**Figure 2 here**
3.2 Kinetics of Ultrasound-assisted Enzymatic Hydrolysis for
In Natura
Seeds
The reactions were performed under the conditions of the optimal values
obtained from Equation 4 (Power 70% for 3 minutes at initial
room temperature, 1.8 B/O, and 0.25 C/S) at pH 4.2. The experimental
data is shown in Figure 3 . As can be seen after 5 minutes the
reaction reaches equilibrium at approximately 88% of FFA (0.75 mol
kg-1). Besides, it has been verified that the
ultrasound equipment heats up beyond usual when a time greater than 7
minutes is used, and, therefore, it is not possible to continue the
reaction with the ultrasound. To evaluate the degree of enzyme
deactivation and the potential of a higher yield of hydrolysis under the
optimal conditions, experiments were carried out by continuing the
reaction after 5 minutes in ultrasound in a refrigerated incubator with
agitation (shaker) at room temperature and 140 rpm. After 30 minutes in
the shaker, a yield of 89.96% was achieved for the hydrolysis reaction,
and after 60 minutes, the yield was 91.57%. The absence of a
significant increase in the hydrolysis yield, expose the deactivation of
the catalyst after the 5 minutes under ultrasonic treatment, which is
consistent with a result reported by Awadallak et al. (Awadallak et al.,
2016), who studied the hydrolysis of soybean oil under ultrasound
treatment, using a phospholipase as catalyst. The authors reported
enzyme inacvation after 10 minutes when using 20% of ultrasound power
(200 W) and after 5 minutes when using 50% of ultrasound power.
To better analyze the influence of employing ultrasound in the reaction,
the same experiment, under the optimal conditions found throughEquation 4, was conducted in the refrigerated incubator (140
rpm) for 3 minutes. The yield obtained was 41.23 ± 2.62%, which is less
than half of the yield obtained under 3 minutes of reaction under
ultrasound treatment, 85.01%, demonstrating the high potential for the
use of ultrasound in the enzymatic hydrolysis of Crambe oil using seeds
in natura.
In Figure 3 , the concentration profiles of MAG, DAG, and TAG
(in mol kg-1) are shown. The decrease in TAG
(Figure 3 (d) ) concentration and increasing concentration of
FFA (Figure 3 (a) ) are the expected behavior and can be seen.
DAG (Figure 3 (c) ) and MAG (Figure 3 (b) ), behave in
the usual way for intermediate compounds of a reaction. All the
concentration profiles obey the principle of mass conservation
(constant , i = TAG , DAG , MAG ,GL , H2O e FFA ). Additionally, inFigure 3, the model results for the concentration profiles of
glycerol (Figure 3 (e) ) and water (Figure 3 (f) ) are
shown. The value of 88% (≈ 0.75 mol kg-1) was reached
after the equilibrium of the enzymatic hydrolysis of the Crambe oil in 5
min.
In Table 2 are presented the values of the estimated parameters
from the fitting of the model, based on the Ping Pong Bi Bi (PPBB)
kinetic mechanism, to the experimental kinetic data. The simulated and
experimental curves are shown in Figure 3 . The coefficient of
determination (R²) values were 1.00, 0.95, 0.88, and 0.99 for TAG, DAG,
MAG, and FFA experimental data, respectively, which demonstrate that the
kinetic model satisfactorily represented the set of experimental data.