Optimization of protein isolate and bioactive peptide production from basil seeds, characterization of isolate and bioactive peptides

In this thesis, enzymatic pretreatment was applied to extract proteins from basil seeds using conventional alkaline extraction and ultrasonic-assisted extraction (UAE) methods. For enzymatic pretreatment, pH (3.0-5.0), temperature (35-50°C), enzyme concentration (0.5-5.0 FBGU/g seed), and time (1-4 hours) were identified as independent variables, and optimization was carried out using the Box-Behnken experimental design. The optimal conditions for enzymatic pretreatment were determined as pH 3.01, temperature 52.11°C, enzyme concentration 3.75 FBGU/g seed, and time 3.70 hours. Under these conditions, the protein yield was calculated as 86.55%, while the reducing sugar content was 164.63 mg/g seed. The coagulated protein percentage, water and oil holding capacities, foaming properties (capacity and stability), emulsifying properties (activity, stability, and capacity), and solubility characteristics of the protein isolates were comprehensively examined. Additionally, structural and thermal properties and amino acid composition of basil seed proteins were determined. The highest coagulated protein percentage (7.0%), water holding capacity (5.2 g/g), and foaming capacity (71%) were observed in the samples produced using enzymatic pretreatment and UAE. Protein solubility was the lowest at pH 4.0 (isoelectric point) and reached its highest level at pH 12.0. Bioactive peptides were produced from the basil seed protein isolate obtained in this thesis study. The enzymatic hydrolysis process for bioactive peptide production was optimized, with the optimal process conditions determined as enzyme:substrate ratio 12.26%, alcalase:flavourzyme ratio 0.63 U/U, and hydrolysis time 157.33 minutes. Under these conditions, the degree of hydrolysis was calculated as 73.73%, and DPPH free radical inhibition as 41.23%. The IC50 values of the produced bioactive peptides were determined as 8.0 µg/mL for DPPH, 15.8 µg/mL for ABTS, 9.15 µg/mL for hydroxyl free radical scavenging activity, and 4.9 mg/mL for chelating activity. For anti-hypertensive activity, the IC50 value was determined as 4.3 mg/mL, while the IC50 values for α-amylase and α-glucosidase inhibition, indicative of anti-diabetic activity, were 3.9 mg/mL and 4.8 mg/mL, respectively. Additionally, the antimicrobial activities of the bioactive peptides were determined, showing efficacy against Gram-positive bacteria. When the gastrointestinal system (GIS) stability of the bioactive peptides was evaluated, antioxidant activity was preserved at approximately 68% during the oral phase but decreased to ~34% in the gastric phase and ~15% in the intestinal phase. Chelating activity decreased by approximately 80% after GIS digestion. Anti-hypertensive activity was retained at 80% during the oral phase but showed an 86% reduction in the intestinal phase. Similarly, anti-diabetic activity decreased by ~84% in the intestinal phase. In conclusion, basil seed protein isolates were found to possess high nutritional value and functional properties. Enzymatic treatment and ultrasonic-assisted extraction methods significantly enhanced the protein yield and functional characteristics. Moreover, the bioactive peptides derived from the isolates demonstrated strong antioxidant, antimicrobial, anti-hypertensive, and anti-diabetic activities, highlighting their potential to offer various health benefits. However, it has been observed that some biological activities are lost during GIS. These findings suggest that basil seed protein isolates and bioactive peptides could be utilized in functional food formulations and health-supportive products.