Nano-particle emulsions were spontaneously prepared using the aqueous titration method (Shafiq et al., 2007; Zhao et al., 2010). The mixture of food grade Tween 80 and Span 80 (Nof, Japan) (1:1, w/w) was mixed with the co-surfactant, propanol (Abbsasi and Radi, 2016), which was purchased from QReC (New Zealand) in different proportions (1-9:1, w/w). The resulting mixture was labeled as S_mix. The ratio of tea seed oil (Forecus, Thailand) and S_mix was thereafter thoroughly mixed in different proportions from 1-9: 9-1. The mixture of oil and S_mix was thereafter mixed with water and visually graded into five categories, A-E (Lourith et al., 2016; Shafiq et al., 2007):

Hydrophilic-lipophilic balance (HLB) of the prepared emulsions was calculated according to the surfactant components, as shown below:

where HLB_T and HLB_S are HLB values, and f _T and f _S are the weight fractions of Tween 80 and Span 80, respectively (Wang et al., 2009).

The emulsions of A grade were stored under ambient conditions for 3 days in order to allow a complete transport processes of the emulsion until its equilibrium was reached (McClements, 2012). Emulsions whose texture remained homogeneous with a clear appearance represent A grade were scaled up for physicochemical characterization and the accelerated stability test by means of heating and cooling for 7 cycles (45 and 4 °C, each 24 h). The droplet size, polydispersity index (PDI) and ζ-potential were determined using the dynamic light scattering and micro-electrophoresis device (Nano-ZS90, Malvern Instruments, UK) with a 50 mV laser. The samples were dispersed in de-ionized water (0.2%). The measurement time was 2 min and each run underwent 12 subruns, performed at a fixed angle of 90° with a sensitivity range between 10 nm to 5 m at room temperature. The stable emulsions were thereafter scaled up and stability was evaluated under 4, 25 and 45 °C, separately, for 3 months. Droplet size, PDI and ζ-potential were determined as usual in addition to pH (Mettler Toledo, s20, Switzerland) and viscosity (25 °C, 160 rpm, spindle no. 2; Brookfield, RVDV2T extra, USA). All of the measurements were done in triplicate (Shafiq et al., 2007).

HLB is the important criteria for an emulsion formation, particularly for spontaneous phase transitions that are driven by thermodynamic effects at a constant temperature with minimal energy consumption. Tween 80 (HLB = 15) and Span 80 (HLB = 4.3) were therefore chosen in a combination of non-ionic surfactants (Polychniatou and Tzia, 2014). Polyols and short chain alcohols were trial used as co-surfactants due to the similarity of these solvents to water in terms of their hydrogen bond formation and high dielectric constants. In addition, they are immiscible in apolar solvents with higher critical micelle concentrations in polar non-aqueous solvents than water, resulting in penetration into the surfactant interface which self-assemble in alcohols (Garti et al., 2001), thus reducing interfacial tensions accordingly (Rao and MacClements, 2012). However, those of propylene glycol and polyethylene glycol afforded none of the A grade emulsions. Therefore, propanol and ethanol were further designed as the co-surfactants. A visual grading using propanol as the co-surfactant resulted in 28 A grade, 33 B grade, 233 C grade and 516 D grade emulsions (Figure 1a). Those of the A grade were included for short-term stability (McClements, 2012) and resulted in 12 systems (P1-P12) that retained their original macroscopic appearance which thereafter were determined as nano-particle emulsions (242 – 426 nm) as shown in Figure 1b, with PDI of 0.1 to 0.4 indicating the narrow size distribution. Although a non-ionic surfactant system is used in this preparation of nano-particles, the emulsions that should be ruled by the free fatty acids of tea seed oil with anionic nature had a negative charge (Li et al., 2012). The observed absolute ζ-potentials greater than 40 mV guide the stability of the formed emulsion with a high energy barrier, protecting coalescence of the internal oil phase (Zhao et al., 2010). The A grade emulsions with the greatest tea seed oil content of 27.27% (P7, 9 and 12) were shown to have a higher mean droplet diameter (414.53 ± 31.80, 426.03 ± 15.19 and 387.47 ± 17.05 nm). The systems with the lower proportions of surfactants and co-surfactant, resulting in a lesser degree of surfactant self-assembly weakly minimized the liquid crystal phase regions (Garti et al., 2001) including a low transportation rate of oil across the water phase (McClements, 2012). The droplet size tends to be bigger accordingly. pH and viscosity (Figure 2b) of the clear emulsions were in the same range of the vegetable oil’s nano-particle emulsion containing non-ionic surfactants (7.04 to 7.32 and 151 to 242 cps) that are commercially produced (Odile et al., 2001; Quemin, 2001). Although ethanol gave A grade emulsions, they were highly viscous (data not shown) and were excluded from this study.

Propanol influences the two-phase area by improving the solubilization capacity of the interface, which is in contrast to a longer chain alcohol (Garti et al., 2001). The HLB of the surfactants was designed at the range of 8 – 16 for a higher possibility of o/w emulsion preparation (Roohinejad et al., 2015). HLB regulates the adsorption of surfactants to o/w interfaces by differentiating the particle size accordingly. An interfacial film is formed and the interfacial tensions are decreased with high HLB. These phenomena facilitate droplet disruption, thereby promoting small droplets and improving long-term stability (Rao and McClements, 2012). The propanolic S_mix system (64.64 to 81.82%) with HLB between 9.22 to 9.41 yielded tea seed oil nano-particle emulsions with 9.09 to 16.67% water. The proportion of surfactant that was higher than 50% was in harmony in the used range for the formation of a stable self-emulsifying delivery system (Tang et al., 2008).

Although the prepared tea seed oil nano-sized emulsions were prepared by means of micro-emulsion, a theoretically thermodynamically stable nano-particle, the stability of the emulsions was assessed to observe the phase behavior which is crucial for an industrial development because the micro-emulsion may be changed due to chemical degradation or microbial contamination (McClements, 2012). The prepared micro-emulsions presented in this context added no preservatives. Accelerated stability tests were first conducted by using 7 heating and cooling cycles (Shafiq et al., 2007). The prepared tea seed oil micro-emulsions were stable with no phase separation, creaming or sedimentation. All of the prepared emulsions maintained their nano-particle characteristics with a smaller droplet size than 500 nm as shown in Figure 1b with low PDI (< 0.4) indicating they were mono-dispersed. The stability in terms of ζ-potential was in agreement. This electrostatic repulsion prevents flocculation, thus limiting sedimentation. This S_mix was less affected by ζ-potential. Although Ostwald ripening was noticed in some systems (P2, 8, 11, 12), the rest would be regulated by the ripening propagating coalescence as the diameters were larger.

Thereafter, long term stability of the prepared micro-emulsion was evaluated as shown in Figures 2 to 4. Following 3 months of storage, the emulsions’ diameters were less than 500 nm. ζ-potential remained negative with an absolute value greater than 40 mV, which suggests stability against coalescence. However, flocculation was noticed and further coalescence in the course of stability assessment was noted at day 30 of the observation, and thereafter ripped into a smaller size, although at a different time interval and at a different temperature. 45 °C was shown to be the most affective followed by ambient condition and 4 °C. However, P11 stored at 4 °C was dramatically changed and phases were separated following day 60 of the study. This system was discontinued for stability studied at 4 °C.

pH and viscosity remained in the same range, although there were some shifts. The acidified nano-particle emulsions were affected, possibly due to the deterioration of fatty acids and other organic substances in the oil as no preservatives were added. A greater viscosity following storage is possible in nano-particle emulsions produced by non-ionic surfactant systems (Nazarzadeh et al., 2013). The two-phase area is supposed to be shrunk with a short chain alcohol that is used as a co-surfactant (Garti et al., 2001) and plays a role in the viscosity accordingly, although this was only slightly observed in this system.