The increasing demand for sustainable, high-quality protein sources has catalyzed a paradigm shift in global dietary patterns, with growing attention directed toward plant-based proteins as viable alternatives to animal-derived proteins. This shift is largely driven by concerns over the environmental footprint of livestock production, including greenhouse gas emissions, excessive water usage, and land degradation, as well as ethical and health considerations (Loveday, 2019; Naghshi et al., 2020; Ozturk & Hamaker, 2023). Leguminous crops, in particular, have garnered interest due to their nutritional adequacy, agricultural viability, and functional versatility in food systems. Among these, kidney bean (Phaseolus vulgaris L.) emerges as a promising candidate owing to its widespread cultivation, cost-effectiveness, and high protein content ranging from 20 % to 30 % on a dry weight basis (Mundi & Aluko, 2012; Singhi & Ozturk, 2025; Srenuja et al., 2023). Direct consumption of whole kidney beans, while nutritionally valuable, presents inherent biochemical and structural limitations that restrict their broader applicability in advanced food systems. The native legume matrix is characterized by a complex composite of starch, non-starch polysaccharides, and anti-nutritional factors (ANFs), including phytohemagglutinin (lectins), tannins, and trypsin inhibitors, that collectively impair protein digestibility, restrict mineral bioavailability, and necessitate intensive thermal pre-processing to ensure safety (Z. Zhang et al., 2023). The isolation of the protein fraction is therefore essential, serving a dual purpose: to enhance nutritional and safety profiles, and to unlock the inherent techno-functional potential of the storage proteins (Roy et al., 2020).

Phaseolin, also known as vicilin or 7S-8S globulin, is the predominant storage protein in kidney beans and accounts for approximately 75–82 % of the total seed protein. It is composed of α-, β-, and γ-subunits with molecular weights ranging from 43 to 53 kDa (Romero & Ryan, 1978; Sathe, 2002). While kidney bean proteins possess a relatively balanced amino acid profile, they are limited in sulfur-containing amino acids such as methionine and tryptophan, which constrains their nutritional completeness (Ahmed et al., 2018). Functionally, kidney bean protein isolates (KBPI), exhibit suboptimal solubility, emulsifying, and foaming properties under varying pH and ionic conditions (Wu et al., 2025). These limitations are further exacerbated by thermal processing, which promotes protein denaturation and aggregation, thereby diminishing dispersibility and reducing their applicability in complex food matrices (Badjona et al., 2024; Peng et al., 2016). To overcome these constraints, various protein modification strategies, including chemical, enzymatic, and physical methods, have been explored to alter protein conformation and enhance functionality (Akharume et al., 2021). Among these, physical non-thermal processing technologies, such as high-pressure homogenization, high hydrostatic pressure, ultrasonication, and microfluidization, have been widely employed to improve the structural and functional characteristics of food biopolymers (Baier & Knorr, 2015).

High-pressure microfluidization (HPM), has emerged as promising nonthermal processing technology that applies very high pressure to force protein suspensions through narrow microchannels (He et al., 2021; L. Zhang et al., 2021). This process generates controlled shear, cavitation, and turbulence, which effectively disrupt protein aggregates, decrease particle size, and alter protein conformation at the secondary and tertiary structural levels (Diana Kerezsi et al., 2024; Ozturk & Mert, 2018). The efficiency of this process is largely governed by parameters such as chamber geometry (Z- or Y-type), pressure, number of passes, temperature, and the nature of the sample (Guo et al., 2021; Ozturk & Turasan, 2022).

Recent studies have highlighted the effectiveness of dynamic high-pressure microfluidization (DHPM) in modifying protein structures and enhancing their functional properties. For instance, treatment of whey protein isolate (WPI) at 100 MPa improved emulsifying performance through conformational rearrangements, reduced aggregation, and smaller particle size, facilitating stronger interfacial adsorption and enhanced antioxidant activity (C. Wang, Li, et al., 2024). In soy protein isolate (SPI), DHPM induced partial unfolding with decreased β-sheet and random coil content, increased β-turns, and greater surface hydrophobicity, thereby improving molecular flexibility (Diana Kerezsi et al., 2024). Similarly, SPI–rutin complexes treated at 120 MPa exhibited enhanced β-sheet content, reduced fluorescence intensity, and stronger non-covalent interactions, leading to improved physicochemical stability (D. Yu et al., 2024). In pea albumin, DHPM reduced particle size and increased solubility (by 18 %), emulsifying activity (by 24 %), and foaming capacity (by 13 %), resulting in more stable Pickering emulsions (H. Wang, Wang, et al., 2024).

Although HPM has been extensively studied in the context of soy, pea, and whey proteins (He et al., 2021; Hu et al., 2022; Koo, Chung, Ogren, et al., 2018), its application to kidney bean proteins remains largely underexplored, revealing a critical gap in the current literature. Owing to their unique globulin-rich composition, extensive inter- and intramolecular interactions, and inherently low techno-functional properties, elucidating the influence of HPM on the structural and functional behavior of kidney bean proteins may provide critical insights for enhancing their techno-functional and nutritional attributes. Moreover, while microfluidization is conventionally employed as a post-extraction treatment to improve the techno-functional behavior of protein isolates (Hu et al., 2022; C. Wang, Li, et al., 2024; D. Yu et al., 2024; L. Zhang et al., 2021; Q. Zhao et al., 2021), its application at the pre-extraction stage has not been widely investigated. Pre-extraction HPM treatment may facilitate cellular disruption and alter the structural organization of intracellular protein matrices, potentially improving extraction yield and purity (Suchintita Das et al., 2023). Conversely, post-extraction application can further modulate protein conformation and intermolecular interactions, enhancing key functional properties for the formulation of plant-based food products (Karabulut et al., 2024). Understanding the differential impacts of pre- and post-extraction HPM treatments on protein structure and behavior is thus essential for the development of optimized processing protocols tailored to specific end-use applications.

In this context, the present study aims to systematically investigate the effects of both pre- and post-extraction high-pressure microfluidization treatments on the structural, physicochemical, and functional properties of kidney bean protein isolates. By elucidating the mechanisms underlying HPM-induced modifications, this study seeks to optimize processing parameters that enhance the techno-functional attributes of KBP, thereby facilitating its effective utilization in next-generation plant-based food systems.