Ls230

Homogenization was validated using a light scattering instrument (Coulter, LS 230, Beckman Coulter, Atlanta, GA, USA) to measure volume‐average droplet diameters. The homogenized milk was frozen (−20°C) and stored for <3 days before subsequent analysis. Samples were thawed (37°C) and vortexed before measurements. Distilled water was used as a background solvent. The refractive indices used for milk fat were 1.46 and 1.33 for the aqueous continuous phase (Ransmark et al., 2019 (link)). Pasteurization was validated using Phosphatesmo MI test strips (Macherey‐Nagel, Düren, Germany) for the determination of alkaline phosphatase (ALP) in milk, according to manufacturer's instructions. A yellow color indicates the presence of functional ALP and a white color indicates inactivation of ALP. Measurements were taken in triplicate.

Particle size of the stigmasterol emulsion was determined using a Particle size analyzer and performed using a laser diffraction particle size analyzer (Beckman-Coulter, LS-230, Miami, FL, USA). The degree of crystallinity was determined using a Rigaku (30 kv/25 mA) Geigerflex D/Mac, C series diffractometer (Tokyo, Japan) with Cu-kα radiation (λ = 1.5406 A°) at room temperature in glancing inclined angle mode.

For the liquid samples before freeze-drying, the particle size and particle size distribution were determined by using a laser scattering size analyzer (LS230^®, Beckman Coulter, Beijing, China). Firstly, the refraction indices of the samples and water were 1.59 and 1.33, respectively. Then, the liquid samples were added into the laser scattering size analyzer. Finally, the particle size and size distribution were recorded by the software. All measurements were tested at least three times in parallel.

The dimensional distribution was carried out with a Laser Light Scattering (LLS) granulometer (Beckman Coulter LS 230, Particle Volume Module Plus, Brea, CA, USA). The MTE raw extract was suspended in distilled water; otherwise, microparticles were suspended in isopropanol. About 200 μL of the suspension was poured into the small volume cell to obtain an obscuration between 8 and 12%. Particle size distributions were calculated using the Fraunhofer model. The results were expressed as d₁₀, d₅₀, and d₉₀, indicating the volume diameters at the 10th, 50th and 90th percentiles, of the particle size distribution. The span is defined as:

Characterizations of the particles were performed by transmission-electron microscopy (TEM) using freeze-fracture preparation technique and by static light scattering (SLS). Sonicated and autoclaved stock solutions of NPs were diluted 1:10 in cell culture medium and cryoprotected by immersion in glycerol solution. Samples were cryofixed into melting Freon 22 and liquid nitrogen. Freeze fracturing took place at −120 °C with a BAF 400 (Bal-Tec, Balzers, Liechtenstein). Freeze-fractured specimens were replicated by application of Pt/C and C by electron-gun evaporation. The replicas were cleaned in concentrated sodium hypochlorite and in acetone and examined with a Tecnai 10 (FEI Company, Hillsboro, OR, USA) transmission-electron microscope operated at 80 kV. Size measurement of single particles and particle aggregations using the TEM images was conducted with the program “Bild-Vermessen 1.0” (CAD-KAS Kassler Computersoftware GbR, Markranstädt, Germany). At least 30 single particles and 20 aggregates of the different NPs were measured. Particle size distribution was measured by SLS using a LS 230 (Beckmann Coulter, Krefeld, Germany). The particle dispersion was dosed into the instrument without special sample preparation. The volume fraction-length mean diameter was measured.