Enhancement of antioxidant and antimicrobial properties of Whey protein isolate and chlorogenic acid complexes through enzymatic and alkaline techniques

Abstract

The interactions of whey protein isolate (WPI) with chlorogenic acid (CQA) using two techniques, alkaline (pH 9) and enzymatic (tyrosinase) were investigated. Complexes, formed between WPI and CQA by alkaline technique (AWPI-CQA) and enzymatic technique (EWPI-CQA), compared to control WPI (CWPI), were characterized in terms of their chemical, structural, emulsifying, antioxidant, and antibacterial properties. Compared to CWPI, both complexation methods significantly reduced free amino groups (CWPI: 588.00 nmol/mg; AWPI-CQA: 409.85 nmol/mg; EWPI-CQA: 412.50 nmol/mg), sulfhydryl groups (CWPI: 68.01 nmol/mg; AWPI-CQA: 18.43 nmol/mg; EWPI-CQA: 48.91 nmol/mg), and tryptophan content (CWPI: 61.21 nmol/mg; AWPI-CQA: 30.12 nmol/mg; EWPI-CQA: 37.64 nmol/mg). Changes in protein structure were examined using internal fluorescence spectra, ultraviolet-visible spectra (UV-Vis) scan, and ultrahigh performance liquid chromatography with electrospray ionization and quadrupole time-of-flight mass spectrometry (UHPLC-ESI-Q-TOF-MS). WPI fluorescence spectra showed that CQA leads to quenching of protein fluorescence. ESI-MS data show that one or more CQA molecules are covalently bound to WPI under both conditions. In addition, AWPI-CQA showed high antioxidative capacity compared to EWPI-CQA and CWPI. On the other hand, EWPI-CQA exhibited notable antimicrobial activity against Staphylococcus aureus LMG 10,147 and MU50 in comparison to AWPI-CQA and CWPI. The development of nutraceutical foods meets the modern consumer needs. Therefore, WPI-CQA complexes can be used as functional components in many food products. Moreover, consumers may benefit from the health-enhancing effects of phenolic compounds.

Introduction

Protein is a vital nutrient essential for the human body playing a key role in both structural composition and energy provision¹. The new technological procedures were used in the dairy industry to exploit and keep bioactive properties of dairy components (Chatterton et al.,². In the past, whey proteins (WP) were often regarded as a byproduct generated during the production of milk and cheese. In recent years, they have been extensively used in the food sector as active ingredients due to their diverse functional and nutritional properties (Jovanović et al.,^3,4). WP constitute approximately 20% of the total protein content in milk and encompass various protein classes with numerous biological effects. β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) are the main classes, which are processed into relatively low-value supplies, such as diverse formulations of whey protein concentrate (WPC) and whey protein isolate (WPI) (Walzem et al.,^2,5,6. Both proteins are a valuable source for essential amino acids, particularly branched-chain amino acids (BCAAs) such as leucine, isoleucine, and valine. WP also contain significant amounts of sulfur-containing amino acids like cysteine and methionine, which are important for antioxidant defense and metabolic functions⁷. The high digestibility and complete amino acid profile of WP make it a superior protein source for supporting muscle synthesis, immune function, and overall health, thereby enhancing its value, and broadening its application prospects in functional foods and nutraceuticals.

Phenolic compounds are considered the largest of secondary metabolites group in plant-based foods. They are characterized by various structures and functions, but generally possessing an aromatic ring bearing one or more hydroxy substituents (Parr and Bolwell⁸). Many phenolic compounds are considered to act as antithrombotic, immunomodulating and even anticarcinogenic. Chlorogenic acid (5-O-caffeoyl-quinic acid; CQA) is an ester formed from caffeic acid and quinic acid. It offers numerous health benefits, including antioxidant, anti-carcinogenic, anti-inflammatory, anti-obesity, cardioprotective, and anti-diabetic effects (Gramazio et al.,⁹). CQA and rosmarinic acid (ROS) incorporate catechol, a compound susceptible to oxidation under alkaline or enzymatic conditions when exposed to oxygen, leading to the formation of quinone. The alkaline method promotes the oxidation of CQA and ROS into reactive quinone structures in the presence of oxygen at elevated pH, which can readily react with nucleophilic amino acid side chains such as –NH₂ and –SH, forming covalent bonds. This method is cost-effective, easy to implement, and commonly used for protein modification due to its high efficiency in generating phenol–protein conjugates. On the other hand, the enzymatic method utilizes tyrosinase, a polyphenol oxidase, to catalyze the hydroxylation and oxidation of phenolic compounds under mild, food-compatible conditions (Bongartz et al.,^10–12).

Phenolic compounds are capable of interacting with proteins through both non-covalent and covalent interactions. Covalent interactions can arise from the oxidation of phenolic compounds, generating radicals or quinones, which can react with proteins as mentioned above. Non-covalent interactions, on the other hand, involve five distinct types of bonds and interactions: electrostatic forces, hydrophobic associations, van der Waals forces, hydrogen bonds, and π- bond interactions. The use of proteins across several applications is closely tied to their chemical, functional, and emulsifying properties (Fu et al.,¹³). Covalent and non-covalent interactions between CQA and proteins such as whey protein isolate, sunflower protein concentrates and soy protein isolate were confirmed through mass spectrometry (UHPLC-ESI-Q-TOF-MS and MALDI-TOF-MS), which directly identified CQA adducts bound to proteins (e.g., increased molecular mass peaks). Concurrently, fluorescence spectroscopy revealed tryptophan quenching and structural changes, indicative of covalent modification. These interactions were also assessed via UV-Vis spectroscopy and fourier transform infrared spectroscopy (FTIR)^14,15, Ali¹⁶, , Ali et al.,^10,17. Protein-phenol interactions offer beneficial effects, such as improvement of protein functional properties and the introduction of beneficial biological activities (Abd El-Maksoud et al.,^18–20,17.

Since the functional performance of whey protein in food systems largely depends on its solubility, improving its solubility is crucial for enhancing its emulsifying capacity and overall applicability in food applications. The chemical binding of phenols can alter several functional properties of proteins, such as solubility, emulsification, foaming capacity, and antioxidant activity, with these modifications being influenced by the interaction forces between proteins and phenolic molecules²¹. The interactions of tannins and sesamol with soy protein isolate and myosin proteins significantly enhanced the protein’s emulsification properties, via hydrophobic interactions and hydrogen bonding^22,23. These interactions are influenced by factors such as the structure and flexibility of the proteins and the structural characteristics of the phenolics. These variations result in distinct interaction mechanisms for different protein-phenolic combinations, which in turn affect the functional properties and structure of proteins, shaping their potential applications in food systems¹⁷. Therefore, in this study, whey protein isolate (WPI) was modified through covalent and non-covalent interactions with chlorogenic acid (CQA) using both alkaline and enzymatic techniques. The objective was not only to characterize the structural and chemical modifications of these complexes, but also to evaluate their antioxidant, antimicrobial, and emulsifying properties. These properties were specifically chosen due to their relevance in the development of multifunctional ingredients for functional food systems. Antioxidant and antimicrobial activities are crucial for improving product stability and safety, while enhanced emulsifying ability broadens the applicability of WPI in emulsified food formulations. By improving these functional attributes, WPI-CQA complexes have the potential to serve as bioactive ingredients in food products with extended shelf life, improved nutritional value, and added health benefits. This approach aligns with the growing consumer demand for natural, health-promoting additives in the food industry.

Materials and methods

Materials

2,4,6-trinitrobenzenesulfonic acid (TNBS), 2,2´-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 8-anilino-1- naphthalenesulfonate (ANS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and chlorogenic acid (CQA) were purchased from Sigma-Aldrich (Seelze, Germany). Mushroom tyrosinase was obtained from Fluka (Steinheim, Germany). The whey protein isolate utilized in this study was sourced from BiPRO (Davisco Foods International, Inc., Eden Prairie, US).

Methods

Preparation of WPI-CQA complexes

WPI-CQA complexes were prepared using two different techniques, alkaline and enzymatic, according to the method of Ali et al.²⁴, with minor changes. For alkaline technique, WPI solutions were prepared using de-ionized water (1 gm: 95 ml) and the pH was adjusted to pH 9. Then 30 mg of CQA, dissolved in 5 ml methanol/water, was added and the pH of mixture was adjusted again to 9. The mixture was stirred at room temperature for 24 h and then dialyzed under the same conditions for an additional 24 h. Finally, the dialysate was freeze-dried to obtain AWPI-CQA complex. The control of WPI (CWPI) was prepared under identical conditions, excluding the addition of CQA. For enzymatic technique, the EWPI – CQA complex and control were prepared with the same method, but the CQA and WPI were dissolved in McIIvaine buffer pH 6.5 and 200 U tyrosinase was added. The enzymatic reaction was terminated by heating the mixture at 85 °C for 10 min to inactivate tyrosinase, followed by dialysis and freeze-drying under the same conditions as above.

Chemical properties of proteins

Covalently and non-covalently bounded CQA

The quantification of covalently and non-covalently bound CQA in WPI was performed following a sequential procedure of Ali and Elsebaie²⁵. After dissolving 4 mg of CWPI and WPI-CQA complexes in 1 mL of 8 M urea, proteins were precipitated using 20% trichloroacetic acid, and the supernatants were discarded. The precipitate was redissolved in 1 mL of 8 M urea. RP-HPLC analysis was conducted on an Agilent 1100 Series HPLC system with a diode-array detector and a PLRP-S column (300 Å, 8 μm, 150 × 4.6 mm, Agilent Technologies, Santa Clara, USA) at 37 °C. Eluents consisted of 0.1% trifluoroacetic acid (v/v) (A) and acetonitrile (B). The gradient program was: 10–18% B over 22 min, 18–80% B over 8 min, 80% B for 3 min, 80 − 10% B over 2 min, followed by 10% B for 7 min, with a total run time of 42 min. A 50 µL injection volume was used at 0.6 mL/min flow rate. Detection wavelengths were 280 nm and 325 nm, with external calibration using CQA standards (10–100 µg/mL).

Free amino and sulfhydryl groups

TNBS and 5,5’-dithio-bis(2-nitrobenzoic acid) reagents were employed to analyze alterations in free amino and thiol groups, following the methodologies outlined by Adler-Nissen^26,27, respectively. The details are mentioned in²⁴.

Structural properties of proteins

Molecular mass by UHPLC-ESI-Q-TOF-MS technique

The UHPLC-ESI-Q-TOF-MS method was employed to examine variations in protein molecular weight, as indicated in²⁰.

Tryptophan fluorescence spectra

A quantity of 1 mg of each protein was solubilized in 1 mL of 8 M urea, and the intrinsic fluorescence emission spectrum was captured utilizing a fluorescence spectrophotometer (Varian Australia PTY Ltd.). The excitation wavelength was established at 290 nm (with a slit width of 2.5 nm), whereas the emission wavelength was monitored between 300 and 700 nm (with a slit width of 5 nm). The assessment of tryptophan content was conducted based on the peak intensity, with adjustments performed by subtracting the intensity measured in an 8 M urea solution. Calibration was performed using tryptophan concentrations ranging from 4 to 20 nM²⁰.

Ultraviolet–visible spectra (UV-Vis) absorption spectroscopy

The diluted CWPI and WPI-CQA protein solutions (0.2 mg/mL) were prepared, and the UV–visible spectrophotometer (Genesys 10 S, Thermo Fisher Scientific, USA) was used to measure the UV spectra in the range from 190 to 700 nm, at scan rate 300 nm/min²⁰.

Surface hydrophobicity

Surface hydrophobicity of CWPI, AWPI-CQA and EWPI-CQA proteins was assessed using the fluorescence probe ANS depending on Hayakawa and Nakai²⁸) method with a minor modification as was described in²⁴.

Particle size and zeta potential

The dimensions of the particles and the zeta potentials of the analyzed proteins were determined utilizing the Zetasizer Nano system (Malvern Instruments Inc., Herrenberg, Germany), and data were processed and reported using Zetasizer Software version 7.13 (https://www.malvernpanalytical.com/en/)¹⁶. Each protein (1 mg) was dissolved in 1 ml of PBS buffer (pH 7) and allowed to stand for at least 1 h. The solutions were transferred to the zeta cells for measurement at 25 °C. All analyses were conducted using 173˚ backscatter detection. The Zetasizer software was utilized to obtain the refractive indices for the protein, recorded at 1.45, and for water, recorded at 1.33, at a temperature of 25 °C.

Isoelectric point (pI)

The pH of studied proteins was adjusted in the range between 3 and 7 and the zeta-potentials of these proteins were determined as mentioned above. Each sample underwent three measurements, and the mean values were subsequently calculated from these results¹⁶.

Antioxidant properties of proteins

The antioxidant capacity of the samples was evaluated utilizing DPPH and TEAC assays in accordance with the methodologies outlined by Binsan et al.^29,30, respectively, with several modifications implemented. The methods were outlined in²⁴. The antioxidant capacities were expressed as Mm TE/100 g protein, using the Trolox for calibration.

Antimicrobial properties of proteins

The broth microdilution method (Cockerill) was used to determine the minimum inhibitory concentration (MIC) of the CWPI and WPI-CQA complexes against Staphylococcus aureus LMG 10,147 and MU50 and Escherichia coli ATCC 8739. The experiments were conducted following the procedure described by Keppler et al.³¹.

Emulsifying properties of proteins

Emulsions Preparation

Oil-in-water emulsions comprised complexes of CWPI and WPI-CQA, with the addition of sunflower oil (10%). These emulsions were formulated by mixing proteins at a concentration of 0.3% with oil at a speed of 500 rpm for a duration of 15 min to produce pre-emulsions. Subsequently, these emulsions were refined using a Bandelin ultrasonic homogenizer, subjected to sonication for 5 min while maintained in an ice bath and operated at an energy input level of 70%³².

Emulsion stability

The stability of freshly prepared emulsions against creaming was evaluated using the method outlined by Khalil et al.³². One mL of each emulsion was placed in a cuvette and centrifuged at 3000 g for 65 min. Absorbance at 500 nm was measured every 5 min.

Particle size and zeta potential of emulsion

The fresh emulsions’ particle sizes and zeta potentials were assessed following the measurement parameters established by Keppler and Schwarz³³. In this study the PBS buffer was used to dilute the emulsions instead of water.

Statistical analysis

All experiments were performed in triplicate (n = 3), and results are presented as mean ± standard deviation (SD). Statistical analyses were carried out using SPSS software (Version 26, IBM Corp., Armonk, NY, USA). One-way analysis of variance (ANOVA) was used to determine significant differences among the groups, followed by Tukey’s post hoc test for pairwise comparisons. The values were considered statistically significant at ≤ 0.05.

Results and discussion

Chemical properties of WPI-CQA complexes

Content of free amino and sulfhydryl groups and Tryptophane

The influence of both covalent and non-covalent interactions between CQA and WPI, under varying conditions, on the alteration of free amino groups (FAG) content was measured and the results are shown in Table (1). The control WPI was found to contain up to 588 nmol/mg of free amine groups. These data are in the line with (Ali¹⁶, . The results showed significant differences between CWPI and the formed WPI-CQA complexes, while no significant differences were found between the AWPI-CQA and EWPI-CQA complexes (Table 1). These findings are in concordance with²⁴. Wang et al.³⁴ found that interaction of β-Lactoglobulin with catechin caused significant decrease in free groups compared to control.

Table 1.

Free amino and sulfhydryl groups, and Tryptophan content of CWPI and WPI-CQA complexes.

Modification conditions	FAG	Sulfhydryl groups	Tryptophan	Total available groups	Total bound groups
CWPI	588.00 ± 21.00a	68.01 ± 2.04a	61.21 ± 1.84a	717.22a	0.0
AWPI-CQA	409.85 ± 6.58b	18.43 ± 0.12c	30.12 ± 1.31c	458.40c	258.82a
EWPI-CQA	412.50 ± 3.75b	48.91 ± 1.56b	37.64 ± 0.08b	499.05b	218.17b

The content of sulfhydryl groups in complexes also showed a significant decrease compared to control: when WPI interacted with CQA under alkaline and enzymatic conditions the sulfhydryl groups content decreased to ⁓ 73% and 28%, respectively. A similar observation was noted for whey protein and myofibrillar proteins when incubated with CQA¹⁵. , Ali¹⁶, , Jie et al.,³⁵. The partial unfolding of the protein structure caused by CQA during its interaction with WPI may have facilitated the conversion of -SH to S-S. Furthermore, CQA possesses the ability to undergo oxidation to form its respective quinone, which may facilitate the transformation of sulfhydryl groups into disulfides, resulting in a subsequent reduction in sulfhydryl levels¹⁵. The notable reduction in sulfhydryl group content in AWPI-CQA compared to EWPI-CQA can be attributed to the deprotonation of free sulfhydryl groups at pH 9, while the enzymatic interaction occurred at a pH of 6.5, which is notably below the pKa value of cysteine. This finding further supports the greater interaction of SH groups with rosmarinic acid (Ros) under alkaline conditions in comparison to the enzymatic conditions published by Ali et al.²⁴.

As also noticed in Table 1, in both complexes, when the proteins interacted with the CQA, the tryptophan content significantly decreased. Therefore, the reactivity of CQA at alkaline conditions was higher than at enzymatic conditions. Similar effects were detected for CQA, epigallocatechin gallate and Ros with β-Lactoglobulin and WPI^36,24,37. A possibility of interactions between various phenolics and amino and thiol groups and tryptophan was reported by^19,34.

Where CWPI is control of WPI; AWPI-CQA are Alkaline WPI and CQA complexes; and EWPI are Enzymatic WPI and CQA complexes. Values presented as the mean ± SD (n = 3). As opposed to different letters in the same row reflecting a significant difference between treatments, the identical letters indicate no significant differences between treatments (p < 0.05).

Finally, the present findings indicate that the CQA-quinone, an oxidized form of phenolic compound formed by alkaline and enzymatic conditions^36,10–12, can interact with nucleophilic amino acid side chains, covalently modifying proteins through NH₂ groups, with an even stronger affinity for SH groups. These findings agree with reports from^16,14,35,38. The high reduction in the content of NH₂ and SH groups and tryptophane in AWPI-CQA compared to EWPI-CQA supports the results of covalently bound CQA content.

Amount of covalently and non-covalently bound CQA

The RP-HPLC method proposed by Ali et al.²⁴ was applied to distinguish between covalently and non-covalently bound CQA. The amount of both covalently and non-covalently associated CQA was calculated as nmol CQA equivalent /mg protein, using free CQA to prepare a standard curve (Fig. 1).

Fig. 1.

Amount of covalently and non-covalently bound CQA, where: CWPI is control of WPI; AWPI-CQA are Alkaline WPI and CQA Complexes; and EWPI are Enzymatic WPI and CQA Complexes. Error bars represent the standard deviation. Values with different letters on the same samples represent statistical differences according to a two-way analysis of variance (p < 0.05).

As a result, the alkaline technique showed a stronger interaction between WPI and CQA. The content of CQA covalently attached to WPI at alkaline conditions was 8.39 nmol CQA / mg protein, while it was 6.03 nmol CQA / mg protein at enzymatic conditions. These findings align with the results of Ali et al.^14,24 who demonstrated that the interactions involving whey proteins, CQA and ROS are more pronounced under alkaline conditions compared to enzymatic conditions. The data presented in Fig. 1 showed that the amount of non-covalently bound CQA was the opposite. The observed results indicating higher CQA attached to WPI at pH 9 compared to binding with PPO align with the decreasing in NH₂ and SH groups and tryptophan contents under alkaline technique compared to enzymatic one (Table 1).

Structural properties of WPI-CQA complexes

Quenching of Tryptophan fluorescence

The quenching of fluorescence intensity can be monitored in relation to the covalent and non-covalent interactions between CQA and WPI. The structure of proteins can be studied using Trp fluorescence spectroscopy, as the photophysical properties of this amino acid are sensitive to the polarity of its environment, such as protein unfolding or interactions (Ghisaidoobe and Chung^39–41, . The fluorescence spectra of CWPI, AWPI-CQA, and EWPI-CQA are shown in Figure (2).

Fig. 2.

The change in tryptophan fluorescence scans of CWPI and WPI-CQA complexes, where: CWPI is control of WPI; AWPI-CQA are Alkaline WPI and CQA Complexes; EWPI are Enzymatic WPI and CQA Complexes.

CWPI exhibited the maximum fluorescence intensity (FI) at 356 nm, suggesting that most of the observed fluorescence is associated with tryptophan residues. In comparison, the AWPI-CQA and EWPI-CQA complexes exposed a minor shift in the maximum FI to 359 nm and 358 nm, respectively. Such a shift may indicate a slight unfolding of the protein structure, resulting in alterations to its conformation and rendering the milieu around tryptophan, the aromatic amino acid, to be relatively more hydrophilic. This occurs as a result of increased exposure to solvents due to interactions with CQA (Ghisaidoobe and Chung^39,34,41). Jiang et al.¹⁵ observed the same results when whey protein and casein incubated with CQA and discussed that the residues of tryptophan and tyrosine were subjected to an environment of greater polarity as a result of both proteins unfolding and the interaction with CQA. The findings presented in Fig. 2 additionally indicated that the tryptophane fluorescence intensity in WPI significantly decreased after the interaction with CQA at both conditions. This observation supports the results of Zhou et al.^34,41, and³⁵ with different proteins and phenolic compounds. Synchronous and three-dimensional fluorescence spectra of soybean 7 S globulin and myofibrillar protein revealed that chlorogenic acid caused quenching of protein fluorescence^35,41.

UV-Vis absorption spectroscopy

UV absorption spectroscopy is an easy method used to analyze changes in the protein structure and identify interactions between substances⁴². The structural alterations in proteins interacted with phenolic compounds, which was observed through UV–Vis spectra. Figure 3 displays the UV spectra of free CQA, CWPI and WPI-CQA complexes. CQA and CWPI showed one peak at 325 and 280 nm, respectively, while WPI-CQA complexes exposed two peaks, 280 and 325 nm. The specific absorption peak at 280 nm is associated with tyrosine and tryptophan residues^10,17. The absorption peaks observed at 325 nm in WPI-CQA complexes indicate an interaction between WPI and CQA, leading to the formation of a new complex. This result is consistent with the findings of Karefyllakis et al.⁴³, Ali¹⁶, Ma et al.,^10,17 who had the same observation after treating whey and sunflower proteins with CQA. Therefore, the UV spectra and fluorescence quenching data verified the formation of WPI-CQA complexes, causing static quenching³⁵.

Fig. 3.

Ultraviolet–visible spectra of CWPI and WPI-CQA complexes. The peak at 280 nm corresponds to the absorbance of aromatic amino acid residues (primarily tryptophan and tyrosine) present in whey protein isolate, while the peak at 325 nm is the characteristic absorption peak of chlorogenic acid (CQA), indicating the successful interaction and formation of the WPI-CQA complex.

UHPLC-ESI-QTOF-MS results

The change in molecular weight (MW) of WPI after treating with CQA at pH 9 and tyrosinase were investigated by the UHPLC-ESI-QTOF-MS apparatus (Fig. 4). The mass spectra obtained from ESI-TOF analysis for each of the proteins, CWPI, AWPI-CQA, and EWPI-CQA, showed two major Beta-Lactoglobulin (β-LG) protein variants (A and B). By analyzing the signal intensities, the signals associated with the charge state of + 17 at m/z 1076.11 (variant B) and at m/z 1081.17 (variant A) were accurately identified. Deconvolution of these signals indicated an average protein mass of 18293.90 Da for variant B and 18379.89 Da for variant A.

Fig. 4.

ESI-MS of CWPI and WPI-CQA complexes, where: CWPI is control of WPI; AWPI-CQA are Alkaline WPI and CQA Complexes; EWPI are Enzymatic WPI and CQA Complexes.

These results align with the previously reported values (Wilde et al.,^44,45). After the incubation at both conditions under study, new peaks were detected, which could account for adding one CQA molecule (⁓ 353 Da) to A and B variants (Fig. 4). The results can be clarified by the oxidation of CQA, leading to the creation of a reactive quinone that forms a covalent bond with the protein. These findings are consistent with the findings in Ali and Elsharkawy⁴⁴ and (Ali¹⁶, . In addition, it was also shown that the molecular weight of proteins increases following their covalent cross-linking with polyphenols⁴⁶. Figures (4) demonstrates that the intensity of two new peaks in AWPI-CQA is higher than EWPI-CQA: this means that the amount of covalently bound CQA to WPI at pH 9 was higher than the enzymatic conditions. These findings support the results for covalently bound CQA.

Surface hydrophobicity

The surface activity of proteins is crucial in various functional applications, such as foaming and emulsification⁴⁷. Hydrophobicity of proteins is influenced by the quantity and configuration of polar functional groups. ANS fluorophore, which noncovalently binds to nonpolar regions of proteins⁴¹, was used to investigate the influence of CQA binding on WPI’s surface hydrophobicity. The results are shown in Table (2). The CWPI and AWPI-CQA complex showed no significant values with 1125.88 and 1121.40, respectively. However, EWPI-CQA hydrophobicity decreased significantly by approximately 27% compared to CWPI, indicating that the WPI surface became more hydrophilic. The reduction in protein hydrophobicity after incubations with phenolic compounds was previously recorded^24,48,47,41. This decrease could be explained by the interaction of FAG of amino acids, such as lysine, with CQA that caused the loss of positive charges, and therefore decreased the amount of potential binding sites for the ANS anion. Moreover, modifications in the protein structure result in variations in the spatial distribution of hydrophilic and hydrophobic areas both internally and on the WPI surface (Zhou et al.,⁴¹.

Table 2.

Hydrophobicity, particle size, and zeta potential of CWPI and WPI-CQA complexes.

Proteins	Hydrophobicity	Particle size (nm)	Zeta potential (mV)
CWPI	1125.88 ± 41.41a	7.20 ± 0.20a	-11.21 ± 0.58ab
AWPI-CQA	1121.40 ± 25.79a	7.17 ± 0.98a	-9.36 ± 0.59b
EWPI-CQA	821.61 ± 11.29b	6.71 ± 0.87a	-12.63 ± 1.47a

Values presented as the mean ± SD (n = 3). As opposed to different letters in the same column reflecting a significant difference between treatments, the identical letters indicate no significant differences between treatments (p < 0.05).

Particle size and zeta potentials

CWPI complexes and particle size (PS) were assessed (Table 2). The average diameter of the CWPI principal fraction was determined to be 7.20 nm. The average particle diameter of WPI was 6.82 nm, as published in¹⁶. After 24 h interaction with CQA at alkaline and enzymatic conditions, no significant differences were recorded. Ali et al.²⁴ reported this observation when WPI interacted with Ros at alkaline conditions. The alterations in WPI charges after interacting with CQA at both conditions was studied by measuring zeta potentials, and the data are presented in Table 2. The zeta potential measurements for all proteins displayed negative values due to their dissolution in PBS buffer at pH 7. Abd El-Maksoud et al.¹⁸ showed that the values were also negative. No significant differences were found between CWPI and EWPI-CQA, although significant differences were found between AWPI-CQA and EWPI-CQA complexes. Ali and Arafa¹⁶ found that WPI zeta potential increased from − 10.97 to -11.13 following the incubation with CQA at pH 9.

Isoelectric point (IEP)

The IEP is closely linked to protein solubility. The zeta potential (ZP) reflects the surface charge of proteins, and, as this charge diminishes near the IEP, proteins lose their solubility and precipitate. The change in IEP (ZP is ~ 0 mV) of proteins was analyzed across measurement of ZP values at different pH levels and the results are shown in Figure (5). The ZP values for CWPI, AWPI-CQA, and EWPI-CQA proteins were positive at pH levels between 3 and 4.5, while they were negative at pH levels ranging from 5 to 7. Figure 3 reveals that the IEP of CWPI and the AWPI-CQA complex are approximately pH 4.87 and 4.88, respectively, whereas the IEP of the EWPI-CQA complex decreased to about pH 4.72, indicating a shift to a more acidic pH. These findings align with Keppler et al.³¹) and Ali and Arafa¹⁶), who observed a decrease in the IEP of WPI following modifications with allyl isothiocyanate and Ros, respectively. A decrease in the IEP of egg white protein after interaction with tea polyphenols may be due to the incorporation of phenolic hydroxyl groups of these compounds⁴⁷).

Fig. 5.

Zeta potentials of CWPI and WPI-CQA complexes at different pH values. Error bars represent the standard deviation.

Additionally, Ali and Arafa¹⁶) explained that the alteration in IEP is attributable to the depletion of charged entities, which occurs due to the interaction of CQA and Ros with available NH₂ groups, ultimately leading to a decrease in the total charge of the protein.

Antioxidant properties

CQA is a natural substance known for its potent antioxidant properties, and its combination with WPI could potentially impart its antioxidant ability through reactive phenolic hydroxyl groups. The antioxidant activities of CWPI, AWPI-CQA, and EWPI-CQA proteins were measured using DPPH and TEAC assays. The results are shown in Table (3). The application of techniques results in a substantial enhancement of WPI’s antioxidant activity. The DPPH results showed that WPI treated with CQA at alkaline conditions recorded antioxidant activity higher than the tyrosinase catalyzed by WPI-CQA complex and control WPI. The TEAC assay exhibited a trend comparable to the results of the DPPH assay (Table 3). These findings are in the line with the literature (Aewsiri et al.,^{18,49,14,17,50}, where it was reported that antioxidative activity of various proteins was enhanced through the covalent bonding with diverse phenolic compounds in comparison to their unmodified proteins. A possible interpretation for the antioxidative properties of the complexes formed between WPI and CQA under both conditions is that the interactions between the proteins and phenolic compounds result in the formation of a protein-phenol conjugate. This conjugate retains the potential for antioxidative activity due to its ability to generate free radicals. Furthermore, the CQA hydroxyl group plays a significant role in enhancing antioxidant activity and interacts with proteins to impart the improved antioxidant properties.

Table 3.

Antioxidant and antibacterial activities of WPI-CQA complexes.

Proteins	Antioxidant assays (mM/100 g)		Staph. aureus		E. coli
	DPPH	TEAC	LMG 10,147	Mu50	ATCC 8739
CWPI	4.40 ± 0.22c	4.52 ± 0.08c	> 4	> 4	> 4
AWPI-CQA	13.30 ± 0.83a	12.68 ± 0.20a	> 4	> 4	> 4
EWPI-CQA	7.36 ± 0.20b	7.33 ± 0.08b	4	4	> 4

Antibacterial activity

The antimicrobial activates of CWPI and WPI-CQA complexes formed using two techniques, enzymatic and alkaline, were measured using the minimum inhibitory concentration (MIC) assays. The tested strains included Staphylococcus aureus LMG 10,147 and MU50, as well as Escherichia coli ATCC 8739. S. aureus LMG 10,147 is a standard reference strain used in food microbiology studies. E. coli ATCC 8739, a well-characterized Gram-negative bacterium, serves as a model organism in antimicrobial evaluations. These strains can be used to illustrate the potential of WPI-CQA complexes as bioactive components in functional foods with antimicrobial effects. The MIC assay results are shown in Table 3. WPI-CQA complex formed by enzymatic technique showed the antimicrobial effect against gram-positive bacteria (Staphylococcus aureus), compared to WPI-CQA complex formed by alkaline technique and control WPI. These findings are comparable to those mentioned by Ali et al.²⁴. The effect of enzymatic technique may be explained by active phenoloxidase that generates quinones, that polymerize to form melanin. These reactive intermediates produced in phenoloxidase-catalyzed reactions showed antimicrobial effect on gram-positive bacteria (Bacillus cereus, B. subtilis, Micrococcus luteus, and Staphylococcus aureus)⁵¹. Moreover, in the presence of phenoloxidase, tyrosine converts to L-3,4-dihydroxyphenylalanin (L-DOPA), which also has an antimicrobial effect.

Where: >4 MIC exceeds the highest concentration tested; and 4 means 4% protein inhibits bacteria growth. Values presented as the mean ± SD (n = 3). As opposed to different letters in the same column reflecting a significant difference between treatments, the identical letters indicate no significant differences between treatments (p < 0.05).

Emulsifying properties

Particle size and zeta potential of emulsions

CWPI and WPI-CQA complexes were utilized as emulsifiers to prepare oil-in-water emulsions and PS and ZP were measured immediately after emulsification and compared with CWPI (Fig. 6A and B). Concerning particle PS, significant differences (at p ≤ 0.05) were observed among all proteins, while for ZP no significant differences were found. complex showed a higher PS of emulsion produced. This may be related to more extensive interactions of AWPI with CQA and the formation of conjugates with CQA compared to EWPI-CQA complex. Similarly, as observed by Ali and Arafa¹⁶, the incubation of WPI with CQA at alkaline conditions caused an increase in PS of oil in water emulsion.

Fig. 6.

Particle size and zeta potential of emulsions prepared with CWPI and WPI-CQA complexes. Error bars represent the standard deviation. Values with different letters on the same samples represent statistical differences according to a two-way analysis of variance (p < 0.05).

Stability of emulsions against creaming

Emulsion stability is a key factor in the food emulsion sector, indicating the capacity of the emulsion to withstand droplet creaming, coalescence, and flocculation. The emulsions, freshly formulated with CWPI, AWPI-CQA, and EWPI-CQA complexes, underwent centrifugation at 3000 rpm for a duration of 70 min to achieve the separation of the serum layer. Subsequently, the creaming stability of these emulsions was evaluated by quantifying the turbidity at 500 nm, with the findings presented in Figure (7). The results revealed that all emulsions were stable against creaming till about 30 min, then they sightly lost stability. Notably, the stability of emulsions emulsified with WPI-CQA complexes was lower than CWPI. Moreover, significant differences were detected in the emulsifying properties of CWP, AWPI-CQA, and EWPI-CQA after 50 min centrifugation, where the emulsion formulated with EWPI-CQA exhibited the least stability among the samples tested. EWPI-CQA complex showed significantly reduced surface hydrophobicity, likely due to the masking of hydrophobic sites by phenolic groups, resulting in weaker interaction at the oil–water interface. The complexation also exhibited increased molecular crosslinking and possibly more aggregation, leading to larger droplet sizes and reduced emulsion stability. All these observations could explain the lower emulsion stability observed with EWPI-CQA complex.

Fig. 7.

Stability of emulsions prepared with CWPI and WPI-CQA complexes against creaming. Error bars represent the standard deviation.

Conclusion

This study highlights the potential of interaction of whey protein isolate (WPI) with chlorogenic acid (CQA) to improve its functional, antioxidant, and antibacterial properties, offering significant advancements for food and nutraceutical applications. Through alkaline and enzymatic techniques, the covalent and non-covalent interactions between WPI and CQA resulted in substantial chemical and structural changes, including reductions in FAG, thiol content, and tryptophan levels, with more pronounced interactions under alkaline conditions. Structural analysis via fluorescence quenching and UV-Vis spectroscopy confirmed the successful formation of WPI-CQA complexes and revealed protein conformational changes. These interactions enhanced WPI’s antioxidative capacity, with the AWPI-CQA complex showing superior performance, while EWPI-CQA demonstrated better antibacterial effects, particularly against Staphylococcus aureus. The emulsifying properties of WPI were also altered, with changes in PS, zeta potential, and stability, reflecting the influence of protein-phenol interactions. These results demonstrate the versatility of phenolic compounds like CQA in enhancing protein-based food systems, offering a pathway to develop innovative functional ingredients tailored to meet consumer demands for health-promoting and bioactive food products. While this study demonstrates the potential of both alkaline and enzymatic techniques for preparing WPI–CQA complexes with enhanced functional and bioactive properties, future research should explore a broader range of CQA concentrations to optimize the modification process and further elucidate the relationship between CQA amount and protein functionality. Additionally, the study was performed at laboratory scale, and further scale-up studies and cost-benefit analyses are recommended to support the industrial adoption of these technologies.

Author contributions

Mostafa Ali and Mohamed Yousef: Writing – review & editing, Validation, Resources, Supervision, Investigation, Conceptualization, Data curation. Mahmoud Khalil and Amira Rizk: Supervision, Investigation, Conceptualization. Yemane H Gebremeskal : Methodology, Investigation, Conceptualization, Writing – review & editing. Fawaz Alzahrani: Investigation, Conceptualization, Data curation. Atef Fathy Ahmed and Aml Abubakr Tantawy: Methodology, Investigation, Conceptualization. Mohamed Said Boulkrane and Denis A Baranenko: Writing – review & editing, Investigation, Conceptualization. Tamer M. El-Messery and Samar Aly: Writing – review & editing, Investigation, Conceptualization.

Funding

The work was funded by the Deanship of Graduate Studies and Scientific Research, Taif University, Kingdom of Saudi Arabia.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.