Evaluation of different dietary polyphenols on conformational, functional changes and lycopene-loaded nanocarriers of walnut protein isolate

doi:10.21203/rs.3.rs-2254263/v1

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Evaluation of different dietary polyphenols on conformational, functional changes and lycopene-loaded nanocarriers of walnut protein isolate

https://doi.org/10.21203/rs.3.rs-2254263/v1

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This study investigated the effects of the interaction of walnut protein isolate (WPI) with epigallocatechin gallate (EGCG), chlorogenic acid (CLA), (+)-catechin (CA), and ellagic acid (EA) on the structural and functional properties of proteins by a free radical grafting method. The results for polyphenol binding equivalents and content of free amino and sulfhydryl groups as well as those from sodium dodecyl sulfate‒polyacrylamide gel electrophoresis confirmed the covalent interaction between WPI and the polyphenols. The binding capacities of the mixtures and conjugates were as follows: WPI-EGCG > WPI-CLA > WPI-CA > WPI-EA. Fourier transform infrared spectroscopy (FTIR) and fluorescence spectrum analysis identified changes in the protein structure. The conjugation process obviously increased the polyphenols’antioxidant properties and the surface hydrophobicity was substantially reduced. WPI-EGCG conjugates had the best functional properties, followed by WPI-CLA, WPI-CA, and WPI-EA. Lycopene (LYC) was loaded into nanocarriers by WPI-EGCG self-assembly. These results indicated that WPI-polyphenol conjugates can be utilized to develop food-grade delivery systems to protect chemically lipophilic bioactive compounds.

Walnut protein isolate

Dietary polyphenols

free radical grafting

Properties

Lycopene

Nanocarriers

Walnut protein isolate (WPI), as a plant protein source, is the byproduct of walnut oil processing (Jin et al., 2020). It is usually underutilized or used as animal feed, which is a waste of an important protein resource (Zhu et al., 2018). Improving the conformational and functional properties might lead to increased use of walnut kernels directly in human foods (Al-Shabib et al., 2020). The interaction of proteins and polyphenols can improve the functional properties of their conjugates, allowing the conjugates to be used as active polymers or active substance delivery materials in recent years. Nanocarriers of natural biopolymers such as proteins have been used for the delivery of lycopene (LYC) due to their amphiphilic properties. LYC is a lipophilic carotenoid widely found in tomatoes, but its application is limited due to its poor water solubility, low stability and bioavailability (Chang et al., 2022). Among the realm of polyphenol compounds, polyphenol conjugates have been proposed as innovative materials that, by combining the advantageous properties of both components, can increase the efficiency of antioxidants and their range of application in nutritional and biomedical fields (Cirillo et al., 2016).

Protein-polyphenol interactions are mainly classified as covalent or noncovalent. Compared with noncovalent complexes, covalent complexes exhibit higher thermal stability, oxidation resistance, and polyphenol protection (Zhou et al., 2020), which provides a strategy for engineering polyphenol-protein complexes (Li et al., 2021). It was reported that a free radical grafting approach (Curcio et al., 2009), which employed a hydrogen peroxide-ascorbic acid pair as the radical initiator system, may be used to prepare biopolymer-polyphenol conjugates (Xu et al., 2019). The advantage of this approach is that the reaction can be performed at room temperature, does not require the presence of metal ions or organic solvents, and exhibits higher antioxidant activity than those prepared using alkaline treatment and physical methods (You et al., 2014). Compared with biopolymers alone, biopolymer-polyphenol conjugates may provide better protection for sensitive bioactive natural compounds used in food delivery systems, which have a wide range of promising applications in the food, pharmaceutical and medical fields. The chemical and functional properties of protein-polyphenol conjugates have been reported in recent years. Others have demonstrated that whey protein isolate with EGCG has the best functional properties, followed by quercetin, apigenin, and naringenin (Liu et al., 2021). Gu et al. fabricated catechin-egg white protein conjugates as novel food-grade antioxidant emulsifiers designed to improve the physicochemical stability of β-carotene emulsions (Gu et al., 2017). Curcumin was loaded into nanocarriers made of self-assembled partially hydrolysed α-lactalbumin (Wang et al., 2022). Sui et al. has mainly discussed changes in protein conformation and function after combination with a single polyphenol (Sui et al., 2018). However, the binding mechanism and the effects of polyphenols on biomacromolecule structure have not been clearly explained, and the covalent interactions of different polyphenols with specific proteins have not been systematically examined. Few researchs have investigated the nutritional and physicochemical properties of different types of protein-polyphenol complexes.

In the present study, the binding capacities, structural changes, antioxidant properties, surface hydrophobicity, and microscopic morphologies of the mixtures and conjugates of WPI with each of four polyphenols, namely, EGCG, CLA, CA, and EA, were determined. The aim of the present study was to provide a reference for research on the application of different protein-polyphenol conjugates to various fields as transport carriers or functional food ingredients.

Materials

Commercial walnuts were purchased from a local market in Aksu (Xinjiang, China) in 2022. The defatted walnut kernels were obtained after removing the oil using a hydraulic oil press (T32, Hengxiang Hydraulic Machinery Factory, Shandong, China). LYC (CAS: 502-65-8, purity > 98%), EGCG (CAS: 989-51-5, purity > 98%), CLA (CAS: 327-97-9, purity > 98%), CA (CAS: 154-23-4, purity > 98%), and EA (CAS: 476-66-4, purity > 98%) were purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). The chemical structures of the four polyphenols are presented in Fig. S1. Hydrogen peroxide (H₂O₂), ascorbic acid, Sodium carbonate (Na₂CO₃),, β-mercaptoethanol, sodium dodecyl sulfate (SDS), L-leucine, 5,5-dithiobis nitro-benzoic acid (DTNB), ethylene diamin etetraacetic acid (EDTA), potassium bromide (KBr), potassium ferricyanide, trichloroacetic acid, FeCl_3, sodium azide, 8-aniline-1-naphthalene sulfonate (ANS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), dimethyl sulfoxide (DMSO), and Folin-Ciocalteu reagent were purchased from Source Biological Technology Co., Ltd. (Shanghai, China). All solutions were prepared using distilled water. All other chemicals or reagents used were of analytical grade.

Preparation Of Wpi- Polyphenol Mixtures And Conjugates

The WPI was prepared as described by Fang et al. with some modifications (Fang et al., 2020). The WPI-polyphenol conjugates were synthesized using a free radical grafting method reported previously with slight modifications (Xu et al., 2019). One gram of WPI was dissolved in 100 mL of phosphate-buffered saline (PBS) (0.02 M PBS, pH 7.2); 1 mL of 5 mol/L H₂O₂ and 0.25 g of ascorbic acid were added and allowed to react in a water bath at 25°C for 2 h. For each of the polyphenols, 5 mmol/L was added to the reaction system, and the mixture was maintained at 25°C for 24 h. Then, the reaction sample was dialyzed for 48 h (molecular weight cut-off: ≥ 10000 Da), with water changed every 6 h to remove any unreacted polyphenols. The dialysate was prefrozen and vacuum lyophilized for 48 h before being placed in a desiccator for later use. A UV-visible spectrophotometer (L6, Inesa Scientific Instrument Co., Ltd., Shanghai, China) was used to track the dialysate until no absorption remained at a specific wavelengths. All unreacted polyphenols were completely removed.

In the procedure above, two different controls were used to establish the impact of conjugation on the properties of the walnut proteins. WPI was prepared as a blank control using the same procedure as used to prepare the conjugates but in the absence of the redox pair and polyphenols. WPI-polyphenol mixtures were prepared using the same procedure used to prepare the conjugates but without adding the redox pair.

Binding Capacities Of The Mixtures And Conjugates

Measurement of polyphenol binding equivalents

The polyphenol binding equivalents were measured using the Folin-Ciocalteu reducing capacity method with slight modifications (Fan et al., 2018). Briefly, 1 mL of the WPI-polyphenol conjugates (1 mg/mL) was mixed with 2.5 mL of 10% (v/v) Folin-Ciocalteu reagent for 5 min. Next, 2 mL of 7.5% (w/v) Na₂CO₃ was added, and the sample was allowed to stand for 2 h in the dark. The absorbance was measured at 765 nm by using the UV-visible spectrophotometer. The phenolic content of EGCG, CLA, CA, or EA in each sample was calculated using the equations obtained from the calibration curves of each polyphenol, and the results were expressed as milligrams of polyphenol per gram of sample.

Measurement Of The Free Amino Group Content

The contents of free amino groups in the protein-polyphenol conjugates were determined following the ortho-phthaldialdehyde (OPA) method with slight modifications (Liu et al., 2015). The OPA reagent was prepared immediately before use by mixing the following reagents: 80 mg of OPA (dissolved in 2 mL of methanol), 50 mL of 0.1 M sodium borate buffer (pH 9.85), 200 µL of β-mercaptoethanol, and 5 mL of 20% (w/v) SDS in distilled water and distilled water was added to reach a volume of 100 mL to create the OPA reagent. Then, 4 mL of OPA reagent and 200 µL of protein solution (4 mg/mL) were thoroughly mixed and reacted in a 35°C water bath for 2 min. After that, the absorbance at 340 nm was measured, and 200 µL of water was added to the OPA reagent as a blank. L-leucine was used as the calibration curve from which the free amino group content (nmol/g) was calculated.

Measurement Of The Sulfhydryl Group Content

Ellman reagent was prepared as described previously with some modifications (Wu et al., 2018). In brief, 4 mg of DTNB was dissolved in 1 mL of 0.086 mol/L Tris-glycine buffer, 0.09 mol/L glycine, and 4 mmol/L EDTA (pH 8.0). Next, 15 mg of the WPI-polyphenol conjugates was dissolved in 5 mL Tris-glycine buffer and vortexed. Thereafter, 50 µL of Ellman reagent was added and mixed rapidly. The suspension was incubated at room temperature for 1 h. The absorbance at 412 nm was measured with WPI used as a blank.

The sulfhydryl group content was calculated using the following Eq. (1):

sulfhydryl group content (µmol/g) = (73.53 × A₄₁₂)/C (1)

where A₄₁₂ is the absorbance measured at 412 nm and C is the protein concentration of the sample (mg/mL).

Sds‒page Analysis

SDS‒PAGE experiments were performed as described previously with slight modifications (Offengenden et al., 2011). The vertical slab gel was 1.00 mm thick. The initial voltage was 100 V. After 30 min, the voltage was increased to 150 V, and the procedure was continued for an additional 90 min. The resolving gel concentration and the stacking gel concentration were 12% and 4%, respectively. The solution of WPI, WPI-polyphenol mixtures or WPI-polyphenol conjugates (3 mg/mL) was mixed with the same volume of loading buffer. The loading volumes of each well and the marker were 10 µL and 5 µL, respectively. To observe the protein following electrophoresis, the gel bands were stained with Coomassie Brilliant Blue R250. After 1–2 h, the dye was removed using a decolorizing solution. The gel bands were then soaked in distilled water overnight. Low-molecular-weight protein standards (10–180 kDa) were used to evaluate the molecular weight of the samples. A gel imager was used in an ImageQuant LAS 4000 digital imaging system (GE Healthcare, Piscataway, NJ, U.S.A.).

Structural Properties Of The Mixtures And Conjugates

FTIR

The FTIR of WPI, WPI-polyphenol mixtures, and WPI-polyphenol conjugates were obtained with the KBr pellet method with some modifications (Dardeer et al., 2022). The 1 mg sample was mixed with 200 mg KBr and ground into a uniform powder in an agate mortar. FTIR spectra were measured using an FTIR spectrophotometer (Perkin-Elmer, UK) from 400 cm^− 1 to 4,000 cm^− 1 with a resolution of 4 cm^− 1. The analysis was performed using PeakFit software (4.12, SeaSolve Software, Richmond, USA). By calculating the second derivative of the region, the main peaks representing the secondary structure of the protein were decomposed into α-helixes (1658 − 1650 cm^− 1), β-sheets (1640 − 1615 cm^− 1), β-turns (1670 − 1660 cm^− 1), and random coils (1650 − 1640 cm^− 1). The Gaussian equation was used to calculate the area of each peak (Karabulut et al., 2022).

Fluorescence Spectroscopy (Dup: Abstract ?)

The WPI, WPI-polyphenol mixtures, and WPI-polyphenol conjugates (0.1 mg/mL) were dissolved in 10 mM PBS (pH 7.0) and subjected to fluorescence spectroscopy (F-7000, Hitachi High-Technologies Corp, Tokyo, Japan) as described previously (Chen et al., 2020). The excitation and emission wavelength settings were 290 and 300–400 nm, respectively, and the slit width was 5 nm. PBS buffer solution without a sample was used as a blank.

Scanning Electron Microscopy (Sem)

To observe the micromorphology of the WPI, WPI-polyphenol mixtures, and WPI-polyphenol conjugates was used a JSM-7001F SEM (JEOL Ltd., Tokyo, Japan) at an accelerating voltage of 20 kV (Zhou et al., 2020). The freeze-dried sample was placed on conductive glue, and excess powder was scraped lightly from a small area. Then, the processed samples were sputter-coated with a gold layer for 30 s and observed under SEM. The SEM images were selected with a magnification of 5.00 k.

Antioxidant Activities

The antioxidant activities of the samples were evaluated by DPPH radical scavenging activity and reducing power. The percentage DPPH radical scavenging activity of the samples (0.5 mg/mL) was investigated by a method reported previously (Jia et al., 2019). Briefly, 1.75×10^− 4 mol/L DPPH solution was mixed with ethanol and stored in the dark. Next, 2 mL of sample solution (0.5 mg/mL) and DPPH solution (2 mL) were added to the same test tube and reacted for 30 min before the absorbance of the sample (${A}_{sample}$) was measured at 517 nm. The DPPH radical scavenging activity was calculated using Eq. (2):

DPPH radical scavenging rate (%) = $\left({A}_{0}-{A}_{sample}\right)/{A}_{0}\times 100$ (2)

where ${A}_{0}$ is the absorbance of DPPH without sample addition and ${A}_{sample}$ is the absorbance of DPPH with sample addition.

The power of the samples (0.5 mg/mL) to reduce Fe³⁺ to Fe²⁺ was determined according to the method reported previously (Yi et al., 2015). Each sample (2.5 mL) was mixed with 1% (w/v) potassium ferricyanide (2.5 mL) and 2.5 mL of phosphoric acid buffer (0.2 mol/L, pH 6.6). Then, the mixture was placed in a water bath at 50°C for 20 min, and 2.5 mL of trichloroacetic acid solution (10%, w/v) was added to stop the reaction. The mixture was centrifuged at 4,000 × g for 10 min at 4°C. The supernatant (5.0 mL) was collected and then mixed with distilled water (5.0 mL) and 0.1% (w/v) FeCl₃ (1.0 mL). Subsequently, the absorbance was measured at 700 nm, and the results are reported as absorbance values.

Surface Hydrophobicity (Dup: Abstract ?)

Surface hydrophobicity was determined using ANS as a fluorescence probe, as described by Dai et al. with slight modifications (Dai et al., 2022). Briefly, 20 µL of 8 mmol L^− 1 ANS solution was added to 4 mL of sample solution (1 mg/mL). The excitation wavelength was set to 390 nm, and the excitation and emission slit widths were set to 5 and 2.5 nm, respectively. The fluorescence intensity was plotted against the protein concentration, and the initial slope was calculated by linear regression analysis, which was the surface hydrophobic value.

Preparation Of Nanocarriers (Lyc)

Preparation of WPI-EGCG nanocarriers and nanocarriers (LYC)

WPI-EGCG (1 g L^− 1) was dispersed in PBS (0.01 mol L^− 1, pH 7.2) at 50°C for 120 min in a water bath and then self-assembled into nanocarriers by sonicating for 5 min. To prepare nanocarriers (LYC), LYC (0.8 g L^− 1) was dissolved in ethanol and added to the above WPI-EGCG nanocarriers (1.0 g L^− 1) at a volume ratio of 1:10 in an ice bath and then rotated at 4°C overnight. The obtained solution was centrifuged at 4000 rpm (HC-2518R, USTC Zonkla Scientific Instruments Co, Ltd, Hefei, Anhui, China) at 4°C for 15 min, and dialyzed overnight to remove free LYC (Wang et al., 2022). Nanocarriers and nanocarriers (LYC) powders were acquired by vacuum freeze-drying.

Characterization Of Wpi-egcg Nanocarriers And Nanocarriers (Lyc)

To verify whether LYC had been successfully combined with WPI-EGCG nanocarriers, the fluorescence of WPI-EGCG nanocarriers and nanocarriers (LYC) was scanned by a fluorescence spectrophotometer (F-7000, Thermo Fisher, USA). The emission fluorescence spectrum of LYC was excited at 488 nm and scanned at 300–800 nm (Chang et al., 2022).

The size distribution (z-average) and zeta potential of WPI-EGCG nanocarriers and nanocarriers (LYC) (1 mg mL^− 1) were dissolved in water (Chang et al., 2022) and measured by dynamic light scattering (DLS) (Nano-ZS 2000; Malvern Instruments, UK) at 25°C with a scattering angle of 90°C.

WPI-EGCG nanocarriers and nanocarriers (LYC) powders were dissolved in water and recorded by a digital camera.

Loading Efficiency Of Lyc

To determine the loading capacity of the nanocarriers (LYC), LYC was extracted from the nanocarriers (LYC) using DMSO at a volume ratio of 9:1 (DMSO: nanocarriers (LYC)). Briefly, the sample was extracted by sonicating for 15 min and then centrifuged at 10000 rpm for 10 min. The amount of nanocarriers (LYC) was calculated through the preacquired standard curve, and the absorbance was recorded at 474 nm. The loading efficiency (LE%) of LYC was determined using Eq. (3):

$$\text{L}\text{E} \left(\text{\%}\right)={\text{m}}_{LYC}/{m}_{nanocarries}\times 100\text{%}$$

where ${\text{m}}_{LYC}$ is the amount of LYC loaded in WPI-EGCG nanocarriers (mg) and ${m}_{nanocarries}$ is the total amount of WPI-EGCG nanocarriers (mg).

Statistical analysis

All experiments were conducted at least in triplicate under the same conditions. The data are expressed as the mean ± standard deviation and were plotted using Origin 8.5 software (Microcal, USA). Statistical analyses were performed using the SPSS 17.0 software (SPSS Inc., Chicago, IL, USA), while the one-way ANOVA test was used to analyze the difference between the means (p < 0.05).

Synthesis of the WPI-polyphenol conjugates

Free-radical grafting, which uses hydrogen peroxide and ascorbic acid as a redox pair of the initiator system, has been proven to be an efficient, simple, and economical strategy for producing protein-polyphenol conjugates (Liu et al., 2016). The binding capacity of the samples was determined through the Folin-Ciocalteu method and was expressed as total phenol equivalents (Feng & Kong, 2022). As shown in Table 1, the total EGCG, CLA, CA and EA contents were 9.22 ± 0.02, 8.99 ± 0.03, 7.65 ± 0.10 and 6.94 ± 0.01 mg/g in the mixtures, respectively. The phenol equivalents of EGCG, CLA, CA, and EA by free-radical grafting were 38.21 ± 2.32, 33.33 ± 1.16, 19.91 ± 1.71, and 9.67 ± 0.66 mg/g, respectively, which confirmed the conjugation of the four polyphenols with WPI. The conjugates induced by free radicals showed higher binding capacity than those fabricated by the physical method because more active sites are generated on the protein surfaces that can interact with polyphenols (Gu et al., 2017). The binding capacity of polyphenols was different; EGCG showed the highest binding capacity, followed by CLA, CA, and EA, which may be ascribed to individual variations in molecular weight, phenolic hydroxyl content, and free radical binding sites (Liu et al., 2021). Liu et al. (Liu et al., 2016) reported that the binding affinity of phenolic compounds with proteins was mainly determined by the amount and position of hydroxyl groups because they had a greater number of binding sites available for interaction with proteins, which was consistent with the results of our research.

Table 1

The contents of polyphenol binding equivalents, free amino groups and sulfhydryl groups in WPI, WPI-polyphenol mixtures and conjugates.
Sample	Polyphenol binding equivalents (mg/g)	Free amino groups (µmol/mg protein)	Sulfhydryl groups (µmol/mg protein)
WPI	------	89.33 ± 0.32^a	48.33 ± 2.88^a
WPI-EGCG conjugates	38.21 ± 2.32^a	47.54 ± 1.31ⁱ	13.87 ± 0.12ⁱ
WPI-CLA onjugates	33.33 ± 1.16^b	50.11 ± 1.36^h	15.29 ± 0.07^h
WPI-CA conjugates	19.91 ± 1.71^c	56.98 ± 2.33^g	19.07 ± 0.62^g
WPI-EA conjugates	9.67 ± 0.66^d	59.83 ± 2.32^f	23.39 ± 0.99^f
WPI-EGCG mixtures	9.22 ± 0.02^e	60.66 ± 1.11^e	25.12 ± 1.73^e
WPI-CLA mixtures	8.99 ± 0.03^f	67.23 ± 1.82^d	31.64 ± 2.66^d
WPI-CA mixtures	7.65 ± 0.10^g	70.44 ± 2.77^c	32.65 ± 2.12^c
WPI-EA mixtures	6.94 ± 0.01^h	75.21 ± 1.56^b	37.84 ± 2.51^b
^a Data are expressed as means ± standard deviations. Values with the different letters in the same column represent a significant difference (p < 0.05). ^b Abbreviations: EGCG; Epigallocatechin gallate, CLA; chlorogenic acid, CA; (+)-catechin, and EA; ellagic acid.

Changes In Free Amino Groups And Sulfhydryl (Sh) Groups

The binding capacity of the mixtures and conjugates was also evaluated through measurement of the free amino and sulfhydryl groups content. As shown in Table 1, the free amino and sulfhydryl group contents of these WPI-polyphenol mixtures and conjugates were all significantly lower than those of WPI. The attachment of polyphenols into WPI was monitored by the decreased contents of free amino and sulfhydryl groups in WPI. It could be noted that the free amino groups of WPI-polyphenol conjugates notably decreased when compared to WPI-polyphenol mixtures. Therefore, we postulated that the reactivity of the phenolic compounds was influenced by the different preparation methods and demonstrated that the free radical grafting method allowed for easier reaction between polyphenols and proteins; the specific principle is mainly related to the reaction mechanism of the two methods. In the reaction induced by the free radical method, the ortho- and para-positions of the hydroxyl group on the aromatic ring were vulnerable to attack by free radicals and combined with the macromolecule to form covalent bonds. It is worth noting that the number of free amino groups was determined in the presence of 1% SDS, which is a denaturant that destroys noncovalent protein interactions, suggesting that the decrease in free amino content may be due to the covalent binding of different polyphenols to WPI (Wu et al., 2018). Meanwhile, in the covalent binding reaction, the reactivity and binding strength with free amino groups ranked in the order of WPI-EGCG > WPI-CLA > WPI-CA > WPI-EA. These results confirmed the influences of the number and position of hydroxyl groups on the reactivity of the phenolic compounds. In addition, the remaining sulfhydryl group content of these conjugates was consistent with the results for the free amino group content (Table 1). The presence of 8 mol∙L^− 1 urea eliminated the interference of disulfide bonds (Prigent et al., 2007). The decreased sulfhydryl group content further confirmed the formation of covalent bonds. As reported, oxidized phenolic compounds can react with nucleophilic groups, such as amino groups, tryptophan, cisterns, methionine, histidine, tyrosine and N-terminal proline, in proteins (Kroll et al., 2003). Our results indicated that the formation of C-N and C-S bonds between the benzene ring of polyphenols and the nucleophilic groups of WPI might be achieved.

SDS‒PAGE

The molecular weights (MWs) of the major components in WPI, WPI-polyphenol mixtures, and WPI-polyphenol conjugates were determined through SDS‒PAGE (Fig. 1). WPI (Lane 1) showed several major bands: ~50, ~ 31–37, and ~ 20–25 kDa. This finding was consistent with those reported by Jin et al. (Jin et al., 2020), who reported the molecular weight of dehulled walnut protein. The SDS‒PAGE profile of the WPI-polyphenol mixtures was similar to that of WPI (Lanes 2, 4, 6 and 8). Due to the weak noncovalent (physical) interactions between polyphenol and WPI, polyphenol will have been removed from the proteins during the dialysis process. In contrast, the polyphenol would have remained more closely attached to the proteins in the WPI-polyphenol conjugates because they were held together by strong covalent (chemical) bonds. As shown in Fig. 1, the SDS‒PAGE profile of the WPI-polyphenol conjugates was considerably different from that of WPI, which demonstrated the increased MWs of the conjugates. This observation was consistent with those reported by Liu et al. (Liu et al., 2021), in which the molecular weight of the walnut protein isolate increased after conjugation with polyphenol. The bands corresponding to WPI-CA (Lane 3) and WPI-EA (Lane 4) did not migrate significantly, perhaps because of a low degree of conjugation. This also indicated that the binding capacities of WPI-CA and WPI-EA were lower than those of WPI-EGCG and WPI-CLA.

FTIR

The WPI-polyphenol mixtures and conjugates were characterized by infrared spectroscopy. FTIR spectroscopy is valuable for monitoring changes in the secondary structure of proteins (Jing et al., 2020). Figure 2 depicts the FTIR spectra of the WPI-polyphenol mixtures and conjugates obtained using (A) the physical methods and (B) free radical methods. The positions and areas of the characteristic absorption peaks of the eight WPI-polyphenol mixtures and conjugates were different from those of WPI, indicating that some chemical changes had occurred. For instance, the absorption bands at 1600–1700 and 1450–1550 cm^− 1 (Liu et al., 2021), which corresponded to the amide I and amide II groups, redshifted to lower wavenumbers, indicating the strong stretching vibration of the C-N covalent bonds in the conjugates. This finding indicated that different polyphenols were conjugated to WPI by C-N covalent bonds. Furthermore, the intensities of the 3200–3700 cm^− 1 peaks increased significantly in all the WPI-polyphenol mixtures and conjugates, which might be due to the introduction of high O-H contents from the polyphenols (Jia et al., 2016).

As mentioned, amide I bands are commonly more sensitive than amide II bands in assessing such secondary structural protein changes. Therefore, the amide I band (1600–1700 cm^− 1) was more useful in the present analysis (Yang et al., 2014). To determine the secondary structure of the protein, Fourier self-deconvolution and a Gaussian function were used to fit the FTIR spectra in this region (Table 1). Table 2 shows that the WPI group contained 7.38%, 79.93%, 5.59%, and 7.10% α-helixes, β-sheets, β-turns, and random coils, respectively. The mixtures and conjugates underwent varying degrees of changes in the secondary structure. Regarding WPI-EGCG conjugates, β-sheets decreased substantially from 79.93–40.81% and β-turns increased from 5.59–21.38%, respectively, through the conjugation process, whereas α-helixes and random coils both increased from 7.38–18.11% and from 7.10–19.71%, respectively; these changes in the secondary structure might result from the covalent interactions between WPI and EGCG. In addition, CLA, CA and EA caused different changes in the secondary structure distribution of WPI, which clearly demonstrated a perturbation that was dependent on polyphenol structure and phenolic hydroxyl group content (Liu et al., 2021).

Table 2

The secondary structure contents of WPI, WPI-polyphenol mixtures and conjugates.
	α-helix(%)	β-sheet(%)	β-turns(%)	Random coil(%)
WPI	7.38	79.93	5.59	7.10
WPI-EGCG conjugates	18.11	40.81	21.38	19.71
WPI-CLA conjugates	9.62	50.33	22.81	17.23
WPI-CA conjugates	17.51	48.64	7.87	25.98
WPI-EA conjugates	20.30	45.49	10.00	24.20
WPI-EGCG mixtures	14.15	42.56	28.09	15.21
WPI-CLA mixtures	22.76	35.64	12.39	29.22
WPI-CA mixtures	21.66	26.50	23.26	28.59
WPI-EA mixtures	12.89	51.43	18.21	17.46
^a Data are expressed as means ± standard deviations. Values with the different letters in the same column represent a significant difference (p < 0.05). ^b Abbreviations: EGCG; Epigallocatechin gallate, CLA; chlorogenic acid, CA; (+)-catechin, and EA; ellagic acid.

Fluorescence Spectroscopy

The fluorescence was attributed to the tryptophanyl and tyrosyl residues (Lu et al., 2018). When the excitation wavelength is set at 290 nm, the luminescent groups are sensitive to their microenvironment, so they are widely used to measure changes in protein tertiary structure (Jiang et al., 2018). As depicted in Fig. 3, the fluorescence intensity of these mixtures and conjugates was clearly lower than that of WPI (606.86 a.u.). The fluorescence intensities of the mixtures (234.21-336.81 a.u.) were obviously higher than those of conjugates (130.26-186.88 a.u.). Specifically, the fluorescence intensities of the WPI-EGCG, WPI-CLA, WPI-CA, and WPI-EA conjugates were 130.26, 155.06, 157.17, and 186.88 a.u., respectively. The decreases in fluorescence intensity indicated that the tryptophan residues of the protein were most likely involved in the cross-linking between the WPI and polyphenol molecules, particularly in the WPI-EGCG conjugates. Similar trends were observed in the free amino and sulfhydryl group contents.

Additionally, the maximum emission wavelength for the WPI was 325 nm; generally, the fluorescence intensity was significantly decreased and the maximum emission was redshifted to 326 nm for the mixtures and conjugates. The redshift indicated that tryptophan residues were more exposed to the solvent or the redshift resulted from the transfer of tryptophan residues into a more hydrophilic environment, which implied that the polarity of the protein environments had increased, causing the protein structures to unfold (Liu et al., 2015). Similar results were also observed from the conjugates of whey protein isolate and EGCG, QC, AG and NG (Liu et al., 2021).

SEM

The surface morphologies of WPI, WPI-polyphenol mixtures and conjugates were characterized through SEM. The SEM images of the samples are depicted in Fig. 4. The surface of WPI showed complex structures comprised of random sheets of various sizes and shapes, which is consistent with previous observations (Jin et al., 2020). The flaky structure was attributed to the association of protein molecules at a high density, which can influence their dispersity and solubility (Yan & Zhou, 2021). In contrast, the WPI-polyphenols showed a rougher surface with small dense concave holes, which was probably due to the impact of polyphenols on the surface hydrophobicity and charges of walnut proteins. These structural features were more prominent in WPI-EGCG conjugates (Fig. 4B) than in other polyphenols (Fig. 4C- I). Changes in the SEM indicated that different polyphenols may effectively act on the protein structure. Similar microstructures have been described in previous works (Zhu et al., 2018).

Antioxidant Capacity

DPPH radical scavenging assays and reducing power were used to determine the antioxidant properties of WPI and the WPI-polyphenol mixtures and WPI-polyphenol conjugates. As shown in Fig. 5A, the antioxidant activity of the WPI-polyphenol mixtures (38.36–63.85%) was slightly higher than that of WPI (18.41%), which may have been because of the presence of polyphenols that were weakly bound to the protein molecules through noncovalent interactions. The DPPH radical scavenging activities of WPI-EGCG, WPI-CLA, WPI-CA, and WPI-EA conjugates were 92.03%, 88.73%, 69.85%, and 64.44%, respectively, which were significantly higher than those of WPI and WPI-polyphenol mixtures. It was reported that the resulting conjugates induced by free radicals presented higher antioxidant activity than those fabricated by the physical method (Gu et al., 2017) because more active sites are generated on the protein surfaces that can interact with polyphenols.

In addition, the reducing power of these mixtures and conjugates was consistent with the results of the DPPH radical scavenging assays (Fig. 5B). The reducing power of the different samples increased in the following order: WPI (0.12) < WPI-polyphenol mixtures (0.23–0.60) < WPI-polyphenol conjugates (0.61–0.81). In WPI-polyphenol conjugates, the WPI-EGCG conjugates had the most antioxidant capacity, followed by CLA, CA and EA, which might be linked to the high content of phenolic hydroxyl groups in the EGCG molecule. In summary, the assays used indicated that covalent attachment of polyphenol to WPI significantly improved the antioxidant activity of WPI, similar to other reports in which the antioxidant activity of egg white proteins was significantly improved by covalent conjugation with tea polyphenol using alkaline/free radical methods (Jing et al., 2020).

Surface Hydrophobicity

Surface hydrophobicity is an index of the number of hydrophobic groups on protein surfaces and contributes critically to proteins’ surface-related functions (Zhan et al., 2018). As depicted in Fig. 6A and 6B, the reaction considerably reduced the fluorescence intensity of all the mixtures and conjugates. The surface hydrophobicity of the mixtures (177.02-205.84) was obviously higher than that of the conjugates (119.26-175.88). Specifically, the surface hydrophobicities of the WPI-EGCG, WPI-CLA, WPI-CA, and WPI-EA conjugates were substantially reduced by 58.88%, 46.66%, 40.88%, and 39.13%, respectively. This might be because WPI-polyphenol conjugates were formed through hydrophobic interactions. When WPI interacts with polyphenols, it undergoes conformational changes and structural opening. Meanwhile, hydrophobic groups such as Trp are exposed and new hydrophilic groups (hydroxyl and carboxyl) are introduced, which decreases surface hydrophobicity (Liu et al., 2021). WPI-EGCG had the lowest hydrophobicity because of the combination of hydrophobic groups and EGCG with a high hydroxyl content. These observations are compatible with soy protein isolate and catechin (Dai et al., 2022).

Characterization Of Lyc-loaded Wpi-egcg Nanocarriers

WPI-EGCG had the best functional properties; for this reason, WPI-EGCG nanocarriers were designed. The average particle size of the nanocarriers was 261.0 ± 1.5 nm; after loading LYC, the average particle size of the nanocarriers was 318.2 ± 2.3 nm (Table S1). The size of the nanocarrier (LYC) was larger (318.2 nm) than that of the nanocarrier (261.0 nm), most likely because the loading of LYC expanded its volume. In addition, the zeta potential of the WPI-EGCG nanocarriers was − 26.7 mV (pH 7.2), indicating that the nanocarriers were negatively charged. The zeta potential of nanocarriers (LYC) increased from − 26.7 mV to -21.87 mV (pH 7.2), suggesting that some LYC might also be adsorbed on the nanocarrier surface, which screened some charges (Chang et al., 2022).

Fluorescence spectroscopy confirmed that LYC was embedded in the nanocarriers (Fig. 7A). The maximum absorption peak for LYC and the nanocarriers (LYC) had the same absorption peak at approximately 491 nm, indicating that LYC was successfully embedded in the nanocarriers (Wang et al., 2022). Liposoluble bioactive compounds can be loaded into the tubular hydrophobic walls of these nanocarriers by hydrophobic interactions (Chang et al., 2022). To characterize the loading ability of nanocarriers, the loading efficiency (LE%) of LYC by WPI-EGCG nanocarriers was 20.22% (Table S1). The powder of nanocarriers (LYC) demonstrated an improved aqueous dispersibility, showing that nanocarriers can greatly improve the aqueous solubility of LYC (Fig. 7B). Therefore, it could be formulated in water-based dairy drinks via encapsulation.

In this research, mixtures and conjugates of WPI and each of the four dietary polyphenols (EGCG, CLA, CA, and EA) were prepared through a physical method and free radical grafting, respectively. In descending order, the binding capacities of the samples were as follows: WPI-EGCG > WPI-CLA > WPI-CA > WPI-EA, regardless of mixtures and conjugates. The increases in the conjugated molecular weights and the reduction in the free amino groups and sulfhydryl groups content in the protein confirmed the formation of covalent bonds between WPI and the polyphenols. FTIR, fluorescence spectroscopy and SEM revealed notable changes in protein structure. Functional properties including antioxidant capacity and surface hydrophobicity were changed and improved to some extent. WPI-EGCG had the best functional properties. WPI-EGCG nanocarriers were prepared by self-assembly, and the hydrophobic sensitive LYC was loaded into WPI-EGCG nanocarriers. This research is warranted to provide a theoretical basis for the application of WPI-polyphenol conjugates in transport systems, and is important for increasing the utilization of walnut protein isolates in the food industry.

Author Contributions Yuzhen Wang: Experiments, Methodology, Software, Investigation, Formal analysis, Writing - original draft. Yu Chen: Data curation, Software, Formal analysis. Jiao Lv: Validation, Formal analysis, Data curation. Chang Li: Validation, Formal analysis, Data curation. Fengjun Wang: Resources, Conceptualization, Supervision, Writing - review & editing, Funding acquisition, Validation.

Funding This research was supported by a Beijing Forestry University Sci-Tech innovation Project (BFUKF202212) and a National Innovation Training program for college students (G202010022091).

Data Availability The authors declare that the data supporting the findings of this study are available within the article. The raw/derived data supporting the findings of this study are available from the corresponding author on request.

Ethics Approval Not applicable.

Consent to Participate Not applicable.

Consent for Publication Not applicable.

Conflict of Interest/Competing Interests Authors declare no confict of interest.

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Evaluation of different dietary polyphenols on conformational, functional changes and lycopene-loaded nanocarriers of walnut protein isolate

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Abstract

Figures

Introduction

Materials And Methods

Materials

Preparation Of Wpi- Polyphenol Mixtures And Conjugates

Binding Capacities Of The Mixtures And Conjugates

Measurement of polyphenol binding equivalents

Measurement Of The Free Amino Group Content

Measurement Of The Sulfhydryl Group Content

Sds‒page Analysis

Structural Properties Of The Mixtures And Conjugates

FTIR

Fluorescence Spectroscopy (Dup: Abstract ?)

Scanning Electron Microscopy (Sem)

Antioxidant Activities

Surface Hydrophobicity (Dup: Abstract ?)

Preparation Of Nanocarriers (Lyc)

Preparation of WPI-EGCG nanocarriers and nanocarriers (LYC)

Characterization Of Wpi-egcg Nanocarriers And Nanocarriers (Lyc)

Loading Efficiency Of Lyc

Statistical analysis

Results And Discussion

Synthesis of the WPI-polyphenol conjugates

Changes In Free Amino Groups And Sulfhydryl (Sh) Groups

SDS‒PAGE

FTIR

Fluorescence Spectroscopy

SEM

Antioxidant Capacity

Surface Hydrophobicity

Characterization Of Lyc-loaded Wpi-egcg Nanocarriers

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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