Response surface optimization and characterization of protein concentrate fraction from Cocoa Bean shell (Theobroma cacao L.) waste with evaluation of its antioxidant activity

Abstract

Cocoa bean shell (Theobroma cacao L.) waste is an underutilized agro-industrial by-product with potential as an alternative plant-based protein source. This study aimed to optimize the extraction of a protein concentrate fraction from cocoa bean shell (CBS) waste using Response Surface Methodology (RSM) and to evaluate its chemical characteristics and antioxidant activity. Protein extraction was performed by alkaline maceration with magnetic stirring varying pH, extraction time, and solvent-to-solid ratio. Optimization was conducted using a Box–Behnken Design (BBD). The quadratic regression model adequately described the experimental data, with an Adjusted R² of 0.9490 and a Predicted R² of 0.9549, while the lack of fit was not significant (p > 0.05). Among the extraction variables, pH showed the most significant effect on protein recovery (p < 0.0001). The optimum conditions were pH 12, extraction time of 90 min, and solvent-to-solid ratio of 0.104 g/mL, resulting in a predicted recovery yield of 1.33% with a desirability value of 0.996. Although the recovery yield was relatively low, the optimized conditions provide baseline information for future process improvement. The resulting protein concentrate fraction contained 1.30 ± 0.11 % yield, 13.67 ± 0.42% protein, 5.98 ± 0.31% ash, 1.88 ± 0.03% fat, and 5.30 ± 0.28% moisture content. SDS-PAGE analysis revealed five protein bands ranging from 15–78 kDa, while FTIR analysis confirmed characteristic protein functional groups and phenolic compounds. The major amino acids identified were tyrosine (6.31 mg/g), glutamate (1.72 mg/g), and aspartic acid (1.68 mg/g). Antioxidant activity was relatively weak, with IC₅₀ values of 1020.38 ± 19.53 ppm and 1820.68 ± 39.12 ppm in the DPPH and FRAP assays, respectively. These findings indicate that CBS waste has potential as a sustainable source of protein-rich ingredients. However, further process intensification and post-extraction modification are required to improve protein recovery and functional properties.