Bromelain excised hydrolysates with potent bioactivity from Bellamya bengalensis soft tissues: Process optimization and characterization

This study addresses the challenge of utilizing underexplored natural and cheap source for antioxidant compounds by optimizing and characterizing protein hydrolysate from the soft tissue of the edible freshwater snail, Bellamya bengalensis, using bromelain enzyme. The aim of this study was to determine the optimal hydrolysis conditions (enzyme substrate ratio, pH, temperature, hydrolysis time) to maximize the degree of hydrolysis and thereby its antioxidant properties using Response Surface Methodology with a three-level Box-Behnken design. The optimal conditions of the hydrolysis were found to be an enzyme-substrate ratio of 0.973, pH 7.5, temperature 53.08 °C, and hydrolysis duration of 6.13 h. Hydrolysates produced at optimized conditions had a higher degree of hydrolysis (47.96 %) and remarkable antioxidant properties (DPPH-54.64 %; FRAP-57.08 mM Fe(II)/gm). Amino acid profiling revealed high concentrations of antioxidative amino acids and hydrophobic amino acids, enhancing the hydrolysates’ functional properties. Fourier transform infrared spectroscopy revealed key absorption peaks at 1644.50 cm⁻1 (Amide I) and 1548.15 cm⁻1 (Amide II). Differential scanning calorimetry showed that the sample was stable up to 60 °C but denatured between 100 °C and 200 °C. Particle size and zeta potential analysis showed uniform particles of 100.4 nm with a zeta potential of −15.7 mV, reflecting good stability. SDS-PAGE confirmed low molecular weight peptides (<25 kDa), indicating effective enzymatic breakdown. This study highlights the potential of B. bengalensis protein hydrolysates as sustainable antioxidant additives for food preservation and other applications in food and non-food industries, offering a sustainable and innovative use of freshwater snail resources.