Enzymatic catalyzed palm oil hydrolysis under ultrasound irradiation: Diacylglycerol synthesis

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Enzymatic catalyzed palm oil hydrolysis under ultrasound irradiation:
Diacylglycerol synthesis
Jamal A. Awadallak
a,
⇑
, Fernando Voll
b
, Marielen C. Ribas
a
, Camila da Silva
b
, Lucio Cardozo Filho
b
,
Edson A. da Silva
a
a
Department of Chemical Engineering, Universidade Estadual do Oeste do Paraná, Toledo, Brazil
b
Department of Chemical Engineering, Universidade Estadual de Maringá, Maringá, Brazil
article info
Article history:
Received 10 March 2012
Received in revised form 21 August 2012
Accepted 30 November 2012
Available online 5 January 2013
Keywords:
Ultrasound irradiation
Diacylglycerol
Palmoil
Lipases
Hydrolysis reaction
Central composite rotatable design (CCRD)
abstract
Diacylglycerol (DAG) rich oils have an organoleptic property like that of regular edible oils, but these oils
do not tend to be accumulated as fat. Palm oil ranks first in the world in terms of edible oil production
owing to its low cost. The aim of this study was to propose a new methodology to produce diacylglycerol
by hydrolysis of palm oil using Lipozyme RM IM commercial lipase as a catalyst under ultrasound irra-
diation. The reactions were carried out at 55 °C with two different methods. First, the reaction system
was exposed to ultrasonic waves for the whole reaction time, which led to enzymatic inactivation and
water evaporation. Ultrasound was then used to promote emulsification of the water/oil system before
the hydrolysis reaction, avoiding contact between the probe and the enzymes. An experimental design
was used to optimize the ultrasound-related parameters and maximize the hydrolysis rate, and in these
conditions, with a change in equilibrium, DAG production was evaluated.
Better reaction conditions were achieved for the second method: 11.20 wt.% (water + oil mass) water
content, 1.36 wt.% (water + oil mass) enzyme load, 12 h of reaction time, 1.2 min and 200 W of exposure
to ultrasound. In these conditions diacylglycerol yield was 34.17 wt.%.
Ó2013 Published by Elsevier B.V.
1. Introduction
Diacylglycerol (DAG) rich oil, with a content of least 80% of 1,3-
DAG, has been used as a functional cooking oil owing to its nutri-
tional properties [1]. Studies on both animals and humans have
shown that although it has similar digestibility and energy value
to regular oil, whose main compound is triacylglycerol (TAG),
DAG oil has the ability to decrease postprandial lipid levels [2,3]
and its consumption has been shown to reduce visceral and
abdominal fat accumulation and body weight [1,4]. DAG can also
be used as an emulsifier and stabilizer in the food, cosmetics and
pharmaceutical industries [5] representing an important class of
chemicals widely used in modern industry [6].
DAGs can be produced by partial hydrolysis, esterification or
glycerolysis through chemical or enzymatic catalysis. The enzy-
matic process is preferred because it requires the lowest tempera-
ture and pressure conditions and uses safe products [7]. Partial
hydrolysis has the advantage of being a one-step reaction, whereas
esterification and glycerolysis have the hydrolyzed oil as a
substratum.
Palm oil ranks first in the world in terms of edible oil production
[8], producing ten times more oil than soy beans for the same area
[9]. In the recent past, this oil has been heavily criticized for its
supposedly unhealthy properties, as its main compound is palmitic
acid, which is a saturated fatty acid [10]; however, some studies
show that its metabolic rate is similar to that of unsaturated fatty
acid catabolism [11], which is beneficial to the human body. This
oil also has a high number of tocopherols and tocotirenols, which
are potent antioxidants [12]. In addition, it is the only oil with a
polyunsaturated to saturated ratio of 1:1 and it is a rich source
of vitamin A, vitamin E and beta carotene [13].
Ultrasound irradiation can reduce mass transfer in enzymatic
reactions [14,15], induce conformational changes in protein and
perturb weak interactions [16,17]. These effects can strongly im-
prove the kinetics of enzymatic reactions [18,19] or contribute to
enzyme deactivation [20].
With regard to biodiesel production, several papers can be
found about the use of ultrasonic irradiation, but only a few can
be found on DAG production, even though the two processes are
very similar. Bath ultrasound was used to improve DAG production
by hydrolysis [7,21] and glycerolysis [22] but no work was found
on hydrolysis of DAG production under ultrasonic probe
irradiation.
1350-4177/$ - see front matter Ó2013 Published by Elsevier B.V.
http://dx.doi.org/10.1016/j.ultsonch.2012.11.017
⇑
Corresponding author. Tel.: +55 45 3379 7001.
E-mail address: awadallak@hotmail.com (J.A. Awadallak).
Ultrasonics Sonochemistry 20 (2013) 1002–1007
Contents lists available at SciVerse ScienceDirect
Ultrasonics Sonochemistry
journal homepage: www.elsevier.com/locate/ultson

This work describes the use of the ultrasonic probe as an ultra-
sound irradiation source to suggest a new way to produce DAG by
enzymatic hydrolysis. Owing to ultrasound deactivation effects on
enzymes, two methods were tested: the entire hydrolysis time was
assisted by ultrasound; and ultrasound was used to promote emul-
sification of the water/oil system, after which the emulsified prod-
uct was used as the hydrolysis substratum. Both methods were
first evaluated in terms of free fatty acid (FFA) content to check
the ultrasound influence on the degree of hydrolysis. In conditions
that led to a high degree of hydrolysis, DAG production was
optimized.
2. Materials and methods
2.1. Lipases
The commercial enzyme Lipozyme RM IM was provided by
Novozyme
Ò
.
2.2. Ultrasound equipment
Ultrasonic irradiation was provided by a UP200 S Hielscher,
200 W ultrasound equipment operating at 23 KHz, and 20% to
100% of the total power. Irradiations were performed at 55 ± 0.1 °C.
2.3. Reaction procedure
2.3.1. Influence of the ultrasound on hydrolysis degree
To evaluate the effect of the ultrasonic irradiation on the sys-
tem, two different methods were performed and compared with
a control reaction (without ultrasound influence). These reactions
were carried out in a 60 ml jacketed glass reactor, at 55 °C for
24 h. Palm oil (15 g) and water (1.5 g) were added to the reactor.
In the first method, enzyme (1.36 wt.% water + oil mass) was
added, the ultrasound probe was inserted into the system to a
depth of about 10 mm, and the power was adjusted to 40 W and
turned on for the entire reaction time. In the second method, the
ultrasound probe was inserted to a depth of about 10 mm into
the water/oil system, the power was adjusted to 80 W and turned
on for 3 min to emulsify the system before being removed, and
then the enzyme (1.36 wt.% water + oil mass) was added while
the solution was mixed by magnetic stirring (300 rpm). The control
reaction was performed by adding the enzyme (1.36 wt.% water + -
oil mass) and mixing by magnetic stirring (300 rpm).
Experimental design reactions were carried out according to the
second method, with changes to the water content (2.0–21.25 wt.%
water + oil mass) and the power [20–100% of total power (200 W)].
The reaction time was 8 h for the first method and 4 h for the
second.
2.3.2. DAG Production
DAG production was investigated in conditions that led to a bet-
ter hydrolysis degree. For these conditions, DAG production can be
increased by reducing the water content to limit the complete
reaction [23]. DAG production reactions were performed using
the optimal conditions of the second method (two-step reaction)
as follows: ultrasound exposure time: 1.2 min; ultrasound power:
100%, temperature: 55 °C; enzyme: 1.36 wt.% water + oil mass.
In these conditions, water influence was evaluated for three dif-
ferent levels: a high level which led to a high FFA degree, an inter-
mediate level, and a low water level. TAG, DAG, MAG and FFA
kinetics were evaluated for 1–24 h for the water level which led
to the best DAG production.
2.4. Experimental design
Two central composite rotational designs (named Design A and
Design B) composed of three and two variables ranging over four
levels were used to maximize the hydrolysis degree and reduce
the requirement for a higher number of experiments. The first
experimental design was used as a preliminary test to estimate
the best reaction conditions. The second experimental design
was used as a sequential strategy of the first one.
The effects of variables such as temperature, enzyme charge and
water content are well known in the hydrolysis process; therefore,
this study focused only on the ultrasound-related variables: expo-
sure time, power and water/oil rate.
Design A was not completely conclusive; therefore, Design B
was evaluated in terms of only ultrasound exposure time and
water/oil rate, with the potency being fixed at 100%.
2.5. Acylglycerol quantification
Assuming that the TAG, DAG and MAG molecules are the result
of the esterification of three, two and one FFA molecule, respec-
tively, (molar mass of 269.25 g/mol) with a glycerol molecule, their
molar mass were calculated as: MMAG = 343.40 g/mol,
MDAG = 594.60 g/mol and MTAG = 845.90 g/mol. The mole frac-
tions of acylglycerols (TAG, DAG and MAG) (free of free fatty acids)
were determined by
13
C nuclear magnetic resonance (NMR) analy-
sis [24], at the NMR laboratory of the Chemistry Department of the
State University of Maringá, under the following conditions: relax-
ation delay = 3.000 s; pulse = 25°; Acq. time = 1.517 s; repeti-
tions = 2048; line broadening = 0.3 Hz; and ambient temperature.
Samples of 0.7 ml (1:3 v/v of oil/CDCl3) were prepared in 5 mm
NMR tubes and stored under refrigeration until analysis. Acylglyce-
rols were identified from the signal corresponding to the G-2 car-
bon of the glyceride skeleton of each acylglycerol. The peaks
related to the G-2 carbon of 1-MAG, 1,2-DAG, 1,3-DAG and TAG
were found on the spectrum at d70.25, d72.1, d68.3 and
d68.9 ppm, respectively [24]. The G-2 carbon peak of the 2-MAG
was found at d74.9 ppm [25]. The NMR spectra were obtained on
a VARIAN (model Mercury Plus 300-BB) spectrometer, operating
at 75.449 MHz for the
13
C analysis.
2.6. Estimation of degree of hydrolysis
The degree of hydrolysis or fatty acid content was determined
by titration of the lipid samples [26–29] with 0.05 M sodium
hydroxide. To each 1 g sample, 25 ml diethylether and 25 ml etha-
nol were added. The sample was then titrated against 0.05 M so-
dium hydroxide. The amount of 0.05 M sodium hydroxide
required to neutralize the acid was noted and the degree of hydro-
lysis was calculated as follows:
Acdð%Þ¼100Vol WM=m
where Acd(m%) is the degree of hydrolysis; Vol is the volume (ml) of
the titrated NaOH solution for the sample; Wis the mean of molec-
ular weight of the fatty acids; Mis the molarity of the NaOH solu-
tion; and mis the weight of the sample (g). All data are the
averages of duplicate samples and were reproducible with 95%
accuracy.
2.7. Statistical analysis
The analysis of the experimental design results was performed
with Statistica 8.0 software (Statsoft, Inc., USA). The significance le-
vel was set as 95% and regression coefficients and associated prob-
abilities were determined by the t-student test. The variance
explained by the model was given by the coefficient of multiple
J.A. Awadallak et al. / Ultrasonics Sonochemistry 20 (2013) 1002–1007 1003

determinations, R
2,
and the mathematical model validation was
determined by the ftest.
3. Results and discussion
3.1. Influence of ultrasound on the degree of hydrolysis
In order to evaluate the effects of ultrasonic irradiation on the
enzymatic hydrolysis, two different methods were investigated.
In the first method, the ultrasonic probe was inserted into the reac-
tion system for the whole reaction time, exposing the enzymes to
ultrasonic waves. The second method was composed of two steps:
(1) the water/oil system was exposed to the ultrasonic waves for a
short period of time to emulsify it; (2) enzymes were added to the
emulsified system and the reaction started without any contact
with the ultrasound. The fatty acid content for both methods is
shown in Fig. 1.
The first method began with a high hydrolysis rate but suddenly
started to decrease, and after 4 h of reaction, it was almost zero,
leading to a poor conversion after 24 h. Unlike ultrasonic baths,
which disperse energy in huge volumes, the probe ultrasound dis-
perses all of the energy just in the reaction system, being highly
efficient at promoting emulsification [30], but can cause enzyme
deactivation at the same time [20]. As the probe achieved high
temperatures (more than 100 °C outside the solution), and close
to the probe, the temperature control system was not so effective,
and two negative effects were found: water evaporation and en-
zyme deactivation.
Very little is known about the actual effect of ultrasound on en-
zyme behavior, and contradictory results of the inactivation and
activation of enzymes upon ultrasound treatment have been re-
ported. The tolerance of the enzymes to ultrasound might depend
on the physiological location of the enzymes on the cell as well as
on its molecular weight [31].
In the second method, effects relating to the ultrasound were
not relevant once the enzymes had no contact with the probe. De-
spite residual evaporation effects, it was less expressive as the
emulsification time was reduced.
3.2. Optimization of ultrasound variables
As the second method led to a higher degree of hydrolysis than
the control reaction, and as the energy cost of three minutes of
40 W emulsification was negligible compared with the 24 h of
temperature control and stirring, an experimental design was used
to optimize this method. The results are described in Table 1 and
Table 2.
There were no similar experiments found to compare the exper-
imental results. Most of the studies on DAG/FFA production under
ultrasonic irradiation have used bath systems [22,24,25,32–35] or
non-1,3-specific enzymes [32]. Two-step reactions, as described
in the second method, have never been tested before for this
system.
Table 1
Experimental Design A and results of CCD. Reaction time: 8 h.
Entry Time (min) Power (%) Water (%) Acd(%)
1 2 35 3.66 9.6
2 7 35 3.66 22.6
3 2 85 3.66 20.5
4 7 85 3.66 8.5
5 2 35 10.87 31.8
6 7 35 10.87 34.1
7 2 85 10.87 39.3
8 7 85 10.87 21.3
9 0.5 60 7.41 34.5
10 8.5 60 7.41 4.2
11 4.5 20 7.41 39.0
12 4.5 100 7.41 34.8
13 4.5 60 0.99 5.9
14 4.5 60 13.04 36.2
15 4.5 60 7.41 25.7
16 4.5 60 7.41 30.6
17 4.5 60 7.41 32.0
Acd(%)
Time (h)
Fig. 1. Free fatty acid content as a function of the reaction time: j, Reaction assisted by ultrasound for the whole reaction time , Two-step reaction (using the ultrasound to
promote emulsification before the reaction) N, Control reaction (without ultrasound influence).
Table 2
Experimental Design B and results of CCD. Reaction time: 4 h.
Entry Time (min) Water (%) Acd(%)
1 0.21 4.58 6.6
2 0.79 4.58 4.2
3 0.21 18.83 9.2
4 0.79 18.83 20.7
5 0.5 12.28 7.4
6 0.5 12.28 6.2
7 0.5 12.28 6.9
8 0.5 21.26 17.7
9 0.5 0.99 7.0
10 0.92 12.28 11.1
11 0.08 12.28 4.9
1004 J.A. Awadallak et al. / Ultrasonics Sonochemistry 20 (2013) 1002–1007

For both Design A and Design B the water content had greater
and more positive effects. In the previous experiments, water evap-
oration was identified as one of the ultrasound equipment compli-
cations and this became clear, when water had this effect on the
Acd(%). In some high power and long-time entries, such as entries
4 and 10 for Design A, it was possible to see the water steam close
to the probe. After the sonication period, the emulsion characteris-
tics were lost and only the oil phase was visible. This high water
dependence derived from overestimated time and power values,
which resulted in negative effects.
For Design A, the second greatest effect was the product
time power or, in other words, energy, which is in agreement
with Hielscher [30], who said that ultrasound reproducibility
experiments depend just on the energy for constant composition
and volume.
Despite the complications, Design A led to good Acd(%) values
and was used as a base for the second experimental design. Energy
was evaluated instead of time and power, and the reaction time
was reduced to 4 h to produce bigger differences among fatty acid
contents.
Unlike the first design, the exposure to ultrasound had positive
effects and showed a high dependence on the exposure time, but
emulsification proved to be highly unstable. Some emulsified sam-
ples lost their characteristics a few minutes after the addition of
enzymes, whereas other samples remained emulsified for the en-
tire reaction time. For the same composition and under the same
exposure time, some samples randomly lost or kept their emulsion.
This effect was more significant for low exposure times than for
longer times, which possibly explains why it was not seen in the
first design. These experiments had to be repeated many times to
ensure that emulsions were not undone in the first reaction
minutes.
Results obtained in both experimental designs were statistically
treated and permitted the validation of an empirical coded model
for FFA conversion in terms of exposure time, power and water
content (or just exposure time and water content for the second
experimental design). The effects of ultrasound variables could
be assessed on the basis of the values of (p) and t-student tests de-
scribed in Table 3.
Design A experimental data were fitted to a quadratic model as
shown in Eq. (1). The empirical model was validated by analysis of
variance (ANOVA).
Acdð%Þ¼6;42 þ2;06:Tþ4;52:Wþ3;14:W
2
þ3;46:T:Wð1Þ
Where Acd(%) is the acid content at the end of the reaction, Tis the
exposure time to the ultrasonic waves and Wis the water content
(wt.% water + oil mass). Table 4 shows the statistical test of models
performed by Fisher’s statistical test for analysis of variance.
The Fvalue calculated for Design B was significantly higher than
the tabulated Fvalue. The coefficients of determination
(R
2
= 0.9949) and correlation (R= 0.9974) were used to check the
model’s reliability. These values imply that Acd(%) can be accu-
rately explained by Eq. (1).
The better conditions from Design B were chosen to evaluate
DAG production, although there were instability issues in these
conditions. As a result, extra time was added to ensure emulsion
stability. DAG production was therefore evaluated as follows:
ultrasound exposure time = 1.2 min; ultrasound power: 100%;
higher water content = 18.83 wt.%).
3.3. DAG production
TAG hydrolysis consists of three serial reactions, in which each
step consumes water [36]. Some studies suggest that the hydroly-
sis rate-limiting step is TAG hydrolysis [23] Thus, the use of ultra-
sound equipment increases the hydrolysis rate and also raises the
DAG production rate, which is then converted into MAG and FFA.
Limiting the water content, reaction equilibrium enables the pro-
duction of more intermediary products and TAG [23,32]. A higher
hydrolysis rate using ultrasound was obtained to the second meth-
od with 100% ultrasound power and 1.2 min of exposure time. In
these conditions, DAG, TAG, MAG and FFA production were evalu-
ated in different lower water levels. Table 5 shows these results for
18.83, 11.20 and 2.00 wt.% (water + oil mass).
These results are agreement with Voll [23] and Yaxuan et al.
[32] works: Decreasing the water content reduces FFA production
and raises the intermediary products. In a lower water level
(2 wt.%), just a little TAG content reacted; therefore, the DAG pro-
duction was limited. An intermediary water level (11.20 wt.%) was
enough to allow a higher TAG conversion, but did not consume it
significantly. A 48 wt.% FFA content reached in higher water con-
tent was not actually higher because Lipozyme RM IM is a specific
enzyme for 1,3 positions in glycerol, thus 2-MAG needs to change
to 1(3)-MAG by acyl migration to be converted into FFA in this
step.
Comparing these results with Gonsalves et al. [5], Babicz et al.
[21] and Cheong et al. [37] showed that to achieve a similar DAG
production, the methodology tested in this work required higher
water content conditions, probably because the emulsification cell
was open and allowed to lose water by evaporation.
The intermediary water level showed the best results for DAG
production; therefore, its conditions were used to evaluate DAG
kinetics for a 1–24 h period. These results are shown in Fig. 2.
NMR spectra for the best and worst DAG production conditions
are shown in Fig. 3.
Table 3
Effect of the parameters estimated for ultrasound influence.
Variable Effect Standard error P-value
Design
A
Design
B
Design
A
Design
B
Design A Design B
Intercept 30.32 6.42 2.66 0.329 <0.0001
a
<0.0001
a
T(L)8.04 4.13 2.51 0.400 0.0150
a
0.0001
a
T(Q)7.69 1.17 2.42 0.460 0.0156
a
0.0522
P(L)2.34 – 2.57 – 0.3944 –
P(Q) 3.86 – 3.01 – 0.2408 –
A(L) 17.08 9.05 2.53 0.404 0.0003
a
<0.0001
a
A(Q)7.75 6.27 2.82 0.477 0.0284
a
<0.0001
a
TP11.28 – 3.30 – 0.0110
a
TA4.17 6.91 3.30 0.565 0.2466 <0.0001
a
PA0.53 – 3.30 – 0.8771 –
a
Statistically significant at 95% confidence level.
Table 4
Variance analysis for validation of mathematical models at 95% confidence level.
Factor Sum of
squares
Degrees of
freedom
Mean
Square
Calculated
F
Tabulated
F
Regression 279.08 5 55.816 174.425 5.05
Residuals 1.6 5 0.32
Total 280.68 10
R
2
= 0. 9949.
Table 5
TAG, MAG, DAG and FFA in different water contents.
Water content (%) TAG (wt.%) DAG (wt.%) MAG (wt.%) FFA (wt.%)
18.83 28,21 21,69 2,10 48
11.20 36,74 32,56 4,30 26,4
2.00 63,35 24,15 1,61 10,9
J.A. Awadallak et al. / Ultrasonics Sonochemistry 20 (2013) 1002–1007 1005

wt (%)
Time (h)
Fig. 2. TAG, MAG, DAG and FFA as function of the reaction time: N, TAG, j, AGL, d, DAG, MAG.
Fig. 3. nmr spectrums for different acylglycerol compositions: (a) 39.24% of TAG, 49.46% of DAG and 11.31% of MAG. (b) 85.37% of TAG and 14.63% of DAG. All data are in
molar basis and Free of FFA.
1006 J.A. Awadallak et al. / Ultrasonics Sonochemistry 20 (2013) 1002–1007

According to Fig. 2, a 34.17 wt.% DAG concentration was ob-
tained after 12 h of reaction, and the reaction almost achieved
chemical equilibrium.
In a similar experiment [23], optimized conditions led to a level
of 35.9% in 72 h and 2.87 wt.% of enzyme. In this work, using ultra-
sound for a 1.2 min emulsification time, a similar conversion was
achieved in just 12 h using half of the enzyme load.
This method of application has great advantages for large-scale
production, as its energy costs are very low and its short emulsifi-
cation time allows the use of reduced continuous ultrasonic equip-
ment to feed large hydrolysis reactors.
4. Conclusion
Ultrasound used to assist the whole reaction time for the tested
conditions led to undesirable effects and a low degree of hydroly-
sis. However, its use before the reaction to promote emulsifications
improved the degree of hydrolysis and its kinetics.
The experimental design has shown the water content to be a
primary influence on fatty acid production once on the emulsifica-
tion process showed water evaporation.
DAG oil with 34.17 wt.% concentration was obtained after 12 h
of reactions exposed to ultrasound for just 1.2 min.
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