In the evolving modern world, there is an increasing paradigm shift from animal proteins to plant proteins among consumers, owing to environmental and sustainability issues related to the consumption of animal proteins (Boukid et al., 2022). Furthermore, substituting animal proteins with plant-based alternatives in existing food systems provides manufacturers with distinct product differentiation advantages in the market. However, the substitution of animal proteins is hindered by the limited functionality of plant proteins, including lower solubility, emulsification, foaming, and gelation properties. Therefore, it is imperative to modify plant proteins to enhance their functionalities. Numerous protein modification techniques exist, such as physical, chemical, and enzymatic methods (Akharume et al., 2021). Physical non-thermal modification techniques are emerging as prominent methods among other approaches. One such technique is microfluidization, also known as Dynamic high-pressure microfluidization (DHPM).

DHPM, initially employed for producing nano-emulsions, nano-particles, and liposomes (Bai et al., 2016) has recently expanded its applications into the realm of food processing particularly noteworthy is its utilization in modifying plant proteins. The actions induced by DHPM on proteins are predominantly mechanical, with minimal temperature fluctuations, typically regulated by circulating cold water around the collection chamber. DHPM harnesses a combination of mechanical forces, including high-velocity impact, sudden pressure drop, high-frequency vibrations, laminar shear, hydrodynamic cavitation, and turbulent forces akin to those observed in high-pressure homogenizers (Sahil Madhumita et al., 2022). Plant proteins commonly exhibit inherent functional limitations, including limited emulsification, foaming, and gelation properties, largely attributed to their poor solubility in solution (He et al., 2021). Mechanical actions during DHPM treatment reduced the protein particle sizes, therefore reducing the protein-protein interactions and favors the protein-water interactions, which enhanced protein solubility. For instance, Moll et al. (2021) reported increased solubility of insoluble pea protein fractions from 23 % to 86 % upon microfluidization at 150 MPa - 5 passes. This enhanced protein solubility increases the interfacial properties of plant proteins (Yang et al., 2018). Several studies exist in the literature on improving functional properties of proteins by DHPM, such as the non-exhaustively improved solubility of Eucommia ulmoides Oliv. seed meal protein (ESMP) (Ge et al., 2021), perilla protein isolate (Zhao et al., 2021); modulation of emulsifying properties of pea globulin (Oliete et al., 2018); gelation properties of soy protein isolate (Zheng et al., 2020).

Controlling variations in pressure and passes constitutes the fundamental parameters during DHPM treatment. Previous research predominantly focused on manipulating only one variable, usually pressure, while keeping the other variable (i.e., passes) constant (Ge et al., 2021; Oliete et al., 2018; Zhao et al., 2021). A notable scarcity of literature addresses the combined influence of both variables (i.e., pressure and passes) during microfluidization on the structure characteristics and functional attributes of proteins. No studies investigating this aspect were found, except for a single study focusing on the variation of pressures (70, 100, and 150 MPa) and passes (1 and 3 passes) during high-pressure homogenization of pea protein (Melchior et al., 2022). Therefore, this study was planned to better understand pressure's effect and pass variables on protein structural, techno-functional, and rheological properties during DHPM treatment.

In this study, Perilla (Perilla frutescens) protein, sourced from the underutilized perilla seed oil press cake, characterized by a protein content of 35–45 % and a balanced amino acid profile with high nutritive values, serves as the model protein (Zhao et al., 2021). Here, cold-pressed perilla oil seed cake-derived protein isolate (PPI) undergoes DHPM treatment at three distinct pressures (100, 150, and 200 MPa) and two different passes (3 and 6 passes). The study aims to assess the impact of increasing both variables, i.e., pressure and passes on the structural attributes of PPI, and elucidate how these structural changes dictate the functional characteristics of protein isolate, providing insights into the structure-functionality relationship of DHPM-modified protein isolate.