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.