*2.3. Particle Size*

The particle size distributions and mean diameters of pea fibers were determined (expressed in % volume) using a Malvern MasterSizer 3000 (Malvern Instrument, Ltd., Malvern, UK) by referencing the method of Chen, et al. [15] with slight modifications. This technique provides information on the equivalent sphere diameter of fiber particles with different geometrical shapes, and the term "diameter" will be used to substitute for "equivalent sphere diameter" for simplicity. The measurement was conducted with the refractive indices of 1.52 and 1.33 for pea fiber and water, respectively, and an absorption index of 0.1 was used. The mean diameters including volume weighted mean diameter D[4,3] (diameter of the sphere of equivalent volume to measured particles) and surfaceweighted mean diameter D[3,2] (particle diameter that has the same specific surface as that of the full distribution) were evaluated. Cumulative percentiles of D10, D50 and D90 were also calculated, which indicated that the size of 10%, 50% and 90% of the particles was below the specified diameter, respectively. The pan of particle size distributions was applied to characterize the width of the particle size distribution, which was calculated according to Equation (1):

$$\text{Span} = \frac{\text{D}\_{\text{90}} - \text{D}\_{\text{10}}}{\text{D}\_{\text{50}}} \tag{1}$$

#### *2.4. Confocal Laser Scanning Microscopy (CLSM) Analysis*

The microstructure of pea fiber was observed using confocal laser scanning microscopy (Carl Zeiss LSM710, Jena, Germany). The preparation of samples were conducted by referencing the method of Huang, et al. [16]. Briefly, pea fibers were mixed with calcofluor white dye at a 50:1 volume ratio, and then the stained sample was added to a culture dish for observation. Images were acquired using a CLSM multiphoton system with a 40× objective lens. The excitation-emission wavelengths of 405–455 nm for calcofluor white and appropriate emission channels were used.

#### *2.5. Scanning Electron Microscopy (SEM) Analysis*

The morphology of pea fiber samples after freeze-drying and grinding was also examined using an environmental scanning electron microscope (Quanta-200, FEI Company, Eindhoven, The Netherlands). ISM-treated PeaF were prepared by sticking them onto double-sided adhesive tape attached to a circular specimen stub, following by sputtering a thin film of gold. The morphology of samples was observed at ×800 magnifications with an accelerating voltage of 5 kV voltage.

#### *2.6. Bulk Density Analysis*

1.8 g of pea fiber powder was accurately weighed and carefully added into a calibrated 25 mL graduated cylinder. Pressure was imposed manually to ensure no further decrease in sample volume. The bulk density was calculated as the volume of sample occupied by per gram dry weight (mL/g) [17].

#### *2.7. X-ray Diffraction (XRD) Analysis*

X-ray diffractograms of pea fiber powders after freeze-drying and grinding were obtained using an X-ray diffractometer (D8 Advance, Bruker, Berlin, Germany) operated at 40 kV and 40 mA with Cu Kα radiation. Before measurements, samples were stored in a desiccator where a saturated solution of NaCl maintained a constant humidity atmosphere (relative humidity = 75%) at 25 ◦C for five days. The scanning angle (2θ) of 5–50◦ with the interval of 0.02◦ was used to obtain XRD patterns. The crystallinity of samples was calculated based on the ratio of the area of the crystalline region to the total area in the XRD spectra using Origin software [18].
