Zetasizer nano zsp equipment

The ζ-potential was determined by electrophoretic light backscattering using Zetasizer Nano ZSP equipment (Malvern Instruments, Malvern, Worcestershire, UK). Milk samples were prepared in the same way as in Section 2.4. Analyses were carried out before and after homogenization, after pasteurization, and during the milk storage period (up to 28 days). Each analysis was performed in triplicate.

Exosomes were characterized by size distribution, zeta potential and the presence of exosomal marker CD63. Size distribution and zeta potential were performed by dynamic light scattering (DLS) using a Zetasizer Nano ZSP equipment (Malvern Instruments, Worcestershire, UK) at room temperature (~25°C), 175 degrees angle and 633-nm laser [4 (link)]. The sample was prepared in a standard Spectrophotometer cuvette (10 mm path length) considering the addition of 10 μL of sample diluted in 900 μL of bi-distilled water to measure particle size. The same diluted sample was used to measure zeta potential using a folded capillary zeta cell. Exosomal marker CD63 was detected in the samples by ExoELISA-Ultra CD63 Kit (System Biosciences, USA) following manufacturer instructions. Briefly, the sample was incubated at 37°C for 1 h on the provided micro-titer plate. After incubation and wash step, CD63 primary antibody was added to each well and incubated for 1 hour. After washing, the same incubation conditions were used for the secondary antibody. Then, super-sensitive TMB ELISA substrate was added and incubated at room temperature for 15 mins with shaking. After the addition of the stop buffer, the presence of CD63 was visually verified.

To determine the size of the fat globules in the milk samples after homogenization by US, the Zetasizer Nano ZSP equipment (Malvern Instruments, Malvern, Worcestershire, UK) was used. In order to analyze only the fat globules and to disrupt the casein micelles, 50 mM ethylenediaminetetraacetic acid (EDTA)/NaOH pH 7.0 buffer (Thermo Fisher Scientific, Waltham, MA; United States) was added in a 1:1 ratio [37 (link)]. Analyses were carried out at a temperature of 20 °C, and values of 1.462 and 1.333 were used for the refractive index of milk and water, respectively [38 (link)]. The z-average value, which derives the mean value from the intensity distribution, was used to compare the different ultrasonic treatments. Analyses were carried out in triplicate.