Comparative Analysis of Protease-Assisted Extraction and Three-Phase Partitioning for Protein Recovery from Thai Black Soybean Okara

Kanthida  Wadeesirisak; Siriluck  Liengprayoon; Supanida  Winitchai; Naiyasit  Yingkamhaeng; Usa  Yonkoksung; Natedao Musigamart

doi:10.55230/mabjournal.v55i3.3718

Authors

Kanthida Wadeesirisak Institute of Food Research and Product Development (IFRPD), Kasetsart University, Bangkok 10900, Thailand
Siriluck Liengprayoon Kasetsart Agricultural and Agro-Industrial Product Improvement Institute (KAPI), Kasetsart University, Bangkok 10900, Thailand
Supanida Winitchai Kasetsart Agricultural and Agro-Industrial Product Improvement Institute (KAPI), Kasetsart University, Bangkok 10900, Thailand
Naiyasit Yingkamhaeng Kasetsart Agricultural and Agro-Industrial Product Improvement Institute (KAPI), Kasetsart University, Bangkok 10900, Thailand
Usa Yonkoksung Cassava and Starch Technology Research Unit, National Center for Genetic Engineering and Biotechnology, Thailand Science Park, Pathum Thani 12120, Thailand
Natedao Musigamart
aapndm@ku.ac.th
Kasetsart Agricultural and Agro-Industrial Product Improvement Institute (KAPI), Kasetsart University, Bangkok 10900, Thailand

Keywords:

Black soybean, okara, soy protein, Sukhothai 3, Three phase partitioning

Abstract

Okara, a by-product of soy-based food production, has gained increasing interest, particularly that derived from black soybean, due to its higher protein and nutrient contents than yellow soybean. However, its utilization remains challenging due to its high perishability. Limited studies have addressed okara bioactive composition, bioactivity, and the environmental impacts of different extraction methods. This study aimed to compare environmentally friendly extraction methods and evaluate their efficiency and functional properties for recovering bioactive proteins from Sukhothai 3 black soybean okara, using protease-assisted extraction (Protease-P6SD & Protease-A2SD) and three-phase partitioning (TPP). Extraction yield, amino acid composition, and antioxidant activity were evaluated. TPP showed a significantly higher extraction yield (26.61 ± 2.24%) than protease-assisted extraction (16.49 ± 0.95% and 15.36 ± 1.63% for Protease-P6SD and Protease-A2SD, respectively) (p<0.05). In contrast, the amino acid composition analysis showed that extracts obtained by protease-assisted extraction contained a broader range of essential amino acids than those obtained by the TPP method. This may be attributed to the higher specificity of proteolytic enzymes toward target proteins compared with chemical extraction methods. Antioxidant assays (DPPH and ABTS) revealed significantly lower IC₅₀values, approximately two-fold lower, in protease-derived extracts compared to TPP (p<0.05), indicating stronger antioxidant activity. These findings demonstrate that a higher extraction yield does not necessarily correspond to superior protein quality. While TPP provides a higher extraction yield, protease-assisted extraction enhances the amino acid profile and bioactivity. Therefore, enzymatic extraction represents a promising green approach for enhancing the utilization of black soybean okara. This study highlights the potential of Sukhothai 3 black soybean okara as a valuable biofunctional ingredient for functional food applications.

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