**Table 7 here**
Higher levels of potency increase the emulsification of the reactants
facilitating the hydrolysis reaction (Özbek and Ülgen, 2000; Wang et
al., 2015), however, the enzymatic deactivation effect may also occur
due to the thermal effect generate through the ultrasonic treatment,
given that the reaction temperature increases during cavitation, besides
of the shear and pressure forces that cause damage to the structure of
the enzyme (Froment et al., 1998; Vercet et al., 2001). The effect of
temperature in the enzymes activities is well know, in one example,
Avelar et al. (Avelar et al., 2013) reported on the influence of
temperature in enzymatic hydrolysis of canola oil employing oil-free
castor seeds as a lipase source; the activity decreased with temperature
increase after 50°C. Cavalcanti et al.(Cavalcanti et al., 2007), shown
similar deactivation of oil-free castor seeds after 40°C. The effect of
deactivation by the ultrasound power was notable after 5 minutes of
reaction, and small or no further activity was detected after this time
when the reaction was continued in a shaker; also it can be noted that
the equilibrium yield for the oil-free seeds was lower, suggesting that
the enzymes may have suffered a more significant deactivation in this
reaction system.
Despite the fact that the lipase concentration is higher on oil-free
seeds, another aspect that allows the use of significantly less amount
of the catalyst mass, is the smaller granulometry of the oil-free seeds,
which provides a higher superficial area favoring the aqueous-organic
interfacial reaction (Pourzolfaghar et al., 2016). Besides, a more
substantial amount of buffer solution was needed for both particle forms
when the reaction was performed in ultrasound since the emulsion of the
system is better, and the aqueous-organic interface increased.
Comparing the yield of reaction at an equivalent time, both systems
exhibit similar behavior under optimal ultrasound conditions. The
oil-free seeds, however, reached equilibrium at a lower yield compared
with in natura seeds, which is resulted from the less stability and
higher deactivation of oil-free seeds, since in previous experiments, it
was demonstrated that after 30 days at 4°C the oil-free seeds had 9% of
deactivation while no deactivation detected for the in natura seeds
(Tavares et al., 2018a). The deactivation can be accelerated by the
ultrasound power used in the reaction. However, notably, the reaction
could be intensified with the use of ultrasound since the time of 4
hours for a yield of 86% (Tavares et al., 2018a) was decreased to 5
minutes of reaction with the in natura seeds. Other intensification
process methods can be performed with microwaves. Nguyen et al. (Nguyen
et al., 2020) reported a green approach to produce FFA from soybean oil
involving autocatalytic hydrolysis employing microwave irradiation. For
this process, however, a surfactant had to be added to the mixture to
reach a satisfactory emulsion; furthermore, the reaction achieves
equilibrium at 96.6% of conversion into FFA after four h at 195 °C.
Therefore, the use of ultrasound for oil hydrolysis catalyzed by vegetal
lipases features a considerable enhancement in intensification of an
eco-friendly process.
The reports in the literature employing castor bean seeds as a source of
lipase for triglyceride hydrolysis reactions use the seeds in the
oil-free form. Coelho et al. (Coelho et al., 2013) used lipase from
oil-free castor seeds (0.075–0.090 mm) to produce FFA by hydrolysis of
vegetable oils (corn and sunflower). The maximum percentage of
hydrolysis of corn oil (84.0%) and sunflower (76.4%) was reached in
60% w/w oil, 0.1 M acetate, pH 4.5, 33°C, and 5% m/m seed particles
after 70 and 80 min of reaction. Performing the enzymatic hydrolysis of
canola oil, Avelar et al., (Avelar et al., 2013), achieved complete
hydrolysis of the oil at 37.5°C, buffer/oil ratio 22.1% after 2 hours,
using castor seeds without oil (2.0% of mass ratio and particles
smaller than 1 mm). Santos et al. (Santos et al., 2013) evaluate
oil-free castor bean seeds (0.075–0.090 mm) and reported the complete
hydrolysis of the soybean after 80 minutes at 44.1% w/w oil/buffer (100
mM sodium acetate pH 4.5), 37°C, 2.0% w/w lipase, and sodium acetate.
The results obtained in the present study, 86% yield after 5 minutes,
using in natura seeds, without process costs for seed preparation, show
impressive enhancement in the processes that are so far described in the
literature.
The oil average extracted in the oil-free catalyst preparation was 47.4
± 1.3%, resulting in 21% of the initial seed mass after sifting. From
the optimum conditions, it was taken that for 10 g of Crambe oil, 6.1 g
of in natura seeds were used, while 1.8 g of oil-free was necessary,
representing 8.6 g of seed. The higher performance of the in natura
seeds can be explained by the presence of components in the lipase that
contribute to its activity. Ory (1969) reported the existence of three
elements in castor bean lipase, a solid fraction, called apoenzyme, a
lipid cofactor, and an activator protein, and concluded that for maximal
lipase activity, all the components are necessary. The use of in natura
seeds, thus, reflects in more enzymatic activity and can be considered
more advantageous for the oil hydrolysis. On the other hand, the use of
oil-free seeds can benefit from the residues of the castor oil industry.
4. Conclusion
To improve the yield of the hydrolysis of Crambe oil using seeds of
castor beans as the catalyst, the use of ultrasound in the reaction was
evaluated using two forms of seeds, in natura and oil-free. The CCRD
factorial design was employed to assess each form of the seed particles,
and optimum conditions were found, reaching approximately 86% yield
after 5 minutes of ultrasound reaction for in natura seed particles and
73% for the oil-free seeds. Mathematical modeling, based on a Ping Pong
Bi Bi kinetic mechanism, was applied for the experimental kinetic data
representation. From unrelated experiments, the FFA concentration values
were predicted using the estimated kinetic constants, demonstrating the
suitability of the model for process optimization and understanding of
the mechanisms in the ultrasound-assisted enzymatic hydrolysis of oils
employing vegetal lipases. The potential of the process of ultrasonic
enzymatic hydrolysis of oil using castor bean seeds as a source of
lipases is highlighted since high conversions were achieved in an
impressive short time. The best yield was obtained with the use of in
natura seeds, which do not undergo any preparation process resulting in
a much lower cost than compared to the use of animal or microbial lipase
enzymes that are usually commercialized.