Ultrasonic pre-treatment modifies the pH-dependent molecular interactions between β-lactoglobulin and dietary phenolics: Conformational structures and interfacial properties

Graphical abstract

Highlights

• •The role of pre-ultrasound on β-LG–phenolic binding at various pH was investigated.
• •Pre-ultrasound at 35% amplitude strengthened the binding affinity of EGCG/CA to β-LG.
• •Pre-ultrasound promoted changes in the protein spatial structure by phenolic binding.
• •Dimer/monomer form of β-LG distinctly affects the complex property under ultrasound.
• •Pre-ultrasound with EGCG binding improved foaming and emulsifying properties of β-LG.

Abstract

There is a need to understand the ultrasound-induced changes in the interactions between proteins and phenolic compounds at different pH. This study systematically explored the role of high-intensity ultrasound pre-treatment on the binding mechanisms of β-lactoglobulin (β-LG) to two common phenolic compounds, i.e., (−)-epigallocatechin-3-gallate (EGCG) and chlorogenic acid (CA) at neutral and acidic pH (pH 7.2 and 2.4). Tryptophan fluorescence revealed that compared to proteins sonicated at 20% and 50% amplitudes, 35%-amplitude ultrasound pre-treatment (ULG-35) strengthened the binding affinities of EGCG/CA to β-LG without altering the main interaction force. After phenolic addition, ULG-35 displayed a similar but a greater extent of protein secondary and tertiary structural changes than the native protein, ascribed to the ultrasound-driven hydrophobic stacking among interacted molecules. The dominant form of β-LG (dimer/monomer) played a crucial role in the conformational and interfacial properties of complexes, which can be explained by the distinct binding sites at different pH as unveiled by molecular docking. Combining pre-ultrasound with EGCG interaction notably increased the foaming and emulsifying properties of β-LG, providing a feasible way for the modification of bovine whey proteins. These results shed light on the understanding of protein–phenolic non-covalent binding under ultrasound and help to develop complex systems with desired functionality and delivery.

1. Introduction

Currently, the interaction mechanisms between dietary proteins and phenolic compounds are gaining substantial interest due to their wide range of applications in the food industry. Milk proteins and phenolic compounds are commonly co-existing food components and studies have documented that dairy proteins can easily interact with phenolics to form mostly non-covalent but also some covalent complexes [21], [40]. Specifically, whey proteins account for about 20% of total milk proteins and have been widely used as a food ingredient due to its high nutritional value and low cost. In addition, whey proteins can be natural vehicles for the interacted phenolics because of several unique characteristics, such as modified protein structure and functionality, and improved stability and bioavailability of the bound substances [4], [10]. As a consequence, the exploration and development of associated systems are now receiving great attention from scientific and industrial perspectives.

β-Lactoglobulin (β-LG) is a main bovine whey protein, making up around 50% by mass of the whey protein fraction of milk. A series of studies have portrayed that the interactions between β-LG and phenolic compounds can induce certain structural changes in proteins, which are closely associated with the functionality, nutritional aspects, and even potential allergenicity of the formed complexes [7], [9], [36], [36]. In particular, (−)-epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea, and chlorogenic acid (CA) is found in coffee and fruits, both of which are commonly consumed phenolics and are shown to have a strong binding affinity for milk proteins, e.g., β-LG [9]. Previous work has unveiled that the interactions between β-LG and phenolic compounds largely depend on the chemical structures of phenolics and surrounding conditions, especially temperature and pH [36], [40]. When pH dropped from physiological pH (~7) to acidic pH (2–3), β-LG was assumed to transfer from the equilibrium dimeric state to monomeric structure due to the strong electrostatic repulsion between two subunits [1], [2], which may prominently affect its binding mechanism with phenolic compounds. In this scenario, albeit equivocal, several studies have explored the interactions between β-LG and phenolics at different pH levels, such as apigenin [41], resveratrol [15], quercetin [18], and ferulic acid [1].

As an effective non-isothermal technology, ultrasound has been applied in many areas of food processing. The high shear stress and mechanical energy generated by high-intensity ultrasound (20–100 kHz) can cause cavitation bubbles and microstreaming currents, resulting in modifications in the physiochemical properties of food components [3]. There is evidence in the literature showing that controlled ultrasound treatment of whey fractions disrupts the particle aggregation and unfolds the protein structures to form a modified interfacial feature [16], [17]. In this regard, molecular spatial structure, flexibility, and hydrophobic-hydrophilic balance as affected by ultrasound are among the key factors that influence the complex formation between proteins and phenolics [40]. Given this, it is expected that the sonication of proteins would have a profound impact on their subsequent interactions with phenolic ingredients. To our knowledge, only a few studies have investigated the role of ultrasound on protein–phenolic binding, such as emodin–micellar casein [38], egg white–gallic acid (GA)/EGCG [5], and myofibrillar protein–GA systems [22]. Despite extensive research on β-LG relating phenolic addition and ultrasound alone, no studies have yet focused on the ultrasound-driven changes in the interactions between β-LG and dietary phenolics, results of which would provide greater insights into the utilization of both techniques in the dairy products.

Therefore, this study aimed to investigate the potential role of ultrasonic pre-treatment on the interactions between β-LG and two phenolics (i.e., EGCG and CA) under neutral and acidic pH. In doing so, multiple spectroscopic and computational approaches, including tryptophan fluorescence, circular dichroism (CD), three-dimensional (3D) fluorescence, UV absorption, and molecular docking were applied to assess the binding mechanisms and associated changes in the conformational and interfacial properties of β-LG. This study contributes to a better understanding of the influences on protein–phenolic binding by ultrasound cavitation and provides motivations for the development of dairy systems with desired property and delivery.

2. Materials and methods

2.1. Materials and chemicals

β-LG from bovine milk (purity ≥ 95%), EGCG (purity ≥ 98%), and CA (purity ≥ 98%) were purchased from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China). 8-anilion-1-naphhalenesulfonic acid ammonium salt (ANS) was a product from Aladdin Industrial Corporation (Shanghai, China). Soybean oil was obtained from Cofco Donghai Grain and Oil Industry (Zhangjiagang) Co., Ltd. (Jiangsu, China). Ultrapure (UP) water purified from a Millipore Milli-Q Advantage A10 Water Purification System (Bedford, MA, USA) was used throughout. All other chemicals and reagents were of analytical reagents and were bought from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China) unless otherwise specified.

2.2. Ultrasonic pre-treatment

To mimic β-LG–phenolic interactions under different pH, two buffering systems were utilized: phosphate buffer saline (PBS, pH 7.2, 10 mM) and disodium hydrogen phosphate-citrate buffer (PCB, pH 2.4, 10 mM). β-LG was dissolved in the respective buffer and vortexed for 2 h at room temperature to ensure uniform hydration. The final concentration of β-LG was calculated spectrophotometrically based on the molar extinction coefficient of 17600 M⁻¹ cm⁻¹ at 280 nm [41]. For the ultrasound pre-treatment, an ultrasonic processor (JY 92-IIDN, Ningbo Scientz Biotechnology Co., Zhejiang, China; 20 kHz; maximum power 900 W) equipped with a 4 mm diameter titanium probe was used. The probe was inserted into 20 mL of β-LG solution (10 μM) in a 50 mL beaker and operated at three different power outputs, i.e., amplitudes of 20%, 35%, and 50% for 30 min per power output. The sonication was set by a pulse duration of on-time 4 s and off-time 2 s. The whole process was immersed in an ice-water bath to avoid heat and protein denaturation. Samples received sonication at 20%, 35%, and 50% amplitudes were labeled as ULG-20, ULG-35, and ULG-50, individually, and the untreated protein was labeled as NLG. Based on the calorimetric method, the power densities were calculated to be 20.34, 65.32, and 106.39 W/cm² at the output powers of 20%, 35%, and 50%, respectively.

2.3. Preparation of β-LG-phenolic complexes

To prepare the stock solution of phenolics, EGCG and CA were respectively dissolved in PBS (pH 7.2) or PCB (pH 2.4) to obtain a final concentration of 10 mM. Then, the untreated or each sonicated β-LG (10 μM, final concentration) was combined with different concentrations of phenolic compounds (EGCG or CA, at pH 2.4 or 7.2) to obtain protein/phenolic molar ratios of 1:0, 1:0.5, 1:1, 1:1.5, 1:2, and 1:3 (volume ratio 1:1), respectively. The pH was readjusted and the mixtures were kept under constant vertexing at 25 °C for 2 h.

2.4. Intrinsic fluorescence quenching analysis

To understand the binding mode of EGCG/CA to β-LG under different circumstances, the intrinsic fluorescence of samples was measured by a Fluromax-4 spectrophotometer (Horiba Scientific, Japan). The excitation wavelength was 280 nm and the emission spectra were recorded from 305 to 500 nm. The slits of excitation and emission were set at 2 nm. Due to the intrinsic fluorescence of EGCG and CA, control samples of each phenolic compound at different concentrations (in PBS, pH 7.2 or PCB, pH 2.4) were also prepared and measured to avoid interference in the fluorescence intensity and maximum emission wavelength (λ_max) in the complexes [26], [37]. The measured fluorescence intensities of EGCG and CA solutions without β-LG were subtracted from the fluorescence intensity of corresponding samples. The quenching mechanism was analyzed by the Stern-Volmer equation as follows [11]; T. [13]:

F₀/F = 1 + K_sv[Q] = 1 + K_qτ₀[Q] (1)

where F₀ and F are the fluorescence intensities in the absence and presence of EGCG or CA; [Q] is the quencher concentration (in this study EGCG and CA); τ₀ is the average lifetime of the unquenched fluorophore (10⁻⁸ s); K_sv is the Stern-Volmer quenching constant, and K_q is the bimolecular quenching rate constant.

For a static quenching process, a double logarithmic equation was presented:

log[(F₀ − F)/F] = logK_a + nlog[Q] (2)

where K_a is the binding constant, and n is the number of binding sites [28].

The thermodynamic parameters for protein–phenolic binding are the main evidence to interpret the interaction forces, and can be calculated by the Van’t Hoff equation [28]:

lnK_a = −ΔH/RT + ΔS/R (3)

ΔG = ΔH − TΔS (4)

where R is the gas constant (8.314 J mol⁻¹K⁻¹); T is the thermodynamic temperature (K); and ΔH, ΔS, and ΔG refer to the enthalpy change (KJ mol⁻¹), entropy change (J mol⁻¹ K⁻¹), and Gibbs free energy change (KJ mol⁻¹), respectively.

2.5. Surface hydrophobicity (H₀) analysis

The surface hydrophobicity was measured by using ANS as a fluorescence probe, as previously described by Chen et al. [4]. The samples were diluted respectively with 10 mM PBS (pH 7.2) or PCB (pH 2.4) to obtain dilutions with protein concentrations from 0.1 to 10 μM. 10 μL of 8 mM ANS was added to 200 μL of each sample solution and incubated in the dark for 15 min at ambient temperature. The fluorescence intensity was then measured by a fluorescence spectrophotometer (RF-5301PC, Shimadzu, Japan) at λ_ex = 390 nm and λ_em = 480 nm. The fluorescence intensity of ANS solution without sample was subtracted from the fluorescence intensity of the samples. The index of surface hydrophobicity (H₀) was obtained by calculating the initial slope of the fluorescence intensity vs. protein concentration plot.

2.6. CD spectroscopy

The CD spectrum of β-LG (10 μM) in the presence of EGCG or CA (25 μM) at different pH with or without ultrasonic pre-treatment (35%) was investigated by a JASCO J-1500 spectropolarimeter (JASCO Corp, Japan). The experiments were performed in the far-UV region from 260 to 190 nm at a 100 nm/min scan rate and 1 nm bandwidth at 25 °C. The CD spectroscopic data was calculated to obtain secondary structural elements by using the online CONTIN program in Dichro Web (http://dichroweb.cryst.bbk.ac.uk).

2.7. 3D fluorescence measurement

The 3D fluorescence spectrum of β-LG (10 μM) complexed with EGCG or CA (25 μM) at different pH with or without ultrasonic pre-treatment (35%) was recorded using a Fluor spectrophotometer (F-7000, Hitachi Ltd, Japan). The excitation wavelength was set from 200 nm to 350 nm and the emission wavelength was recorded between 250 and 500 nm. The increments of excitation and emission were both fixed as 5 nm and the scan speed was 2400 nm/min. The experiments were conducted at 25 °C.

2.8. In silico molecular docking (MD)

MD simulations were performed to investigate the possible binding modes of EGCG/CA on β-LG at different pH. The 3D crystal structures of β-LG (PDB: 2Q2M and 1BEB) were extracted from the Protein Data Bank (http://www.rcsb.org). The chemical structures of EGCG and CA were retrieved from the PubChem Compound database (https://pubchem.ncbi.nlm.nih.gov). Both phenolics and protein molecules were prepared with AutoDock Tools 1.5.6 [19], and docking calculations were conducted using AutoDock Vina [33]. To calculate all potential binding pockets, the grid space was set covering the entire surface of each tested protein. One hundred different runs were automatically conducted for each ligand and the pose with the lowest binding energy was selected. Visualization of the protein–ligand interactions, including binding sites, interacting amino acid residues and forces was acquired by Discovery Studio 2019 (Dassault Systèmes Biovia Corp®).

2.9. Determination of techno-functional properties

The solubility of β-LG complexed with two phenolics under different conditions was determined as previously described [4]. In brief, each sample solution (1 mg protein/mL, protein/phenolic molar ratio 1:1.5) was centrifuged at 8000 r/min for 20 min at 4 °C in a high-speed refrigerated centrifuge (Allegra X-30R, Beckman, Germany). With proper dilution, the protein concentrations in the supernatant and in the original sample were measured by a reducing agent compatible Bradford assay kit (Thermo Scientific). The protein solubility (%) was reported as a percentage of protein in the supernatant over the total protein content.

Changes in the foaming property of β-LG induced by phenolic addition and ultrasound pre-treatment were analyzed based on the method of Chen and Ma [5]. Briefly, 20 mL of sample solution (1 mg protein/mL, protein/phenolic molar ratio 1:1.5) was poured into a graduated glass cylinder and foamed at 8,000 r/min for 1 min by a high-speed homogenizer (XHF-D, Ningbo Scientz Biotechnology Co., Zhejiang, China). The volumes of the foam were recorded at 2 (V₂) and 30 min (V₃₀), individually. The foaming ability (FA) and foaming stability (FS) were calculated with the following equations:

FA (%) = V₂/20 × 100 (5)

FS (%) = V₃₀/ V₂ × 100 (6)

The emulsifying property was determined by using the turbidimetric method of Pan et al. [23] and Pearce and Kinsella [24], with minor modifications. To generate emulsions, sample solution (10 mg protein/mL, protein/phenolic molar ratio 1:1.5) was combined with soybean oil at a volume ratio of 3:1 and homogenized at 12,000 r/min for 2 min. Then, 50 μL of the fresh emulsion was pipetted at 0 and 30 min and diluted 100× times with 0.1% sodium dodecyl sulfate solution. The mixtures were recorded for their absorbance at 500 nm with a UV–Vis spectrophotometer (UV-2600, Shimadzu, Japan). The emulsifying activity index (EAI) and emulsion stability index (ESI) were calculated using Eqs. (1), (2):

$$\mathit{EAI}(m^2/g)=\frac{2\times 2.303\times A_0\times N}{10000\times \mathrm{\Phi }\times C}$$ (7)

$$\mathit{ESI}(\%)=\frac{A_{30}}{A_0}\times 100\%$$ (8)

where A₀ and A₃₀ are the respective absorbance of the emulsion at 0 and 30 min; N is the dilution factor; C is the protein concentration (g/mL), and Φ is the volume ratio of oil (0.25).

2.10. Emulsion characteristic analysis

The emulsions were prepared as indicated in Section 2.9. Analysis of the partition of protein in the aqueous continuous phase and at the interface of formed emulsions was measured base on the protocol of Cheng et al. [6], with slight modifications. In brief, the serum and cream layers were obtained by centrifuging the emulsions at 10,000 r/min for 30 min, after which the serum layer was carefully separated by a 0.45 mm syringe filter. The protein concentration in the aqueous phase was determined by a modified Bradford assay kit as earlier indicated. The partition coefficient (PC) of β-LG in the aqueous phase was calculated based on the following equation:

$$\text{PC}=\frac{V_w}{V_l}\times \left(\frac{W_t}{W_w}-1\right)$$ (9)

where V_w and V_l are the respective volumes of the aqueous phase and oil phase. W_t and W_w are the concentrations (mg/mL) of total protein and the protein in the aqueous phase, respectively. $\frac{W_w}{W_t}$ × 100 refers to the percentage of protein in the aqueous phase. The content of EGCG/CA in the aqueous phase was also tested by the Folin-Ciocalteu method, and the levels of interfacial EGCG/CA (F_p) were expressed as the proportion of interfacial EGCG/CA to the total amount of phenolics in the emulsions.

2.11. Statistical analysis

All experiments were performed thricely at least, and data were expressed as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA), followed by Duncan’s multiple range test was used to compare mean values at p < 0.05. IBM SPSS Statistics Version 22.0 (SPSS Inc., Chicago, IL, USA) was applied for all statistical analysis.

3. Results and discussion

3.1. Fluorescence quenching analysis

To unveil the possible role of ultrasound treatment in the modification of dairy protein–phenolic interactions, fluorescence quenching analysis was first applied to understand the binding mode of EGCG/CA to β-LG under different circumstances. The protein intrinsic fluorescence is mainly induced by the fluorescence emission of Tyr and Trp residues when excited at 280 nm, and a reduction in the protein fluorescent intensity implies certain quencher-fluorophore interactions [11]). As shown in Fig. 1, the native β-LG showed a maximum emission wavelength (λ_max) at 337 nm, being characteristic of Trp emission. Ultrasound treatment uniquely affected the emission spectra of β-LG (Supplementary Fig. S1): taking ULG-35 as an example, at neutral pH, sonication increased the fluorescent intensity of β-LG ascribed to protein conformational rearrangement and exposure of burried hydrophobic domains [17]; while at acidic pH, the fluorescence intensity was slightly decreased, which could be due to the intermolecular aggregation induced by a combined effect of extreme pH and ultrasonic actions [14]. Albeit in different degrees, the presence of EGCG/CA quenched the fluorescence intensity of β-LG in a dose-dependent manner, indicating that the interactions with two phenolics influenced the protein fluorophore. Of note, in β-LG–EGCG systems, no obvious shift in the λ_max was observed, whereas CA-addition induced a notable red-shifting in the λ_max of β-LG, particularly in NLG–CA group at neutral pH (337 to 347 nm). This implied that CA-interaction modified the protein’s polypeptide backbone, inducing the microenvironment of Trp residues to a more polar region, being capable to interact with water molecules [30]. In addition, it was noted that the fluorescence intensity of samples at pH 2.4 was higher than those at pH 7.2, which can be explained by the consequence of dimerization leading to self-quenching of the Trp19 residue, as observed in previous studies [1], [31].

Fig. 1.

Fluorescent emission spectra of 10 μM β-LG non-covalently bound to CA or EGCG in the absence and presence of ultrasonic pre-treatment at pH 7.2 (A) and 2.4 (B). CA, chlorogenic acid; EGCG, epigallocatechin gallate; NLG, native β-LG; ULG-35, β-LG treated with ultrasound at 35% amplitude.

The Stern-Volmer equation is widely applied to interpret the quenching mechanism between proteins and small molecules, and a linear plot generally represents a single quenching process [11]. The representative Stern-Volmer plots of β-LG bound to EGCG or CA are given in Supplementary Fig. S2 and the quenching parameters are presented in Table 1. As seen, the calculated K_q were within 0.58 × 10¹²–4.25 × 10¹² L mol⁻¹s⁻¹ for all samples and the values were appreciably higher than the maximum diffusion-limited quenching constant (2 × 10¹⁰ M⁻¹ s⁻¹). This implies that the quenching process of β-LG with both EGCG and CA is dominated by a static, instead of a dynamic quenching mechanism, in consistence with previous reports [9]). For a static quenching mechanism, the double logarithmic equation was further applied and results are tabulated in Table 1. The calculated K_a values were from 0.36 to 7.61 × 10⁴ L mol⁻¹ and from 0.56 to 5.76 × 10⁴ L mol⁻¹ for interactions with EGCG and CA, respectively. EGCG showed a higher binding ability to native β-LG at pH 2.4 than pH 7.2, which can be attributed to the higher amount of stabilizing forces with the surrounding residues in the monomer form, comparable to previous data in β-LG–ferulic acid/cocoa polyphenol systems [1], [31]. To gain deeper insights into the binding modes between EGCG/CA and two forms of β-LG, MD simulation was applied and the interaction details are given in Fig. 2. It was clearly shown that both EGCG and CA preferly bound to the interface of the two subunits in the dimer form (Fig. 2A, while the binding locations for the monomeric β-LG lay within the calyx shaped β-barrel region (Fig. 2B). EGCG and CA shared quite a few interacting amino acid residues (Fig. 2C), including those of the residues 134–141 (α-helix), 145–150 (I β-strand), and 23–24 (A β-strand) in the dimer form and 20–22 (disordered), 59–66 (C-D β-strand), 123–126 (disordered), and 151–152 (disordered) in the monomer form. The Trp 19 residue, which counts for the bulk of Try fluorescence is also involved in the interactions between EGCG and monomer β-LG.

Table 1.

Quenching constants, binding constants, and thermodynamic parameters for the binding of EGCG/CA to β-LG under different circumstances.

pH	Phenolics	Samples	T (K)	Ksv (104 L mol−1)	Kq (1012 L mol-1s−1)	Ka (104 L mol−1)	n	R2	ΔH (KJ mol−1)	ΔG (KJ mol−1)	ΔS (KJ mol−1 K−1)
7.2	EGCG	NLG	298	2.00	2.00	0.65	0.90	0.996	64.48	−21.75	0.29
			310	2.64	2.64	1.77	0.96	0.993		−25.22
		ULG-20	298	1.45	1.45 2.13	0.86	0.86	0.992	17.98	−22.45	0.14
			310	2.13		1.14	0.89	0.993		−24.08
		ULG-35	298	4.25	4.25 3.95	1.82	0.92	0.993	91.50	−24.32	0.39
			310	3.95		7.61	1.06	0.989		−28.98
		ULG-50	298	3.60	3.60	0.36	0.79	0.987	30.29	−20.28	0.17
			310	3.62	3.62	0.57	0.83	0.992		−22.32
	CA	NLG	298	1.52	1.52	1.57	1.00	0.991	33.90	−23.95	0.19
			310	2.34	2.34	2.67	1.01	0.989		−26.28
		ULG-20	298	1.85	1.85	1.34	0.97	0.987	51.94	−23.56	0.25
			310	1.99	1.99	3.01	1.04	0.997		−26.60
		ULG-35	298	2.14	2.14	1.48	0.97	0.991	76.84	−23.80	0.34
			310	2.17	2.17	4.90	1.08	0.992		−27.85
		ULG-50	298	2.32	2.32	0.56	0.87	0.992	66.52	−21.41	0.29
			310	2.43	2.43	1.59	0.96	0.998		−24.95
2.4	EGCG	NLG	298	0.58	0.58	1.13	1.06	0.988	46.65	−23.15	0.23
			310	0.53	0.53	2.35	1.14	0.994		−25.96
		ULG-20	298	0.74	0.74	0.98	1.06	0.992	55.87	−22.78	0.26
			310	0.81	0.81	2.34	1.10	0.989		−25.95
		ULG-35	298	0.62	0.62	1.12	1.06	0.993	102.32	−23.11	0.42
			310	0.78	0.78	5.52	1.14	0.995		−28.15
		ULG-50	298	0.68	0.68	0.94	1.03	0.995	32.56	−22.68	0.19
			310	0.65	0.65	1.56	1.08	0.996		−24.90
	CA	NLG	298	1.59	1.59	1.38	0.99	0.995	62.52	−23.64	0.29
			310	1.73	1.73	3.68	1.07	0.996		−27.11
		ULG-20	298	1.68	1.68	1.08	0.97	0.992	88.20	−23.02	0.37
			310	1.62	1.62	4.28	1.11	0.989		−27.50
		ULG-35	298	1.67	1.67	1.06	0.96	0.994	108.75	−22.96	0.44
			310	1.96	1.96	5.76	1.10	0.992		−28.27
		ULG-50	298	1.55	1.55	0.93	0.95	0.997	90.67	−22.66	0.38
			310	1.53	1.53	3.85	1.09	0.991		−27.22

CA, chlorogenic acid; EGCG, epigallocatechin gallate; NLG, native β-LG; ULG-35, β-LG treated with ultrasound at 35% amplitude.

Fig. 2.

Examples of the best binding poses (A-B) and interacting residues (C) for EGCG/CA docked on the β-LG dimer and monomer. CA, chlorogenic acid; EGCG, epigallocatechin gallate.

Following ultrasound pre-treatment, the binding behaviors between β-LG and two phenolic compounds were variously affected: in β-LG–EGCG system, pre-sonication at 20% and 35% amplitudes minorly changed or slightly enhanced the binding constants between β-LG and EGCG; whereas, pre-exposed to 50% amplitude unexceptionally slashed the K_a values at both pHs. A similar tendency was observed for β-LG–CA binding, where the highest K_a was obtained in ULG-35 interacted with CA at 310 K. Moderate ultrasound irradiation can properly unfold the protein structure to expose more reactive groups to facilitate the binding process; whereas high ultrasound amplitude disrupted the intermolecular collision by driving unfavored aggregation of protein molecules [16], [39].

To further unveil the interaction mechanisms between β-LG and two phenolics as influenced by the pH and ultrasound, thermodynamic parameters were calculated and presented in Table 1. The ΔG values were negative for all samples, confirming that the binding process was spontaneous. Based on the theory of Ross and Subramanian [29], it can be speculated that, the interactions between β-LG and EGCG or CA were primarily through hydrophobic interactions as ΔH > 0, ΔS > 0. For an endothermic process (ΔH > 0), the K_a values increased with increasing temperatures, as evidenced in Table 1. Comparably, the main interaction forces between tea polyphenols, rutin, and β-LG at neutral pH were dominated by hydrophobic interactions, which is similar to our results [2], [9]. These data collaborated well with previous data showing that ultrasound may strengthen or weaken the interactions between dietary protein and phenolics, but would not alter the type of major driving forces [38]. Overall, ULG-35 showed the highest K_a and ΔH values when interacting with EGCG/CA, thus was selected in the following experiments.

3.2. Protein conformational structural modifications

The surface hydrophobicity is an index reflecting protein conformational changes and is known to have a profound impact on the interfacial properties of involved proteins. As shown in Fig. 3, attributed to the exposure of hydrophobic groups, ULG-35 showed an H₀ value of 108.6 and 171.2 at pH 7.2 and 2.4, which are prominently higher than that of the respective NLG. At neutral pH, the addition of EGCG/CA gradually decreased the H₀ of both NLG and ULG-35, except for an opposite slight increase at a higher phenolic/protein ratio (3:1). In comparison, at pH 2.4, the values of H₀ saw a reduction first and then a steep increase at phenolic/protein ratios of 2:1 and 3:1. The addition of phenolics at high concentrations may induce unfolding of protein structures to expose previously hidden hydrophobic regions [25]. In most cases, a more pronounced change in the H₀ upon phenolic addition was observed in ULG-35 than NLG, which can be explained by the higher level of hydrophobic regions in ULG-35 to promote intermolecular stacking interactions. Similar results have been portrayed in pre-sonicated egg white–GA/EGCG systems [5].

Fig. 3.

The surface hydrophobicity of β-LG interacted with EGCG or CA with or without ultrasound pre-treatment at pH 7.2 (A) and 2.4 (B). CA, chlorogenic acid; EGCG, epigallocatechin gallate; NLG, native β-LG; ULG-35, β-LG treated with ultrasound at 35% amplitude.

The secondary structural profile of β-LG as affected by ultrasound and phenolic binding was further analyzed by CD spectroscopy and results are shown in Supplementary Fig. S3 and Table 2. The NLG displays a typical β-sheet rich secondary structure pattern, which is in line with previous reports [1], [9], [17]. For NLG–phenolic interactions at 1:1.5 (protein/phenolic ratio), a pH-dependent conformational change was observed: at pH 7.2, EGCG/CA binding induced a decrease in the levels of α-helix and β-sheet with an expansion of the β-turn and random coil; while at pH 2.4, an increase in the contents of β-elements at the cost of α-helix and random coil was noticed, implying a stabilizing effect on the β-barrel of the protein molecule in the monomeric form [1]. Following ultrasound treatment, minimization of the β-turn and unordered structure at the expense of α-helix and β-sheet was caused [16]. With the aid of pre-sonication, the addition of EGCG/CA induced a similar but a greater extent of protein structural rearrangement, as evidenced by the higher values of element increments or decrements when compared to NLG–phenolic binding. Ultrasound pre-treatment was beneficial for the hydrophobic stacking interactions between β-LG and phenolics, and the form of β-LG (dimer/monomer) played a crucial role in the spatial structure of complexes due to the distinct binding locations [1], [8]. A similar phenomenon of ultrasound-driven protein structural changes by phenolic binding was witnessed in EGCG/GA–egg white [5] and emodin–micellar casein systems [38].

Table 2.

The secondary structural profiles of β-LG bound to EGCG or CA with or without ultrasonic pre-treatment at pH 7.2 and 2.4.

pH	Samples	α-helix (%)	β-sheet (%)	β-turn (%)	Random coil (%)
7.2	NLG	19.5	30.8	22.5	27.3
	NLG-EGCG	18.8	29.1	23.4	28.9
	NLG-CA	17.7	30.1	24.1	28.1
	ULG-35	20.1	32.2	21.6	26.1
	ULG-35-EGCG	18.9	25.7	25.1	30.4
	ULG-35-CA	16.8	26.6	23.4	33.2
2.4	NLG	24.0	28.8	20.5	26.7
	NLG-EGCG	22.7	30.7	21.4	25.2
	NLG-CA	22.3	30.1	21.7	25.9
	ULG-35	26.3	31.5	19.9	22.3
	ULG-35-EGCG	23.7	35.2	23.3	17.8
	ULG-35-CA	24.1	34.7	22.8	18.4

CA, chlorogenic acid; EGCG, epigallocatechin gallate; NLG, native β-LG; ULG-35, β-LG treated with ultrasound at 35% amplitude

To obtain more detailed information on the conformational and microenvironmental changes in β-LG, 3D fluorescence spectroscopy was applied. The 3D spectra are shown in Fig. 4 and relevant fluorescent parameters are summarized in Table 3. Peak A (λ_ex = 275 nm, λ_em = 335 nm) represents the spectral features of Tyr and Trp residues. At pH 7.2, ultrasound treatment increased the intensity of peak A and subsequent EGCG/CA binding quenched the peak fluorescence to a large extent with a red-shift in the λ_max (from 335 to 340 nm). In comparison, at pH 2.4, sonication slightly decreased the intensity of peak A and phenolic addition further induced an intensity reduction, where the extent of decrement at pH 2.4 was smaller than pH 7.2, suggesting a stronger binding affinity between EGCG/CA and β-LG in the dimeric form. In addition to peak A, peak B (λ_ex = 230 nm, λ_em = 335 nm) reflects the characteristic band of the protein polypeptide backbone (C O) [9], [28]. Under neutral pH, the area of this region became larger after sonication; in the presence of EGCG or CA, the peak was blurred and the region area was decreased, indicating that phenolic addition induced unfolding and loosening of the protein polypeptide chain [8], [9]. However, this peak was invisible at acidic pH, which can be explained by the fact that β-LG is a monomer retaining most of its secondary structure while losing its native tertiary structure at pH 2.4 [31]. These data agreed well with the results of protein intrinsic fluorescence and CD spectra.

Fig. 4.

The 3D fluorescence spectra of β-LG complexed with EGCG or CA with or without ultrasound pre-treatment at pH 7.2 (A) and 2.4 (B). CA, chlorogenic acid; EGCG, epigallocatechin gallate; NLG, native β-LG; ULG-35, β-LG treated with ultrasound at 35% amplitude.

Table 3.

The 3D fluorescence parameters for β-LG in the presence of EGCG or CA and absence and presence of ultrasound recorded at pH 7.2 and 2.4.

pH	Peak	Samples
		NLG		ULG-35		ULG-35 + EGCG		ULG-35 + CA
		Peak position λex/λem(nm/nm)	Intensity F0	Peak position λex/λem(nm/nm)	Intensity F0	Peak position λex/λem(nm/nm)	Intensity F0	Peak position λex/λem(nm/nm)	Intensity F0
7.2	A	230/335	221.8	230/335	230.2	230/335	95.42	230/335	154.7
	B	275/335	414.7	275/335	528.6	275/340	268.4	275/340	367.0
2.4	A	–	–	–	–	–	–	–	–
	B	275/335	541.0	275/335	470.0	275/335	327.1	275/340	340.3

CA, chlorogenic acid; EGCG, epigallocatechin gallate; NLG, native β-LG; ULG-35, β-LG treated with ultrasound at 35% amplitude.

3.3. Changes in protein techno-functional properties

Previous studies have documented that both ultrasound and phenolic complexation are promising approaches in the modification of protein techno-functionalities [27]. Here, to investigate the potential synergistic effect of sonication and phenolic addition on modifying β-LG attributes, protein solubility, foaming, and emulsifying properties were determined and results are given in Fig. 5.

Fig. 5.

Techno-functional properties of β-LG interacted with EGCG or CA with or without pre-sonication at different pH: protein solubility (A), foaming ability (B), foaming stability (C), and emulsifying properties (D). CA, chlorogenic acid; EGCG, epigallocatechin gallate; NLG, native β-LG; ULG-35, β-LG treated with ultrasound at 35% amplitude; EAI, emulsifying activity index; ESI, emulsion stability index. Bars with different letters indicate significant difference (p < 0.05, Duncan’s test).

3.3.1. Protein solubility and foaming property

The protein solubility serves as a key parameter of desired functional attributes. Compared to NLG, ultrasonic irradiation increased the solubility of β-LG by 6.2–6.7% (Fig. 5A). Ultrasound cavitation generates strong shear and mechanical forces to dissociate insoluble aggregates by disrupting the hydrogen bonds and hydrophobic interactions, thus enabling strong protein-water interactions [20]. Under both pH, the presence of EGCG/CA statistically diminished the solubility of NLG and ULG-35 (p < 0.05), which can be explained by a less polar microenvironment and the formation of larger complex particles [5], in line with previous reports in egg white–EGCG [5] and myofibrillar protein–GA samples [22]. With the aid of ultrasound irradiation, compared to NLG–phenolic groups, phenolic addition witnessed a 3.2%–10.1% increment in the solubility of β-LG, except for a 11.6% reduction in ULG-35–CA system at pH 2.4.

The foaming property indicates the ability of protein molecules to absorb on the gas–water interface, providing steric and electrostatic stabilization of the foam bubbles [10]. As seen in Fig. 5B-C, due to the higher flexibility of monomeric β-LG, NLG and ULG-35 showed a greater foaming property at pH 2.4 than pH 7.2. Following ultrasound treatment, ULG-35 had an improved foaming ability but a reduced foaming stability in comparison to NLG, similar to the results in egg white [5]. The presence of EGCG/CA adversely affected the foaming ability of NLG, which can be explained by the reduced protein solubility (Fig. 5A) and lower molecular flexibility of formed complexes. Intriguingly, a notably higher foaming ability was noted in ULG-35–EGCG at pH 7.2, which can be due to the appropriate unfolding of proteins to form highly flexible molecules to be transferred to the air–water interface. For the foaming stability, interactions with EGCG/CA sees a 12.9–25.7% increase in the foaming stability of both NLG and ULG-35 (p < 0.05); despite a reduction after sonication, ULG-35–EGCG/CA showed a comparable foaming stability to the NLG–phenolic groups. Ultrasound treatment was shown to increase the foaming ability but reduce the foaming stability of induced proteins, whereas phenolic addition was proven to have a positive effect on the foaming stability [10], [32]. The pronounced increment in the foaming stability of ULG-35 by phenolics can be related to the higher extent of protein unfolding by co-added phenolics to promote protein expanding at the air–water interface, as witnessed in Table 2. The combined use of ultrasound and phenolic addition (particularly EGCG) can be an efficient tool to improve the foaming property of dairy proteins.

3.3.2. Emulsifying property and partition behavior in the emulsions

The emulsifying property of each treated sample was further analyzed and results are given in Fig. 5D. Considering that NLG–EGCG/CA groups showed a reduced solubility and foaming ability than NLG, only ULG-35–EGCG/CA samples were determined. As shown, similar to the results of foaming property, ultrasound induced a higher EAI and ESI value in ULG-35 than NLG due to an improvement in the interfacial layer. At neutral pH, phenolic addition decreased the EAI and ESI values of ULG-35 by 3.0–10.3% and 3.5–9.3%, respectively; by contrast, at pH 2.4, the addition of CA, but not EGCG significantly reduced the emulsifying attributes of ULG-35 (p < 0.05). The CA-treated group showed a greater EAI and ESI value than the EGCG-induced counterparts at pH 7.4, while an opposite trend was noted at pH 2.4. This pH-dependent variance in the interfacial properties of protein–phenolic aggregates can be related to the protein solubility (Fig. 5A) and pH-induced changes in the adsorption of complexes at the oil–water interface, which warrants investigation in the future study.

To further understand the role of ultrasound and phenolic binding on the interfacial adsorption behaviors of β-LG, the partition of protein in the emulsions was analyzed and results are shown in Table 4. It was demonstrated that emulsions with a higher content of proteins at the oil–water interface can form a more flexible membrane leading to a higher physical stability [12], [23]. ULG-35 showed significantly lower levels of proteins in the aqueous phase and higher values of PC than the respective NLG at both pHs (p < 0.05), supporting our previous inferring that ultrasound cavitation promoted the transfer of proteins to the oil–water interface. Compared to ULG-35, non-covalent interactions with EGCG/CA statistically reduced the interfacial partition of β-LG, which can be explained by the competitive adsorption between β-LG and phenolic compounds. A small amount of ULG-35 originally adsorbed on the interface can be partially replaced by the phenolics or the formed aggregates were of weaker capacity to adsorb to the surface of oil droplets. Similar phenomenon was portrayed in the emulsions formed by α-lactalbumin–EGCG and rice protein hydrolysates–CA complexes [23], [34]. These results indicate that due to the ultrasound acoustic cavitation, β-LG exposes more reactive groups to bind with EGCG and CA, while the modified protein functionality largely depends on the specific protein–phenolic interaction and the structure of proteins in the complexes.

Table 4.

Partition of proteins in the aqueous phase vs. interfacial membrane in oil-in-water emulsions prepared with β-LG containing EGCG or CA with or without ultrasonic pre-treatment at pH 7.2 and 2.4.

pH	Samples	Percentage of protein in the aqueous phase (%)	PC	Fp (%)
7.2	ULG	83.29 ± 0.77ab	0.60 ± 0.03bc	–
	ULG-35	78.68 ± 1.24c	0.81 ± 0.06a	–
	ULG-35 + EGCG	82.33 ± 1.36b	0.64 ± 0.06b	12.18 ± 1.22a
	ULG-35 + CA	85.23 ± 0.69a	0.52 ± 0.03c	7.22 ± 1.00b
2.4	ULG	73.51 ± 0.64b	1.08 ± 0.04c	–
	ULG-35	68.45 ± 1.00d	1.38 ± 0.06a	–
	ULG-35 + EGCG	71.26 ± 0.83c	1.21 ± 0.05b	27.53 ± 0.85a
	ULG-35 + CA	76.25 ± 0.73a	0.93 ± 0.04d	6.08 ± 0.29b

Different letters in the same column indicate significant difference (p < 0.05, Duncan’s test).

PC, partition coefficient of protein; Fp, interfacial phenolic fraction; CA, chlorogenic acid; EGCG, epigallocatechin gallate; NLG, native β-LG; ULG-35, β-LG treated with ultrasound at 35% amplitude.

4. Conclusion

In this study, the potential role of ultrasound pre-treatment combined with EGCG/CA binding on the physiochemical, conformational and interfacial properties of β-LG was investigated. Results showed that moderate ultrasound irradiation (i.e., 35% amplitude) promoted protein structural unfolding and reactive groups exposure to interact with both phenolics without changing the major driven force. In the presence of phenolics, ULG-35 showed a greater tendency in protein conformational changes than NLG, which can be explained by the ultrasound-driven hydrophobic stacking between ingredients. The primary form of β-LG at different pH (dimer/monomer) played a crucial part in the spatial structures of treated proteins, thus having a profound effect on the techno-functional properties of formed complexes. The combined use of ultrasound pre-treatment and EGCG binding showed a positive impact on the foaming and emulsifying properties of β-LG. Future work should focus on the optimal conditions of treatments to extend the applications of ultrasound with phenolic addition in the modification of dairy proteins.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article: