3. Results and discussion
3.1. Melting and solidification temperatures in supercritical CO2
The Tm and Ts data for beeswax and lipid mixtures were divided into two regions referred to region 1 and region 2 (Figure 2). In region 1, both temperatures decreased with increasing pressure, while in region 2 the melting and solidification temperatures increased with the increase in pressure. In the transition between the two regions (at 150 bar) each sample presented a different behavior. For the beeswax, the Ts increased but the Tmcontinued to decrease. For the beeswax-avocado oil mixture, Ts remained constant and Tm increased, and for the beeswax-Brazil nut oil mixture, Ts decreased and Tm remained constant.
According to de Sousa et al. (2006), this behavior can be explained by two distinct phenomena: the increase in the melting temperature of the compounds due to the increase in pressure and the decrease in the melting temperature by CO2 dissolution in the sample.
The plotted points of Tm and Ts (Figure 2) for each sample were used to determine the depression at the melting and solidification points, respectively, at a given pressure compared to atmospheric pressure. Figure 3A introduces Tm depression with increasing of pressure. The melting temperature depression for both mixtures of edible oil and beeswax were very similar, but the depression of melting temperature of pure beeswax was noticeably lower. This phenomenon may indicate that the fatty acid composition of the samples has a substantial effect on the Tm depression in pressurized CO2 because beeswax has longer chain fatty acids than the other oils studied. Ciftci and Temelli (2014) observed a greater Tm depression for lipids containing shorter chain fatty acids. In Figure 3B, which represents the Tsdepression for pressures up to 100 bar, all samples showed similar Ts depression; however, for higher pressures the Ts depression of the beeswax-avocado oil mixture was higher than that observed for beeswax-Brazil nut oil mixture; that of pure beeswax was the lowest, a fact that can be justified by the same argument used for the Tm depression, which is lipids or mixture of lipids that have heavy chain fatty acids have a lower Ts depression.
On the basis of the analysis of the Tm and Ts, the studied lipid mixtures could be used as carrier materials for particle formation with supercritical CO2at a temperature of 60 °C and pressures from 150 to 300 bar. This process temperature can ensure a safety margin for maintaining the lipid mixture in liquid state during the period of contact with supercritical CO2.
3.2. Volumetric expansion in supercritical CO2
The volumetric expansion of lipids in response to increased pressure varied for each sample, as shown in Figure 4. In an attempt to investigate the relationship between volumetric expansion and melting point depression, the Ve of lipids in pressurized CO2 was studied at points referring to region 2 of the melting curve and at intermediate pressures to those points. However, the volumetric expansion of lipids increased with increasing pressure at 60 °C temperature (Figure 4). Typically, the solubility of CO2 in the liquid lipid phase increases with pressure to a certain level and then reaches a plateau (Jenab and Temelli, 2012); however, in this study this phenomenon was only observed for the pure beeswax and beeswax-brazil nut oil mixtures in the pressure range used. The smallest Ve was observed for beeswax, and the largest Ve varied depending on the pressure range. For pressures below 225 bar, the beeswax-Brazil nut oil mixture showed the greater Ve. At 225 bar the volumetric expansions of this mixture and pure Brazil nut oil were similar, and for pressures above this the Ve of Brazil nut oil was high due to the fact that the binary mixture had reached the plateau. The behavior of the beeswax-avocado oil mixture and pure avocado oil were similar, with the Ve of the mixture slightly above 150 bar. For the other studied points, the Ve of pure avocado oil was slightly higher.
Comparing Figures 2 and 4, it can be seen that for the studied region there is a simultaneous increasing of volumetric expansion and melting temperature. According to Ciftci and Temelli (2014), there is a possibility of a slight increase in the Tm of fats and oils saturated with CO2 for relatively high pressures, which is attributed to the concurrent effects of the increase in the Tm due to a higher hydrostatic pressure and lower Tm due to CO2 dissolution.
3.3. Solubility of supercritical CO2 in lipids
The increase in volume observed in the volumetric expansion tests is due to the dissolution of CO2 in the studied lipids; therefore, determining the solubility of CO2 in the lipids and in the liquid lipid mixture can be useful to interpret the behavior of the volumetric expansion of the oil in equilibrium with supercritical CO2.
As shown in Figure 5, the solubility of CO2 in the studied lipids increased with increasing pressure. This phenomenon probably occurs by two mechanisms. The first is that with the increase in pressure the density of CO2 increases, becoming more similar with the density of the studied lipids; the second mechanism is that the increase in pressure facilitates the entry of CO2 into the tube containing the lipids so that there is regulation of internal and external pressure to the tube.
A study carried out by de Sousa et al (2006) with Precirol® ATO5, Compritol® 888 ATO and Gelucire® 43-01 as lipid carriers, also verified the solubility of CO2 in the lipids, varies by changing pressure, a behavior similar to that observed in this work.
3.4. Solubility of lipids in supercritical CO2
The solubility of the studied lipid mixtures in supercritical CO2 at 60 °C and different pressures (150, 200 and 250 bar) and contact time (1, 2 and 3 hours) ranged from 1.84 ± 0.27 g/kg of CO2 to 6.51 ± 0.02 g/kg of CO2 for the beeswax-avocado oil mixture and from 0.79 ± 0.10 g/kg of CO2 to 3.87 ± 0.25 g/kg of CO2 for the beeswax-Brazil nut oil mixture (Table 1). The solubility of lipid mixtures in supercritical CO2 increased when pressure increased. Studies by McHugh and Krukonis (2013) show that the solvent power of a supercritical fluid is related to its density, which is reinforced by Rad, Sabet and Varaminian (2019) when they stated that an increase in pressure leads to an increase in density and solvation power of supercritical CO2. Based on this, we can infer that the solubility of lipid mixtures in supercritical CO2 probably increased when the pressure increased because the fluid density increased, becoming more similar to that of the lipid mixtures.
Solubility studies carried out by Rodrigues and collaborators (2005) with Brazil nut oil did not show a high correlation between the BNO solubility in supercritical CO2 and the increase in pressure, contrary to what was observed in our experiments; for pressure variation also between 150 and 250 bar the solubility measured by the authors ranged from 2.56 to 2.64 g/kg at 50 °C and from 2.14 to 2.79 g/kg at 70 °C. The authors did not perform measurements at 60 ° C.
These results revealed that particle formation with supercritical CO2 at 60°C using the studied lipid mixtures can be conducted in a short contact time to minimize the degradation of heat sensitive bioactive compounds, which makes these mixtures good carriers for solid lipid particles. However, it must be considered that the solubility of beeswax-avocado oil mixture in supercritical CO2 at 60 °C and 200 bar was also affected by the static contact time.
3.5. Fatty acid profile
Total ion chromatogram of fatty acid found present in avocado oil, Brazil nut oil and beeswax are represented in Figure 6. It can be seen in the total ion chromatogram that several peaks presented by the samples have similar retention times, which indicates the presence of similar fatty acid or with similar functional groups. The qualitative fatty acid profiles of studied lipids are reported on Table 2.
Methyl elaidate, methyl 11-octadecenoate and linoleic acid ethyl ester were identified in both edible oils, and methyl margarate was identified in all studied lipids, including beeswax.
The differences on fatty acid profile probably has an influence on the other results obtained, such as the elevated solubility of the beeswax-avocado oil mixture in supercritical CO2. The fatty acid profile affects the solubility of the lipids in supercritical CO2 due to the different polarity and density of each fatty acid directly affecting the characteristics of the whole lipid. In addition, beeswax, which is a wax and solid at room temperature, has a more complex fatty acid profile than that found for edible oils with only a similar fatty acid, which probably justifies its different behavior.