemical constituents have been intensively studied (3, 4).
The main olive oil phenols are oleuropein, tyrosol, and
hydroxytyrosol. It has been reported that the tyrosol has
scavenging effects on ONOO– (5) and O2– (6). In addition to
its antioxidative effects, it has been shown that tyrosol inhibits
lipopolysaccharide (LPS)-induced cytokine release from human
monocytes (7) and the activity of the leukocyte 5-lipoxygenase
release in human mononuclear cells (8). Furthermore, previous
studies have suggested that tyrosol has an anti-inflammatory
effect in vitro (9), fully protected Caco-2 cells against the
cytotoxic/apoptotic effects of oxLDL (10), and a neuroprotective
effect in the stroke rat model (11). Recently, it has been
* Corresponding author. Prof. Youssef Gargouri, Laboratoire de
Biochimie et de Génie Enzymatique des Lipases, ENIS route de Soukra,
3038 Sfax-Tunisia., Tel/ Fax: + 21674675055, E-mail: ytgargouri@
yahoo.fr.
† Laboratoire de Biochimie et de Génie Enzymatique des Lipases.
‡ Laboratoire des Bioprocédés.
§ Laboratoire de Chimie Industrielle II.
10.1021/jf071685q CCC: $37.00
2007 American Chemical Society
Published on Web 11/15/2007

Optimization of Acetylated Tyrosol Synthesis
J. Agric. Food Chem., Vol. 55, No. 25, 2007 10299
Table 1. Three-Variable Box-Behnken Experimental Design
and its lipophilic derivatives with little hydrophobic character
showed a good protective effect against H2O2-induced oxidative
DNA damage (20) and very good DPPH-radical scavenging
activity (21). Furthermore, it was reported that the acetylated
derivatives of tyrosol were more potent inhibitors of PAF-
induced rabbit platelet aggregation than tyrosol, indicating that
the addition of an acetyl group on the tyrosol structure results
in phenolic compounds that are more potent inhibitors by 2
orders of magnitude (22).
run
X1
X2
X3
1
2
3
4
5
6
7
8
-1.0000
1.0000
-1.0000
1.0000
-1.0000
1.0000
-1.0000
1.0000
0.0000
0.0000
0.0000
0.0000
0.0000
-1.0000
-1.0000
1.0000
1.0000
0.0000
0.0000
0.0000
0.0000
-1.0000
1.0000
-1.0000
1.0000
0.0000
0.0000
0.0000
0.0000
0.0000
-1.0000
-1.0000
1.0000
1.0000
The present work focuses on the reaction parameters that
affect the enzymatic acetylation of tyrosol with ethyl acetate
by an immobilized Staphylococcus xylosus lipase. In addition,
the aim of this study was to determine, by means of response
surface methodology (RSM), the optimal conditions of tyrosol
acetate ester synthesis. A Box Behnken design (23–25) is set
up to establish the relationship between the conversion yield of
tyrosol acetate and the reaction factors (ethyl acetate/hexane
volume ratio, enzyme amount, and temperature). The enzymatic
synthesis yields were compared to the chemical methods.
9
-1.0000
-1.0000
1.0000
1.0000
0.0000
10
11
12
13
parallel. After 48 h of reaction, aliquots of the mixture reaction were
withdrawn; the immobilized enzyme was removed by centrifugation
at 8000 rpm for 5 min, and the supernatant was used for HPLC analysis.
A calibration curve based on different concentrations of tyrosol was
employed using benzoic acid as the internal standard. The conversion
yield of tyrosol acetate was calculated as the ratio of the number of
moles of tyrosol used per total number of tyrosol.
MATERIALS AND METHODS
Materials. The tyrosol, the deuterated chloroform (CDCl3), and the
2,2-diphenyl-1-picrylhydrazyle (DPPH) were purchased from Fluka
(Suisse). The n-hexane, the acetone, the pyridine, and the acyl chlorure
were purchased from Prolabo (Paris, France).The carbonate of calcium
(CaCO3) and the ethyl acetate were purchased from Pharmacia (Uppsala,
Sweden).
Tyrosol (1). Colorless amorphous solid. MS: mass calculated 138
for C8H10O2 found m/z 161 [M + Na]+, 120 [M – H2O]+, 93[M-CH2-
CH2-OH]+. IR (liquid) cm-1: 3323 (m), 1590 (s). 1H NMR (300 MHz,
CDCl3): 7.1 (2H, d, J ) 8 Hz), 6.8 (2H, d, J ) 8 Hz), 4.8 (1H, s,-OH),
3.8 (2H, t, J ) 7 Hz, -CH2CH2OH), 2.8 (2H, t, J ) 7 Hz, -CH2CH2OH),
1.6 (1H, s,-OH). 13C NMR (75 MHz, CDCl3): 38.62 (C-7), 64,24 (C-8),
115.84 (C-3, C-5), 130.57 (C-2, C-6), 130.81 (C-1), 154.64 (C-4).
Monoacetylated Tyrosol (2). Colorless oil. After 48 h of incubation,
the monoacetylated tyrosol was purified by preparative HPLC under
the same conditions as the analytic one. LC-MS analysis showed a
fragment at m/z 181 [M + H]+, 202, 7 [M + Na]+; 120 [M – H2O]+,
93 [M-CH2-CH2-OH]+ ([M]+, calculated for 180, 2004 C10H12O3). IR
(liquid) cm-1: 3323 (m), 1726 (m), 1620 (m), 1250 (s). 1H NMR (300
MHz, CDCl3): 7.1 (2H, d, 7.8 Hz), 6.8 (2H, d, 7.8 Hz), 2.06 (3H, s,
COCH3), 4.2 (2H, t, 2.7 Hz, -CH2-CH2-OCOCH3), 2.8 (2H, t, 2,7 Hz,-
CH2-CH2-COCH3), 1.26 (1H, s, -OH). 13C NMR (75 MHz, CDCl3):
21.1 (C-10), 34.1 (C-7), 65.6 (C-8), 115.84 (C-3, C-5), 130.1 (C-2,
C-6), 129.4 (C-1), 154.6 (C-4), 172.1 (C-9, CdO).
Instrumentation. HPLC Analysis. The identification of tyrosol and
its acetylated derivatives was carried out by HPLC analysis. It was
performed on a Shimadzu apparatus composed of an LC-10ATvp pump
and an SPD-10Avp detector. The column was a C-18 (4.6 × 250 mm;
Shimpack VP-ODS), and its temperature was maintained at 40 °C. The
flow rate was 0.5 mL/min. The mobile phase used was 0.1% phosphoric
acid in water (A) versus 70% acetonitrile in water (B) for a total running
time of 50 min, and the following proportions of solvent B were used for
the elution: 0–30 min, 20–50%; 30–35 min, 50%; and 35–50 min, 50–20%.
LC–MS Analysis. The LC–MS experiments were carried out with
an Agilent 1100 LC system consisting of degasser, binary pump,
autosampler, and column heater. The column outlet was coupled to an
Agilent MSD Ion Trap XCT mass spectrometer equipped with an ESI
ion source. Data acquisition and mass spectrometric evaluation were
carried out on a personal computer with Data Analysis software
(Chemstations). For the chromatographic separation, a Zorbax 300 Å
Extend-C-18 column (2.1 × 150 mm) was used. The column was held
at 95% solvent A (0.1% formic acid in water) and 5% solvent B (0.1%
formic acid in ACN) for 1 min, followed by an 11 min step gradient
from 5% B to 100% B; then, it was kept for 4 min with 100% B;
finally, the elution was achieved with a linear gradient from 100% B
to 5% B in 2 min. The flow rate was 200 µL min-1, and the injection
volume 5 µL.
Diacetylated Tyrosol (3). Colorless oil; after 1 h of incubation, the
diacetylated tyrosol was purified by preparative HPLC under the same
conditions as the analytic one. LC-MS analysis showed a fragment at
m/z 223 [M + H]+, 245 [M + Na]+, ([M]+, calculated for 222, 2371
C12H14O4).
Experimental Design. Optimization of the experimental conditions
of the synthesis of a phenolic acid ester was achieved by using response
surface methodology (RSM). Recall that RSM is a technique consisting
of (i) design of experiment to provide adequate and reliable measure-
ments of the response, (ii) development a mathematical model having
the best fit to the data obtained from the experimental design, and (iii)
determination of the optimal values of the independent variables that
produce maximum or minimum value of the response (23–25).
In this paper, RSM was applied to establish and exploit the
relationship between the response studied (conversion yield of the
tyrosol acetate) and the three selected experimental variables (ethyl
acetate/hexane volume ratio, enzyme amount, and temperature). In this
regard, we have postulated a quadratic polynomial model represented
by the following equation:
NMR and IR Experiments. NMR spectra were recorded on JNM
A-300 spectrometer (JEOL). IR spectra were recorded on FT/IR-410
(JASCO).
Production and Immobilization of Lipase. Staphylococcus xylosus
lipase was produced as described by Mosbah et al. (26). The enzyme
immobilization was made onto CaCO3 as described by Ghamgui et al.
(27). The activities of the immobilized lipases were measured titri-
metrically with a pH-stat, under the standard assay conditions described
previously by Gargouri et al. (28) using olive oil emulsion as the
substrate. One international unit (UI) of lipase activity was defined as
the amount of lipase that catalyzes the liberation of 1 µmol of fatty
acid from olive oil per minute at pH 8.5 and 37 °C.
∧
2
2
y ) b0+ b1X1+ b2X2+ b3X3+ b11X1 + b22X2 +
2
b33X3 + b12X1X2 + b13X1X3 + b23X2X3 (1)
Alcoholysis Reactions. The transesterification reactions were carried
out in screw-capped flasks containing 12 mM tyrosol added to various
ethyl acetate to n-hexane volume ratios (6 mL of total volume), followed
by different amounts of immobilized lipase. The mixture reaction was
incubated at different temperatures with shaking (200 rpm). A reaction
under the same conditions without adding enzyme was realized in
with y ) yˆ + e, where e represents deviation between measured
response (y) and estimated response (yˆ); b0, bj, bjk, and bjj are estimated
model coefficients; and Xj represents coded variables related to the
corresponding natural variable Uj by the equation Xj ) (Uj
and
high
Uj
) high and low levels of Uj).
low

10300 J. Agric. Food Chem., Vol. 55, No. 25, 2007
Table 2. Range of Variables for the Experimental Design
Aissa et al.
Table 3. Experimental Conditions of the Box-Behnken Design and the
Corresponding Experimental Responses
level
run
ethyl acetate/hexane
enzyme (UI)
temperature (°C)
yielda%
variable
-1
0
+1
1
2
3
4
5
6
7
8
0.20
1.00
0.20
1.00
0.20
1.00
0.20
1.00
0.60
0.60
0.60
0.60
0.60
0.60
0.50
0.60
0.66
50
50
50
50
50
50
40
40
60
60
40
40
60
60
50
50
40
60
50
77.07
65.12
94.79
83.39
74.12
59.69
77.22
62.79
62.20
67.66
41.40
80.00
83.55
80.71
74.00
79.53
82.11
U1: ethyl acetate/hexane volume ratio (v/v)
U2: enzyme amount (UI)
U3: temperature (°C)
0.20
50
40
0.60
275
50
1.00
500
60
500
500
275
275
275
275
50
To estimate the model coefficients, a three-variable Box Behnken
design, requiring 13 experiments (Table 1), is carried out in a cubic
experimental domain. The experimental points are located in the middle
of a cube ridges (12 experiments) and at the center of the cube (1
experiment) (23–25). The model parameters are estimated by a least-
squares fitting of the model to experimental results obtained in the
design points. The adequacy of the model is tested using four check
points (24, 25).
The fitted model is used to study the relative sensitivity of the
response to the variables and to look for the optimal experimental
conditions. In this paper, the relationship between the response and
the experimental variables is illustrated graphically by plotting the
response surfaces and the isoresponse curves (23). The canonical
analysis is used to determine the best experimental conditions, which
permitted the maximization of the yield conversion of the tyrosol acetate
synthesis yield. It consists of rewriting the fitted second-degree equation
in a form in which it can be more readily understood. This is
accomplished by a rotation of axes that remove all cross-product terms
bjk, Xj, and Xk, while keeping the initial origin at the center point. This
step is suitable when the stationary point is outside the experimental
domain.
9
10
11
12
13
14
15
16
17
500
50
500
275
250
500
400
500
a Estimated standard deviation is equal to SD ) ✓(38.80/3) ) 3.60.
Table 4. Analysis of Variance
source of
degrees of mean
variation sum of squares freedom
square F-ratio prob > F signif
a
regression
residuals
total
R2
2196.33
38.80
2235.13
0.983
9
3
244.036 18.869
12.933
1.71
*
12
In this study, the generation and the data treatment of the Box-
Behnken design are performed using the experimental design software
NemrodW (29).
a * indicates significant at the level 95%.
Table 5. Validation of the Model with the Check-Points
DPPH Radical-Scavenging Effect. The DPPH radical-scavenging
effect was evaluated according to Bouaziz et al. (30). A 4 mL volume
of methanolic solution of varying sample concentration was added to
10 mL of DPPH methanol solution (0.15 mM). After mixing the two
solutions gently and leaving for 30 min at room temperature, the optical
density was measured at 520 nm, using a spectrophotometer. The
antioxidant activity of each sample was expressed in terms of IC50
(micrograms per milliliter required to inhibit DPPH radical formation
by 50%) and calculated from the log-dose inhibition cuve.
Chemical Synthesis of Acetylated Tyrosol. Acetylation was gener-
ated by a modified method according to Capasso et al. (31). Briefly, acetic
anhydride (160 µL) was added to tyrosol (0.145 mmol). The reaction was
left at 0 °C overnight. After that, 150 µL of sulfuric acid were added and
kept for 4 h at 0 °C. The reaction mixture was treated to neutrality with
NaHCO3, and the ester was extracted by ethyl acetate. Acetylated
derivatives was concentrated in Vacuo to dryness at 40 °C, dissolved in
ethyl acetate, and analyzed by HPLC and LC-MS.
run
yi
yˆi
e ) yi – yˆi
t exp.
signifiance
14
15
16
17
80.71
74
79.53
82.11
82.382
66.484
75.84
-1.672
7.516
3.69
-0.330
1.569
0.830
N.S.
N.S.
N.S.
N.S.
88.074
-5.964
-1.329
conditions of the Box-Behnken design with the corresponding
measured responses. Four additional experiments (runs no 14
to 17) are included in order to check the validity of the fitted
model.
Estimated Models. The observed responses (runs 1 to 13)
are used to compute the model coefficients by the least-squares
method (23, 24) using the NemrodW software. The resulting
estimated model, expressed in coded variables, is
yˆ ) 83.55 ((3.60) - 6.53 ((1.27)X1 + 10.01 ((1.27)X2 -
2
0.28 ((1.27)X3 + 1.09 ((2.38)X1 - 4.55 ((2.38)X22 - 16.19
RESULTS AND DISCUSSION
2
((2.38)X3 + 0.14 ((1.80)X1X2 + 0.00 ((1.80)X1X3 + 8.29
To prepare the acetylated derivative of tyrosol, immobilized
Staphylococcus xylosus lipase was used. Several conditions were
tested, including different amounts of enzyme, various temper-
atures, and ethyl acetate/hexane volume ratio. In a preliminary
study, we showed that the addition of hexane to the reaction
medium was necessary to improve the stability of the im-
mobilized Staphylococcus xylosus lipase. Also, we found that
the addition of water at the beginning of the reaction or a crude
molecular sieve 4 Å (5%, w/w) failed to improve the conversion
yield. In light of these results, the ethyl acetate/hexane volume
ratio (U1), the amount of enzyme (U2), and the temperature (U3)
are chosen as the most effective operating variables on the
response.
((1.80)X2X3
Analysis of Variance and Validation of the Models. The
fit quality of the yield model is attested with analysis of the
variance (ANOVA) as revealed in Table 4. Indeed, this table
shows that the regression sum of squares is statistically
significant when using the F-test at a 95% probability level,
which suggested that the variation accounted for by the model
was significantly greater than the unexplained variation. Like-
wise, the fit value, the coefficient of multiple determination of
the polynomial model, termed R2, was equal to 0.983, which
indicated that 98.3% of the variability in the response could be
explained by the second-order polynomial prediction equation
given above. On the other hand, we used the four check-point
results to validate the fitted model. It can be seen, from Table
5, that the measured values yi are very close to those calculated
Experimental Design Data. To carry out the Box-Behnken
experimental design, a high and a low level were chosen for
each factor (Table 2). Table 3 shows the real experimental

Optimization of Acetylated Tyrosol Synthesis
J. Agric. Food Chem., Vol. 55, No. 25, 2007 10301
Figure 1. Contour plots and response surface plot showing the effect of enzyme amount, ethyl acetate /hexane volume ratio, and their mutual interaction
on tyrosol acetate synthesis at constant temperature equal to 50 °C.
Figure 2. Contour plots and response surface plot showing the effect of enzyme amount, temperature, and their mutual interaction on tyrosol acetate
synthesis at ethyl acetate/hexane volume ratio fixed at 0.6.
(yˆi) using the model equation. Besides, the differences between
calculated and measured responses are not statistically significant
when using the t test at a 95% probability level. We can then
conclude that the second-order model is adequate to describe
the response surfaces, and it can be used as a prediction equation
in the studied domain.
of Klibanov (32), who stated that the enzyme activity is strongly
affected by the choice of organic solvent. In fact, the use of
hydrophilic solvents with log P < 2.5 have the capability of
stripping off even the essential water from the enzyme surface,
leading to an insufficiently hydrated enzyme molecule and
consequently to a decrease of the enzyme activity (33). The
solvents with log P > 4 show better reaction rates. Therefore,
the hydrophobic solvents preserve the catalytic activity without
disturbing the microaqueous layer of the enzyme.
Figure 2 shows the effect of varying the enzyme amount
(50–500 UI) and the reaction temperature (40–60 °C) on
alcoholysis at a constant solvent volume ratio (U1 ) 0.6). It
can be seen that, at low enzyme amount and temperature, the
conversion yield was strongly decreased, whereas high reaction
yields were obtained when using high enzyme level and
moderate temperature. In fact, high temperature (above 55 °C)
dramatically decreased the conversion yield at any given enzyme
amount. This phenomenon is probably due to the inactivation
of the enzyme at high temperature. Krishna et al. (34) suggested
that high temperature can reduce the operational stability of the
enzyme. Chiang et al. (35) had observed that an increase of the
temperature up to 55 °C resulted in a lower alcoholysis yield at
Graphical Interpretation of the Response Surface Model.
Following the validation of the model, the response surface and
the isoresponse curves are drawn by plotting the response
variation against two of the factors while the third is held
constant at its mean level.
Figure 1 shows the effect of enzyme amount (50–500 UI)
and ethyl acetate/hexane volume ratio (0.2–1) on the conversion
yield of tyrosol acetate at 50 °C.We have observed that the
conversion yield enhances essentially by increasing the amount
of enzyme or lowering the ethyl acetate/hexane volume ratio.
Indeed, the contour plots in Figure 1 show that a high tyrosol
acetate synthesis yield (95%) can be obtained by using a high
amount of enzyme (over 400 UI) and low ethyl acetate/hexane
volume ratio (under 0.3). This result can be explained by the
negative effect of the solvent ratio on the enzyme activity, which
greatly affects the reaction yield. These data agree with those

10302 J. Agric. Food Chem., Vol. 55, No. 25, 2007
Aissa et al.
Figure 3. Contour plots and response surface plot showing the effect of ethyl acetate /hexane volume ratio, temperature, and their mutual interaction
on tyrosol acetate synthesis with enzyme amount fixed at 0.6.
any given amount of enzyme because of the inactivation of the
lipase at high temperatures. We can also conclude, from this
figure, that the reaction conducted with a high amount of enzyme
(over 300 UI) at 50 °C led to a relatively high conversion yield
(85%). However, with an amount of lipase higher than 500 UI,
the difference almost leveled off (data not shown). The amount
of enzyme is probably the rate-determining factor of the reaction
up to 500 UI. This phenomenon may be related to the fact that
the active sites of the enzyme molecules present in large excess
would not be exposed to the substrates due to possible protein
aggregation. As a result, lipase molecules can associate with
each other, masking the active sites that cannot accommodate
the substrate. Agglomeration using immobilized lipases has been
reported by Foresti and Ferreira 36. Figure 3 shows the variation
of the reaction yield in the plane solvent volume ratio (0.2–1)
versus temperature (40–60 °C) at a constant level of enzyme
Figure 4. Curvature of yield response versus Zj.
(500 UI). This figure confirms the negative effect of the ethyl
acetate /hexane volume ratio on the tyrosol acetate yield already
in the corresponding direction. The constant ys is the calculated
shown in Figure 1. It also illustrates that, at constant enzyme
response value at the stationary point. The interpretation is easier
level, the tyrosol acetate synthesis carried out at a moderate
by analyzing each response along every Zj-axis separately. Using
reaction temperature (approximately 50 °C) and the lowest
the variable transformation equations (not shown), we obtained
solvent volume ratio (0.2) gave a high conversion yield (90%).
the following canonical form of the model:
To further analyze the experimental results and to obtain a more
precise estimation of the optimum synthesis conditions, we
applied the canonical analysis.
Canonical Analysis. The second-order polynomial model is
a conic function and it can be analyzed by canonical analysis.
This function has a stationary point S where the partial derivative
of predicted response with respect to each of the variables is
2
^
2
2
2.853Z2 - 15.218Z3 (3)
These data allow us to determine the features of the response
surface in each direction of the experimental domain. When
analyzing the response surface along each of the three directions
OZ1, OZ2, and OZ3, the equation of the response is reduced to
the following equations, respectively:
zero ∂y ∂X ) 0;∂y ∂X ) 0;∂y ∂X ) 0 . This point could
/
/
/
1
2
3
be a maximum, a minimum, or a saddle point.
In the present study, the coordinates of the stationary point S
are X1 ) 2.721; X2 ) 1.505; and X3 ) 0.419. It corresponds to
a maximum of yˆ. This point is situated outside the experimental
domain at a distance from the center equal to 3.14. In this case,
the canonical analysis requires only a rotation of the Xj axes in
such a way that they become parallel to the principal axes Zj of
the contour system. Under these conditions, the canonical model
is of the form
The corresponding curves are represented in Figure 4. From
these curves and the variable transformation equations, we can
conclude that the maximization of the yield requires a low level
of X1 (X1 ) -1), a high level of X2 (X2 ) +1), and the mean
level of X3 (X3 ) 0). This corresponds to the following settings
of the natural variables: ethyl acetate/hexane volume ratio U1
) 0.2; enzyme amount U2 ) 500 UI; and temperature U3 in
3
3
yˆ ) y +
b Z +
λ Z2
(2)
∑
∑
s
j
j
jj j
j)1
j)1
The λj (i ) 1, 2, 3) will describe the curvature of the response,
while the linear coefficient bj will describe the slope of the ridge

Optimization of Acetylated Tyrosol Synthesis
J. Agric. Food Chem., Vol. 55, No. 25, 2007 10303
Figure 5. Contour plots and response surface plot showing the effect of enzyme amount, temperature, and their mutual interaction on tyrosol acetate
synthesis at ethyl acetate/hexane volume ratio fixed at 0.6.
Figure 6. Production of tyrosol acetate during alcoholysis reaction.
Reaction conditions: ethyl acetate/hexane volume ratio of 0.2, 500 UI
enzyme amount, and temperature of 54 °C. The tyrosol acetate level
was estimated using the HPLC system.
the vicinity of 50 °C. As the results of the canonical analysis
agree with those of the contour plot study, we can conclude
that there is no a masked optimum: the one predicted by a few
sections of contour plot analysis represents a real optimum for
the whole experimental domain.
Result Confirmation. In order to confirm the obtained result,
two independent replicates were carried out under the following
conditions: ethyl acetate/hexane volume ratio equal to 0.2;
enzyme amount equal to 500 UI; and temperature equal to 54
Figure 7. HPLC chromatogram of the reaction medium in the optimal
conditions. The separation was made on C18 reverse-phase HPLC. Flow
0.6 mL/min, and UV detection was at 280 nm. A. Time ) 0. B. Time )
48 h. 1. Tyrosol. 2. Internal standard. 3: Monoacetyltyrosol.
°C. The temperature value is indicated by the contour plots
represented in Figure 5 and generated from the fitted model
by keeping constant the solvent mixture ratio (U1 ) 0.2). These
contour plots indicated that, under these conditions, the predicted
conversion yield was 96.86%. Moreover, we showed how
sensitive the estimated response is to movements away from
the optimum. The conversion yields obtained at 48 h of reaction
time are indicated in Figure 6, together with the value calculated
from the model equation. These results clearly showed that the
experimental response values agree with those calculated. Once
again, this verification revealed a high degree of accuracy of
the model under the investigated conditions.
°C) was presented in Figure 6. The level of tyrosol acetate
increases rapidly to reach its maximal value after 48 h. The
experimental conversion yield of tyrosol acetate (95.36%) was
very close to the predicted value estimated (96.86 ( 3.70) after
a reaction time of 48 h.
Structure Determination of Enzymatic Acetylated Deriva-
tive. Tyrosol contains two hydroxyl groups in its structure, and
therefore three acetylated derivatives were expected: two isomers
with two monoacetylated derivatives and one diacetylated
derivative. HPLC analysis of the reaction mixture after 48 h
(Figure 7) showed a new peak which corresponds to the
acetylated derivative. The purified compound was characterized
by spectroscopic methods.
The time course of the alcoholysis reaction between the
tyrosol and the ethyl acetate by immobilized S. xylosus lipase
onto CaCO3 under optimal conditions (acetate/hexane volume
ratio of 0.2; 500 UI enzyme amount, and temperature of 54
To establish the structure of acetylated derivatives, a positive
mass spectrum was performed. Standard tyrosol gave two peaks

10304 J. Agric. Food Chem., Vol. 55, No. 25, 2007
Aissa et al.
combination with results obtained for monoacetylated tyrosol
lead to the conclusion that peak 3 is the diacetyl derivative of
tyrosol. This compound has previously been chemically syn-
thesized and identified by the use of ESI-MS in the negative
mode (22).
DPPH Radical Scavenging Effect of Tyrosol and Mono-
acetylated Tyrosol. The DPPH (2,2 diphenyl-2-picrylhydrazyl)
radical scavenging effect of both tyrosol and monoacetylated
tyrosol was measured and compared to that of BHT (1 µg/mL).
Tyrosol exhibited IC50 values of 10.9 µg/mL and in the same
ring compared to that of monoacetylated tyrosol (10.25 µg/mL).
These findings suggest that the scavenging activity of tyrosol
is not notably influenced by the presence of an acyl group. It
has been reported that the antioxidant activity was related to
the number and nature of the hydroxylation pattern on the
aromatic ring. It is generally assumed that the ability to act as
a hydrogen donor and the inhibition of oxidation are enhanced
by increasing the number of hydroxyl groups in the phenol ring
(30). Other results are in line with our findings and show that
the antiradical activity of hydroxytyrosol was not affected by
the presence of an acyl group at C-1 (20). In addition,
Fragopoulou et al. (22) reported that the monoacetylated
derivatives of tyrosol were more potent inhibitors of PAF-
induced rabbit platelet aggregation than tyrosol. Meanwhile, the
diacetylated derivative induced washed rabbit platelet aggrega-
tion and inverted the biological activity from inhibition to
aggregation.
In this study, the evaluation of the effect of the ethyl acetate/
hexane volume ratio (0.2–1), enzyme amount (50–500 UI), and
temperature (40–60 °C) on acetylated tyrosol yield was studied.
The optimization of synthesis conditions was performed using
response surface methodology. A Box-Behnken design permits
determination of the optimal conditions (reaction temperature
of 54 °C, enzyme amount of 500 UI, and volume ratio of ethyl
acetate/hexane of 0.2) leading to a high conversion yield
(95.3%). The product obtained under these conditions was
identified, using spectroscopic methods, to be a monoacetylated
derivative of tyrosol which exhibited the same radical scaveng-
ing activity as tyrosol. Chemical acetylation produced mono-
and diacetylated tyrosol, which provides evidence of the
chemoselective properties of the biocatalyzed acetylation. The
existence of lipophilic derivatives of phenols via esterification
of the hydroxyl functions with aliphatic molecules can be used
as a tool to increase their lipophilicity and therefore improve
their intestinal absorption and cell permeability. Moreover, this
lipophilic analogue could be used as food antioxidant in oil-
based formulae or cosmetic fields.
Figure 8. HPLC chromatogram of the reaction medium of the chemical
acetylation of tyrosol. The separation was made on C18 reverse-phase
HPLC. Flow was 0.6 mL/min, and UV detection was at 280 nm. 1. Tyrosol.
2. Monoacetyltyrosol. 3. Diacetylhydroxytyrosol.
at m/z 121 [M – H2O]+ and 161 [M + Na]+. The acetylated
derivative showed almost identical positive mass spectrum as
its native form. The predominant peaks were at m/z 203 [M +
Na+] and 181 which corresponds to the molecular weight of a
tyrosol molecule plus an acetyl group. In order to locate the
position of the acetylation, IR and NMR experiments were
carried out. IR spectra showed a broad band at around 3323
cm-1 corresponding to characteristic hydrogen-bonded phenolic
O–H vibration and a characteristic band at around 1726 cm-1
attributed to the ester function CdO. These data provide
evidence of the existence of acetyl groups in the molecule and
the perseverance of the hydroxyl group. The presence of an acyl
group linked to the tyrosol and its position were determined by
1
comparison of H NMR and 13C NMR spectra between the
acetylated tyrosol and its native form. With regard to 13C NMR
data, the spectrum of the acetylated tyrosol showed signals at
δ 172 ppm and δ 21, 1 ppm, which were attributed to a carbon
ester function (C-9) and the methyl function (C-10), respectively
(Figure 7). By 1H NMR, we were able to locate the position of
the acetylated hydroxyl. Data showed a chemical shift of triplet
2H at 4.2 ppm attributed to the protons of carbon C8 for the
acetylated derivative, whereas the chemical shift of the same
protons in the native form was detected at 3.8 ppm, suggesting
that the acetyl group was linked to the methylene group (C8).
For the aromatic protons of the tyrosol and its monoacetylated
form, no modification of chemical shift was observed. These
results led to the conclusion that the tyrosol was monoacetylated
on the primary hydroxyl group. Similar results have been
reported by Grasso et al. (20) during the chemical acetylation
of hydroxytyrosol by fatty acids. However, to our knowledge,
no reports on the enzymatic acetylation of tyrosol have been
reported.
ABBREVIATIONS USED
Chemical Acetylation. A mixture composed of acetic acid
anhydride and sulfuric acid or pyridine represents the usual
acetylating agents, described in many studies for the acetylation
of polyphenols (31). HPLC analysis (Figure 8) of the reaction
mixture showed two peaks attributed to acetylated derivatives.
Under the above conditions, a conversion yield of 94.08% was
obtained. To establish the structures of the synthesis products,
LC-MS analysis was performed. The positive ion mass spectrum
of peak 2 gave an identical positive mass spectrum to that of
the monoacetylated tyrosol obtained by the enzymatic method,
indicating that the predominant peak 2 corresponds to the
monoacetylated tyrosol. The positive ion mass spectrum of peak
3 gave molecular peaks at m/z 223 [M + H]+ as well as at m/z
245 [M + Na]+, corresponding to the molecular weight of
tyrosol plus two acetyl groups corresponding to the molecular
weight of tyrosol plus two acetyl groups. These data in
SXL: Staphylococcus xylosus lipase; SXLi, immobilized
Staphylococcus xylosus lipase; UI, enzymatic international unit;
CaCO3, calcium carbonate; CDCl3, deuterated chloroform;
HPLC, high performance liquid chromatography; LC-MS, high
performance liquid chromatography coupled to mass spectrom-
etry; IR, infrared; NMR, nuclear magnetic resonance; DPPH,
2,2-diphenyl-1-picrylhydrazyle.
ACKNOWLEDGMENT
We are grateful to Mr. A. Gargoubi (technician, CBS), Mr.
H. Auissaoui (engineer, CBS), Mrs. E. Dabbabi (engineer,
FSM), and Mrs. A. Kbadou (assistant professor, FSS) for their
technical assistance. Our thanks are due to LPRAI - Marseille
Company for supplying us with the software package NemrodW.

Optimization of Acetylated Tyrosol Synthesis
J. Agric. Food Chem., Vol. 55, No. 25, 2007 10305
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Received for review June 8, 2007. Revised manuscript received August
3, 2007. Accepted August 6, 2007. This work received financial support
from the Ministry of Higher Education, Scientific Research and
Technology in Tunisia.
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