Fingerprinting of Semi-Refined Flaxseed Protein Using Raman Spectroscopy and Multivariate Analysis: Pulsed Electric Field (PEF)-Assisted Alkali and Aqueous Extraction Methods Alter Composition and Protein Conformation

This study investigated the structural modifications of semi-refined flaxseed protein extracts (SRFPE) during pulsed electric field (PEF)-assisted extraction at varying energy levels in both alkaline and neutral conditions. Raman spectroscopy coupled with principal component analysis (PCA) was employed to analyze spectral band markers to monitor alterations in secondary and tertiary protein structures, with complementary radar chart visualization providing rapid, at-a-glance assessment of structural modifications. Compositional fingerprints revealed distinct Raman bands and changes: the presence of diverse amino acids (C–H stretch at ~2933 cm ⁻¹); increased phenolic compounds (COO stretch at 1604 cm ⁻¹ and aromatic ring stretch at 1633 cm ⁻¹) with PEF treatments applied between 347 and 534 kJ/kg in alkaline conditions; and minor contributions from carbohydrates and lipids. PCA effectively distinguished between α-helical structures (1659 cm ⁻¹) predominant in aqueous extracts and β-sheet structures (1671 cm ⁻¹) in alkaline extracts, for SRFPE obtained without PEF treatment or those PEF-treated with up to 139 kJ/kg. Protein tertiary structure analysis revealed tryptophan residues predominantly in hydrophilic environment (I ₁₃₆₀/I ₁₃₄₀ ratio <1), increased solvent exposure of tyrosine residues (I ₈₅₀/I ₈₃₀ >1), varying hydrogen bonding strengths from W17 frequencies (878–883 cm ⁻¹), and disulfide bond conformations (500–550 cm ⁻¹) showed t-g-t conformer predominant in PEF treated (347–534 kJ/kg) alkali extracts, while g-g-g and g-g-t conformers predominated in SRFPE obtained without PEF treatment or those PEF-treated with no more than 139 kJ/kg. Raman spectroscopy with PCA enabled rapid and non-destructive monitoring of extraction-induced molecular changes by distinguishing and evaluating protein structures and non-protein components in complex samples.