Effect of ultrasound treatment on interactions of whey protein isolate with rutin

Highlights

• •The WPI-R complex was prepared by pH-driven method (pH-DM) combined with ultrasonic treatment.
• •The interaction between whey protein and rutin is covalent, and appropriate ultrasonic treatment could promote degree of polyphenol binding and grafting between whey protein isolate and rutin.
• •The ultrasonic treatment increased the WPI-R complex's solubility while decreasing its surface hydrophobicity and free sulfhydryl.
• •The secondary structure of the complex was improved with ultrasonic treatment, which results in three-dimensional honeycomb structures with small and uniform pore sizes.

Abstract

Rutin is a biologically active polyphenol, but its poor water solubility and low bioavailability limit its application to the food industry. We investigated the effect of ultrasound treatment on the properties of rutin (R) and whey protein isolate (WPI) using spectral and physicochemical analysis. The results revealed that there was covalent interaction between whey protein isolate with rutin, and the binding degree of whey isolate protein with rutin increased with ultrasound treatment. Additionally, solubility and surface hydrophobicity of WPI-R complex improved with ultrasonic treatment, and a maximum solubility of 81.9 % at 300 W ultrasonic power. The ultrasound treatment caused the complex to develop a more ordered secondary structure, resulting in a three-dimensional network structure with small and uniform pore sizes. This research could provide a theoretical reference for studying protein–polyphenol interactions and their applications in food delivery systems.

1. Introduction

Rutin (R) is a natural polyphenol in tea, orange peel, and tomatoes, with excellent antioxidant, anticancer, and anti-inflammatory properties [[1], [2], [3], [4]]. The low bioavailability and solubility in water of rutin limit its application in the food processing and pharmaceutical industries. Rutin was found to be significantly enhanced in water solubility and bioavailability when encapsulated by proteins or polysaccharides such as chitosan, cyclodextrin and other molecules [[4], [5]]. In addition, the rutin-chitooligosaccharide complex reduced bitterness while increasing antioxidant and antibacterial activity [6].

Dietary protein–polyphenol coexistence is quite common in foods, the interactions between polyphenols and proteins could improve polyphenol stability, protein solubility and emulsification properties, and polyphenol antioxidant properties [[7], [8], [9], [10], [11], [12]]. Therefore, the interaction between polyphenols with proteins and their applications have become a current research hotspot [[13], [14], [15], [16], [17], [18]]. The interactions between polyphenols and proteins are mainly non-covalent and covalent, where the non-covalent interactions are mainly influenced by non-covalent forces such as hydrogen bonding, hydrophobic interactions, and electrostatic interactions. Covalent interactions are mainly dominated by environmental pH, enzymatic reactions, and free grafting. pH is a critical process parameter in processing, influencing the structure and properties of protein and polyphenol substances and providing an effective means of modification. Polyphenols are oxidized to active electrophilic o-quinone structures under alkaline conditions, where they react with nucleophilic groups on protein side chains, such as amino or sulfhydryl groups, to form irreversible covalent bonds. [19].

Since polyphenols are unstable and sensitive to light and heat, gentle methods are desirable for their processing. Ultrasound is a mild physical method whose cavitation can improve and promote food quality [[20], [21], [22], [23], [24], [25]]. Researchers have demonstrated that applying ultrasound to protein molecules causes more reactive groups to be exposed, strengthening the protein–polyphenol cross-linking, improving the properties of the resulting gels, and reducing the porosity and particle size of the gel network structure.

The special molecular structure of proteins provides a variety of functional groups and high affinity hydrophobic binding sites, binding ligands, and functionally active substances, making proteins an ideal carrier of functionally active substances. Whey protein is a by-product of cheese production, and its rich nutrition and excellent gelation properties are often used as a food additive as a carrier and stabilizer for bioactive molecules [[26], [27]]. Moderate modifications of whey protein isolate to give them structural and functional properties suitable for specific food systems could broaden the applications.

Thus, this work aims to investigate the effects of ultrasound treatment on the physicochemical characteristics of the WPI-R complex. The modifications were characterized using surface hydrophobicity, free sulfhydryl groups, UV spectra, fluorescence, and microstructure of WPI-R complex to provide a theoretical foundation for strengthening the synergistic mechanism of food functional components.

2. Materials and methods

2.1. Materials

Whey protein isolates (Hilmar™ 9410, 92 %purity) was purchased from Hilmar Cheese Company (MN, USA). Rutin (≥95.0 %, BR,) was obtained from Solarbio life sciences (Beijing, China). Other reagents are analytically pure or higher grade.

2.2. Methods

2.2.1. Preparation of WPI-R complex

Complex of WPI-R were prepared using the pH-driven method (pH-DM) as described by Wang [25]. Rutin dispersion (60 mg/mL(w/v)) and a 40 g/L of whey protein isolate solution were prepared and stirred separately, and placed in a refrigerator at 4 °C overnight. The whey protein isolate solution was adjusted to pH 12.0 and labelled as WPI pH12. After that, WPI pH12 was mixed 1:1(v/v) with rutin solution, stirred for 10 min before ultrasound treatment. The ultrasonic power was 0, 150 W, 300 W, 450 W, and 600 W. The samples were sonicated for 20 min and named as 0, 150 W, 300 W, 450 W, and 600 W respectively. The samples were treated at 0 °C during sonication, and then the solution was adjusted to pH 7.0. The above-treated sample solution was dialyzed and freeze-dried for 48 h for further use.

2.2.2. Determination of UV scanning spectra

The UV absorption spectra of different WPI samples (1.0 mg/mL) were recorded in 250–400 nm range with a sampling interval of 0.2 nm at a moderate scanning speed using a UV spectrophotometer (UV1800, Shimadzu, Japan).

2.2.3. Determination of endogenous fluorescence spectra

The spectral emission variations of sample solutions (0.2 mg/ml) were scanned in the 300–450 nm spectral range using a fluorescence spectrophotometer (Shimadzu RF5300, Shimadzu, Japan). During scanning, the excitation and emission slit widths were kept at 10 nm, the excitation wavelength was 280 nm, and the scanning speed was 240 nm/min.

2.2.4. Fourier transform infrared spectroscopy (FTIR)

The samples were lyophilized and powdered. ART-FTIR spectrometer (Nicolet-6700, Thermo Fisher Scientific, Waltham, MA, USA) was used for scanning, and the conditions were set to a spectral range of 500–4000 cm⁻¹ with 32 scans and a resolution of 4 cm⁻¹.

2.2.5. Scanning electron microscopy (SEM)

The samples were freeze-dried, coated with gold, and then observed using a scanning electron microscope (EVO MA-15, ZEISS ltd, Berlin, Germany).

2.2.6. Determination of degree of grafting

The covalent interaction is mainly between the free amino and mercaptan groups of proteins and polyphenols, so the grafting rate of free amino groups is expressed to measure the interaction of polyphenols with proteins. The free amino content was determined by the O-phthalaldehyde (OPA) method [28]. The degree of grafting of the protein–polyphenol complex was determined and calculated as:

$$\text{Degree of grafting (\%)=}\frac{{\mathrm{C}}_0\text{-}{\mathrm{C}}_1}{{\mathrm{C}}_0}\times 100\%$$

where C₀ is the total amount of free amino groups in the untreated mixture; C₁ is the total amount of free amino groups after sonication.

2.2.7. Determination of changes in free sulfhydryl contents

Free sulfhydryl content in proteins was determined using Ellman's reagent (DTNB). Diluted protein samples (0.5 mL of free sulfhydryl content on the surface) were added to 2.5 mL of Tris-glycine buffer (0.086 M Tris, 0.09 M glycine, 0.004 M EDTA, pH 8.0). Add 0.02 mL of DTNB (4 mg/mL dissolved in Tris-glycine buffer) and incubate for 15 min at room temperature before measuring the absorbance value at 412 nm (A412).

The free sulfhydryl contents ($\mathrm{\mu }\mathrm{m}\mathrm{o}\mathrm{l}$ SH/g) were calculated as:

$$\delta (\mu mol/g)=\frac{{10}^6\times A_{412}\times D}{1.36\times {10}^4\times C}$$

where δ is the free sulfhydryl content, μmol/g protein; A₄₁₂ is the measured absorbance value, C is the protein concentration (mg/mL), and D is the dilution multiple; 1.36 × 10⁴ is the molar absorbance coefficient.

2.2.8. Determination of surface hydrophobicity

The fluorescent probe ANS was used to determine the surface hydrophobicity of proteins. The protein solution was diluted with ultrapure water into five concentration gradients (25 to 200 ug/L(w/v)). The relative fluorescence intensity of the samples was measured after 0.02 mL ANS solution (8 mM dissolved in 10 mM phosphate buffer saline, pH 7.2) was added to 4 mL sample solution. The wavelengths of excitation and emission were 390 nm and 470 nm, respectively. The initial slope of fluorescence intensity over protein concentration represents the protein's surface hydrophobicity.

2.2.9. The binding capacity of polyphenols

The Folin-Ciocalteau method determined the total phenolic content and the binding degree of rutin with WPI. The complex was dialyzed in purified water for 12 h at 4 °C. 4 mL dialysis extra fluid was mixed with 4 mL distilled water and 4 mL Folin-Ciocalteu phenol reagent and set aside for 5 min. A 1.5 mL solution of 20 wt% sodium carbonate was added and thoroughly mixed. The mixture was then incubated for 2 h at room temperature in the dark. The absorbance was measured at 765 nm, and the degree of polyphenol binding was calculated as follows:

$$\text{Degree of polyphenol binding\%=}\frac{\text{total rutin - rutin outside the dialysate}}{\text{total rutin}}\times 100\%$$

2.2.10. Measurement of particle size and Zeta potential

The particle size (z-average size) and Zeta potential of the WPI-R complex was determined using a Malvern nanoparticle sizer (Zetasizer Nano-ZS, Malvern Instruments ltd., Worcestershire, UK) at 25 °C. The refractive index of the protein particle and the dispersed phase was 1.46 and 1.33, respectively.

2.2.11. Determination of turbidity and solubility

The absorbance of samples was measured at 600 nm, and turbidity was expressed as the absorbance.

The solubility of the complex was determined by Coomassie bright blue method. WPI (10 mg/mL) was centrifuged at 6010 g for 15 min. Centrifuged supernatant (0.1 mL) was well-mixed with Coomassie stain solution and left for 10 min. The absorbance was measured at 595 nm. The protein concentration of the WPI-R complex was calculated from the standard protein concentration equation (y = 0.0096x + 0.0567, R² = 0.9929).

2.3. Statistical analysis

All the experiments were done in triplicate. The data were processed using SPASS 24, peakfit 4.12 and then plotted using Origin9.

3. Results and discussion

3.1. UV scanning spectral analysis

The characteristic UV peak of WPI pH12 (Fig. 1) was at 280 nm, and the absorption peak of rutin was at 268 nm. However, the characteristic peak of the WPI-R complex purple-shifted to 273 nm, demonstrating that covalent binding of whey protein isolate and rutin occurred [29]. The intensity of UV absorption of WPI-R complex increased as ultrasonic power increased, indicating that ultrasonic cavitation altered the microenvironment of aromatic amino acids in the complex. The highest ultrasonic power discovered was 300 W, indicating that this power condition was most favorable for unfolding whey protein isolate molecular structure.

Fig. 1.

Ultraviolet scanning spectrum of complex and rutin.

3.2. Endogenous fluorescence spectroscopy

The interaction of rutin with whey protein isolate was studied using endogenous fluorescence of protein molecules with various sonication treatments. The fluorescence emission peak of the WPI-R complex (Fig. 2) showed a red shift at 335 nm when compared to the whey protein isolate, suggesting that the ultrasound treatment affected the protein's secondary structure, causing the protein structure to unfold. A hydrophilic environment increases the polar environment of aromatic amino acids and tryptophan. Interestingly, for all ultrasound powers above 150 W, the fluorescence intensities were higher than those of the un-sonicated WPI-R complex, except for the fluorescence intensity at 150 W, which was lower than that of the un-sonicated complexe. This phenomenon needs to be further investigated. This result suggested that the ultrasound power significantly affects the structure of WPI-R complex, probably because the ultrasound could promote the unfolding of the protein molecular structure [30].

Fig. 2.

Endogenous fluorescence spectrum changes of WPI-R and WPI.

3.3. FTIR spectral analysis

The stretching and bending vibrations of the peptide backbone in the amide I and II bands in the FTIR spectrum may represent different secondary structures. The absorption bands of the protein at 1600–1700 cm⁻¹ and 1450–1550 cm⁻¹ are the amide I and amide II groups. The amide I band (C O stretching vibration) of the WPI-R complex shifts from 1633 cm⁻¹ to 1634 cm⁻¹, and the amide II band (N—H, C—N) changes from 1530 cm⁻¹ to 1534 cm⁻¹(Fig. 3), indicating that rutin caused a change in the secondary structure of whey protein isolate [31]. The WPI-R complex did not change in the amide I band before and after the ultrasound. However, the amide II band shifted from 1534 cm⁻¹ to 1526 cm⁻¹, indicating that ultrasound affected hydrogen bonding and other interactions within the WPI-R complex [[32], [33]].

Fig. 3.

FTIR of complex with ultrasound treatment.

FTIR spectra of this region were fitted using Fourier self-inverse convolution and Gaussian functions to detect the protein's secondary structure (Table 1), and it was found that the secondary structure of the WPI-R complex changed with ultrasound treatment. Conversely, rutin increased the WPI-R complex's β-sheet structure to 48.12 % while decreasing the unordered coil structure to 10.24 %. (Table 1). Both the α-helix and β-sheet structures of the complex increased after sonication, and the unordered coil structure was reduced to 7.72 %, indicating that sonication improved the secondary structure of whey protein isolate.

Table 1.

Secondary structure content.

Group	α-helix (%))	β-sheet (%)	β-turn (%)	Unordered coil (%)
WPI	18.11 ± 0.20a	37.51 ± 0.59b	24.00 ± 1.19a	20.38 ± 0.45a
0 W WPI-R	18.08 ± 0.61a	48.12 ± 0.88a	23.57 ± 0.48a	10.24 ± 0.49b
150 W WPI-R	19.41 ± 1.15a	50.60 ± 1.33a	22.69 ± 0.65a	7.30 ± 0.27c
300 W WPI-R	19.26 ± 0.61a	49.08 ± 0.89a	23.35 ± 0.60a	8.32 ± 0.65b
450 W WPI-R	19.13 ± 0.85a	49.05 ± 1.34a	23.53 ± 0.75a	8.29 ± 1.03b
600 W WPI-R	18.18 ± 0.57a	49.74 ± 0.90a	23.82 ± 0.54a	8.25 ± 0.73bc

3.4. Analysis of surface morphology of complex

The surface morphology of WPI-R complex with ultrasound treatment was characterized by scanning electron microscopy, and the results were shown in Fig. 4. The WPI pH12 possessed irregular lamellar structures, while the WPI-R complex displayed stable lamellar structures and a three-dimensional network structure emerged between the lamellar layers. The complex with 150w ultrasound treatment showed a particular three-dimensional structure with large pores on its three surfaces. The structure of WPI-R complex with 150w ultrasound treatment revealed that almost all of the lamellar junction sections were dense honeycomb structures with uniform size and distribution. The differences in complex structures suggest that ultrasonic treatment is an effective method for improving the complex structure.

Fig.4.

SEM of WPI-R and WPI with ultrasound treatment.

3.5. Determination of degree of grafting

Polyphenols are oxidized to quinones under alkaline conditions, which easily react with free amino and thiol groups in whey proteins [28]. Free amino group detection can be used to evaluate the grafting rate of polyphenols covalently binding to proteins. Table 2 showed that the grafting rate of covalent attachment of whey protein isolate with rutin was 28.72 %. The grafting rate increased significantly after ultrasound treatment, meaning that ultrasound facilitates protein binding to rutin.

Table 2.

Degree of polyphenol binding and grafting of the complex.

Group	0 W	150 W	300 W	450 W	600 W
Degree of polyphenol binding（%)	79.89 ± 0.40d	81.05 ± 0.08c	83.02 ± 0.23a	81.96 ± 0.08b	83.58 ± 0.17a
Degree of grafting (%)	28.72 ± 2.32d	43.43 ± 2.08b	39.96 ± 1.26c	51.56 ± 1.51a	30.91 ± 2.38d

Note: Data in the table are mean ± standard deviation; In the same column, the same letter from a to d means that there is no significant difference (p > 0.05), but there is a significant difference between different letters (p < 0.05).

3.6. The degree of polyphenols binding

The structure of whey protein isolate was partially extended under alkaline conditions, facilitating the binding affinity of rutin to whey protein isolate. The degree of polyphenols binding between whey protein isolate and rutin increased with ultrasound treatment (table 2). The binding affinity increased with ultrasonic treatment, illustrating that ultrasound could promote the binding of rutin to whey protein isolate. The highest degree of binding was achieved at 600 W due to the thermal effect of high ultrasonic power, which increased rutin solubility and facilitated the reaction between rutin and whey protein isolate.

3.7. Free sulfhydryl contents analysis

Sulfhydryl groups in proteins, especially free cysteine sulfhydryl groups, have very active chemical properties that influence the redox state and functional properties of proteins, changing the food structure. The addition of rutin increased the protein's free sulfhydryl content, as shown in Fig. 5, indicating that rutin interacts with whey protein isolate. The above results were attributed to rutin reacting with nucleophilic protein groups like amino, tryptophan, cysteine, and histidine to form C—N or C—S bonds, exposing free sulfhydryl groups inside the whey protein isolate molecule [34]. With ultrasound treatment, the free sulfhydryl groups of WPI-R decreased slightly, possibly due to the oxidation of exposed free sulfhydryl groups by the free hydroxyl groups (OH) (Fig. 6) generated by ultrasonic cavitation, resulting in a decrease in free SH group content [[35], [36]].

Fig. 5.

Changes of free sulfhydryl group content of WPI-R and WPI with ultrasound treatment.

Fig. 6.

Surface hydrophobicity change of WPI-R and WPI with ultrasound treatment.

3.8. Surface hydrophobicity analysis

Surface hydrophobicity is closely related to protein emulsification, foaming, and gelation ability because it is an essential indicator of protein tertiary structure [20]. The surface hydrophobicity of the complex decreased after adding rutin (Fig. 9), due to the hydrophilic hydroxyl group (–OH) of rutin, which could enhance protein hydration. The decreased surface hydrophobicity caused by ultrasound treatment could be attributed to the increased binding of whey protein isolate to polyphenols.

Fig. 9.

Solubility changes of WPI-R and WPI.

3.9. Particle size and zeta potential analysis

The formation of WPI-R complex was studied by measuring the change in particle size (Fig. 7). The Z-average size of the WPI-R complex increased when compared to the WPI. Moreover, the ultrasonically treated complex had the largest Z-average size increase at 150 W. The particle size of the complex decreased significantly as the ultrasonic power increased, with the smallest particle size discovered at 600 W. This was due to the cavitation and shearing effects of high-intensity ultrasound, which destroyed interparticle interactions and thus reduced particle size.

Fig. 7.

Z-average size and Zeta potential changes of complex.

Intermolecular electrostatic interactions can also be analysed using the zeta potential. The absolute Zeta potential values of the treated WPI-R complex increased, indicating that the ultrasound treatment facilitated protein molecule unfolding and increased intermolecular repulsive forces.

3.10. Turbidity analysis

The primary cause of turbidity is the formation of aggregates between protein molecules due to a certain degree of intermolecular aggregation caused by the combined effect of electrostatic interaction, hydrophobic interaction, and disulfide bonds. The turbidity of whey protein isolate crosslinked with rutin was significantly reduced in Fig. 8, indicating that the binding of whey protein isolate and rutin changed the charge distribution on the protein surface, disrupting intermolecular protein aggregation and thus improving solubility. whey protein isolate and rutin combined benefited from ultrasonic cavitation and thermal effect, resulting in moderate protein dissociation in the complex. The high-power ultrasound reduced the particle size of the complex, increased solubility, and reduced solution turbidity.

Fig. 8.

Turbidity changes of WPI-R and WPI.

3.11. Solubility analysis

Solubility is regarded as the most practical index for evaluating the functional properties of proteins. Increasing solubility increases protein properties; thus, good solubility is required for using proteins as emulsifiers.

As shown in Fig. 9, the solubility of WPI-R complex was all higher than whey protein isolate, indicating that ultrasound enhanced protein solubility. The solubility of WPI-R complex increased with ultrasonic treatment, reaching 84.3 % at 300 W. This indicated that rutin could cause the internal structure of whey protein isolate molecules to unfold partially, exposing the internal hydrophilic groups. Moreover, the hydroxyl group (–OH) in rutin can form hydrogen bonds with water molecules, which improves protein hydration and solubility; additionally, the cavitation effect of ultrasonication can break non-covalent interactions such as hydrogen bonds and hydrophobic interactions of protein, exposing more hydrophilic amino acid residues and thus improving solubility. However, excessive sonication power harms the hydrophilic and hydrophobic interactions within the protein, reducing its solubility.

4. Conclusion

Rutin grafting and sonication can significantly alter whey protein isolate properties. Rutin covalently interacted with whey protein isolate to form a WPI-R complex under alkaline conditions, causing changes in the secondary structure of whey protein isolate. Ultrasound treatment increased the binding of whey protein isolate to rutin, which improved protein solubility and free sulfhydryl content, decreased surface hydrophobicity, and produced a gel with a three-dimensional homogeneous honeycomb network structure. This research benefits the synergistic mechanism of functional food factors and the health food industry.

CRediT authorship contribution statement

Na Guo: investigation, Writing-original & editing. Shuang Ye: investigation. Ganghua Zhou: Validation. Yimeng Zhang: Validation. Fangyan Zhang: Software. Xu Jingjing: Methodology. Shenyu Pan: Validation. Guilan Zhu: Writing - review & editing, Formal analysis. Ziying Wang: Conceptualization.

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.