Plant proteins have recently garnered attention for their nutritional benefits and environmental sustainability (Gatto & Chepeliev, 2024). The potential for enhancing functional properties of plant-derived proteins has become a focal point in addressing the increasing demand for sustainable protein sources (Duluins & Baret, 2024; Kew et al., 2023). Perilla protein isolate, derived from the nutrient-rich seeds of Perilla frutescens plant, presents itself as a promising candidate for such advancements. It contains a significant amount of protein (∼30–40%) with essential amino acids such as histidine, phenylalanine, and tryptophan therefore can also be used as a source of bioactive peptides in pharmaceutical applications (Chumphukam et al., 2018; Kim, Liceaga, & Yoon, 2019; Zhao, Wang, Hong, Liu, & Li, 2021). However, the realization of its full functional potential remains constrained by challenges such as limited solubility, functional and colloidal stability. This requires innovative approaches to modify and enhance the protein's functional attributes.

Dynamic high-pressure microfluidization/Microfluidization (DHPM), is a novel, thermo-mechanical protein modification method. During DHPM, the intensifier pump forces the liquid protein dispersion to move through narrow sized (usually <100 μm) interaction chambers, which increased dispersion velocity up to 400 m/s. While passing through micro-sized interaction chambers at such high velocity, the globular plant proteins experience various mechanical forces such as laminar shear, turbulent, hydrodynamic cavitation, high velocity impact, high frequency vibration, and instantaneous pressure. The collective mechanical action of these above forces brought the structural and functional changes to plant protein (Sahil, Prabhakar, & Kumar, 2024) This disruption mechanism can enhance solubility and reduce particle size, thereby influence colloidal properties, which can contribute to improved techno-functionalities in food manufacturing processes (Moll, Salminen, Schmitt, & Weiss, 2021). Previously, various plant proteins have been modified using microfluidization such as pea protein (He et al., 2021), oat protein (Cheng et al., 2022), potato protein isolate (Hu, Xiong, Xiong, Chen, & Zhang, 2021), peanut protein isolate (Hu et al., 2021), and Eucommia ulmoides Oliv. seed meal proteins (Ge et al., 2021). Furthermore, enzymatic hydrolysis is a well-known method from the past, known for modulating functional properties of protein. These techniques selectively cleaved peptide bonds, based on the specific enzymes action site, and required proper control over environment condition for optimum enzymatic action. In the past, it has been used to modify various proteins such as soy protein (Lamsal, Jung, & Johnson, 2007), pea protein (Konieczny, Stone, Nickerson, Tanaka, & Korber, 2020), peanut protein isolate (G. Zhao, Liu, Zhao, Ren, & Yang, 2011), pulse proteins (Vogelsang-O'dwyer, Sahin, Arendt, & Zannini, 2022), rice endosperm protein (Nisov, Ercili-cura, & Nordlund, 2020), chickpea protein isolate (Mokni Ghribi et al., 2015), walnut protein (Moghadam et al., 2020), Elaeagnus mollis oil meal protein (Guo, Zhao, Yang, Li, & Yu, 2022), and lentil protein isolate (Avramenko, Low, & Nickerson, 2013).

To date, perilla protein has been extracted and modified using various approaches, including phosphorylation (Zhao, Hong, Fan, Liu, & Li, 2022b)), enzymatic hydrolysis (Kim & Yoon, 2020), high-intensity ultrasound (Zhao et al., 2022a), and microfluidization (Zhao, Yan, Liu and Li, 2021). However, these studies focused on single technique modifications rather than combined ones. In the past, some literature explored the concept of dual modification, demonstrating improved efficiency compared to single modifications (Zhao, Zhang, Li, & Dong, 2018). For instance, the work of Zhao et al. (2018) demonstrated that different pre-treatments, i.e., thermal treatment (90 °C for 10 min) and HPH treatment (30 MPa, 3 cycles) significantly improved the SPI's degree of hydrolysis (%) and DPPH radical-scavenging activity (%) compared to untreated SPI, with HPH pre-treatment outperforms the thermal pre-treatment. These studies have shown positive effects on the functional properties of rice dreg proteins (Zhang et al., 2021), oat bran (Rosa-Sibakov, de Carvalho, Lille, & Nordlund, 2022), and soy proteins (Chen, Chen, Yu and Wu, 2016a). However, these studies exclusively addressed the dual impact and did not compare it with the individual effects of DHPM or EH. Therefore, based on these considerations, this study aims to comparatively investigate the effects of DHPM, EH, and sequential DHPM-EH on the structural and functional characteristics of PPI.

DHPM can alter the surface charge of the proteins, which can consequently affect the structure and enhance protein functional properties, while enzymatic hydrolysis can cleave peptide bonds, preferably from the sites that can attract hydrophobic and hydrophilic groups (Cheng et al., 2022; Liu et al., 2022; Sharma, Sahil, Madhumita, Kumar, & Prabhakar, 2023; Vogelsang-O'dwyer et al., 2022). The study hypothesized that DHPM pre-treatment would increase enzymatic hydrolysis efficiency, resulting in significant modifications to protein structure. These modifications were expected to positively impact functionality and nutritional properties. Hence, the principal aim of this investigation was to examine the impact of dynamic high-pressure microfluidization, enzymatic hydrolysis treatment, and their synergistic application on the alteration of the structural, physicochemical, functional, thermal, nutritional, antioxidant, and rheological attributes of perilla protein isolate (PPI). This knowledge can be further used to develop PPI in food and pharmaceutical applications.