The increasing global demand for sustainable protein sources has spurred research into proteins from unconventional yet abundant sources. In this context, green leaf waste, often regarded as agricultural byproducts, has emerged as a promising protein source (Kaur & Bhatia, 2022; Møller et al., 2021; Patil, Gendley, & Patil, 2021), as their use addresses both global protein demand and environmental concerns about waste disposal. Green leaf waste from the leaves of vegetables, particularly Brassica oleracea (e.g., cauliflower, cabbage, broccoli, Brussel sprouts) that are conventionally used in non-food applications such as feedstock, composting and energy production, presents a major challenge in modern agriculture. The sheer volume of this waste is a logistic concern for farmers and waste management system, and a contributor to environmental issues such as methane emission from landfills (Prade et al., 2021; Tang et al., 2020). Thus, Pérez-Vila, Fenelon, O'Mahony, and Gómez-Mascaraque (2022) reported that green leaves represent a promising sustainable source of protein for food applications, with RuBisCO being a major component offering good nutritional and functional properties.

Cauliflower (Brassica oleracea var. botrytis L) was chosen as the focal point for this study due to its global availability (25 million tonnes annually), substantial leaf biomass generation and its botanical similarity to other important cultivars of Brassica oleracea. These vegetables share a taxonomic lineage and similarities in leaf structure and composition (Smith & King, 2000). Therefore, insights gained from the study of cauliflower leaves can potentially be applied to other Brassica crops, expanding the scope of impact. Cauliflower leaves exhibit unique biochemical properties driven by their Brassica specific traits and phytochemical profiles, setting them apart from other leafy biomass. Cauliflower leaves comprise 35–40 % of total vegetable weight and are predominately discarded during post-harvest processing. These leaves are an excellent source of fibre, protein, vitamins, essential fatty acids and are particularly rich in glucosinolates, phenolic compounds, polyamines, and high-quality proteins, including bioactive peptides and sulphur-rich amino acids (Mythili, Rajeswari, Bosco, & Kamatchi Alias Rajalechumi, 2021; Xu et al., 2017)). The use of cauliflower LPC thus offers a novel, sustainable approach to valorizing agricultural by-products while delivering nutritional advantages and supporting the development of functional foods.

In the pursuit of sustainable protein sources, this research delves into the extraction of proteins for human food formulations. The process involves pressing green leaves to extract juice containing water soluble proteins and other components from plant leaves. This technique has the advantage of simplicity and minimal use of additives, making it a straightforward method for the initial extraction process. Another commonly used protein extraction process involves homogenizing leaves in deionised water or weak buffers, resulting in cell lysis and concurrent release of intracellular proteins as result of hypotonic impact (Soo, Samad, Zaidel, Jusoha, & Muhamada, 2021). Several biochemical and enzymatic reactions occur due to cell disruption, exposing intracellular contents to oxygen, enzymes and other reactive compounds. These reactions can affect protein stability, colour and overall quality. Protein degradation occurs rapidly after cell lysis due to the release of protease enzymes, necessitating the use of various additives to stabilise organic compounds in the leaf juice and also prevent enzymatic oxidation. Common antioxidants include sodium metabisulphite (Ali, Yuwono, Istianah, & Putri, 2019; Nieuwland et al., 2021), and sodium sulphite (Tanambell, Møller, Corredig, & Dalsgaard, 2022) inhibit polyphenol oxidase preventing enzymatic browning. Chelators of metals such as magnesium and calcium (e.g., ethylenediaminetetraacetic acid, EDTA) (Tsugama, Liu, & Takano, 2011) can be included to inhibit, lipoxygenase and lipid oxidation thus reducing rancidity and protein degradation (Saleem et al., 2020). Additives, such as NaCl, can be added to increase protein solubility (Hu et al., 2017). The stabilizing agents can be added into the extracting solvent or added to the leaf juice after pressing to improve protein extraction and preserve leaf juice quality and stability.

Mechanical pre-treatment of green leaf juice with help of sieve filtration before extraction has not been examined widely. The use of sieve filters can improve protein extraction by eliminating interference from large particles such as fibres, cell debris and other particulate matter that may hinder extraction (Andrade, Fetzer, dos Passos, & Ambye-Jensen, 2025; Bray & Humphries, 1978; Echavarría Vélez, Torras, Pagán, & Ibarz, 2011).By removing solids, sieve filters allow better contact between the extraction buffer and the leaf juice, facilitating the release of proteins from the plant cells (Demoulin et al., 2023). This can improve the overall efficiency and yield of protein extraction. It is important to select an appropriate mesh size or screen type for the sieve filters based on the desired filtration level and the characteristics of the green juice.

Various methods are used for protein precipitation and concentration, such as heat coagulation (Chowdhury, Lashkari, Jensen, Ambye-Jensen, & Weisbjerg, 2018; Koschuh et al., 2004), addition of flocculants (Baraniak, 1990), alkaline-acid precipitation (Nissen et al., 2021; Rawdkuen & Roger, 2020; Soo et al., 2021; Zhang, Sanders, & Bruins, 2014) and bacterial fermentation (Santamaría-Fernández et al., 2017), lactic acid fermentation (Domokos-Szabolcsy et al., 2022). Alkaline-acid precipitation and heat coagulation are the most widely used method demonstrating high protein yield and functional properties across the biomass (Chowdhury et al., 2018; del Mar Contreras et al., 2019). However, alkaline-acid precipitation is often preferred over the heat coagulation method due to milder processing conditions, preserving protein structure and functionality, while minimising partial thermal denaturation and interaction with other components (Balfany, Gutierrez, Moncada, & Komarnytsky, 2023). Understanding the nutritional value of leaf protein concentrates (LPCs) is crucial for developing value-added products and alternative protein sources. Additionally, the techno-functionality of LPCs play a vital role in food development, as they can impact consumer acceptance, organoleptic properties, and overall product quality (Ogunwolu, Henshaw, Mock, Santros, & Awonorin, 2009; Sekhon & Bhatia, 2021).

Cauliflower is a little studied biomass source for LPC extraction but commercially significant due to high volume, food sustainability, low cost and nutrient rich, while being representative of physicochemical characteristics of the important Brassica oleracea group of plants, adding further translational impact to potential findings (Xu et al., 2017). The novelty of this study lies in its systematic evaluation of how different concentrations of stabilizing solutions and sieve filtration sizes, when integrated with the alkaline-acid precipitation method, influence the yield and quality of LPC from cauliflower leaves. Stabilizing solution was employed and studied to address the fast oxidizing and colour changing reactions of leaf juice occurring straight after juicing, while sieve filtration is aiming to separate large fibre rich insoluble particles of leaf juice.

While prior studies have primarily focused on Brassica species such as cabbage and broccoli, this study investigates cauliflower leaves and introduces a dual-optimization approach combining sieve filtration and stabilizing agents, to investigate improved protein recovery and quality. The selection of variables was based on the findings of preliminary studies confirming the instability of cauliflower leaf juice and the presence of large particles. The structural and physicochemical properties of the LPC were investigated to assess their potential use as a functional food ingredient.