2. Materials and methods
2.1. Material
Beeswax from a farm located in Pirassununga, state of São Paulo, Brazil was used as solid lipid material. Edible oils of interest were commercial avocado oil (Hass, Bauru, Brazil) and Brazil nut oil extracted from commercial Brazil nuts (Bertholletia excelsaH.B.K.) from Pará (Brazil). The extraction of Brazil nut oil was performed with supercritical CO2 (99%; Linde, Sertãozinho, Brazil) at 70 °C and 350 bar with solvent-to-feed ratio of 38.77 g CO2/g sample. Lipid mixtures (1:1 w/w) consisting of beeswax with avocado oil or Brazil nut oil were prepared at 90 °C by manual mixing. These lipid mixtures were studied to discover their possible application as a new carrier material for particle formation.
2.2. Lipid behavior in supercritical CO2 observed in phase monitor
A phase monitor (SFC/RESS, Thar Instruments Co./Waters, Pittsburgh, USA) (Figure 1A) was used for measurement of properties of lipids in supercritical CO2 such as Tm, Ts, volumetric expansion (Ve) and solubility of supercritical CO2. In these experiments, a height scale was placed inside the high-pressure vessel to measure the increase of the sample level due to the expansion with pressurized CO2. The sample was melted at 90 °C to eliminate crystal memory, and about 0.2 g of each sample was placed in a clear glass tube (0.3 cm in diameter and 4.0 cm in height) fixed to the autoclave using adhesive tape and near the sapphire window. The tube was placed into the high pressure cell (5.5 cm in diameter and 5.8 cm in height) in a position that allowed its observation using a camera through the sapphire window.
2.2.1 Melting and solidification temperatures in supercritical CO2
First, the cell was heated until the entire sample melted at atmospheric pressure. For Ts identification, the cell was cooled down slowly (about 0.5 °C/min) until sample solidification was visually observed. After Ts indentification, the sample was cooled down for extra 3 °C above Ts, awaited 10 minutes for equilibration and then the cell was heated slowly (about 0.5 °C/min) until the first brightness was visually observed, this temperature was identified as Tm. This process was repeated for all the pressures studied that ranged from 0 bar to 300 bar. The beeswax, beeswax-avocado oil mixture and beeswax-Brazil nut oil mixture were the studied lipids. The Tm and Ts were used to define and recommend a process temperature for particle formation. Thus, volumetric expansion, solubility of supercritical CO2, and solubility of lipids in supercritical CO2 were determined and carried out at this temperature.
2.2.2. Volumetric expansion in supercritical CO2
The scale placed inside the high-pressure cell was calibrated by adding different volumes of distilled water into the glass tube at ambient conditions.
The sample volume of lipids in supercritical CO2 was measured at pressures that ranged from 150 to 300 bar after 10 min at each pressure to clearly observe the lipid-CO2interface. Volumetric expansion was expressed as the percentage increase in volume relative to the initial volume at atmospheric pressure. In the case of solid samples at atmospheric pressure, the initial volume was recorded as the observed volume slightly above its Tmand at atmospheric pressure. Then, the cell was gradually pressurized to 150 bar and cooled to reach process temperature, maintaining the sample in liquid state to allow the measurement. The beeswax, avocado oil, Brazil nut oil, beeswax-avocado oil mixture and beeswax-Brazil nut oil mixture were the lipids studied. All measurements were done in duplicate.
2.2.3. Solubility of supercritical CO2
The solubility of supercritical CO2 in lipids (g of CO2/kg of lipid) was calculated from volumetric expansion data. The variation of the volume of the lipid material at a fixed pressure was considered as the volume corresponding to the CO2 mass dissolved in the known mass of lipid material. The CO2 density in each pressure was used to calculate the CO2 mass from the variation of the volume.
2.3. Solubility in supercritical CO2
The static solubility of lipid mixtures in supercritical CO2 (g/kg of CO2) was determined based on the methodology described by Cornelio-Santiago et al. (2017) and carried out at the Laboratory of High Pressure Technology and Natural Products (LTAPPN) of the Faculty of Animal Science and Food Engineering of the University of São Paulo (FZEA / USP) (Pirassununga, Brazil). Figure 1B contains the schematic of the equipment used. The experiment initially consisted of the contact between the lipid mixture (5 g) dispersed in glass beads (sufficient amount to completely fill the equilibrium cell) and supercritical CO2 under fixed conditions of pressure (150, 200 or 250 bar) and temperature (60 °C) in a balance cell (300 cm3). After a fixed contact time (1, 2 or 3 hours), a sample of the mixture, consisted by lipids solubilized in supercritical CO2, was moved to a high-pressure collector of known volume (8.58 cm3).
For this purpose, the CO2 inlet valves for the balance cell and the solution outlet from the balance cell for the high-pressure manifold were opened simultaneously to maintain constant pressure inside the balance cell as a variation in pressure could destabilize the solution.
The solution sample contained in the high-pressure collector was depressurized into a collection bottle. After depressurization, the high-pressure collector and the depressurization line were washed with pressurized ethanol (about 75 mL) to remove possible remaining materials and then it was dried by CO2 flow from another cylinder, all the ethanol used in this cleaning was also collected in the collector bottle. Finally, the mixture of lipid material and ethanol was placed in an oven at 70 °C for complete evaporation of ethanol until a constant weight was reached and the mass of lipid solubilized by CO2 could be measured.
The temperature control of both the equilibrium cell and the high pressure collector of known volume was performed by immersion in a thermostatic bath (Suprilab, Campinas, Brazil) and the pressure control by a high pressure pump (Eldex AA100S, Napa, CA, USA ). All experiments were done in duplicate. The values of the density of CO2under different conditions of temperature and pressure were calculated using the empirical equation of Huang et al. (1985). The means were compared using Tukey’s test.
2.4. Fatty acid profile
Both edible oils and beeswax were evaluated in terms of their fatty acid (FA) profile. The oils were prepared in the form of fatty acid methyl esters according to the 969.33 AOAC method (AOAC, 2005) and then analyzed by gas chromatography (GC) to determine the FA composition. The beeswax was prepared by dilution in chloroform followed by an intense mechanical shake until total dissolution, following the methodology described by Svečnjak et al. (2019).
The GC analysis was carried out on a gas chromatograph coupled with a mass spectrometer (GC/MS) (QP 2010 Plus, Shimadzu, Japan) with automatic injector (AOC-5000, SWI). A non-bonded poly (biscyanopropyl siloxane) phase (100 m × 0.25 mm x 0.2 μm) capillary column (Supelco SPTM2560, Bellefonte, USA) was used. The injection temperature was 250 °C, and the column temperature ranged from 100 °C to 195 °C with a temperature ramp of 5 °C/min and from 195 °C to 250 °C with a temperature ramp of 2 °C/min. The interface and ion source temperature was 250 °C, with a solvent cut time of 10 min. The injected volume was 1 μL with a split ratio of 12.5; helium was used as the carrier gas. The mass spectra were obtained by electron impact using 70 eV ionization energy as the quadrupole analyzer from 40 and 350 m/z. The fatty acid methyl esters were identified by comparison with mass spectra using GC/MS solutions v. 2.5 software, which has as database the NIST 11 and NIST 11 s libraries.