Protein extraction from hazelnut meal and characterization of hazelnut protein

Global population growth, climate change, and the accelerated depletion of natural resources have dramatically increased the urgency of identifying sustainable and functional protein sources to meet future food security demands. According to FAO projections, by 2050, the world population will surpass 9 billion, placing unprecedented pressure on existing food systems. Animal-based proteins, while nutritionally valuable, are increasingly criticized for their environmental impacts, including high greenhouse gas emissions, extensive land and water use, and contribution to biodiversity loss. In addition, ethical considerations regarding animal welfare and health concerns over red meat consumption have strengthened the drive toward alternative protein sources. This context has led to growing interest in plant-based proteins, especially those derived from underutilized agro-industrial byproducts. Valorizing such side streams aligns with circular economy principles and reduces food waste while creating valueadded ingredients for the food industry. Oilseed meals (press cakes) are particularly promising due to their high protein content, but they are often underexploited or used primarily as low-value animal feed. Türkiye is the world's largest producer of hazelnuts, accounting for over 60% of global supply. After oil extraction, hazelnut press cake remains as a protein-rich byproduct, with protein contents reaching approximately 54% on a dry basis. Despite this high nutritional potential, its utilization in human food systems is limited, primarily due to challenges in extracting functionally intact proteins. Conventional alkaline extraction methods are widely used because they effectively solubilize proteins and deliver high yields. However, they also cause significant denaturation, altering secondary and tertiary structures. This structural damage can diminish solubility, emulsifying capacity, and foaming properties, making the resulting isolates less suitable for many food applications. Moreover, the use of strong alkalis raises environmental concerns due to wastewater treatment requirements and chemical hazards. To address these issues, there is growing interest in developing green extraction techniques that minimize environmental impacts and preserve protein functionality. One such approach involves the use of Natural Deep Eutectic Solvents (NADES). NADES are mixtures of natural components (e.g., sugars, organic acids, amino acids, and polyols) that form eutectic systems through hydrogen bonding. They have emerged as promising green solvents due to their biodegradability, low toxicity, tunable physicochemical properties, and ability to disrupt plant cell walls and solubilize biomolecules without harsh conditions. In this thesis, three different NADES systems were designed for the extraction of proteins from hazelnut press cake. These systems were based on choline chloride (ChCl) combined with glycerol (DES-GLY), sorbitol (DES-SOR), or glucose (DESGLU), all of which are food-grade, biodegradable, and relatively inexpensive. Extraction experiments were conducted both with and without ultrasonic assistance. Ultrasound is a well-known green intensification technique that enhances mass transfer, disrupts plant matrices, and can reduce extraction times and solvent consumption. For comparison, conventional alkaline extractions were also performed at pH 8.0, 9.0, and 10.0, using a fixed solid-to-liquid ratio (1:20 w/v). The resulting protein isolates were analyzed comprehensively for yield, solubility, emulsifying and foaming properties, particle size, zeta potential, and structural characteristics using FTIR spectroscopy, SDS-PAGE electrophoresis, intrinsic fluorescence spectroscopy, and scanning electron microscopy (SEM). Key findings of this study revealed nuanced trade-offs between yield and functional quality. The highest protein yield was observed for alkaline extraction at pH 10 (ALK10), achieving approximately 260 mg/g with 63% extraction efficiency. DESGLY and DES-SOR systems produced somewhat lower yields (~200–220 mg/g) and extraction efficiencies (50–55%). This difference is expected, given that strong alkalis solubilize proteins more aggressively. However, it is hypothesized that the lower yields in DES systems can be mitigated through process optimization—such as fine-tuning solvent composition, applying controlled pH shifts for protein precipitation, or integrating sequential extraction steps. In terms of solubility, a crucial functional property for food applications, DESextracted proteins significantly outperformed alkaline-extracted proteins. Solubility values ranged between 70–76% for DES isolates versus 58–63% for alkali isolates. This superior solubility is attributed to the milder extraction conditions of NADES systems, which reduce protein denaturation and aggregation. High solubility is essential for applications in beverages, dairy analogues, and other liquid foods. Emulsifying properties were also improved in DES-extracted proteins. For example, the emulsion activity index (EAI) reached up to 15 m²/g for DES-GLY extracts, while DES-SOR maintained emulsion stability for approximately 60 minutes. These results align with previous studies showing that NADES extraction can improve protein surface activity by preserving flexible, partially unfolded conformations conducive to interface adsorption. Such properties are valuable in designing plantbased creams, dressings, and meat analogues.In contrast, foaming capacity was higher for alkaline-extracted proteins (37–50%) compared to DES-extracted proteins (10–25%). Alkaline extraction can induce partial unfolding and exposure of hydrophobic residues, which can stabilize air– water interfaces during whipping. While this may be advantageous in certain bakery or confectionery applications, it comes at the cost of other functional properties and environmental sustainability. Structural analyses supported these functional findings. FTIR and intrinsic fluorescence spectroscopy indicated more extensive unfolding and secondary structure disruption in alkali-extracted proteins. SDS-PAGE analyses under reducing conditions revealed similar band patterns between DES and alkaline extracts, suggesting that polypeptide subunit composition remained broadly conserved despite extraction method. SEM imaging highlighted morphological differences: alkaline-extracted proteins exhibited compact and smooth surfaces, while DES-extracted proteins showed porous, irregular structures—a feature that may promote better water binding and hydration behavior in food matrices. Zeta potential analyses indicated that alkaline extracts had higher negative surface charges (−45 to −47 mV) compared to DES extracts (−29 to −35 mV), which may influence colloidal stability in formulations. Overall, this thesis demonstrates that NADES systems represent a sustainable and functional alternative to conventional alkaline extraction for valorizing hazelnut press cake proteins. While alkaline methods excel in yield and foaming capacity, NADES systems deliver superior solubility and emulsifying properties, along with a greener, more environmentally friendly process profile. Glycerol- and sorbitol-based NADES systems, in particular, appear to weaken protein–protein interactions and enhance surface activity, creating protein ingredients well-suited for diverse food applications. Ultrasound-assisted extraction further improved NADES performance by enhancing mass transfer and extraction efficiency. This aligns with broader trends in green process intensification, supporting more efficient and scalable extraction solutions. Future research directions should focus on: • Optimizing NADES formulations at the molecular level to maximize extraction yield while preserving or enhancing functional properties. • Investigating the digestibility and bioavailability of NADES-extracted proteins using in vitro and in vivo models. • Evaluating sensory properties and consumer acceptance of foods formulated with NADES-extracted proteins. • Assessing economic feasibility and life cycle impacts of NADES-based extraction at industrial scale. In conclusion, valorizing hazelnut press cake as a high-value protein source using NADES systems can support more sustainable, circular, and clean-label food production, aligning with both industry goals and global sustainability targets.