Hydro 2000

The particle size distribution of the risk husk powder and wood flour powder were measured by a laser diffraction particle size analyzer Hydro 2000S (Malvern Instruments Ltd., UK) with distilled water/ethylic alcohol solution (50:50 in volume) to allow good powder dispersion whilst avoiding swelling. The results are expressed using the Mie method with a refraction index of 1.53 corresponding to those of sawdust particles (Malvern, 2007 ). The results are generated in volume, based on the assumption that the particles are spherical. Five replicates were carried out for each biomass and the mean average distribution is reported.

Each freshly prepared emulsion and reconstituted emulsion (1 g powder in 4 mL Milli-Q water) was placed in a Mastersizer 2000 laser light scattering instrument (Malvern Instruments Ltd., Worcestershire, UK) equipped with a Hydro 2000S sample handling unit at room temperature (21–23 °C) as described by Can Karaca et al. [47 (link)] to measure the average droplet size and distribution. The relative refractive index of emulsion was 1.112, which was calculated as the ratio of the refractive index of flaxseed oil (1.479) to the refractive index of water (1.33). The particle size was reported as surface-average diameter (D_3,2) and volume-average diameter (D_4,3) expressed using Equations (2) and (3), respectively.
$${\mathrm{D}}_{3,2}=\frac{{{\displaystyle \sum }}_{\mathrm{i}=1}{\mathrm{N}}_{\mathrm{i}}{\mathrm{d}}_{\mathrm{i}}^3}{{{\displaystyle \sum }}_{\mathrm{i}=1}{\mathrm{N}}_{\mathrm{i}}{\mathrm{d}}_{\mathrm{i}}^2}$$
$${\mathrm{D}}_{4,3}=\frac{{{\displaystyle \sum }}_{\mathrm{i}=1}{\mathrm{N}}_{\mathrm{i}}{\mathrm{d}}_{\mathrm{i}}^4}{{{\displaystyle \sum }}_{\mathrm{i}=1}{\mathrm{N}}_{\mathrm{i}}{\mathrm{d}}_{\mathrm{i}}^3}$$
where N_i is the total number of particles of diameter (d_i) [32 ]. All measurements were performed in triplicate, and two measurements were made for each replicate. The average value of two measurements in each replicate was collected and reported as the mean ± one standard deviation (n = 3).

Particle-size distribution of freeze-dried FBA-P samples was measured using a Mastersizer 2000 laser static light scattering instrument (Malvern Instruments Ltd., Malvern, UK) equipped with a Hydro 2000S system for handling samples. FBA-P (1.47 g) was suspended in 100 mL of water and stirred for 5 min prior to analysis.

The size distribution of the vaccine particles was determined by laser diffraction using a Hydro2000S wet sample dispersion unit and Mastersizer 2000 (Malvern Instruments Ltd). The parameters were: for wavelengths, 633 nm and 455 nm; for obscuration, 5 to 7%; for recirculation speed, 2000 rpm; and the dispersant was 150 mM NaCl. Mie theory was applied with the following optical parameters: the real refractive index of the particles was 1.65; and the imaginary refractive index of the particles was 0.01.

The size of the lipid droplets in the oil-in-water emulsion was determined by laser diffraction using a Mastersizer2000 coupled to a Hydro2000S (Malvern Instruments, Worcestershire, UK) according to manufacturer’s instruction. The emulsion was diluted directly in recirculating water (2000 rpm) reaching an obscuration of 12–15%. Refractive index was 1.445 for CO and 1.462 for RO.
In connection with oxidation status measured as peroxide value (PV, meq O2/kg oil), the emulsions stabilized by the highly saturated MPL, were highly stable, while the PV in the emulsion containing a relatively high concentration of PUFA (SL/RO) increased significantly over time. Regarding the particle size of lipid droplets, these were homogeneous and similar between all three emulsions over time ranging from 0.04–3 µm with a mean of around 0.5 µm, thus similar to lipid droplets in infant formulas^{38 (link)}. Details from stability studies are found in Supplementary data (Figure S1 and S2).

Size distributions of TA microcrystals and MSs were determined by Mastersizer MS 2000 (Malvern Instruments Ltd., Worcestershire, UK) equipped with a Hydro 2000 S automatic dispersion unit. Prior to analysis, MSs powder was suspended in the aqueous medium consisting of 1 w/v % PEG 4000 and 0.5 w/v % polysorbate 20. The suspended samples were then dropwise added to an automatic dispersion unit to obtain a 10–15% range of obscuration. Sample and background measurement times were set to 5 s and 10 s, respectively, and 5 runs were conducted for each measurement. Mie theory was applied to calculate the size distribution by volume with the refractive index value of 1.52. The resultant particle sizes of the three batches were averaged and presented as mean ± standard deviation (SD) (n = 3). The d_0.5, d_0.9, and d_0.1 indicated the median value defined as the diameter where 50%, 90%, and 10% of the population were below this value, respectively. SPAN value was an indicator representing the homogeneity of the particle size and was calculated by dividing the difference of d_0.9 and d_0.1 by d_0.5 [21 (link)].

Droplet size was measured by laser diffraction using Mastersizer^® 2000 with Hydro 2000s (Malvern Panalytical Ltd., Malvern, UK). Therefore, 200 µL of the emulsion was added to about 10 mL of purified water and mixed manually. The mixture was put in Hydro 2000s under stirring (2000 rpm) until an obscuration between 10% and 20% was reached. After 60 s the measurement was started. One measurement cycle recorded the signal three times for 30 s. For each emulsion three aliquots were measured. For each aliquot three repetitions were performed.