2.3. Emulsion Characterization

Particle size and distribution of emulsions were measured in the fresh samples after UHPH or CH treatments using a Mastersizer laser diffraction 2000 analyzer (Malvern Instruments Ltd., Worcestershire, UK). Emulsion were added directly to the recirculating measuring cell containing distilled water or 0.5% SDS solution until 5–9% obscuration was achieved. The optical model based on the Mie theory of light scattering by spherical particles was used. The optical model used were a refractive index of 1.460 for BM, a refractive index of 1.332 for water, and absorption of 0.01. Results were expressed as the volume-weighted mean diameter (d_4.3, μm) and Span index. Measurements were made separately for water and SDS as dispersing media. Span is a parameter which indicates the homogeneity of the particle size distribution and was calculated using Equation (1).

where d_x (μm) is the size point below which x% of the particles is contained.

The zeta-potential of emulsions was measured using a Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK). Emulsions were diluted 1:100 with ultrapure water and allowed to equilibrate at 25 °C for 120 s in the cuvette prior to analysis. The measurement was performed on the same day of homogenization using an automatic voltage selection. Zeta-potential was calculated using the Smoluchowski model using the software provided by Malvern Instruments.

Flow curves were performed on fresh emulsions (24 h after treatment) at 20 °C with a controlled stress rheometer (Haake Rheo Stress 1, Thermo Electron Corporation, Karlsruhe, Germany). A concentric cylinders probe was used. Samples were loaded into the probe for 5 min before starting the test in order to reach equilibrium.

Flow curves were obtained in ascending and descending shear rates in the range of 0.1 and 100 s⁻¹ for 60 s, respectively. Ostwald de Waele rheological model (Equation (2)) were fitted for descending curves, and the rheological parameters (K, n) were obtained. From the difference between the area under the ascendant and descendant curves, the hysteresis was calculated as indicative of thixotropic behavior.

where s is the shear stress (Pa), K is the consistency index (Pa.sⁿ), g is the shear rate s⁻¹, and n is the flow behavior index (n = 1 indicates Newtonian behavior n ≠ 0 indicates non-Newtonian behavior).

Colloidal stability of the emulsions was evaluated by using Turbiscan MA 2000 optical analyzer device (Formulaction, Toulouse, France). Emulsions were transferred into borosilicate glass tubes of 27.5 mm dimeter up to 40 mm height, and sodium azide (0.04%) was added to prevent microbial growth. Three tubes of each sample were prepared and stored at 20 °C for 8 days. The evolution of stability was analyzed at 0, 1, 4, 6, and 8 days. This equipment provides a powerful technique for characterization of dispersions, detecting variations in stability phenomena by measuring backscattering (BS) along the sample tubes. Stability index (TSI, Formula 3) and creaming layer thickness evolution was determined by using the software (Turbisoft 2.3.1.125 version) provided by the manufacturer.

where scan_i (h) is mean BS for each i of measurement, scan_i−1 (h) is mean BS for i−1 measurement, and H is the height of a sample. Higher TSI indicates stronger destabilization caused by particle aggregation and/or dynamic migration.

A confocal laser-scanning microscope (Leica TCS SP5, Leica Microsystems GmHB, Mannheim, Germany) was used to observe the structure of fresh emulsions (24 h after production). The protein and oil were fluorescently labelled together and separately from phospholipid components of the samples. Proteins were labelled by Fast Green FCF (Sigma-Aldrich, St. Louis, MO, USA), prepared at concentration of 1% in distilled water and subsequent addition of 10% to the emulsion, excited by a 633 nm laser, and detected at 650–750 nm. Oil was labelled by Nile Red (5H-Benzo-phenoxazine-5-one, 9-diethylamino; Sigma-Aldrich, St. Louis, MO, USA), by dissolving 1 mg/mL in acetone. About 100 μL of Nile Red solution was added to 1 mL of emulsion. Excitation was made at a 488 nm laser and detected at 500–600 nm. Phospholipids were labelled by Liss Rhod PE (1,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-lissamine rhodamine B sulfonyl; 1 mg/mL; Avanti Polar Lipids Inc., St. Louis, MO, USA), by adding 40 μL to 1 mL emulsion, excited by a 561 nm laser and detected at 575–630 nm. All fluorescently labelled samples were mounted on cavity plates and examined at room temperature with a 100× oil immersion objective.