Stabilization of Pickering Emulsions with Legume Proteins : Structural Modification of Legume Proteins through Physical Processing and Polyphenol Complexation = 콩류 단백질을 활용한 피커링 에멀젼의 안정화 : 물리적 처리 및 폴리페놀 복합화로 변형된 콩류 단백질 활용

다국어 초록 (Multilingual Abstract)

Legume proteins, such as faba bean protein isolate (FBPI) and pea protein isolate (PPI), have emerged as sustainable materials for emulsion stabilization owing to their intrinsic amphiphilicity and interfacial activity. However, their inherent molecular compactness and limited solubility often restrict their colloidal dispersion and interfacial adsorption. To overcome these limitations, this research employed two complementary approaches, physical restructuring via sequential heat-ultrasound (HU) treatment and molecular complexation with polyphenols, to modulate the interfacial functionality and stabilization efficiency of legume proteins in Pickering emulsion systems. In the first study, the ultrasonication time dependent restructuring of heat-treated legume proteins was investigated, focusing on how HU treatments influence the interfacial adsorption and stabilization of FBPIs and PPIs in high internal phase Pickering emulsions (HIPPEs). Heat treatment (90 °C, 2 h) unfolded protein aggregates, while subsequent ultrasonication (20 kHz, 120 W, 5-20 min) altered particle morphology and molecular interactions. Controlled sonication (5-10 min) produced smaller, more uniform particles with enhanced solubility and flexible secondary structures, which facilitated the formation of cohesive interfacial films and viscoelastic network structures in HIPPEs. Conversely, prolonged sonication (20 min) led to partial reaggregation, weakening interfacial adsorption and droplet uniformity. In the second study, FBPI was complexed with dual polyphenols, gallic acid (GA) and rutin, to examine how different phenolic structures regulate protein conformation and Pickering emulsion stabilization. GA, a low molecular weight hydrophilic phenolic acid, facilitated protein dispersion and promoted subtle conformational rearrangements toward a more solvated state. Conversely, rutin, a bulkier flavonoid containing multiple aromatic rings, promoted hydrophobic association and interfacial affinity, yet excessive incorporation led to intermolecular aggregation and structural heterogeneity. These molecular effects were reflected in the emulsion microstructure, where GA dominant complexes generated smaller and more uniform oil droplets with cohesive interfacial layers, whereas excessive rutin caused heterogeneous droplet aggregation and reduced stability. The dual incorporation of GA and rutin synergistically regulated interfacial organization, and the formulation with an intermediate rutin:GA ratio of 0.4:1.0 exhibited the most coherent and continuous interfacial network. Structural analyses revealed that GA enhanced ordered secondary structures, while rutin improved flexibility and film continuity, leading to stable and elastic Pickering emulsions.

국문 초록 (Abstract)

콩 단백질은 양친매성특성과 우수한 계면활성으로 인해 피커링 에멀젼의 지속 가능한 안정화 소재로 주목받고 있다. 그러나 단백질의 고유한 분자적 조밀성과 낮은 용해도는 콜로이드 분산성과 계면 흡착성을 제한하는 주요 요인으로 작용한다. 본 연구에서는 이러한 한계를 극복하기 위하여, 열-초음 파 처리를 통한 물리적 구조 재편성과 폴리페놀 복합체 형성을 통한 분자 적 상호작용 조절이라는 두 가지 상보적 접근법을 적용하여, 콩 단백질의 계면 기능성과 피커링 에멀젼 안정화 효율을 향상시키고자 하였다. 첫 번째 연구에서는 열처리된 콩 단백질의 초음파 처리 시간에 따른 구 조적 재배열을 조사하여, 연속적인 열 및 초음파(HU) 처리가 잠두 분리단백 (FBPI)과 완두 분리단백(PPI)의 고내상 피커링 에멀젼(HIPPE)에서의 계면 흡 착 및 안정화 거동에 미치는 영향을 규명하였다. 열처리(90 °C, 2 h)는 단백질 응집체를 부분적으로 전개시켰으며, 후속 초음파 처리(20 kHz, 120 W, 5-20 min)는 입자 형태와 분자 간 상호작용을 변화시켰다. 특히, 5-10분의 제어된 초음파 처리는 입자 크기 감소, 균일한 분산, 그리고 유연한 이차구조 형성 146 을 유도하여, HIPPE 내에서 응집력 있는 계면막과 점탄성 네트워크구조를 형성하였다. 반면, 20분의 장시간 초음파 처리는 재응집을 초래하여 계면 흡 착력과 유화 입자 균일성을 저하시켰다. 두 번째 연구에서는 FBPI를 갈릭산(GA) 과 루틴 두 가지 폴리페놀과 복 합화하여, 상이한 페놀 구조가 단백질의 구조, 계면 조립, 그리고 피커링 에 멀젼 안정화에 미치는 영향을 분석하였다. GA는 소수성이 낮은 저분자 페놀 산으로서, 전기적 및 수소결합 상호작용을 통해 단백질의 용해도 증가와 부 분적 재접힘을 유도하였다. 반면, 다환 구조를 지닌 고분자형 플라보노이드 인 루틴은 소수성 상호작용과 계면 흡착을 강화하였으나, 과량 첨가 시 입 자 간 응집을 촉진하였다. 이러한 분자적 상호작용은 에멀젼의 미세구조에 도 반영되어, GA가 우세한 복합체는 균일하고 작은 오일 방울과 응집력 있 는 계면층을 형성한 반면, 루틴이 과도한 경우 불균일한 응집 및 안정성 저 하를 보였다. 두 폴리페놀 간의 상호 균형은 오일–수상 계면에서 단백질–폴 리페놀 복합체의 조직화를 결정하였으며, 그중 중간 비율의 루틴:GA 0.4:1.0 조건에서 가장 응집력 있고 연속적인 계면막이 형성되었다. 분광학적 분석 결과, GA는 α-helix 및 β-sheet 등의 규칙적 이차구조 형성을 촉진하였으며, 루틴은 구조적 유연성과 필름 연속성을 향상시켜 안정적인 피커링 에멀젼 을 구현하였다.

목차 (Table of Contents)

• Chapter I. Ultrasonication time dependent structuring of heat-treated legume proteins: Interfacial adsorption and stabilization of faba bean and pea protein isolates in high internal phase Pickering emulsions 1
• 1. Introduction 1
• 2. Materials and methods 8
• 2.1. Materials 8
• 2.2. Physical modification of legume protein isolates 8
• 2.3. Properties of HU-treated legume protein isolates 9
• 2.3.1. Particle size 9
• 2.3.2. ζ-potential 10
• 2.3.3. Solubility 10
• 2.3.4. Turbidity 11
• 2.3.5. Visual appearance 11
• 2.3.6. Optical microscopy 11
• 2.3.7. SDS-PAGE 12
• 2.3.8. Surface hydrophobicity 12
• 2.3.9. Fourier transform infrared spectroscopy 13
• 2.3.10. Circular dichroism spectroscopy 13
• 2.4. Preparation of high internal phase Pickering emulsions 14
• 2.5. Droplet size 14
• 2.6. ζ-potential 15
• 2.7. Optical microscopy 15
• 2.8. Confocal laser scanning microscopy 15
• 2.9. Transmission electron microscopy 16
• 2.10. Adsorbed protein at the interface and interfacial protein concentration 16
• 2.11. Rheological properties 17
• 2.12. Statistical analysis 18
• 3. Results and Discussion 19
• 3.1. Physicochemical changes in FBPIs and PPIs by heat-ultrasound treatment 19
• 3.1.1. Particle size and surface charge 19
• 3.1.2. Solubility and turbidity 23
• 3.1.3. SDS-PAGE 29
• 3.1.4. Surface hydrophobicity 32
• 3.1.5. Fourier transform infrared spectroscopy 36
• 3.1.6. Circular dichroism spectroscopy 39
• 3.2. Stabilization of high internal phase Pickering emulsions by heat-ultrasound modified FBPIs and PPIs 42
• 3.2.1. Macroscopic and colloidal features 42
• 3.2.2. Microscopic characterization of Interfacial adsorption 48
• 3.2.3. Interfacial protein adsorption and packing behaviors 56
• 3.2.4. Viscoelastic properties 64
• 4. Conclusion 67
• Chapter Ⅱ. Enhancing Pickering emulsion stability through faba bean protein isolate-dual olyphenol complexation 69
• 1. Introduction 69
• 2. Materials and methods 75
• 2.1. Materials 75
• 2.2. Fabrication of FBPI-dual polyphenol complex nanoparticles 75
• 2.3. Properties of FBPI-dual polyphenol complex nanoparticles 78
• 2.3.1. Particle size 78
• 2.3.2. ζ-potential 78
• 2.3.3. Polyphenol binding content 78
• 2.3.4. Solubility 79
• 2.3.5. Determination of exposed sulfhydryl contents 79
• 2.3.6. Determination of exposed free amino group contents 80
• 2.3.7. SDS-PAGE 81
• 2.3.8. Fourier transform infrared spectroscopy 81
• 2.4. Preparation of Pickering emulsions 82
• 2.5. Droplet size 82
• 2.6. ζ-potential 83
• 2.7. Optical microscopy 83
• 2.8. Transmission electron microscopy 83
• 2.9. Confocal laser scanning microscopy 84
• 2.10. Backscattering profiles and Turbiscan stability index 84
• 2.11. Adsorbed protein at the interface and interfacial protein concentration 85
• 2.12. Statistical analysis 86
• 3. Results and discussion 87
• 3.1. Physicochemical properties of FBPI-dual polyphenol complex nanoparticles 87
• 3.1.1. Particle size and ζ-potential 87
• 3.1.2. Polyphenol binding content 91
• 3.1.3. Solubility 94
• 3.1.4. Exposure of functional groups upon polyphenol complexation 97
• 3.1.5. SDS-PAGE 101
• 3.1.6. Fourier transform infrared spectroscopy and secondary structure 103
• 3.2. Pickering emulsion stabilization by FBPI-dual polyphenol complex nanoparticles 107
• 3.2.1. Droplet size and ζ-potential 107
• 3.2.2. Microscopic characterization 112
• 3.2.3. Backscattering profiles and Turbiscan stability index 116
• 3.2.4. Adsorbed protein at the interface and interfacial protein concentration 121
• 4. Conclusion 126
• References 127
• Abstract (in Korean) 146