Over the years, the role of proteins as a source of amino acids has expanded to include their functional roles through bioactive peptides. These small fragments of protein (2–20 amino acids) are classified based on their bioactivities e.g., antioxidative, antihypertensive, antidiabetic, immunomodulator, antimicrobial, opioid (Minkiewicz, Iwaniak, and Darewicz, 2019). The BIOPEP-UWM™ database currently records over 4600 bioactive peptides. What makes these peptides particularly appealing is their minimal side effects in humans (Zou, He, Li, Tang, and Xia, 2016), making them potential ingredients in functional foods or therapeutic agents.

The bioactive peptides exhibit their bioactivities only when they are released from their parent proteins. Hence, the selection of a parent protein is a pivotal step in bioactive peptide production. According to Udenigwe and Aluko (2012), parent proteins are mainly selected on the basis of two criteria: value addition to protein-rich materials from underutilized sources or by-products, and using proteins with specific sequences which exhibit desired bioactivities. The velvet bean (Mucuna pruriens) is suitable candidate for meeting these criteria. This tropical legume is cultivated as cover crop in Africa, South America, and Asia. Although it is rich in protein (23–35%), it is underutilized as food (Janardhanan, Gurumoorthi, and Pugalenthi, 2003; Lampariello, Cortelazzo, Guerranti, Sticozzi, and Valacchi, 2012; Segura-Campos, Tovar-Benítez, Chel-Guerrero, and Betancur-Ancona, 2014). Velvet bean has a high amount of hydrophobic amino acids, which are known properties of bioactive peptides (Indrati, 2021; Kalidass and Mahapatra, 2014; Sánchez and Vázquez, 2017). Velvet bean protein hydrolysates are reported to exhibit angiotensin-converting enzyme (ACE) inhibitory activity (i.e., low IC50 of 0.9 × 10−3 μg/mL) and antioxidative properties (Chalé et al., 2014; Segura-Campos et al., 2014; Segura-Campos, Espadas-Alcocer, Chel-Guerrero, and Betancur-Ancona, 2013; Tuz and Campos, 2017).

Various processes can be performed to release bioactive peptides from parent proteins, including in vivo hydrolysis via digestive enzymes, in vitro hydrolysis via microbial or plant enzymes, and microbial fermentation (Rutherfurd-Markwick, 2012). Among these methods, in vitro enzymatic hydrolysis is often preferred due to mild conditions for the reaction and feasibility for the scale-up (Yang et al., 2021). Additionally, combination of methods (i.e., fermentation and enzymatic proteolysis) can be applied to enhance the release of smaller peptides (Peredo-Lovillo, Hernández-Mendoza, Vallejo-Cordoba, and Romero-Luna, 2022).

In an enzymatic hydrolysis, two approaches can be used: batch or continuous reactions. However, batch reactions are considered unproductive due to their time-consuming start-up and end-procedures (Prazeres and Cabral, 1994; Sitanggang, Drews, and Kraume, 2016). Furthermore, batch-to-batch variations in quality pose a challenge to meeting requirements for consistent product quality (Yu, 2008). Therefore, a continuous system is a preferred. In this study, we attempted to produce bioactive peptides continuously from protein concentrate isolated from fermented velvet bean, using an automated membrane reactor system. The continuous system facilitated by the membrane separation is a promising alternative to the major reason for the delay in bioactive peptide commercialization, which is the lack of scalable and consistent production methods (Chakrabarti, Guha, and Majumder, 2018). Moreover, this study offers a safer method of producing bioactive peptides using fermentation and membrane technology. By achieving optimal operating conditions, we anticipated that the permeate would display high and consistent antioxidant and ACE inhibitory activity for further biochemical characterization.