Integrated Enzyme- and Ultrasound-Assisted Extraction of Fennel Seed Proteins: Process Optimization, Structure, and...

In this study, proteins were extracted from fennel seeds (Foeniculum vulgare) using an integrated enzyme-assisted and ultrasound-assisted extraction approach. Enzymatic pretreatment was optimized to maximize protein extraction yield while promoting cell wall degradation, as reflected by reducing sugar release. Following optimization, ultrasound-assisted extraction was applied as an alternative to classical alkaline extraction to shorten processing time and enhance extraction efficiency. The optimized enzymatic pretreatment conditions were determined as pH 4.20, 48.95°C, enzyme dosage of 1.91 FBGU/g seed, and a treatment time of 2.60 h. Under these conditions, protein extraction yield increased from ~61% in the control sample to ~92% after enzymatic pretreatment followed by classical alkaline extraction, which required 1 h of processing. Notably, within only 10 min of ultrasound-assisted extraction following enzymatic pretreatment, a slightly higher yield (~93%) was achieved. Functional characterization revealed that enzyme-ultrasound-treated protein isolates exhibited improved solubility, emulsifying, foaming, and water/oil-holding properties compared to control samples. Molecular weight profiling (SDS-PAGE), structural features assessed by FT-IR spectroscopy, and thermal properties evaluated by differential scanning calorimetry collectively indicated that the applied treatments did not cause noticeable degradation of protein subunits, while subtle conformational modifications were associated with improved functional performance and enhanced thermal stability, as reflected by the increase in denaturation temperature from 143.28°C to 176.23°C. Amino acid profiling demonstrated a balanced essential amino acid composition, highlighting the nutritional quality of fennel seed protein isolate. The results demonstrate that the combination of enzymatic pretreatment and ultrasound-assisted extraction is an effective and sustainable strategy for producing functional fennel seed protein isolates suitable for food applications.