Raman Spectroscopic Analysis of SPI

The 10 g of samples of SPI at different storage periods were solubilized 100 mL of deionized water at a concentration of 100 mg/mL for Raman spectroscopy analysis. The Raman spectroscopy analysis was determined according to the method of Zhu et al. [16 (link)], using PerkinElmer Raman Station 400 F dispersive Raman spectrometer equipped with a 785 nm diode laser under 80 mW of laser power. The laser was focused on the samples on glass slides. For each sample, the spectrum measurement with 60 s exposure time was repeated 10 times scan for each sample under 2 cm⁻¹ resolution, and the spectral range was set to 400~2000 cm⁻¹. Each sample was scanned three times, and the average spectrum was used for a representative spectrum for each sample.
Quantitative analysis on the secondary structure of SP under specific conditions was performed by Gaussian fitting using the Peakfit 4.12 software (Seasolve Software, Framingham, MA, USA). Raman spectra (400–2000 cm⁻¹) were plotted as relative intensity (arbitrary units) against Raman shift (wavenumber (cm⁻¹)).