Potential of Fluorescence Spectroscopy and Principal Component Analysis for the Characterization of Antioxidant Capacity of Soy Protein Hydrolysate Fractionation by Sequential Membrane Ultrafiltration and Nanofiltration

Many biological compounds, such as peptides, possess the ability to decrease unfavorable effects of oxidative free radicals in biological and food systems, known as antioxidant capacity. Current antioxidant capacity assays are time-consuming and unable to distinguish the contributions of specific species to the antioxidant capacity. In this study, we have evaluated the potential of intrinsic fluorescence and principal component analysis for its use as a rapid method for antioxidant capacity determination of protein hydrolysates during their fractionation by membrane operations.  To demonstrate this potential, soy protein hydrolysate fractions with different antioxidant capacity were produced by sequential ultrafiltration (UF) and nanofiltration (NF). Crude soy protein hydrolysates (SPH) were obtained by enzymatic hydrolysis (pepsin and pancreatin mixtures) of soy protein isolate thermally treated at 95 ^oC for 5 min and subjected to sequential UF (10 kDa MWCO step) and NF (2.5 kDa MWCO step at pH 4 and 8). Collected UF and NF fractions were evaluated for antioxidant capacity with the Oxygen Radical Absorbance Capacity (ORAC) and Folin Ciocalteau Reagent (FCR) assays, and their fluorescence excitation-emission matrices (EEM) were obtained. Principal component analysis (PCA) of fluorescence EEM data for UF and NF fractions revealed two principal components (PC) that captured significant variance in the fluorescence spectra and could be related to tryptophan (PC₁) and tyrosine (PC­₂) amino acid residues based on their excitation and emission properties. Multi-linear regression models were developed to obtain relationships between the antioxidant capacity and the two PCs associated with the fluorescence EEM of the fractions.  These models generated fluorescence and PCA-captured ORAC (ORAC_FPCA) and FCR (FCR_FPCA) estimates of UF and NF fractions. The ORAC_FPCA and FCR_FPCA antioxidant capacities for NF samples displayed strong, linear relationships (R²>0.99). A clustering effect that separated UF and NF peptide fractions with respect to ORAC_FPCA and FCR_FPCA was observed, due to their differences in molecular weight, charge, and antioxidant capacities, indicating different roles of UF and NF membrane processes that were in play for improving the antioxidant capacities. The significance of this study hinges on the ability of fluorescence EEM and PCA to identify individual and combined contributions of tryptophan and tyrosine residues in SPH fractionated by sequential UF and NF to their antioxidant capacities that were not directly identified by ORAC and FCR assays. Therefore, the proposed method could be potentially developed as a rapid, non-destructive, online tool during membrane fractionation of protein hydrolysates with antioxidant capacity.