Effect of Air Classification-Based Dry Fractionation on Structural, Thermal, and Functional Properties of Chickpea Protein Concentrate for Nanoemulsion Applications

Summary

Dry-extracted chickpea protein concentrate exhibited high protein content, strong foaming properties, and effective stabilization of nanoemulsion at a 3.0% w/v concentration. Structural and thermal analyses revealed an amorphous-dominant organization with low residual crystallinity and enhanced molecular flexibility, supporting its suitability as a sustainable, plant-based functional ingredient for food colloid systems.

Abstract

Chickpea protein concentrate (CPC) was obtained via a dry extraction approach and systematically characterized in terms of its compositional, functional, and structural properties to evaluate its suitability for nanoemulsion (NE) applications. The dry-extracted CPC possessed a protein content of 44.8% and demonstrated superior functional performance, including a foaming capacity of 61.1% and a high foam stability of 94.7%, reflecting efficient interfacial adsorption and cohesive film formation. NE formulation prepared using 3.0% w/v CPC as the emulsifying agent achieved a significant reduction in droplet size, yielding a Z-average droplet diameter of 152.7 nm and a low polydispersity index (0.30), indicating a fine, relatively uniform droplet size distribution and kinetically stable colloidal stability. Comparative differential scanning calorimetry (DSC) analysis of chickpea flour (CF) and CPC revealed two main endothermic transitions. While both samples exhibited a similar low-temperature transition at 68.4 °C associated with the release of bound water, CPC showed a substantially lower enthalpy change, indicating partial disruption of native molecular order following dry extraction. The X-ray diffraction (XRD) profile of the CPC utilized in the NE systems exhibited a broad amorphous halo within the 2θ range of 10°-30°, interspersed with limited low-intensity sharp reflections. These findings confirm an amorphous-dominant structure with partial crystalline regions, with the overall crystallinity of the concentrate estimated at approximately 15%. This study describes a reproducible and solvent-free protocol for the production of oil-in-water NEs using dry air-classified CPC, suitable for instructional use and potential industrial scale-up.

Introduction

Legume-derived proteins have gained significant attention as multifunctional ingredients for the design of structurally stable food emulsions. Among these sources, chickpea (Cicer arietinum) is particularly notable due to its high global production (the 3^rd most widely cultivated legume crop worldwide), substantial protein content (18%-24%), hypoallergenic nature, and balanced essential amino acid profile^1,2. Typically, protein isolates contain a higher protein concentration (80%-90%) as they undergo additional processing to remove carbohydrates and fats, whereas CPC (50%−75% protein)....

Protocol

Sample preparation
Protein separation from chickpea flour (CF)
Kabuli chickpeas (Cicer arietinum L.), harvested during the 2025 growing season and sourced from southeastern Türkiye, were processed using an impact-based air classifier milling system, in which particles were subjected to centrifugal forces and repeated impacts against the grinding disc and ring gear. Following an initial pre-milling step, coarse grits were further milled to produce CF using an air classifier mill. During impact milling, an airflow rate of 40-45 m³/h, a classifier speed of 7,000-8,000 rpm, and a feed rate of 200 kg/h were applied, in accordanc....

Results

Nutritional and physicochemical characterization of CF and CPC
Compositional parameters
The compositional parameters of the CF and CPC are summarized in Table 1. The CPC contained 44.8% protein, 5.8% crude fat, and 45.9% total carbohydrates, demonstrating a substantial enrichment of protein relative to CF as a result of the dry fractionation process. The increased protein content observed in CPC confirms the effectiveness of air classification in concentrating protein-rich fracti.......

Discussion

In this study, CPC was obtained by air classification and characterized in terms of its compositional, functional, structural, and colloidal properties, with a particular focus on its application as a plant-based nanoemulsifier. Non-thermal methods, such as air classification, can yield a marked enrichment of protein content relative to CF, yielding a CPC with a protein content of 44.8%³. Compared with the predominantly wet-extraction-based CPC reported in the literature, the present work extends .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
50 mL conical tubes	Corning	352070	Polypropylene centrifuge tubes
Air classifier mill		CSM1250	Air classifier mill used for protein fractionation
Analytical balance	Sartorius	BCE224I-IS	Analytical balance (max. capacity 120 g)
Automatic pipettes	Brand	3123000055	Adjustable pipettes (200 μL, 1 mL, 5 mL)
Centrifuge	Eppendorf	5810R	Refrigerated centrifuge
Chickpea sample		—	Food-grade chickpeas
Colorimeter	HunterLab	A60-1014-593	Instrument for colour analysis
Differential scanning calorimeter	NETZSCH	—	DSC analysis
Digital thermometer	Hanna Instruments	HI-98501	Temperature monitoring
Dipotassium hydrogen phosphate (Na2HPO4)	Sigma-Aldrich	S7907	Analytical grade
Fat analyzer	C. Gerhardt GmbH & Co. KG	13-0005	Soxhlet-based fat analysis
High-speed homogenizer	VELP	SA20900010	Homogenization of coarse emulsions
Hydrochloric acid (HCl)	Sigma-Aldrich	320331	Analytical grade
Hydrochloric acid solution (0.1 N)	Sigma-Aldrich	H1758	pH adjustment
Kjeldahl catalyst tablets	C. Gerhardt GmbH & Co. KG	12-0328	Kjeldahl analysis
Magnetic stirrer	IKA	3339000	Sample mixing and hydration
Medium-chain triglyceride oil	Ataman Kimya A.	https://www.atamanchemicals.com /medium-chain-triglycerides-mct _u30009/?lang=TR#:~:text =Orta%20zincirli%20trigliseritler %20(MCT)%2C%20hindistance vizi%20ve%20hurma%20%C3 %A7ekirde%C4%9Fi%20ya%C 4%9Flar%C4%B1nda,g%C3%B Cne%C5%9F%20kremleri%20i% C3%A7in%20%C4%B1slat%C4% B1c%C4%B1%20ajand%C4%B1r.	Food and pharmaceutical grade oil
Moisture analyzer	Mettler Toledo	HC103	Moisture determination
Particle size and zeta potential analyzer	Malvern Instruments	ZEN3600	DLS and electrophoretic mobility
pH meter	Mettler Toledo	MET-30671567	pH measurement
Plastic centrifuge tubes	ISOLAB	078.02.003	Disposable plastic tubes
Porcine pepsin	Sigma-Aldrich	P7012	Enzyme (1:10,000)
Potassium dihydrogen phosphate (KH2PO4)	Sigma-Aldrich	P5379	Analytical grade
Probe sonicator	Bandelin	—	Emulsion sonication
Protective goggles	Baymax	BX-2500	Laboratory safety equipment
Protein analyzer	C. Gerhardt GmbH & Co. KG	12-0520	Protein determination
Protein digestion system	C. Gerhardt GmbH & Co. KG	12-0700	Kjeldahl digestion unit
Sodium chloride (NaCl)	Sigma-Aldrich	S9625	Analytical grade
Sodium hydroxide (NaOH)	Sigma-Aldrich	S5881	Analytical grade
Spatula	—	https://www.blabmarket.com/ meta-etiket/plastik-spatul?srsltid =AfmBOoqsvXR_3rQgU1n8QBn DyR_P9D52v2Hahy27RLOH7Zg VbFzzcbsb	Plastic spatula
Ultrapure water system	ELGA Labwater	PC110COBPM1	Water resistivity 18.2 MΩ·cm
Volumetric flask	Schott Duran	2120117	100 mL volumetric flask
Water bath	Memmert	WTB24	Temperature control (20–25 °C)
X-ray diffractometer (Cu–Kα)	PANalytical	STEM-LE-0294-LC	XRD analysis (45 kV, 40 mA)