Fractionation and encapsulation of copaiba (Copaifera sp.) oleoresin through supercritical technologies

Author: José Luis Pasquel Reátegui

Abstract. This work was divided in three stages. Stage (I) consisted in the formation of emulsions containing oleoresin from copaiba (Copaifera officinalis), using modified starches as stabilizers. The effects of modified starch concentration (g/L), oleoresin concentration (mg/mL) and oleoresin/water ratio (%, v/v) in the average diameter of the droplets were investigated. Sonication time and ultrasonic power were fixed at 6 min and 480 W, respectively. The formulation that provided the lowest droplet diameter was selected for a second planning, varying ultrasonic power and the sonication time. The emulsions with higher kinetic stability and lowest droplet diameter were subjected to drying by freeze-drying and spray-drying. Both drying techniques produced amorphous particles with different sizes and low humidity. Also, high encapsulation efficiency and high stability against thermal degradation were achieved. The morphological analysis indicated that the nature of the modified starches did not affect its microstructure, but drying techniques produced particles with different external microstructures. The CLSM (Confocal Laser Scanning Microscopy) analysis confirmed the encapsulation of copaiba oleoresin within the polymer matrix. Stage II of this work was the encapsulation of copaiba oleoresin by Supercritical Fluid Extraction of Emulsions (SFEE), from the emulsion selected in Stage I (lower droplet diameter and higher kinetic stability). Initially, CO₂ and emulsion flow rates were evaluated, maintaining temperature, pressure and solvent extraction time constant. The diameter of the coaxial nozzle used to inject the emulsion into SFEE system was 177.8 μm. After selecting the condition of suspension with the lowest residual ethyl acetate concentration, another study was carried out, varying the solvent extraction time, to maximize the rate of organic solvent removal from the suspension in SFEE. The suspension with the lowest concentration of ethyl acetate was subjected to drying. The results showed a reduction in the ethyl acetate concentration from 79588 (ethyl acetate concentration at the start of the emulsion) to 1484.5 ppm, thus reaching the level allowed by the FDA (Food and Drug Administration), which is 5000 ppm per day. Also, it was found that the lower the concentration of ethyl acetate in the suspension, the lower the recovery of β-caryophyllene and the greater the loss of oleoresin in the collection flask. The size of the suspended particles showed little variation from the droplet size of the emulsion injected into the SFEE system. It was also verified that the dried particles showed similar characteristics to the particles characterized in Stage (I). Finally, Stage III of this thesis consisted in the fractionation of copaiba oleoresin with supercritical CO₂, to concentrate sesquiterpenes and diterpenic acids. First, the effects of pressure and adsorbent material on the composition of the recovered fractions were evaluated. Next, to recover the oleoresin retained in the adsorbent materials used in SFF (Supercritical Fluid Fractionation), a Soxhlet extraction was performed using ethyl acetate followed by ethanol. The results showed that pressure and adsorbent materials influenced the fractionation kinetics and the chemical composition of the fractions. The production of purified fractions of β-caryophyllene with zeolite 13X at 9 MPa was verified. This condition also allowed concentrating and purifing copalic acid, which remained adsorbed even after desorption with supercritical CO₂ and was recovered by Soxhlet extraction with ethyl acetate.

Keywords: Modified starch, Nanoemulsion, Ultrasound, Microparticles, Supercritical Fluid Extraction of Emulsions, Fractionation.