Effect of Phenolic Hydroxyl Group Number on Regulation of the Self-Assembly Behavior of Edible Dock Protein and Catechins

Abstract

To investigate the effect of phenolic hydroxyl group number on the interaction between catechins and a plant-derived protein carrier, four catechins with varying hydroxyl numbers—epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC), and epigallocatechin gallate (EGCG)—were investigated. The new plant-derived edible dock protein (EDP) was selected as a carrier matrix. EDP, when employed as a protein delivery carrier, possessed a hydrophobic amino acid content of 45%. This structural feature enabled it to provide more hydrophobic cavities for small molecule compounds, thereby facilitating better binding with them. The results indicated that the order of loading capacity of catechins within EDP was EGCG (9.7%) > ECG (9.1%) > EGC (8.8%) > EC (7.1%). This sequence was consistent with the number of hydroxyl groups in catechin: EGCG (8) > ECG (7) > EGC (6) > EC (5). Among the four catechins, EGCG had the highest binding constant (Ka = 2.6 × 10³ L/mol), leading to the largest quenching of EDP. During self-assembly, hydrogen bonding, hydrophobic and electrostatic interactions were the main driving forces, and the interaction between EGCG and EDP was the strongest. This study indicated that the hydroxyl group number of polyphenolic compounds can determine its binding affinity with proteins.

1. Introduction

Catechin and its derivatives are the main functional components of tea and account for 12–24 wt% of the dry weight of tea [1]. As food-derived functional factors with a multiple hydroxyl structure, catechins have anti-inflammatory, antioxidant, anti-tumor, anti-atherosclerosis and other biological activities [2,3]. However, catechins are susceptible to environmental factors such as light, oxygen and heat, which can cause them to lose their original physiological effects [4]. In addition, before reaching the small intestine, the phenolic hydroxyl groups of catechins are easily destroyed by stomach acid, microorganisms and enzymes, resulting in the loss of their physiological activity [5]. These factors limit the efficient application of catechins in functional foods. Therefore, developing a steady-state, controlled-release, and efficient absorption delivery system is significant for improving the biological activity and nutritional health benefits of catechins.

Protein is an ideal material for designing delivery carriers due to its good structural variability, compatibility and degradation [6]. Nevertheless, since the per capita demand for plant and animal protein worldwide is increasing year by year, the development of new proteins has important strategic significance. Edible dock (Rumex patientia L. × Rumex tianschanicus A.LOS) is a perennial herbaceous species belonging to the Polygonaceae family [7]. Due to its easy cultivation, high yield, high nutrition and easy processing characteristics, edible dock was identified as a new food raw material by China’s National Health Commission in 2021 [8]. Edible dock protein (EDP) is a new plant protein resource with a protein content exceeding 36 wt%. Moreover, EDP contains more than 45.52% of hydrophobic amino acids and thus has a good binding ability with bioactive compounds [9]. Accordingly, it can be speculated that EDP has broad application prospects in the development of delivery systems for enhancing the stability and bioactivity of active substances.

It has been reported that protein delivery systems can improve the stability and bioavailability of catechins and enable them to better exert their nutritional value and health benefits [8]. For instance, casein can bind with catechin (C), epicatechin (EC), epigallocatechin (EGC), and epigallocatechin gallate (EGCG) through non-covalent interactions, enhancing their stability and bioavailability. However, these substances have different affinities for casein: EGCG > EGC > EC > C [10]. Lysozyme can self-assemble with epicatechin gallate (ECG), EGC, and EGCG through interactions such as hydrogen bonding, hydrophobic forces, and π-π stacking to form compounds, improving their stability and bioactivity. Among these compounds, EGCG exhibited the best stabilizing and enhancing effects [11]. The same proteins exhibit significant differences in their binding ability, as well as in their stabilizing and enhancing effects, toward different catechins and their derivatives.

Non-covalent interactions such as hydrogen bonds, hydrophobic interactions, electrostatic interactions, and van der Waals forces are the primary interactive forces between proteins and catechin molecules [12]. The number of phenolic hydroxyl groups in catechins is the main factor affecting their non-covalent interactions with protein molecules. The phenolic hydroxyl group of catechins interacts with the carboxyl, hydroxyl, amino, and other functional groups of protein molecules to mainly form hydrogen bonds. Hydrophobic interaction is mainly formed by the interaction between the hydrophobic regions of proteins and the non-polar aromatic ring of catechins. Moreover, the spatial hindrance of catechins can be influenced by the number of hydroxyl groups, which in turn affects the way they interact with proteins and the strength of these interactions [13]. Accordingly, researchers speculate that the number of phenolic hydroxyl groups in catechins may be the main factor affecting the protein’s loading capacity and enhancing stability. However, it is still unclear how the number of phenolic hydroxyl groups affects the self-assembly and molecular interactions of proteins and catechins.

In the present study, EC, EGC, ECG, and EGCG were chosen as representative catechins with disparate numbers of phenolic hydroxyl groups, while edible dock protein (EDP) was chosen as the carrier material. Taking intermolecular interactions as the starting point, the regulation mechanism of the number of phenolic hydroxyl groups on the microenvironment and microstructure of the EDP–catechin compounds was investigated. Furthermore, the self-assembly mechanism between EDP and catechins regulated by their phenolic hydroxyl group number was elucidated. This study will provide scientific guidance for efficiently improving the stability and bioavailability of catechins.

2. Materials and Methods

2.1. Materials

Harbin Sancao Agricultural Technology Co., Ltd. (Harbin, China) provided the edible dock powder. Epicatechin (EC, ≥98%), epigallocatechin (EGC, ≥98%), epicatechin gallate (ECG, ≥98%), and epigallocatechin gallate (EGCG, ≥98%) were purchased from ChemFaces Co., Ltd. (Wuhan, China). Folin reagent was purchased from Solarbio Technology Co., Ltd. (Beijing, China). The remaining analytical-grade chemical reagents were all purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The EDP used in this study was prepared from edible dock powder by alkaline extraction followed by isoelectric precipitation. Briefly, 100 g of edible dock powder was mixed with 1000 mL of 95% ethanol and stirred for 1 h to remove lipids and pigments. After centrifugation at 5000 rpm for 20 min, the resulting precipitate was collected and dispersed in phosphate buffer (pH 8.0) at a solid-to-liquid ratio of 1:20. The pH of the dispersion was adjusted to 11.0 using 0.5 M NaOH, and the mixture was stirred at room temperature for 3 h. The supernatant obtained after centrifugation (5000 rpm, 20 min) was adjusted to pH 4.0 ± 0.1 to precipitate EDP, and this process was repeated once. The precipitate was then washed three times with distilled water to remove soluble salts, followed by freeze-drying to obtain EDP with a protein content of 85.3% (w/w).

2.2. Preparation of the EDP–Catechin Compounds

One hundred milligrams of EDP was dissolved in 40 mL of phosphate buffer solution, and the pH was adjusted to 7.0 to obtain a 2.5 mg/mL EDP stock solution. To create a catechin stock solution (7 mg/mL), 14 mL of each type of catechin was dissolved in 2 mL of phosphate buffer solution [7]. Ten milliliters of the EDP stock solution was mixed with 0.4 mL of each catechin solution. To enable non-covalent binding between the catechins and EDP, the mixture was magnetically agitated for one hour at 25 °C in the absence of oxygen. EDP-EC, EDP-EGC, EDP-ECG, and EDP-EGCG compounds were produced by dialyzing the samples for 24 h at 4 °C to eliminate unbound free catechins. These were then placed in brown bottles for storage [14].

2.3. Dynamic Light Scattering (DLS)

After the EDP–catechin compounds were diluted to an appropriate concentration with ultrapure water, the zeta potential, particle size, and polydispersity index (PDI) were measured by a DLS (ZSU3100, Malvern Instruments Ltd., Malvern, UK) instrument. All measurements were performed in triplicate to ensure the accuracy and reproducibility of the obtained data.

2.4. Encapsulation Efficiency (EE) and Loading Capacity (LC) of Catechins

Following the methodology of [15] with a minor adjustment, 1 mL of the EDP/EDP–catechin compounds was mixed with 1 mL of Folin reagent (1.0 M). After shaking, the mixtures were stored at 25 °C for 5 min in the dark. The mixed solution was then mixed with 4 mL of Na₂CO₃ solution (7.5%, w/v), and it was left to react for 2 h in the dark. EDP was used as the blank control, with its absorbance deducted to correct for protein interference. Standard solutions of the catechin and its derivatives were used to plot a standard absorbance vs. concentration curve. The absorbance was measured at 760 nm using a microplate reader, and the Na₂CO₃ solution was the blank control.

$$\mathrm{EC}:\mathrm{Y}=0.0124\mathrm{X}+0.0838,{\mathrm{R}}^2=0.9984$$

(1)

$$\mathrm{EGC}:\mathrm{Y}=0.0087\mathrm{X}+0.0593,{\mathrm{R}}^2=0.9991$$

(2)

$$\mathrm{ECG}:\mathrm{Y}=0.0092\mathrm{X}+0.0588,{\mathrm{R}}^2=0.9998$$

(3)

$$\mathrm{EGCG}:\mathrm{Y}=0.0105\mathrm{X}-0.0563,{\mathrm{R}}^2=0.9997$$

(4)

The encapsulation efficiency (EE) and loading capacity (LC) of EC, EGC, ECG, and EGCG were calculated according to Equations (1)–(6), where W₁ is the mass of the measured catechin and its derivatives, W₂ is the total mass of the catechin and its derivatives, and W₃ is the mass of the compounds [16].

$$\mathrm{EE}\left(\%\right)={\displaystyle \frac{{\mathrm{W}}_1}{{\mathrm{W}}_2}}\times 100$$

(5)

$$\mathrm{LC}\left(\%\right)={\displaystyle \frac{{\mathrm{W}}_1}{{\mathrm{W}}_3}}\times 100$$

(6)

2.5. Transmission Electron Microscopy (TEM)

The EDP–catechin compounds were diluted with distilled water to an appropriate concentration; then, the diluted EDP–catechin compounds were dropped onto a copper mesh using a pipette and dried at 37 °C. The micromorphology of the EDP–catechin compounds was examined using TEM (HT-7700, JEOL Ltd., Tokyo, Japan). TEM images of each sample were obtained at an acceleration voltage of 120 kV [9].

2.6. Determination of Interface Properties

The surface characteristics of the compounds were analyzed through contact angle measurements (SDC-200S, Shengding Precision Instrument Co., Ltd., Dongguan, China). The tilting plate method was used to measure the contact angle at 25 °C for a 0.5 μL liquid droplet volume. The hanging drop method was used to measure the surface tension at intervals of 0.5 s [7].

2.7. Low-Field Nuclear Magnetic Resonance (LF-NMR)

The distribution of bound and free water in the samples was determined using an LF-NMR (MesoMR23-060H-I Niumai Analytical Instruments Co., Ltd., Suzhou, China). Parameters: the resonance frequency was 18 MHz, the spectral width was 100 kHz, the echo time was 0.2 ms, the waiting time was 6000 ms, and the radiofrequency delay time was 0.2 ms. The transverse relaxation periods for bound and free water were determined to be T1 and T2, respectively, and the corresponding area proportions were determined to be PT1 and PT2 [8].

2.8. Magnetic Resonance Imaging (MRI)

Referring to the method of [17] with slight modifications, the water distribution inside the compounds was observed using MRI (MesoMR23-060H-I Niumai Analytical Instruments Co., Ltd., Suzhou, China). The parameters were two slices, each measuring 5.0 mm in thickness and 0.5 mm in spacing.

2.9. Fourier Transform Infrared (FTIR) and Ultraviolet (UV) Spectra

FTIR spectroscopy was employed to characterize the molecular structure of the lyophilized compounds materials (NicoletTMiS™ 50, Thermo Fisher Scientific, Waltham, MA, USA). The freeze-dried compounds were pressed into thin sheets. The number of scans for the sample spectra was fixed at 32, and the wavenumber range was set from 4000 to 400 cm⁻¹. Peak fit 4.12 software was used to conduct the analysis [18]. With respect to the UV (752N Jingke Instrument Co., Ltd., Shanghai, China) spectrum, after the samples was diluted to an appropriate concentration, the absorbance was measured in the wavelength range of 200–500 nm. The slit width was set at 10 nm, and the sampling interval was 1 nm. Blank protein samples were used as the control group [19].

2.10. X-Ray Diffraction (XRD)

XRD (Rigaku Smart Lab SE, Rigaku Corporation, Tokyo, Japan) with Cu-Kα radiation (λ = 0.1542 nm) was used to examine the EDP–catechin compounds’ crystalline structure. Before measurement, the scanning rate and spectrum collecting range were set as 5°/min and 5–90° (2θ), respectively.

2.11. Fluorescence Spectroscopy

The fluorescence spectra were measured using an FL 6500 fluorescence spectrophotometer (6500, PerkinElmer, Hopkinton, MA, USA). The EDP–catechin compounds of appropriate concentration were prepared. The excitation and emission slit widths were both set at 5 nm [20], the excitation wavelength was set at 280 nm, and the emission wavelength ranged from 300 to 500 nm. The excitation and emission wavelength ranges for the three-dimensional fluorescence spectra measurement were 200–400 nm and 200–500 nm, respectively.

2.12. Determination of Fluorescence Spectroscopy

Referring to the methodology with minor adjustments [14], the EDP–catechin compounds were incubated at 293 K, 303 K, and 313 K for 1 h. In the final combinations, the amounts of catechin and its derivatives were 25, 50, 100, 200, and 300 μmol/L. Fluorescence measurement parameters: the excitation was at 280 nm, the emission range was 300–500 nm, and the excitation/emission slit width was 5 nm. The Stern–Volmer approach was employed to investigate fluorescence quenching dynamics:

$${\mathrm{F}}_0/\mathrm{F}=1+\mathrm{Kq}\mathsf{\tau }0\left[\mathrm{Q}\right]=1+\mathrm{Ksv}\left[\mathrm{Q}\right]$$

(7)

where F₀ and F represent the protein solution’s fluorescence intensity prior to and following the addition of catechin chemicals, respectively. The parameters include: [Q] (catechin concentration), Ksv (Stern–Volmer constant), τ0 (unquenched fluorophore lifetime, ~10⁻⁸ s for biological macromolecules), and Kq (quenching rate constant).

Equation (8) describes the binding constant (Ka) and site number (n) characterizing protein–quencher complexes in static quenching:

$$\lg \left[\left({\mathrm{F}}_0-\mathrm{F}\right)/\mathrm{F}\right]=\mathrm{lgKa}+\mathrm{nlg}\left[\mathrm{Q}\right]$$

(8)

Equations (9) and (10) below were utilized to quantify the thermodynamic characteristics. ΔG represents free energy, ΔS represents entropy, and ΔH represents enthalpy change:

$$\ln \mathrm{K}\mathrm{a}=-\Delta \mathrm{H}/\left(\mathrm{RT}\right)+\Delta \mathrm{S}/\mathrm{R}$$

(9)

$$\Delta \mathrm{G}=\Delta \mathrm{H}-\mathrm{T}\Delta \mathrm{S}$$

(10)

where T is the test temperature, R is the gas constant [8.314 J (mol K)⁻¹], and Ka is the protein–quencher binding constant.

2.13. In Vitro Digestion

The EDP–catechin compounds (10 mL) were first blended with 10 mL of simulated salivary fluid (SSF), which is composed of NaCl, KCl, CaCl₂, NaH₂PO₄, urea, Na₂S, etc., and the mixture was digested at 37 °C for 2 min. Subsequently, the above mixture (10 mL) was added to 10 mL of simulated gastric fluid (SGF), which is composed of sodium chloride, dilute acid, and pepsin. After incubation at 37 °C for 1 h, 10 mL of the mixture was added to an equal volume of simulated intestinal fluid (SIF), which is composed of phosphates, trypsin, etc., for intestinal digestion at 37 °C for 2 h. The EDP–catechin compounds were collected at each digestion time, and the retention rates of EC, EGC, ECG, and EGCG were calculated according to Section 2.4 [21].

2.14. The Stability of EDP–Catechin Compounds

Storage stability: The EDP–catechin compounds were refrigerated at 4 °C for a three-month period. The particle size, zeta potential, PDI, and retention rate of the four EDP–catechin compounds were measured. These indexes were determined using Section 2.3 and Section 2.4.

UV stability: The EDP–catechin compounds were exposed to UV light at 365 nm for 60 h to assess the UV stability of the encapsulated catechin and its derivatives. Samples were taken at 0, 6, 12, 24, 36, 48, and 60 h [8]. The retention rate of catechins was determined based on Section 2.4.

Thermal stability: A specific quantity of the freeze-dried EDP–catechin compound powder was put in an aluminum crucible to test the thermal stability. Thermogravimetric analysis (TGA) was performed on the material in an air atmosphere. The TGA’s temperature was programmed to rise at a rate of 10 °C per minute from 30 °C to 600 °C [22].

2.15. Statistical Analysis

All the experiments were conducted in triplicate, and the obtained data were presented as the mean ± standard deviation (SD). Graphs were made utilizing Origin 2021 (Origin Lab, Northampton, MA, USA). The significance was evaluated using the Statistical Program Social Sciences 13.0 (SPSS, Chicago, IL, USA). The significance was set at p < 0.05.

3. Results and Discussion

3.1. Physicochemical Characteristics of the EDP–Catechin Compounds

The particle size and PDI of the EDP–catechin compounds are shown in Figure 1A. The EDP-EC, EDP-EGC, EDP-ECG, and EDP-EGCG compounds had particle diameters of 208.1 nm, 200.5 nm, 257.2 nm, and 211.1 nm, respectively. The particle size of these four compounds was noticeably larger than that of EDP (176.5 nm). This increase in hydrodynamic particle size was not due to simple volume expansion of EDP nanoparticles but mainly attributed to the non-covalent binding of catechins to EDP (hydrogen bonding, hydrophobic and electrostatic interactions), which induced the conformational unfolding of EDP and the mild aggregation of EDP nanoparticles with catechins as molecular bridges (as confirmed by TEM results in Figure 1C). In addition, the higher number of phenolic hydroxyl groups in catechins led to stronger intermolecular interactions, resulting in different degrees of particle size increase. After the addition of catechins, the PDI of the EDP–catechin compounds did not change significantly (p < 0.05). The EDP nanoparticles had a negative charge on their surface (Figure 1A). This is because the pH of the solution was higher than that of the protein’s isoelectric point (pI ≈ 4.0) [7]. After adding catechins, the negative charge of the compounds was further increased, resulting in molecular electrostatic repulsion. EDP-EGCG exhibited the greatest absolute zeta potential value (29.32 mV) among the four compounds. This was mainly due to EGCG having the highest number of phenolic hydroxyl groups, which not only formed the most hydrogen bonds with EDP but also enhanced the electrostatic interaction between the negatively charged hydroxyl groups of EGCG and the positively charged amino groups of EDP (pH 7.0 > pI of EDP ≈ 4.0). Meanwhile, the galloyl group in EGCG increased the hydrophobic interaction with EDP’s hydrophobic regions, and the synergistic effect of multiple non-covalent interactions resulted in the strongest electrostatic repulsion of EDP-EGCG composites.

In the EDP–catechin compounds, the encapsulation efficiency of EC, EGC, ECG, and EGCG was 70.5%, 82.8%, 90.6%, and 96.5%, respectively, with corresponding loading ability of 7.1%, 8.8%, 9.1%, and 9.7% (Figure 1B). The loading capacity order was EC < EGC < ECG < EGCG. By analyzing the molecular structures, it was found that the phenolic hydroxyl group numbers in these compounds were five for EC, six for EGC, seven for ECG, and eight for EGCG. The order of the phenolic hydroxyl group numbers of the four catechins was in agreement with the loading capacity of catechins within EDP. Consequently, catechins with more phenolic hydroxyl groups formed more hydrogen bonds with EDP, thereby strengthening their interactions. This was one of the reasons for the change in loading capacity of catechins within EDP. Due to the presence of aromatic amino acids, the Folin–Ciocalteu reagent may react with the protein, resulting in a higher protein embedding rate or loading amount. Using a blank EDP as the control and measuring under the same conditions, the absorbance value is subtracted from all sample readings to correct for protein interference. Although this correction method can reduce interference, it still has certain limitations.

As shown in Figure 1C, the microscopic morphological and aggregation of EDP and EDP–catechin compounds were observed using TEM. The EDP was initially uniformly dispersed as small particles without forming large aggregates. After adding EC, EGC, ECG, and EGCG, the EDP and catechins combined through hydrophobic interactions and hydrogen bonds. The catechins served as molecular bridges that facilitated the mild aggregation of EDP particles, resulting in the formation of a loose network structure (TEM images showed that the particle size of composites was less than 300 nm with no large agglomerates). This mild aggregation was beneficial to the stability of the composite system, which was further confirmed by the subsequent storage stability tests showing that the EDP-EGCG composites with the most obvious mild aggregation had the lowest particle size increase and highest catechin retention rate after 90 days of storage. A similar phenomenon was also observed when EGCG was added to soy protein, and this aggregation was beneficial to the stability of the compound system [14].

3.2. Low-Field Nuclear Magnetic Resonance and Magnetic Resonance Imaging

LF-NMR can effectively distinguish different states of water by measuring the relaxation times (T1 and T2) of water in products. As shown in Figure 2A, two distinct water distributions representing bound water (T1) and free water (T2) were observed at 10–25 ms and 1000–3500 ms, respectively. After binding with catechins, the relaxation time of T1 indicated that the distribution of water linked to the protein had obviously changed. The encapsulation of catechins in EDP significantly increased the percentage of T2 peak area (PT2) and decreased the percentage of T1 peak area (PT1). This shift suggested an elevation in the quantity of free water and a reduction in the quantity of bound water associated with the protein. For instance, upon encapsulation of EGCG by EDP, the proportion of bound water declined from 7.96% to 4.06%, while there was an increase in free water from 92.04% to 95.94% (

Table 1. The peak area proportion of the EDP and EDP–catechin compounds.

Sample	T1 (ms)	PT1 (%)	T2 (ms)	PT2 (%)
EDP	15.70 ± 1.05 b	7.96 ± 0.32 a	1644.68 ± 110.30 a	92.04 ± 0.32 b
EDP-EC	18.04 ± 1.21 a	4.75 ± 0.42 b	1534.368 ± 102.90 b	95.25 ± 0.42 a
EDP-EGC	15.70 ± 1.05 b	4.12 ± 1.23 b	1644.676 ± 123.74 a	95.88 ± 1.23 ab
EDP-ECG	16.83 ± 2.04 ab	3.95 ± 0.98 b	1534.368 ± 77.96 b	96.05 ± 0.98 a
EDP-EGCG	16.83 ± 1.21 ab	4.06 ± 1.22 b	1534.368 ± 123.74 b	95.94 ± 1.22 ab

Note: The data are presented as the mean ± standard deviation (n = 3). Different lowercase letters in the same column indicate statistically significant differences between samples. T1 and T2 are the relaxation times. PT1 and PT2 are the relative peak area percentages.

). These results might be ascribed to the hydrophilic phenolic hydroxyl groups of catechins forming more hydrogen bonds with water molecules, thus enhancing the hydration ability of the composites [23]. In addition, the mild aggregation of EDP nanoparticles induced by catechin binding may also lead to the redistribution of water within the aggregates, resulting in an increase in free water proportion. Therefore, the encapsulation of catechins was beneficial to the conversion of bound water to free water in the protein matrix, which was a comprehensive result of enhanced hydration and water redistribution.

Magnetic resonance imaging (MRI) is an imaging technique based on hydrogen proton signals. Due to the intuitive and visual advantages, it was widely used in the food industry to evaluate the distribution and changes in moisture content [17]. Figure 2B shows the MRI images of EDP and EDP–catechin compounds. The proton density of the samples was quantified, and the results showed that the proton density of the complex was about twice that of pure EDP. The increased red areas in MRI images were consistent with the quantified proton density results, indicating that the water content and proton density of EDP were significantly increased after complexation with catechins. This could be due to the formation of hydrogen bonds between the phenolic hydroxyl groups of catechins and EDP molecules, thereby enhancing water-binding capacity. Among the four compounds, EDP-EGCG had the highest red areas, which was related to EGCG having the highest number of phenolic hydroxyl groups. The addition of catechins caused varying degrees of increase in proton density in EDP, ultimately forming compound network systems with different densities. Therefore, the changes in red areas in MRI images reflected the changes in water content and proton density [24], which were related to the addition of catechins and their interactions with EDP.

3.3. Contact Angle and Surface Tension

Contact angle and surface tension can reflect the interaction ability between the surface of the EDP–catechin compounds and liquids, as well as their surface properties and dispersibility. A lower surface tension was associated with improved interfacial properties of aqueous dispersion, which was conducive to the formation and dispersion stability of the EDP–catechin compounds [25]. As shown in Figure 3(A1,A2), the surface tension and contact angle of EDP were 388.8 mN/m and 84.6°, respectively. There were notable alterations in the interfacial characteristics when EC, EGC, ECG, and EGCG were encapsulated in EDP (p < 0.05). The contact angles of EDP-EC (70.1°), EDP-EGC (67.3°), EDP-ECG (64.4°), and EDP-EGCG (59.4°) were all reduced in comparison to EDP (Figure 3(B1–E1)). Among the four compounds, EDP-EGCG showed the lowest contact angle, suggesting that EGCG has a greater tendency to self-assemble with EDP to form compounds. Figure 3(B2–E2) shows that the surface tension of EDP dramatically dropped after binding with catechins (p < 0.05). As amphiphilic molecules, EDP has both hydrophobic and hydrophilic regions. When catechins bound to the hydrophobic regions of EDP through hydrophobic interactions, the hydrophobic regions of EDP underwent reorganization, and the hydrophilic regions were exposed to the aqueous phase, thus reducing the surface tension of the composite dispersion. However, the correlation between surface tension and composite formation/stability was not absolute, and the final stability of the composite was determined by the synergistic effect of surface tension, zeta potential and particle size distribution [9]. Furthermore, the hydrogen bonding interactions between EDP and catechins were stronger when the number of phenolic hydroxyl groups in catechins was higher. This is the main reason why EGCG, which has the most phenolic hydroxyl groups, binds to proteins to form complexes with the lowest interfacial tension.

3.4. FTIR, UV and XRD

FTIR spectroscopy was employed to analyze the interactions and structural changes between EDP and catechin and its derivatives. Figure 4A–D show the FTIR spectra of EDP, catechin and its derivatives, and EDP–catechin compounds. A broad and intense peak of EDP appeared at 3300 cm⁻¹ due to the stretching vibrations of intermolecular hydrogen bonds between O-H and N-H [26]. EC, EGC, ECG, and EGCG all exhibited distinctive peaks in the FTIR spectra. However, these distinctive peaks disappeared in the EDP–catechin complexes, suggesting that the catechins were successfully incorporated into the EDP molecule. The observed shift in the O-H stretching band in EDP–catechin compounds’ spectra demonstrated hydrogen bonding interactions between catechins and EDP. Similar shifts were also observed between SPI and EGCG, indicating the presence of hydrogen bonding [14]. The stretching vibrations of C-O and C-N produced the amide I band (1650 cm⁻¹), whereas the bending vibrations of N-H and the stretching vibrations of C-N produced the amide II band (1540 cm⁻¹) [27]. After the addition of EC, EGC, ECG, and EGCG, the peaks of the amide I band shifted from 1637 cm⁻¹ to 1618 cm⁻¹, 1619 cm⁻¹, 1629 cm⁻¹, and 1620 cm⁻¹, respectively. Meanwhile, the peaks of the amide II band shifted from 1542 cm^–1 to 1524 cm⁻¹, 1520 cm⁻¹, 1525 cm⁻¹, and 1540 cm⁻¹, respectively. These shifts in the amide I and amide II bands indicated changes in the chemical environment of C-O, N-H and C-N groups of EDP, which is indirect evidence of hydrophobic interactions between catechins and EDP.

The FTIR spectra in the 1600–1700 cm⁻¹ region were fitted to examine the secondary structure alterations of EDP [28]. As illustrated in Figure 4E, EDP had 21.82% α-helix, 42.11% β-sheet, 21.58% β-turn, and 14.49% random coil. When EC, EGC, and ECG were combined with EDP, the α-helix content decreased to 15.23%, 18.37%, and 15.27%, respectively, while the β-turn content increased to 29.36%, 25.75%, and 27.77%, respectively. The results indicated that the encapsulation of catechin and its derivatives transformed the α-helix of EDP into β-turn. When EC and ECG were encapsulated in EDP, the β-sheet decreased to 38.21% and 36.15%, respectively, while the random coil increased to 17.2% and 20.8%. This phenomenon indicated that EC and ECG promoted the transition of β-sheet to random coil in EDP, resulting in a more loosely structure protein. The α-helix content dropped from 21.82% to 19.64%, the β-sheet dropped from 42.11% to 41.18%, the random coil dropped from 14.49% to 13.22%, and the β-turn rose from 21.58% to 25.96% when EGCG interacted with EDP. These modifications showed that the α-helix, β-sheet, and random coil in EDP changed to β-turn when EGCG was added. Similar phenomena were also observed in the interaction between ovalbumin and EGCG [29]. Overall, the addition of catechins induced conformational changes in EDP (e.g., the conversion of α-helix to β-turn), resulting in the loosening of the internal structure of EDP and the exposure of more hydrophobic and hydrophilic groups, which provided more binding sites for intermolecular non-covalent interactions [30].

Protein conformational changes and the creation of complexes between active ingredients and proteins can be investigated using UV spectroscopy [31]. The absorbance of EDP dramatically increased with the addition of catechin and its derivatives (Figure 4F). This suggested that the catechin and its derivatives disrupted the EDP structure and formed hydrophobic interactions with the protein’s hydrophobic amino acid residues. Additionally, the catechins may form new conjugated systems with the Trp and Tyr residues in EDP through π-π stacking, leading to π-π electron transitions and an increase in absorbance [32]. The conformational unfolding of EDP may expose more aromatic amino acid residues, which also contributes to the increase in UV absorbance, and the above two factors may synergistically affect the UV spectrum of the composites.

XRD is often used to analyze the crystalline structure of materials. As shown in Figure 4G, EDP exhibited two broad peaks around 8.2° and 28.5°, suggesting that this protein had an amorphous structure. Catechin and its derivatives exhibited a series of sharp peaks in the range of 5° to 40°, indicating their crystalline structure. After interaction with EDP, the characteristic peaks of the four catechins and their derivatives no longer appeared. This indicated that EDP molecules successfully encapsulated the catechins, and the crystalline structure of catechins was converted to an amorphous state due to non-covalent interactions, resulting in the disappearance of their characteristic diffraction peaks. A similar amorphous transformation was observed in zein–EGCG composites, which was a typical structural change of polyphenol–protein composites [33].

3.5. Fluorescence Spectra

The intrinsic fluorescence of proteins mainly originates from their aromatic amino acid residues, including Trp, Tyr, and Phe. Among them, tryptophan residues are the most sensitive to changes in microenvironmental polarity. Therefore, the endogenous fluorescence of proteins has been widely used to assess changes in their tertiary structure [18]. It can be seen in Figure 5A–D that all catechins quenched the fluorescence of EDP. The quenching order of EDP fluorescence intensity by catechin was: EGCG > ECG > EGC > EC. We discovered that the order of quenching corresponded to the number of hydroxyl groups in catechins that are phenolic: EGCG (8) > ECG (7) > EGC (6) > EC (5). This is mainly because ECG and EGCG contain a galloyl group and have a higher number of phenolic hydroxyl groups; the galloyl group not only provides additional hydrogen bonding sites but also increases the steric hindrance and hydrophobic interaction with EDP, and the synergistic effect of more hydroxyl groups and galloyl groups exerts a greater influence on the spatial structure of EDP, thus leading to a stronger quenching effect on the intrinsic fluorescence of EDP. The maximum emission wavelength of EDP changed when catechins were added. All catechins caused a red shift in the maximum emission wavelength of EDP molecules. This suggested an increase in the polarity of the microenvironment surrounding Trp and Tyr residues in EDP. Similar phenomena were also observed by [10], who found that the binding order of milk caseins with tea increased with the number of -OH groups, EGCG > EGC > EC > C.

As depicted in Figure 5(E1–I2), the three-dimensional fluorescence spectrum of EDP displayed two distinct peaks. Peak a represents the spectral characteristics of Trp and Tyr residues, indicating the alterations in the protein’s tertiary structure. In contrast, Peak b primarily reflects the fluorescence behavior of the polypeptide backbone, with its intensity correlating to the protein’s secondary structure. The fluorescence intensity of Peak a in EDP significantly decreased, demonstrating a change in the spatial structure of the protein. The order of the decrease in Peak a is also consistent with the results of the fluorescence spectra in Figure 5A–D. This further suggests that the number of phenolic hydroxyl groups in catechins significantly affected their interaction with the protein. The fluorescence intensity of Peak b in EDP was also remarkably decreased, which could be attributed to catechin altering the main chain structure of the peptide [34].

3.6. Molecular Fluorescence Analysis

Intrinsic fluorescence spectroscopy is a technique used to study the interactions between proteins and polyphenols [35]. Trp, an aromatic amino acid, exhibits intrinsic fluorescence at specific excitation wavelengths and is highly sensitive to its surrounding microenvironment. The fluorescence spectra of EDP and EDP–catechin compounds are shown in Figure 6A–D. The Stern–Volmer equation was used to determine Ksv and Kq. The fluorescence quenching was confirmed to be static for EDP-EC, EDP-EGC, EDP-ECG, and EDP-EGCG as Ksv decreased with increasing temperature, and all Kq values exceeded the maximal dynamic quenching constant of 2.0 × 10¹⁰ L·mol⁻¹·s⁻¹. The order of Ksv and Kq values at the ideal binding temperature of 293 K was EGCG > ECG > EGC > EC, suggesting that the strongest interaction occurred between EGCG and EDP, followed by ECG, EGC, and EC. It should be noted that potential inner-filter effects were not corrected in this analysis; therefore, the calculated Stern–Volmer parameters should be considered as approximate estimates. According to these results, catechin and its derivatives caused conformational changes in the protein structure, exposing hydrophobic groups that were previously hidden to a more hydrophilic polar environment [36].

The structural differences among catechin and its derivatives significantly affected their ability to bind to EDP. EGCG and ECG contain galloyl groups and thus can provide additional interaction sites (e.g., hydrogen bonds and hydrophobic interactions), resulting in more stable compounds. EC and EGC had fewer binding functional groups, leading to less stable compounds. These results demonstrated that different types of catechins interacted with EDP. When ΔH < 0 and ΔS < 0, hydrogen bonds and van der Waals forces were the primary driving forces; when ΔH > 0 and ΔS > 0, hydrophobic interactions were the dominant force; and when ΔH < 0 and ΔS > 0, electrostatic forces were predominant. As shown in

Table 2. Quenching constants, number of binding sites, and thermodynamic parameters for the interaction between EDP and catechins at different temperatures.

Sample	T (K)	Ksv (104 L·mol−1)	Kq (1012 L·(mol·s)−1)	Ka (103 L·mol−1)	n	ΔH (KJ·mol−1)	ΔG (KJ·mol−1)	ΔS J·(mol·K)−1
EDP-EC	293	0.0690	0.0690	0.0158	0.5155	−36.85	−6.73	−102.81
	303	0.0694	0.0694	0.0096	0.4279		−5.7
	313	0.0623	0.0623	0.0060	0.3786		−4.67
EDP-EGC	293	0.1240	0.1240	0.0436	0.5670	−54.30	−8.73	−155.51
	303	0.0893	0.0893	0.0116	0.4226		−7.18
	313	0.0771	0.0771	0.0106	0.4268		−5.62
EDP-ECG	293	0.5114	0.5114	1.8155	0.8718	−45.03	−18.09	−91.92
	303	0.4497	0.4497	0.7822	0.7788		−17.18
	313	0.3661	0.3661	0.5601	0.7513		−16.26
EDP-EGCG	293	0.6628	0.6628	2.6149	0.8828	−38.43	−19.26	−65.44
	303	0.5624	0.5624	1.7464	0.8489		−18.61
	313	0.6074	0.6074	0.9516	0.7675		−17.95

, ΔH < 0 and ΔS < 0, indicating that the interaction between EDP and catechins was primarily driven by hydrogen bonds and van der Waals forces. It should be noted that the van ’t Hoff analysis was carried out over a narrow temperature range (293–313 K), and the obtained thermodynamic parameters only reflect the main driving forces of the interaction; in addition, the contribution of weak electrostatic or hydrophobic interactions cannot be completely excluded. Among them, EDP-EGCG had the highest binding constant (Ka = 2.6 × 10³ L/mol) due to having the highest number of hydroxyl groups. Investigation found that ΔG < 0, indicating a spontaneous binding interaction between EDP and catechin molecules. Similar findings have been reported in the interaction mechanisms between soy protein and tea polyphenols [18].

3.7. Stability Assessment of Compounds

The UV stability of the EDP–catechin compounds can reflect the degradation of catechins during processing and storage. As shown in Figure 7A, after UV treatment for 60 h at 365 nm, the residual amounts of free EC, EGC, ECG, and EGCG were 12.3%, 19.6%, 24.3%, and 26.7%, respectively. Upon encapsulation, the retention rates of these catechins and their derivatives in the EDP were significantly enhanced, reaching 50.9% for EC, 52.6% for EGC, 59.5% for ECG, and 65.7% for EGCG. This corresponds to a 2.5- to 4.1-fold improvement in UV stability compared to the free forms, demonstrating that complexation with EDP provides substantial protection against UV-induced degradation. Moreover, catechins with a higher number of phenolic hydroxyl groups showed stronger UV stability after interaction with EDP. This enhanced UV resistance suggests that EDP encapsulation could be a promising strategy for improving the light stability of catechins in applications where UV exposure is unavoidable.

The thermal gravimetric analysis (TGA) of the samples was evaluated. Figure 7B shows that the EDP and EDP–catechin compounds had substantial mass loss within the temperature range of 30–150 °C, which was predominantly attributed to the evaporation of free water molecules. The minimal mass change observed for catechin and its derivatives suggested that these samples contained relatively low levels of water. A second phase of significant mass loss occurred as the temperature increased from 200 °C to 350 °C. This stage of mass loss was primarily due to the additional decomposition of bound water, facilitated by the interaction between the carboxyl (COOH) and amine (NH₂) groups present in the samples. Concurrently, the decomposition of the polymer backbone and the cleavage of glycosidic bonds also contributed to the overall mass reduction in this temperature range [37]. At the final temperature of 600 °C, the residual mass (TG%) of free catechins was as follows: EC, 47.31%; EGC, 49.82%; ECG, 40.78%; and EGCG, 43.76%. In contrast, the EDP–catechin complexes exhibited significantly higher residual mass: EDP-EC, 61.63%; EDP-EGC, 63.74%; EDP-ECG, 60.93%; and EDP-EGCG, 62.34%. The residual mass of the complexes was 1.3 to 1.5 times higher than that of the free forms. This suggested that the interaction between catechin and its derivatives and EDP enhanced the thermal stability of catechins to a certain extent [22].

The storage stability of EDP–catechin compounds was evaluated by keeping them in a refrigerator at 4 °C for 90 days. As shown in Figure 7C–F, an increase in the particle size of the four compounds was observed after storage, which was attributed to the aggregation of compounds. Concurrently, the polydispersity index (PDI) of the compounds was significantly increased, indicating the deteriorated dispersibility and reduced stability of the solution system. In addition, the absolute value of the zeta potential of the EDP–catechin compounds was decreased. The zeta potential absolute value of the EDP-EC compound showed the largest reduction, which was presumably related to EC having the fewest phenolic hydroxyl groups. Consequently, the electrostatic repulsion between EDP molecules and catechin and its derivatives diminished. This could be the pivotal factor contributing to the aggregation and decreased stability of the compounds [16]. However, the retention rate of catechin and its derivatives in EDP remained above 60% (Figure 7F), with EDP-ECG and EDP-EGCG achieving retention rates exceeding 75%. These findings showed that EDP molecules enhanced the storage stability of catechin and its derivatives.

With respect to digestion stability, Figure 7G shows that both free and encapsulated catechins had substantial retention rates after digestion in SSF. The retention rates of free EC, EGC, ECG, and EGCG decreased to 39.7%, 40.9%, 43.5%, and 39.4%, respectively, following further digestion in SGF, whereas the retention rates of EC, EGC, ECG, and EGCG in EDP were 74.3%, 75.5%, 81.6%, and 82.5%, respectively. These findings demonstrate that EDP encapsulation delayed degradation in the stomach of catechin molecules, resulting in enhanced digestive stability. About 20% of the free catechin and its derivatives were present after the combinations were digested in SIF, whereas the EDP encapsulation retained more than 40% of the catechin and its derivatives, wherein the encapsulated EGCG reached 46.4%. Polyphenolic compounds had good stability under acidic conditions, but the simulated digestive environment in the stomach was not simply acidic. Gastric peroxidase systems and metal ion catalysts were also present, so free catechins were easily degraded during gastric digestion. These results indicate that the interaction between catechins and EDP enhanced the stability of catechins during simulated gastrointestinal digestion, with EGCG receiving the most significant protection. Similar results were also found when EGCG was loaded by sodium caseinate [38]. It should be noted that although the in vitro digestion model is widely used and standardized, it cannot fully replicate the complex physiological conditions of the human digestive tract.

3.8. Correlation Analysis

Through Pearson correlation analysis, this study further explored the relationships between the number of phenolic hydroxyl groups in different catechins and the respective response variables. The heatmap results showed significant positive correlations between the phenolic hydroxyl group content and encapsulation efficiency, loading capacity, and storage stability (p < 0.001), indicating that the more hydroxyl groups there are in the catechin structure, the stronger their binding ability with the EDP carrier (Figure 8). This may be due to the additional binding sites or enhanced hydrogen bonding interactions provided by the increased number of hydroxyl groups. Meanwhile, the phenolic hydroxyl group content was significantly negatively correlated with zeta potential (p < 0.001), indicating that as the number of hydroxyl groups increased, the negative charge of the EDP–catechin complexes was further enhanced, thereby increasing the electrostatic repulsion between molecules. Additionally, a certain positive correlation trend was observed between phenolic hydroxyl group content and storage retention rate, suggesting a positive impact on the storage stability of the complexes.

4. Conclusions

In this study, a novel plant protein was selected as the carrier material to construct a delivery system for encapsulating catechins with different numbers of phenolic hydroxyl groups (EC, EGC, ECG, and EGCG). Among the four catechins tested, EGCG within EDP had highest loading capacity and stability due to having the greatest number of phenolic hydroxyl groups. During the self-assembly, hydrogen bonds, hydrophobic interactions and electrostatic forces were the main driving forces between EDP and catechins. Moreover, the interaction strength was strongest between EDP and EGCG. In addition, EDP encapsulation significantly enhanced the UV stability, thermal stability, storage stability, and digestive stability of catechins. The present study suggests that the phenolic hydroxyl group number of catechins can determine their binding affinity with proteins.