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. 2025 May 15;28:102563. doi: 10.1016/j.fochx.2025.102563

Green development of whey protein-based complex system for the intestinal delivery of curcumin: Focus on formation mechanism, stability, and in vitro digestive characteristics

Qingguan Liu a,⁎, Wanqing Huang c, Chunxia Zhou a, Sichen Yang a, Jiaqi Hong a, Ailin Xie a, Qiang Cui b,⁎
PMCID: PMC12152569  PMID: 40502415

Abstract

This study delves into the feasibility of developing an intestinal delivery system for curcumin using whey protein as the wall material, employing a simple and eco-friendly (without using organic solvents) pH-induced protein-polyphenol co-assembly strategy. The results indicate that stable complex formed under curcumin concentrations ranging from 0.1 to 0.4 g/L (screened by the preliminary experiment) with particle size between 269 and 287 nm, zeta potential around −38 mV, and curcumin loading capacity ranging from 3.61 to 9.14 mg/g. Curcumin primarily interacts with whey protein through hydrophobic interactions and hydrogen bonds, as confirmed by FT-IR spectroscopy and molecular docking analysis. Curcumin has a different affinity to proteins ranked as bovine serum albumin (BSA) > β-lactoglobulin (β-LG) > α-lactalbumin (α-LA). Curcumin exists in an amorphous state within the protein, which also increases the surface hydrophobicity of the protein (from 565 to 687 AU) and leads to quenching intrinsic fluorescence of the protein. The binding of curcumin causes a transition from random coil to β-sheet in the protein's secondary structure but has a limited impact on its tertiary structure. Stability analysis shows that the complex remains stable in environments with 100–500 mM NaCl and heat treatment at 60–90 °C. In vitro digestion results indicate that the complex aggregates during simulated gastric fluid digestion and decompose during simulated intestinal fluid digestion, effectively enhancing the bioaccessibility of free curcumin by 60 % (P < 0.05). This study provides a theoretical basis for the green processing of protein-based oral delivery systems for lipophilic compounds.

Keywords: Curcumin, Whey protein, Complex, Stability, Bioaccessibility

Graphical abstract

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Highlights

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    Green preparation of curcumin-loaded whey protein complex by pH-induced protein-polyphenol co-assembly.

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    Hydrophobic interactions and hydrogen bonds were involved in complex formation.

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    The affinity of the proteins for curcumin is ranked as BSA > β-LG > α-LA.

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    Curcumin modified the protein's secondary structure but had limited impact on the tertiary structure.

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    Complex can deliver curcumin to the intestinal with improved bioaccessibility.

1. Introduction

Polyphenolic natural products are important functional components in food, possessing various health-promoting physiological and pharmacological activities to animals and humans (Choudhary & Tahir, 2023; Khalifa et al., 2023), which have attracted widespread attention from researchers in fields such as phytochemistry and food science. Curcumin (Cur) is a typical hydrophobic polyphenolic compound containing a large number of hydrophobic groups, conjugated double-bond systems and intramolecular hydrogen bonds, widely found in the rhizomes of plants from the ginger family or the arum family (Stanton & Grabrucker, 2023). Curcumin exhibits an appealing bright yellow color, which changes with variations in pH, and is commonly used as a natural dye (Suwannateep et al., 2011) in the chemical industry, as well as an acid-base indicator (Mali & Pandey, 2024). Importantly, research in modern medicine and pharmacology has confirmed that curcumin possesses multiple physiological and pharmacological activities beneficial to human wellness, including anti-inflammatory, antibacterial, antioxidant, and tumor-suppressing effects (Liu et al., 2022; Yang et al., 2020). These activities highlight the significant research and application value of curcumin in functional foods and pharmaceuticals. However, curcumin is a lipophilic compound with extremely low water solubility (Chen et al., 2014), and its stability is easily affected by light (cleavage of weak bonds), heat (breaking of chemical bonds), alkaline environments (formation of phenoxide anions), and gastrointestinal digestion (Liu, Loo, Zhang, & Ng, 2024), leading to reduced bioavailability in the body. This severely restricts the application of curcumin in the food and pharmaceutical industries.

Research in the field of micro-nano technology indicates that using appropriate carriers to load lipophilic compounds is an effective method to enhance their water solubility, stability, absorption, and bioavailability (Sabet, Rashidinejad, Melton, & McGillivray, 2021). These carriers can be composed of one or more substances such as proteins, polysaccharides, and lipids. Among them, protein-based carriers are widely used for loading lipophilic polyphenols like curcumin due to their high nutritional value, easy availability, multifunctionality, biocompatibility, and biodegradability. Effective delivery systems for curcumin have been successfully developed using whey protein (Ye et al., 2021), casein (Liu et al., 2023), egg white protein (Wang, Zhang, Wang, Xu, & Zhou, 2020), zein (Zhang et al., 2024), rice bran protein (Wang et al., 2024), soy protein (Li et al., 2022), and pea protein (Ren et al., 2024) to address the challenges of curcumin's industrial application.

Whey protein (WP) was one of the first proteins studied for the loading of lipophilic compounds due to its advantages over other proteins, including high nutritional value, ease of separation and preservation, excellent processing characteristics, and wide application scenarios. Whey protein is a byproduct of cheese production, primarily composed of α-lactalbumin, β-lactoglobulin, bovine serum albumin, and immunoglobulins (Sabet et al., 2021). It possesses many outstanding functional properties and is widely used in food processing as a thickener, gelling agent, stabilizer, emulsifier, and microencapsulation wall material (Sabet et al., 2021). Numerous studies have reported on the use of whey protein to load curcumin. Emulsification processes can achieve the loading of curcumin in heat-treated whey protein microgels, enhancing curcumin's antioxidant activity and enabling its controlled release in vitro (Mohammadian, Salami, Momen, Alavi, & Emam-Djomeh, 2019). Ethanol can facilitate the dissolution of curcumin, and ultrasonic homogenization can produce whey protein‑iron-curcumin ternary composite particles, where curcumin effectively inhibits the oxidation of iron ions (Ding, Chen, Zheng, & Xi, 2024). Emulsification-evaporation combined with ultrasonic homogenization can create glycosylated whey protein particles loaded with curcumin, significantly improving its bioavailability (Li, Jiang, & Shi, 2024). It should be noted that bio-availability refers to the quantity and rate at which active molecules enter cells and the body. However, bio-accessibility refers to the degree to which lipophilic bioactive molecules are micellized in the intestine after in vitro digestion. However, current research on whey protein-loaded curcumin still faces many challenges before industrialization. The use of organic solvents to promote curcumin dissolution is not environmentally friendly, and the subsequent removal of organic solvents can lead to high costs (Liu et al., 2024, Luo, Xie, Li, Zhou and Liu, 2024). Complex preparation processes introduce considerable uncertainty regarding product stability, and techniques like ultrasonic homogenization cannot achieve large-scale production (Liu, Liu, & Ban, 2025). Therefore, there is a need to develop simple, efficient, and environmentally friendly processing methods for preparing whey protein-based curcumin encapsulation systems and to achieve scalable production of these products.

Co-assembly refers to the process in which two or more kinds of molecules form complexes through intermolecular interactions. The pH-induced protein-polyphenol co-assembly is a recently popular strategy for developing protein-based polyphenol loading systems. It primarily utilizes the pH-dependent dissociation and reassembly effects of proteins and polyphenols to achieve the loading of polyphenols by proteins. Using this method, systems for loading curcumin have been developed with pea protein (Ren et al., 2024), soy protein hydrolysates (Yuan et al., 2021), and ovalbumin (Li et al., 2024). However, there have been no reports on the preparation of whey protein-based curcumin loading systems using this method, nor on the in-depth study of their loading characteristics, formation mechanisms, stability, and digestion properties. Therefore, the aim of this study is to prepare curcumin-loaded whey protein complex using the pH-induced protein-polyphenol co-assembly process and to investigate the loading characteristics, formation mechanisms, stability, and in vitro digestion properties. This research will provide ideas and references for the green preparation of functional milk-based materials, and promote the application of whey protein-based curcumin loading systems in the field of functional foods.

2. Materials and methods

2.1. Materials

Whey protein (purity >80 %) and curcumin (purity of 95 %) were purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). Pepsin (3000 units/mg), trypsin (200 units/mg), pig bile salts, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were obtained from Sigma-Aldrich ((Sa. Louis, USA). All other chemicals and reagents were of analytical grade.

2.2. Preparation of curcumin-whey protein complex

The preparation method was modified based on the study by Liu et al. (2022). Six grams of whey protein powder were added to 300 mL of deionized water and stirred with a magnetic stirrer (MS7, IKA, Germany) to dissolve. The mixture was hydrated overnight at 4 °C, and the pH was adjusted to 7 using 1 mol/L NaOH solution. For each 50 mL of protein solution, 5 mg, 6.67 mg, 10 mg, and 20 mg of curcumin were added, resulting in curcumin concentrations of 0.1, 0.13, 0.2, and 0.4 g/L in the four sample groups, respectively. The pH was then adjusted to 12 using 1 mol/L NaOH solution, and the mixture was stirred continuously (800 rpm) for 30 min to ensure complete dissolution. Finally, the pH was adjusted back to 7 using 1 mol/L HCl solution to complete the sample preparation. The prepared samples were either used directly for analysis or freeze-dried into a powder for later use. It should be noted that when the pH value is 12, the short-term treatment mainly promotes the dissolution of curcumin and has a limited effect on the decomposition of curcumin. At the same time, the subunits of whey protein will dissociate to form proteins in the molten globule state, which will lead to a certain degree of protein denaturation. In fact, the commercial whey protein we purchase is prepared by spray drying and is also a partially denatured protein.

2.3. Measurement of particle size and zeta potential

The measurement of particle size and zeta potential of the complex was modified according to the method described by Liu et al. (2024). The samples were diluted 20 times with deionized water to avoid multiple scattering effects during instrument analysis. The average particle size, polydispersity index (PDI), and zeta potential were measured using a laser particle size analyzer (NANO ZS90, Malvern Instruments, UK).

2.4. Measurement of encapsulation efficiency (EE) and loading capacity (LC)

The EE and LC of curcumin in the complex were determined based on the method described by Liu, Jing, Han, Zhang, and Tian (2019). Freshly prepared samples were centrifuged at 8000 g for 5 min. The supernatant was appropriately diluted with anhydrous ethanol to extract curcumin (has been confirmed a feasible method to extract curcumin from particles by the above reference), and the absorbance of the samples was measured at 428 nm using a UV spectrophotometer (T9, PUXI, China). The curcumin content was calculated based on the standard curve y = 0.1135× + 0.0422 (R2 = 0.9995). The EE and LC were calculated using the following formulas:

EE%=Encapsulated content ofCurAdded content ofCur×100 (1)
LCmg/g=Encapsulated mass ofCurWhey protein mass (2)

among them, the Encapsulated content of Cur represents the concentration of curcumin detected in the supernatant, the added content of Cur represents the concentration of curcumin added to the sample, the Encapsulated mass of Cur represents the product of the EE and the added mass of curcumin, the Whey protein mass represents the mass of whey protein added to the sample.

2.5. Measurement of surface hydrophobicity

The surface hydrophobicity of the samples was determined using the method described by Xie et al. (2024). The samples were diluted with deionized water to 0.5 mg/mL (protein concentration). Four milliliters of the diluted sample were mixed with 20 μL of ANS (8.0 mmol/L, pH 7.0) and scanned using a fluorescence spectrophotometer (F-7000, Hitachi, Japan) to obtain the fluorescence spectrum. The excitation wavelength was set at 370 nm, with the emission wavelength ranging from 400 to 700 nm, and the slit width was set to 5 nm.

2.6. Measurement of fluorescence spectroscopy

The fluorescence spectrum of the samples was measured with slight modifications based on the method described by Li, Hong, et al. (2024). The samples were diluted with deionized water to 0.2 mg/mL (protein concentration). A fluorescence spectrophotometer (F-7000, Hitachi, Japan) was used to scan the samples at an excitation wavelength of 280 nm to obtain the intrinsic fluorescence spectrum of the protein. The emission wavelength was set between 285 and 450 nm (results also prove that the effective wavelength range is 290–450 nm), with both the excitation and emission slits set to 2.5 nm.

2.7. Determination of circular dichroism (CD) spectroscopy

The samples were diluted with deionized water to a protein concentration of 0.2 mg/mL. CD spectrometer (J-810, Jasco, Japan) was used to record the spectra of the samples in the far ultraviolet region (190–260 nm) at a detection temperature of 25 °C, with a cuvette path length of 1.0 mm. Finally, the secondary structure content of the protein was calculated using the CD website (http://dichroweb.cryst.bbk.ac.uk) based on the CONTIN algorithm.

2.8. Determination of fourier transform infrared (FT-IR) spectroscopy

Four groups of samples were prepared for infrared spectroscopy testing: curcumin, whey protein, curcumin-whey protein mixture, and curcumin-whey protein complex (0.2 g/L). The samples were freeze-dried into a powder and mixed with potassium bromide (KBr) at a mass ratio of 1:100, then ground and pressed into transparent pellets. The infrared spectra of the samples were measured using an infrared spectrometer (NICOLET iS10, Thermo Fisher, USA) by scanning from 500 to 4000 cm−1 for 64 scans, with a resolution set to 4 cm−1. Perform baseline correction through polynomial fitting, and conduct normalization processing on the spectrum using maximum value normalization.

2.9. Determination of differential scanning calorimetry (DSC)

The thermal properties of the samples were determined using a thermal analyzer (DSC 3, METTLER TOLEDO, Switzerland), based on the method described by Liu, Tan, et al. (2024). Five milligrams of each sample—curcumin, whey protein, curcumin-whey protein mixture, and curcumin-whey protein complex (0.2 g/L)—were placed in crimped aluminum pans. The aluminum pans were then heated from 25 to 260 °C at a rate of 5 °C/min under a nitrogen purge at 50 mL/min.

2.10. Molecular docking analysis

Molecular docking was employed to simulate the 3D structures of the macromolecule (WP) and the receptor (Cur). Crystal structures of α-LA (PDB ID: 1F6R), β-LG (PDB ID: 3NPO), and BSA (PDB ID: 4F5S) were retrieved from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (http://www.rcsb.org). The 3D structure of Cur was sourced from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and was pretreated using PyMOL software (Version 2.3.0, DeLano Scientific LLC, USA) and AutoDockTools (v1.5.6). The molecular docking analyses between proteins and Cur were performed using the AutoDock Vina 1.1.2 docking program after energy minimization treatment. Discovery Studio 2019 was utilized to analyze the interaction mode of the docking results.

2.11. Scanning electron microscopy (SEM) observation

Powder samples (curcumin, whey protein, curcumin-whey protein complex, SGF-digested complex, and SIF-digested complex) were cast onto a metal substrate and sprayed with a layer of 10 nm gold nanoparticles. Scanning electron microscope images of the samples were then taken under vacuum conditions using a scanning electron microscope (Quanta 200, FEI, Netherlands) at an accelerating voltage of 10 kV with a magnification of 1500 times. The concentration of curcumin in the complex was 0.2 g/L.

2.12. Stability testing of the complex

2.12.1. Ionic stability

Samples of curcumin at a concentration of 0.2 g/L were divided into six groups, to which different amounts of NaCl were added to prepare NaCl concentrations of 0, 100, 200, 300, 400, and 500 mM. After stirring and dissolving, the average particle size, polydispersity index (PDI), and zeta potential of the samples were measured using a laser particle size analyzer. No obvious precipitation or phase separation was observed.

2.12.2. Thermal stability

Samples of curcumin at a concentration of 0.2 g/L were divided into five groups and subjected to heating at temperatures of 0, 60, 70, 80, and 90 °C. After 30 min, the samples were cooled to room temperature, and the average particle size, polydispersity index (PDI), and zeta potential were measured using a laser particle size analyzer.

2.13. Digestion characteristics testing

2.13.1. Digestive stability

Based on the method of Yan et al. (2019) with minor modifications. Briefly, 5 mL of the sample (curcumin concentration of 0.2 g/L) was mixed with 45 mL of HCl solution (0.1 mM) and shaken for 10 min at 37 °C and 100 rpm. Subsequently, 20 mg of pepsin was added, and the pH was adjusted to 2.0 using 1 M HCl for simulated gastric fluid (SGF) digestion. After 60 min, 250 mg of bile salts were added, the pH was adjusted to 7.0 using 1 M NaOH, and the mixture was shaken for 10 min at 37 °C and 100 rpm. Then, 20 mg of trypsin was added for simulated intestinal fluid (SIF) digestion, and the mixture was shaken for 2 h at 37 °C and 100 rpm. Samples were taken at each digestion stage to determine the particle size and zeta potential referring to the method in section 2.3. The activity of the enzyme used has been verified before use.

2.13.2. Bioaccessibility measurement

Curcumin-water solution, curcumin-whey protein mixture, and curcumin concentration of 0.2 g/L complex samples (with equal mass of curcumin in all three samples) were digested according to the method in section 2.13.1. At the end of the SIF digestion, 2 mL of the sample solution was centrifuged (8000 rpm, 10 min) in a centrifuge tube to obtain the mixed micellar phase. The supernatant was collected, and absorbance was measured at 428 nm using a UV spectrophotometer to calculate the concentration of curcumin. The curcumin bioaccessibility was calculated according to the following equation (Liu, Wang, McClements, & Zou, 2018):

Bioaccessibility%=C1C2×100 (3)

where C1 is the concentration of curcumin in the micelle sample and C2 is the concentration of curcumin in the total digestate sample.

2.14. Statistical analysis

Data were obtained from three replicate experiments, and the average and standard deviation were calculated. The data were analyzed using one-way ANOVA with the statistical software package SPSS 25.0, and the level of significance between variables was set at P < 0.05.

3. Results and discussion

3.1. Particle size and zeta potential analysis

The effect of curcumin concentration on the particle size and zeta potential of the complex is shown in Fig. 1. As the concentration of curcumin increases, the particle size exhibits an increasing trend (from 269 nm to 285 nm) (Fig. 1A), and the PDI does not exceed 0.3 (Fig. 1B). Meanwhile, the absolute value of the zeta potential in the samples ranges between 36 and 39 mV (Fig. 1C). These results indicate that the complexes prepared with curcumin concentrations not exceeding 0.4 g/L have a smaller particle size, a uniform size distribution, and a high surface charge, allowing for strong electrostatic repulsion that maintains the stability of the complex. It should be noted that this electrostatic repulsive force is the interaction force between different complexes, rather than the main force for the binding between curcumin and the protein. Liu, Sun, Cheng, and Guo (2022) found that an appropriate amount of soy isoflavone added (with a mass ratio of soy isoflavone to protein not exceeding 1:40) does not induce a decrease in the stability of the complex particles, which is consistent with our results.

Fig. 1.

Fig. 1

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Particle size (A), PDI (B), and zeta potential (C) of curcumin-whey protein complex with different curcumin concentrations. Different letters indicate significant differences (P < 0.05).

3.2. EE and LC analysis

The EE is a key indicator for evaluating the effectiveness of delivery carriers for bioactive compounds. The effect of curcumin concentration on EE is shown in Fig. 2A. As the concentration of curcumin increases from 0.1 to 0.4 g/L, the EE significantly decreases from 72.2 % to 45.7 % (P < 0.05). This reduction in encapsulation efficiency indicates that as the curcumin concentration increases, the binding sites on the protein for curcumin become saturated, resulting in excess curcumin that cannot bind to the protein and remains in a free crystalline state. The trend in EE aligns with our previous data on the encapsulation of curcumin using zein-based nanoparticles (Liu et al., 2019). The effect of curcumin concentration on LC is shown in Fig. 2B. As the concentration of curcumin increases from 0.1 to 0.4 g/L, the LC significantly increased from 3.61 to 9.14 mg/g (P < 0.05). This indicates an improvement in the loading capacity of whey protein for curcumin. Chen, Zhong, and Chen (2022) also observed a significant increase in LC as the concentration of quercetin increased when studying the encapsulation of quercetin by whey protein. This is also consistent with the results of our study.

Fig. 2.

Fig. 2

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Encapsulation efficiency (A) and loading capacity (B) of curcumin in curcumin-whey protein complex with different curcumin concentrations. Different letters indicate significant differences (P < 0.05).

3.3. Surface hydrophobicity analysis

Surface hydrophobicity is an important indicator affecting colloidal stability, with higher surface hydrophobicity often indicating the occurrence of colloidal aggregation. ANS can bind to hydrophobic groups in proteins, so fluorescence spectroscopy is commonly used to detect it, with fluorescence intensity reflecting the degree of surface hydrophobicity (Wang et al., 2024). As shown in Fig. 3A, the surface hydrophobicity of whey protein is the lowest (565 AU). As the concentration of curcumin increases, the surface hydrophobicity of the complex gradually rises to 687 AU. These results indicate that the addition of curcumin leads to an increase in the surface hydrophobicity of the complex, which in turn causes an increase in particle size (Fig. 1A). This may be due to some hydrophobic curcumin binding to the protein surface, resulting in enhanced surface hydrophobicity of the protein.

Fig. 3.

Fig. 3

Fig. 3

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Surface hydrophobicity (A), fluorescence spectra (B), far-ultraviolet CD spectra (C), and protein secondary structure content (D) of curcumin-whey protein complex with different curcumin concentrations.

3.4. Fluorescence spectroscopy analysis

Fluorescence spectroscopy was used to study the binding of curcumin to whey protein, with results shown in Fig. 3B. Whey protein exhibited the highest fluorescence intensity (678 AU) at an emission wavelength of 336 nm, which is primarily associated with the tryptophan and tyrosine residues in the protein. The binding of curcumin resulted in a dose-dependent decrease in the fluorescence intensity of the complex, while the maximum emission wavelength of the protein did not show significant shifts. This indicates that the binding of curcumin to the tryptophan and tyrosine residues of the protein leads to fluorescence quenching, but there is little change in the protein's tertiary structure. Mohammadian et al. (2019) also observed a similar phenomenon of fluorescence quenching in whey protein when encapsulating curcumin using whey protein microgels, which is consistent with our findings.

3.5. CD spectroscopy analysis

The CD spectroscopy is one of the most widely used methods for determining the secondary structure of proteins and is often employed to study conformational changes in proteins during interactions with small molecules. The far-ultraviolet CD spectra of whey protein under different curcumin concentrations is shown in Fig. 3 A. WP exhibits two negative peaks at 217 nm and 208 nm (Fig. 3A), which correspond to the β-sheet and α-helical structures in β-LG and α-LA (Liu et al., 2017). After interaction with different concentrations of curcumin, the ellipticity of these two characteristic peaks in the complex protein increases (Fig. 3A), indicating that curcumin binding alters the secondary structure of whey protein. Specifically, the changes in protein secondary structure content are shown in Fig. 3B. The binding of curcumin leads to a conversion of some of the random coil structures in WP to β-fold structures (Fig. 3 B), inducing a more ordered protein conformation. In a previous study, we found that the binding of soy isoflavones to whey protein resulted in a decrease in α-helices and an increase in β-sheets, which may be attributed to the differing properties of the polyphenols (Liu, Sun, Cheng, Zhang, & Guo, 2022). The modification of protein structure by polyphenols depends on the types and properties of both the protein and the polyphenols (Baba, McClements, & Maqsood, 2021).

3.6. FT-IR spectroscopy analysis

The FT-IR spectroscopy was used to analyze four groups of samples to characterize the molecular interactions between curcumin and whey protein. As shown in Fig. 4A, curcumin exhibits an O—H stretching vibration peak at 3512.05 cm−1, along with a series of characteristic peaks in the range of 812.37–1599.02 cm−1 (Liu, Han, Tian, & Liu, 2020). In the spectrum of whey protein, the vibration peaks at 3295.43, 2923.77, 1656.02, and 1535.17 cm−1 correspond to O—H stretching vibrations, C—H stretching vibrations, C Created by potrace 1.16, written by Peter Selinger 2001-2019 O (amide I) stretching vibrations, and N—H (amide II) bending vibrations, respectively (Ramos et al., 2013). After the formation of the curcumin-whey protein complex, many characteristic peaks that originally belonged to curcumin disappeared, indicating that curcumin is encapsulated by the protein. Additionally, there were shifts in the O—H vibration peak of whey protein (from 3295.43 to 3297.71 cm−1) and in the amide bands (amide I shifted from 1656.02 to 1658.3 cm−1, and amide II shifted from 1535.17 to 1542.01 cm−1). However, no shift in the amide I band was observed in the physical mixture. This suggests that curcumin primarily interacts with whey protein through hydrogen bonding (shifts in the O—H vibration peak) and hydrophobic interactions (shifts in the amide II). Li, Jiang, and Shi (2024) found that curcumin mainly binds to glycosylated whey protein through hydrogen bonds and hydrophobic interactions. Furthermore, our previous research also confirmed that hydrogen bonding is one of the driving forces for the binding of curcumin to zein-based particles (Liu et al., 2020). These results support our current research conclusions.

Fig. 4.

Fig. 4

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FT-IR Spectra (A) and DSC thermograms (B) of curcumin, whey protein, curcumin-whey protein complex, and curcumin-whey protein mixtures. The concentration of curcumin in the complex was 0.2 g/L. The mass of curcumin in the mixtures was equal to that in the complex.

3.7. DSC analysis

The DSC was further employed to confirm the encapsulation of curcumin and to study the transformation of curcumin's crystalline structure. As shown in Fig. 4B, curcumin exhibits an endothermic peak at 183.5 °C, indicating a melting peak. Whey protein shows a broad endothermic peak at 84.5 °C, representing the denaturation of β-LG and α-LA (Liu et al., 2022). After the formation of the curcumin-whey protein complex, the melting peak of curcumin could not be detected. Meanwhile, the endothermic peak of whey protein shifted from 84.5 °C to 86.01 °C. However, a weak endothermic peak for curcumin was still observed in the physical mixture at 185.37 °C. These data suggest that curcumin is encapsulated by whey protein in the complex, leading to its crystalline structure being transformed into an amorphous state. Hu et al. (2024) also observed a similar disappearance of the curcumin endothermic peak when using zein/cyclodextrin complex for curcumin loading, which is consistent with our findings.

3.8. Molecular docking analysis

Molecular docking techniques were further employed to investigate the binding sites (amino acid residue) and interaction mechanisms between curcumin and whey protein, as shown in Fig. 5 and Table 1. Binding energy is a key indicator for assessing the strength of interactions between proteins and ligands. Generally, when the binding energy is less than −1.2 kcal/mol, it is considered that the receptor and ligand can form a stable complex (Liang et al., 2024). According to the results in Fig. 5 and Table 1, the binding energies of curcumin with α-LA, β-LG, and BSA are −6.6 kcal/mol, −7.3 kcal/mol, and − 7.4 kcal/mol, respectively. Therefore, curcumin can strongly interact with all three proteins and form stable molecular conformations. Moreover, the affinity of the proteins for curcumin is ranked as BSA > β-LG > α-LA.

Fig. 5.

Fig. 5

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The 3D bonding model, 2D bonding schematic diagrams, interaction modes, and best docking positions between curcumin and α-LA, β-LG, and BSA using molecular docking.

Table 1.

The predicted binding energies and binding modes of curcumin as well as the amino acid residues involved in hydrogen bonds and hydrophobic contacts.

Binding energy (kcal/mol) Binding pose Residues involved in hydrogen bonds Residues involved in hydrophobic contacts
β-LG(3NPO) −7.3 Image 1 Asn90, Ser116 Leu39, Ile71, Asn109
α-LA(1F6R) −6.6 Image 2 His32, Asn56, Tyr103 Gln54, Tyr103
BSA (4F5S) −7.40 Image 3 Val342, Glu443, Pro446, Asp450
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Curcumin is rich in phenolic rings, double bonds, and hydroxyl groups, which serve as interaction sites with proteins (Cheng et al., 2023). The 3D bonding model (Fig. 5) shows that curcumin can penetrate the hydrophobic pocket of β-LG and bind with amino acid residues (Leu39, Ile71, and Asn109) through hydrophobic interactions. Additionally, the hydroxyl groups of curcumin can form hydrogen bonds with Asn90 and Ser116 of β-LG, stabilizing the conformation of the complex. Curcumin can enter the cavity of α-LA and bind with Gln54 and Tyr103 in the cavity through hydrophobic interactions. Furthermore, curcumin can also bind with amino acid residues of α-LA (His32, Asn56, and Tyr103) through hydrogen bonding. Similarly, curcumin can bind with amino acid residues in the hydrophobic region of BSA (Val342, Glu443, Pro446, and Asp450) through hydrophobic interactions. In summary, the molecular docking results indicate that curcumin primarily binds to the amino acid residues of whey protein through hydrophobic interactions and hydrogen bonds, which is consistent with the FT-IR spectroscopy analysis (Fig. 4A). In fact, many studies have shown that the primary driving forces for the binding of lipophilic polyphenols to whey protein are hydrophobic interactions and hydrogen bonds, such as with galangin (Ma & Zhao, 2019), pterostilbene (Liang et al., 2024), and curcumin (Baba et al., 2021), which strongly supports our research findings.

3.9. SEM analysis

The SEM was used to observe the microstructure of the curcumin-whey protein complex formation. Curcumin exhibits a spherical crystalline structure (Fig. 6A), while whey protein displays a spherical particle structure with a smooth surface (Fig. 6B). In contrast, the formed complex presents a flake-like structure, completely different from the structures of the two individual components (Fig. 6C). This indicates that curcumin and whey protein underwent structural remodeling driven by pH adjustment. Additionally, some granular crystalline structures can still be observed on the surface of the complex, suggesting that curcumin was not completely encapsulated (Fig. 2), resulting in some curcumin crystals remaining on the surface of the complex.

Fig. 6.

Fig. 6

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SEM images of curcumin (A), whey protein (B), curcumin-whey protein complex (C), the simulated gastric fluid digestion products of the complex (D), and the simulated intestinal fluid digestion products of the complex (E). The concentration of curcumin in the complex was 0.2 g/L.

3.10. Stability analysis

3.10.1. Ionic stability analysis

In beverage processing, ion concentration is a crucial factor affecting the stability of colloidal particles. Therefore, exploring the ionic stability of colloids is essential for their application in the beverage industry. Different NaCl concentration on the stability of the curcumin-whey protein complex is shown in Fig. 7. As the NaCl concentration increases, the particle size of the curcumin-whey protein complex gradually increases, but does not exceed 400 nm (Fig. 7A), and the polydispersity index (PDI) remains below 0.3 (P > 0.05) (Fig. 7B), indicating that there is no significant aggregation and that the complex maintains a uniform distribution within the 500 mM NaCl concentration range. However, the zeta potential results show a significant decreasing trend (P < 0.05) (Fig. 7C), suggesting that sodium ions bind to the surface of the complex, leading to the shielding of the negative charge on the complex's surface. This weakens the electrostatic repulsion between the complexes, resulting in an increase in particle size (Fig. 7A). It should be noted that the current test results are only the detection results on the day after the addition of NaCl, and the long-term stability has not been monitored. Therefore, as the electrostatic repulsion between the complexes decreases, it also indicates a decrease in their long-term stability, and there may be a further increase in particle size or the phenomenon of precipitation.

Fig. 7.

Fig. 7

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Particle size (A), PDI (B), and zeta potential (C) of curcumin-whey protein complex at different NaCl concentrations. The concentration of curcumin in the complex was 0.2 g/L. Different letters indicate significant differences (P < 0.05).

3.10.2. Thermal stability analysis

Heat treatment is a commonly used sterilization method in beverage processing. Thus, it is crucial to investigate the effect of heat treatment on colloidal stability. The impact of heat treatment on the stability of the complex is shown in Fig. 8. The particle size of untreated samples is around 300 nm, and as the heat treatment temperature increases, the particle size of the complex gradually decreases (P < 0.05) (Fig. 8A). Furthermore, heat treatment has a limited effect on the PDI (Fig. 8B) and zeta potential (Fig. 8C) of the complex (P > 0.05). These results indicate that during the heating process at 60–90 °C, the particle size decreases, enhancing stability. The reduction in particle size due to heat treatment may be attributed to the unfolding and re-aggregation of protein molecules, leading to a decrease in hydrodynamic radius. Shen, Fang, Gao, and Guo (2017) also observed a reduction in the hydrated particle size of whey protein due to heat treatment, which is consistent with our findings.

Fig. 8.

Fig. 8

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Particle size (A), PDI (B), and zeta potential (C) of curcumin-whey protein complex at different temperatures. The concentration of curcumin in the complex was 0.2 g/L. Different letters indicate significant differences (P < 0.05).

3.10. Simulated gastrointestinal digestion characteristics analysis.

3.10.3. Digestion stability analysis

The digestive characteristics of bioactive compound delivery systems are crucial for the effective intestinal delivery of active compounds. Ideally, we want the delivery system to maintain structural stability throughout the gastrointestinal digestion process, with the loaded active substances not being released, allowing for direct delivery to the small intestine for absorption. However, it is challenging for carriers made from biopolymers to remain completely intact throughout the digestive tract. Additionally, due to compactness and the diffusion effect of small molecules, some core materials may be released from the carrier. Specifically for protein-based carriers, we aim to prevent structural disintegration in the stomach to minimize the release of core materials, ensuring that more core substances reach the intestinal absorption sites. Therefore, we employed a simulated gastrointestinal digestion model to evaluate the digestion stability of the complex. As shown in Fig. 9A, digestion in simulated gastric fluid (SGF) significantly increased the particle size of the complex from 278 nm to 334 nm (P < 0.05). Subsequent digestion in simulated intestinal fluid (SIF) resulted in a significant decrease in particle size to 236 nm (P < 0.05). The trend in the polydispersity index (PDI) mirrored that of the particle size (Fig. 9B). This indicates that the complex aggregated during SGF digestion, while it was hydrolyzed by trypsin during SIF digestion. Zeta potential analysis results (Fig. 9C) show that SGF digestion changed the zeta potential of the complex from −38 mV to 8 mV, and SIF digestion further increased the absolute value of the zeta potential (−39 mV). The transition of the zeta potential from negative to positive and then back to negative during digestion is attributed to the pH changes of the digestive fluids. Moreover, the lower zeta potential in SGF digestion weakened the electrostatic repulsion between the digested particles, which is consistent with the observed increase in particle size (Fig. 9A). Further examination using SEM revealed changes in the microstructure of the complex during digestion. After SGF digestion, the complex displayed block-like aggregates (Fig. 6D), while after SIF digestion, it transformed into smaller, more uniformly dispersed particles (Fig. 6E). The SEM analysis supports the findings of the particle size and zeta potential analyses, indicating that SGF digestion led to the aggregation of the complex, while SIF digestion resulted in its decomposition. Hu, Kou, Chen, Li, and Zhou (2019) also observed that whey protein nanoparticles loaded with mandarin peel extracts aggregated during SGF digestion, and subsequent SIF digestion led to particle hydrolysis.

Fig. 9.

Fig. 9

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Particle size (A), PDI (B), zeta potential (C), and curcumin bioaccessibility (D) of the curcumin-whey protein complex after simulated gastrointestinal digestion. The concentration of curcumin in the complex was 0.2 g/L. The mass of curcumin in the free state and in the mixture is equal to that in the complex. Different letters indicate significant differences (P < 0.05).

3.10.4. Curcumin bioaccessibility analysis

Bioaccessibility refers to the proportion of bioactive compounds that can be absorbed by the small intestine after passing through the gastrointestinal tract. This metric is crucial for the absorption of active compounds and their physiological effects in the body. The bioaccessibility results of curcumin before and after encapsulation, following simulated gastrointestinal digestion, are shown in Fig. 9D. The bioaccessibility of free curcumin is 59.78 %, which is relatively high, possibly due to its binding with enzymes and bile salts in the digestive tract. The bioaccessibility of the mixture of curcumin and whey protein is higher than that of free curcumin (63.96 %), likely because some curcumin binds with whey protein digests. However, the bioavailability of the curcumin-whey protein complex reaches 90.26 %, representing an increase of 60 % and 41 % compared to free curcumin and the curcumin-whey protein mixture, respectively. This indicates that using whey protein encapsulation is an effective strategy to enhance the bioaccessibility of curcumin.

The primary reason protein-based carriers improve the bioavailability of lipophilic polyphenols is that the carriers aggregate in gastric fluid, inhibiting the release and degradation of lipophilic polyphenols, allowing more of these compounds to enter the intestine. Under the action of digestive enzymes and bile salts, they form mixed microgels with protein digests, which are then absorbed by the intestinal epithelial cells (Liu, Sun, Zhang, Zhang, et al., 2022). Research by Li and Zhong (2024) has shown that using casein-based carriers can effectively enhance the bioaccessibility of curcumin in in vitro digestion models, consistent with our findings. In the future, animal experiments should be conducted to verify the synergistic effect of whey protein encapsulation on the in vivo bioavailability of curcumin, providing a more reliable theoretical basis for the application of curcumin-whey protein complexes as functional foods.

4. Conclusions

Curcumin-loaded whey protein complex can be successfully prepared using a pH-induced protein-polyphenol co-assembly process. The amount of curcumin added significantly affects the complex stability and the encapsulation characteristics of curcumin. Curcumin primarily binds to whey protein through hydrophobic interactions and hydrogen bonds, leading to a transformation of its crystalline structure into an amorphous state while inducing an ordered transformation of the protein's secondary structure. The complex exhibits high ion stability and thermal stability. The loading of whey protein can effectively deliver curcumin to the small intestine and significantly enhances its bioaccessibility. The findings provide a theoretical basis for the green processing and application of protein-based lipophilic polyphenol oral delivery systems in nutrient supplement, functional dairy products, and functional protein beverages.

CRediT authorship contribution statement

Qingguan Liu: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Funding acquisition, Conceptualization. Wanqing Huang: Software, Methodology, Investigation. Chunxia Zhou: Software, Formal analysis, Data curation. Sichen Yang: Formal analysis, Data curation. Jiaqi Hong: Formal analysis, Data curation. Ailin Xie: Formal analysis, Data curation. Qiang Cui: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition.

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.

Acknowledgements

This project was financially supported by Scientific Research Start-Up Funds of Guangdong Ocean University (060302042317), Guangdong Ocean University College Students Innovative Training Program (010403042411), and the Science and Technology Department of Sichuan Province (2024NSFSC1254).

Contributor Information

Qingguan Liu, Email: liuqingguansdk@163.com.

Qiang Cui, Email: cuiqiangwx@163.com.

Data availability

Data will be made available on request.

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Data Availability Statement

Data will be made available on request.

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