Peakfit version 4

An FTIR spectrometer (Bruker Tensor 27, Bruker Optics, Ettlingen, Germany) was employed to determine the spectra of CSPI undergoing various F–T cycles. After mixing with KBr (1:100), CSPI was compressed as a thin tablet to scan at wavelengths of 4000–400 cm⁻¹. Peakfit Version 4.12 (SPSS Inc., Chicago, IL, USA) software was adopted to analyze the FTIR spectrum data and the protein secondary structure.

The Raman spectra were recorded on a Thermo DXR Raman microscope using the 532 nm laser excitation and constant laser power of 8 mW, grating with 900 lines/mm, and a spectrograph aperture of 50 μm pinhole. All samples were recorded under the same experimental conditions. The 30 wt% OVA solution (5 µL), and corresponding hydrogel (~10 mg) were placed on golden plates (Gold EZ spot micro, Thermo Fisher Scientific, Waltham, MA, USA), while the solid sample (~5 mg) was measured on a glass microscope slide. After acquisition, fluorescence background was subtracted from the Raman spectra using fifth order polynomial fit (OMNIC for Dispersive Raman 9.2.41 software). The selected spectral region was deconvoluted by applying the PeakFit™ version 4.12 software (SPSS Inc).

The Fourier-transform infrared (FT-IR) spectra were obtained using a Nicolet 6700 FT-IR spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) at room temperature. Each spectrum was recorded at the absorbance between 400 to 4000 cm⁻¹ at a resolution of 4 cm⁻¹ using an average of 60 scans by Nicolet Omnic 32 software (Thermo Fisher Scientific Inc., Waltham, MA, USA). The measurements were made directly for freeze-dried powder samples for milk proteins, whey protein, sodium caseinate 180, polyphenol extract, and casein-, whey protein-, and milk-based blackcurrant samples. Tests were carried out in triplicate with three measurements for each replicate. To determine the effects of polyphenols on the secondary structure of milk proteins, the spectral region between the wavenumbers of 1600 cm⁻¹ and 1700 cm⁻¹ was selected. The full bandwidth at half height (FWHH) was adjusted to 13 cm⁻¹, and the resolution enhancement factor was set to 2.4. The selected spectra were smoothed with 2 points, and the second derivative calculations were used to obtain the percentage of secondary structural elements by the PeakFit Version 4.12 software (SPSS Inc., Chicago, IL, USA).

FTIR spectra could reflect the secondary structure of proteins [30 (link)]. The MP samples (dry form) were measured by FTIR spectra with some modifications based on the reported method by Sun et al. [31 (link)]. The samples were mixed with bromatum Kalium, and the mixture was ground and pressed into a disc. FTIR spectra were obtained in the wavenumber range from 400 to 4000 cm⁻¹. Detection was carried out at 20 °C, and the infrared image of each sample was a superimposed image of multiple scans. The results were analyzed by EZ-Ominic software and Peak Fit Version 4.12 software (SPSS Inc., Chicago, IL, USA).