Insighting the effect of ultrasound-assisted polyphenol non-covalent binding on the functional properties of myofibrillar proteins from golden threadfin (Nemipterus virgatus)

Graphical abstract

Highlights

• •The structure change of Myofibrillar protein through ultrasound assisted polyphenols addition was analyzed by the multispectral analysis.
• •Ultrasound assisted polyphenols addition reduced the Oil/Water interfacial tension of Myofibrillar protein.
• •Ultrasound assisted polyphenols addition improved the emulsification, foaming and antioxidant properties of Myofibrillar protein.

Abstract

In this study, the effect of ultrasound-assisted non-covalent binding of different polyphenols (tannins, quercetin, and resveratrol) on the structure and functional properties of myofibrillar proteins (MP) from the golden threadfin (Nemipterus virgatus) was investigated. The effect of ultrasound-assisted polyphenol incorporation on the structure and properties of MP was evaluated by multispectral analysis, interfacial properties, emulsification properties and antioxidant properties et al. The results revealed that the protein–polyphenol interaction led to a conformational change in the microenvironment around the hydrophobic amino acid residues, resulting in an increase in the equilibrium of the MP molecules in terms of affinity and hydrophobicity. Ultrasound assisted polyphenols addition also led to a significant decrease of the oil/water interfacial tension (from 21.22 mN/m of MP to 8.66 mN/m of UMP-TA sample) and a significant increase of the EAI (from 21.57 m²/g of MP to 28.79 m²/g of UMP-TA sample) and ES (from 84.76 min of MP to 124.25 min of UMP-TA). In addition, ultrasound-assisted polyphenol incorporation could enhance the antioxidant properties of MP, with the DPPH and ABTS radical scavenging rate of UMP-TA increase of 47.7 % and 55.2 % in comparison with MP, respectively. The results demonstrated that the noncovalent combination with polyphenols under ultrasound-assisted conditions endowed MP with better functional properties, including solubility, emulsification, foaming, and antioxidant properties through structure change. This study can provide innovative theoretical guidance for effectively preparing aquatic protein–polyphenol non-covalent complexes with multiple functions and improving the processing and utilization value of aquatic proteins.

1. Introduction

Golden threadfin is one of the fish with the highest economic value in China's South China Sea seafood. Compared with other seawater fish, golden threadfin has an absolute advantage in maintaining the relative stability of resources and catch. The flesh of golden threadfin is rich in protein, of which myofibrillar protein (MP) accounts for > 65 % of the total protein of the fish flesh, which is mainly composed of myosin and actin [1]. However, the characteristics of MP such as solubility, poor dispersibility, and easy oxidization and deterioration, directly limited its application in the food industry [2]. Therefore, how to modify the structure of MP proteins to improve their functional properties has become one of the current research hotspots of the golden threadfin.

Ultrasonic wave is a new food processing technology, which has been widely applied in the treatment and modification of food industry. Ultrasonication has attracted much attention from researchers by promoting the unfolding of protein structures through cavitation and mechanical effects, thereby increasing the binding capacity of proteins with small molecules such as polyphenols. Li et al. reported that the emulsification activity index (EAI) and emulsion stability (ES) of chicken myofibrillar proteins increased significantly after ultrasonication, and myofibrillar proteins could form more stable emulsions [3]. Tian et al demonstrated that ultrasound has a significant effect on conformational and physicochemical properties, facilitating the degradation of protein aggregates by disrupting the hydrogen bonding and hydrophobic interactions that sustain proteins [4].

Phenolic compounds are widely found in fruits and herbs. They have good antioxidant properties and positive effects on human health. Therefore, they are usually incorporated into food matrices to improve food quality and stability [5]. Polyphenols are unique phenolic compounds containing a large number of hydroxyl and carboxyl structures, which can be combined with proteins to form complexes and change the structural and functional properties of proteins. The binding between proteins and polyphenols can be divided into covalent binding and non-covalent binding. Covalent binding of polyphenols to proteins was found to have undesirable effects such as reducing protein digestibility and bioavailability et al [6]. Compared with covalent binding, non-covalent modification has received increasing research attention due to its greater flexibility in altering protein structure. Hao et al have investigated the non-covalent interactions of pea isolate protein (PPI) with epigallocatechin-3-gallate (EGCG), chlorogenic acid (CA), and resveratrol (RSV). It was found that the emulsification, foaming and in vitro digestibility of PPIs were significantly increased upon binding with phenolic compounds [7]. Chen et al. have used the non-covalent binding of tannic acid with ovalbumin to prepare ovalbumin-tannic acid complexes and found that its interfacial activity was reduced and the emulsification stability at the isoelectric point was significantly increased compared with ovalbumin [8].

Both ultrasonic treatment and non-covalent binding of polyphenols have the potential to improve and promote the functional properties of proteins. However, the studies on the properties of MP from golden threadfin (Nemipterus virgatus) through combining ultrasonic preconditioning with polyphenol addition are still limited. In this study, the effect of ultrasonic-assisted pretreatment combined with polyphenols (tannins, quercetin, and resveratrol) non-covalent binding on the structural and functional properties of MP was investigated. The multi-scale structure characterization of MP was carried out through the combination of multiple spectroscopies (such as FT-IR, CD, endogenous fluorescence, and synchronous fluorescence et al.) and the functional properties (such as emulsification, foaming, and antioxidant properties et al.) were also measured. This study will clarify the mechanism how ultrasonic pretreatment combined with polyphenols non-covalent binding on the functional properties of MP through the joint analysis of its multi-scale structure. The results of this study will also provide a reference for the utilization of ultrasonic pretreatment and polyphenols addition in the marine fishery processing industry.

2. Materials and methods

2.1. Materials

The frozen surimi of olden threadfin was purchased from LDT Qingdao Sheng teng Seafood Co., Ltd (September 2023 batch, AAA grade, protein content 19.50 %, moisture content was 74.20 %). Tannic acid (TA), quercetin (QR), and resveratrol (RSV) were purchased from Zhejiang Yi wu Yicheng Bio-technology Co, Ltd. soybean oil was purchased from Yi hai Jiali Foods Marketing Co, Ltd. Potassium bromide (spectroscopic grade) was purchased from Shanghai McLean Biochemical Technology Co. All other reagents used in this study were of analytical grade.

2.2. Extraction of myofibrillar proteins

MP was extracted from Goldline fish following the method of Wang et al [9]. First, take an appropriate amount of frozen golden fillet surimi, ground the meat, add 5 times the volume of 10 mmol/L, pH 7.0 phosphate buffer mix thoroughly, centrifuge at 9000 g for 15 min, and collect the precipitation. Then, take the last precipitation, add 5 times the volume of 10 mmol/L phosphate buffer (0.6 mol/L NaCl, pH 7.0), and ground the precipitation. After standing in the ice bath for 1 h, centrifuge at 9000 g for 15 min and filter with three layers of gauze. Finally, take the supernatant with a measuring cylinder to take the volume, add 10 times 4 ℃ distilled water, 9000 g centrifuge for 10 min, the resulting precipitation was myofibrillar protein, stored at 4 ℃ for use.

2.3. Ultrasound-assisted preparation of myofibrillar protein −polyphenol-complexes

Goldfish myofibrillar proteins were taken and a 1 % (w/v) concentration protein solution was prepared. The samples were ultrasonicated for 20 min (300 W, 2 s between treatments), and 0.05 % (w/v) tannic acid (TA), 0.05 % (w/v) quercetin (QR), and 0.05 % (w/v) resveratrol (RSV) were added, respectively. Then, the samples were magnetically stirred for 1 h under the condition of ice bath. Among them, the goldfish myofibrillar protein solution was labeled as MP; the sonicated pretreated protein solution was labeled as UMP; the sonicated pretreated combined tannic acid Yan sample was labeled as UMP-TA; the sonicated pretreated combined quercetin sample was labeled as UMP-QR; and the sonicated pretreated combined resveratrol sample was labeled as UMP-RSV.

2.4. Characterization of myofibrillar protein–polyphenol complexes

2.4.1. Protein solubility

The determination of protein solubility was according to the method of Wang et al, with minor modification [10]. 20 mL of myofibrillar protein sample (2 mg/mL) under different conditions was taken in a 50 mL centrifuge tube, centrifuged (9000 g, 15 min), and the supernatant was taken, and the protein concentration in the supernatant was determined by Lowry's method. The formula for calculating solubility is as follows:

$$\mathrm{s}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{c}{c_0}\times 100$$ (1)

Where: C₀ is the concentration of protein before centrifugation, 2 mg/mL; C is the concentration of protein in the supernatant after centrifugation, mg/mL.

2.4.2. Zeta potential

The Zeta potential test was conducted using a nanoparticle size analyzer. The method of determination referred to Zhang et al, with appropriate modification [11]. To avoid multiple scattering, solutions of sonication-assisted polyphenol-bound MP were diluted 200-fold using distilled water at the corresponding pH, and zeta potentials were measured at room temperature using a Nano Brook Omni Zeta Potentiometer. The sample equilibration time was 120 s.

2.4.3. SDS-PAGE

SDS-PAGE was applied by the method of Zheng et al, with a slight modification [10]. First, the protein concentration was adjusted to 1.5 mg/mL, and a 100 μL sample was added to a 50 μL loading buffer. Then, 5 % concentrated glue and 12 % separation glue were used for electrophoresis, and the loading amount was 10 μL. In addition, the voltage of the sample in the concentrated glue was constant at 80 V, and the voltage was adjusted to 120 V after entering the separation glue. Finally, the electrophoresis was stopped when the blue strip was 3–5 mm away from the bottom of the adhesive.

2.4.4. Total sulfhydryl content

The determination method for total sulfhydryl group content was based on Bian et al.'s method with slight modification [12]. Using the total sulfhydryl content assay kit, 10 mg of reduced glutathione (GSH) was added to 1.3 mL of distilled water before use and configured to 25 μmol/mL, and a standard curve was established based on the concentration of the standard tube (x, μmol/mL) and the absorbance ΔH standard (y, ΔH standard). Based on the standard curve, the curve equation was measured as y = 0.036x + 2.332 (R² = 0.998). The ΔH measurement (y, ΔH measurement) was brought into the equation to calculate the sample concentration. The total sulfhydryl content (μmol/mg) was equal to the sample concentration calculated from the standard curve divided by the sample protein concentration.

2.4.5. Ultraviolet spectra

The UV spectra determination was based on Bian et al, with slight modification [12]. Solutions of MP, UMP, UMP-TA, UMP-QR, and UMP-RSV (1.0 mL) were mixed with PBS (2.0 mL) and the UV–visible spectra were measured using a UV–visible spectrophotometer in the range of 200–400 nm. The blank control was PBS.

2.4.6. FT-IR spectroscopy

FT-IR spectra of samples were based on Hao et al, with slight modification [7]. The freeze-dried five samples were thoroughly ground by mixing them with potassium bromide in the ratio of 1:100 and then this mixture was loaded into a metal mold. After that, the samples were compressed into tablets by a tablet press at 15–20 MPa for 30 s. FT-IR spectra were obtained by scanning the samples 32 times in the wave number range of 4000–400 cm⁻¹.

2.4.7. Far-UV circular dichroism (CD)

The secondary structure of protein samples was measured according to the method of Zhang et al [11]. Myofibrillar protein emulsion and desorbed interfacial protein were diluted to 0.1 mg/mL with pH buffer and scanned by circular dichroic chromatography. The wavelength range was 190–260 nm, the sample volume was 0.2 mL, the resolution was 0.5 mm, the scanning speed was 100 nm/min, the sensitivity was 20 mdeg, the response time was 0.25 s, and the buffer was used as blank. The secondary structure content was calculated using CDNN software.

2.4.8. Fluorescence spectroscopy

The fluorescence intensity was determined by a fluorescence spectrophotometer and slightly modified by Wang et al [13]. The samples were diluted to a protein concentration of 0.1 % (w/v) with ultrapure water. Emission wavelengths from 295 nm to 450 nm were collected and the slit width and excitation wavelength were set to 5 nm and 290 nm, respectively. In addition, Δλ was set to 15 nm and 60 nm, respectively, and the excitation and emission wavelength slit widths were both 5 nm, and the synchronized fluorescence spectra were recorded in the synchronized scanning mode. Finally, the fluorescence spectra of the complexes were recorded by fluorescence spectrophotometer at an excitation wavelength of 295 nm. Protein fluorescence bursts caused by energy transfer from tryptophan (Trp) and tyrosine (Tyr) residues to ligands can directly respond to ligand–protein binding. Quantitative analysis of burst data based on the Stern-Volmer equation:

$$\frac{F_0}{F}=1+K_{\mathit{sv}}\left[\mathrm{Q}\right]=1+K_q{T}_0[Q]$$ (2)

where F₀ and F denote the fluorescence intensity of the UMP with unbound and bound polyphenols, respectively, and [Q] and K_sv were the concentration and quenching constant of the polyphenols, respectively. K_q and T₀ were the fluorescence quenching constant and the fluorescence lifetime in the absence of a quencher, respectively, which was typically 10^-8 s for biological macromolecules. When K_q was greater than the maximum dynamic diffusion quenching constant (2.0 × 10¹⁰ L mol⁻¹ s ^-1), it is a static quenching, otherwise it is considered a dynamic quenching.

2.4.9. Surface hydrophobicity index (H₀)

protein surface hydrophobicity was determined by the ANS fluorescent probe method，according to the method of Han et al [14]. The protein solution was diluted to four concentrations (0.5, 0.25, 0.125, 0.0625 mg/mL), 4 mL of different concentrations were taken and 20 μL of ANS solution (8.0 mmol/L ANS, 0.01 mol/L Tris-HCL, pH 7.0) were added respectively, mixed well, and then left to stand for 10 min away from the light. excitation wavelengths, emission wavelengths, excitation wavelength, emission wavelength, and slit width were 374 nm, 485 nm, and 5 nm, respectively. The relative fluorescence intensity (RFI) value of the protein was obtained by subtracting the fluorescence intensity value of the sample to which the probe was added from the endogenous fluorescence intensity value of the corresponding sample. The RFI was plotted against the protein concentration and the slope of its initial segment was used as an indicator of the surface hydrophobicity of the protein (H₀).

2.4.10. Dynamic interfacial tension

Dynamic interfacial tension was measured using the method described by Peng et al, with minor modification [15]. The sample concentration was adjusted to 0.1 mg/mL. Measurements were made with an automated contact angle goniometer and data were collected programmatically using DROP image software. The DROP image software requires the input of component phase densities to calculate interfacial tension from droplet shape analysis. The pendant drop method is used to assess interfacial tension. A drop of solution equivalent to 200 μL was hung on a needle in a water-saturated environmental chamber to prevent evaporation. A digital camera captured images of the droplets, and automatic image analysis software calculated the surface tension every 2 s over a 600 s period. Experiments were performed at room temperature (22 ± 2 °C) and instruments were calibrated daily using the spherical portion of the Precision Combined Calibration Device. Surface tension experiments were completed in duplicate.

2.4.11. Emulsifying activity index (EAI) and emulsifying stability (ES)

The emulsification activity index (EAI) and emulsification stability (ES) of MP were determined by the turbidity method and were modified concerning the method of Wang et al [10]. 6.0 mL of surimi particulate protein solution with different pH values and 2.0 mL of soybean oil were taken into 50 mL plastic measuring cups and homogenized at 12000 g for 1 min, and 20 μL of the freshly prepared emulsion was immediately taken and dispersed in 4 mL of SDS solution containing a mass concentration of 0.1 %. The optical density at 500 nm was quickly read and noted as A₀. After the samples were allowed to stand for 10 min, 20 μL was sampled again at the same location and the optical density was recorded as A₁₀ by repeating the above steps. EAI and ESI were calculated according to the following formulas, respectively:

$$\mathrm{E}\mathrm{A}\mathrm{I}(\mathrm{m}2/\mathrm{g})=\frac{2\times 2.303\times A_0\times N}{C\times \varnothing \times A_{10}}$$ (3)

$$\mathrm{E}\mathrm{S}(\mathrm{m}\mathrm{i}\mathrm{n})=\frac{A_0}{A_0-A_{10}}\times \mathrm{\Delta }t$$ (4)

Where A₀ was the initial optical density value of the sample; A₁₀ was the optical density value of the sample after being placed for 10 min; N was the dilution multiple, N = 200; C was the protein concentration, C = 0.5 %; Ф=0.2; Δt = 10 min.

2.4.12. Foaming activity index (FC)and foaming stability (FS)

The determination of MP foamability was based on the method of Wang et al, with appropriate modification [10]. Each protein sample was configured as a 0.2 % (w/v) protein solution, stirred for 1 h, and then homogenized for 1 min at 20,000 g. The volume of the resulting foam was read and counted as V₀. The homogenized sample was allowed to stand for 30 min and the foam volume was re-read and counted as V₃₀. Foaming capacity (FC) and foaming stability (FS) were calculated using the following equations:

$$\mathrm{F}\mathrm{C}({\mathrm{c}\mathrm{m}}^3/\mathrm{g})=\frac{V_0}{m}$$ (5)

$$\mathrm{F}\mathrm{S}(\%)=\frac{V_{30}}{V_0}\times 100$$ (6)

Where m is the mass of protein used (g).

2.4.13. DPPH radical scavenging capacity

The determination of DPPH radical scavenging capacity was based on the method of wang et al [10]. 1 mg/ml DPPH solution was prepared with ethanol, while the sample was re-dissolved in distilled water to 0.5 mg/mL. 2 mL of sample solution was mixed with 2 mL of DPPH solution, and the mixture was protected from light for 1 h at room temperature, then the absorbance was measured at 517 nm. The DPPH free radical scavenging capacity was calculated as follows:

$$DPPHfreeradicalscavengingcapacity\left(\%\right)=\left[1-\frac{\left(A_0-A_1\right)}{A_0}\right]\times 100\%$$ (7)

Where: A₁ is the absorbance of the sample mixed with DPPH; A₂ will be the absorbance of 2 mL of DPPH mixed with 2 mL of deionized water; A₃ is the absorbance of 2 mL of anhydrous ethanol mixed with 2 mL of sample.

2.4.14. ABTS radical scavenging capacity

The determination of ABTS radical scavenging capacity was based on the method of Yan et al [16]. The ABTS solution was configured: 18 mg of ABTS and 4 mg of K2S2O8 were dissolved in 4 mL of distilled water and mixed thoroughly Then the mixture was placed in the refrigerator for 16 h, and the reaction took place away from light to obtain the ABTS stock solution. Take 3 mL of the stock solution and add 100 mL with distilled water to obtain the ABTS working solution. Then, 1 mL of sample solution (0.5 mg/mL) was mixed with 3 mL of ABTS working solution and reacted for 1 h. Then the absorbance was measured at 734 nm. Distilled water was used as a blank for zeroing. the ABTS free radical scavenging capacity was calculated as follows:

$$ABTSfreeradicalscavengingcapacity\left(\%\right)=\frac{A_0-A_1}{A_0}\times 100\%$$ (8)

Where: A₀ is the absorbance without sample; A₁ is the absorbance of the sample.

2.4.15. FRAP reducing power

The determination of FRAP Reducing Power was based on the method of De Gregorio et al [17]. Samples were prepared in phosphate buffer solution. 2 mL of sample were mixed with 1 mL potassium ferricyanide solution (1 %, w/v) and soaked at 50 °C for 20 min. Then, the solution was removed from the water bath and 1 mL of trichloroacetic acid (10 %) was added and mixed thoroughly. Take 1 mL of the above solution, add 3 mL of distilled water, then add ferric chloride (0.4 %) 0.1 mL, let it stand for 5 min, and test the absorbance at 700 nm. In this case, the three polyphenols were used as reference compounds in the assay, and an increase in the absorbance of the reaction mixture implies an increase in reducing power.

2.5. Statistic analysis

The experimental data were plotted using Origin 2023 software and significant differences between the data were analyzed using SPSS Statistics 25.0 software, all experiments were repeated three times and the level of significance between the data was set at P < 0.05.

3. Results and discussion

3.1. Solubility analysis

Solubility is an important factor in proteins and determines many of their functional properties [18]. As shown in Fig. 1A, the solubility of MP in the untreated group was 46 % and the solubility of UMP, UMP-TA, UMP-QR and UMP-RSV were increased to 51.66 %, 55 %, 51 %, and 62.77 %, respectively. The increased solubility of the protein samples after sonication pretreatment as well as polyphenol binding might be due to sonication cavitation can disrupt the hydrogen bonding and hydrophobic interactions, which allows for strong protein–polyphenol interactions and better solubility. Liu et al found that ultrasonic treatment effectively improved the solubility and dispersion stability of MP in water [19]. Liu et al. found that the addition of polyphenols significantly enhanced the solubility of wheat germ protein (WGP) with chlorogenic acid in the range of 25–150 μmol/g protein, and the solubility of WGP increased with polyphenol concentration increased. This might be because the binding of chlorogenic acid to WGP decreases its surface hydrophobicity, and leads to an increase in protein solubility [20].

Fig. 1.

Solubility (A); SDS-PAGE spectra (B); Zeta potential (C). Note: in Fig. 1 (B), M is the bovine serum protein standard, and 1–5 are MP, UMP, UMP-TA, UMP-QR and UMP-RSV, respectively.

3.2. SDS-PAGE analysis

The distribution of the SDS-PAGE bands reflects the molecular weight changes of the protein and can be used to characterize the binding between the protein and polyphenols [21]. As can be seen in Fig. 1B, there were four different bands on the MP lane, mainly including myosin heavy (MHC), α-actinin (AC), pro-myosin (TM), and myosin light chain (MLC), which is consistent with the report that myosin and actin are the main components of myofibrillar proteins [22]. Compared with MP, the MLC and AC bands in the spectra of the ultrasonicated and polyphenol-binding samples became fainter, and the MLC bands in the spectra of the UMP, UMP-TA and UMP-QR samples were darkened, implying degradation of the proteins, which may be due to the cavitation effect of ultrasound [23]. Typically, degradation of protein molecules manifests itself in the form of blurring, weakening, and dilation of higher molecular weight bands, as well as new bands or darkening of bands. The results showed that compared with untreated MP, the degradation of myosin heavy chain and actin in MP samples by ultrasonic pretreatment and polyphenol binding was obvious, as evidenced by a first increase and then a decrease in the intensity of the bands. This is mainly due to the dissociation of these protein aggregates by the high pressure and shear forces induced by sonication.

3.3. Analysis of zeta's potential

Zeta potential indicates the surface charge of the suspended particles, which is an important parameter for protein stabilization. A higher absolute zeta potential implies a lower tendency to protein aggregate, while a lower absolute zeta potential implies a higher tendency to protein aggregate [24]. As shown in Fig. 1C, ultrasound-assisted polyphenol incorporation had a significant effect on the potential of MP from golden threadfin. The zeta potential of untreated MP was −15.85 mV, while, after ultrasound treatment, the absolute potential of the UMP was increased to (−17.16 mV), and the potential values of the samples with the addition of polyphenols (TA, QR and RSV) were −19.09 mV, −18.22 mV, and −13.44 mV, respectively. Goldfish myofibrillar proteins and polyphenols have electrostatic interactions in which the homologous charges repel each other and therefore are more useful for promoting the stability of the solution system. Liu et al found that the zeta potential values of wheat alcohol-soluble proteins showed the same phenomenon of increase after the addition of tannic acid treatment, and increased with the content of tannic acid. This might be related to the cavitation effect of ultrasound and the non-covalent interaction of polyphenols [20].

3.4. Total sulfhydryl content

Sulfhydryl groups are often considered to be a key indicator of conformational alternation in protein molecules. Typically, the total sulfhydryl content includes not only free sulfhydryl groups on the surface but also sulfhydryl groups buried inside the protein molecules [25]. The results of ultrasound-assisted polyphenol binding on the total sulfhydryl content of MP are shown in Fig. 2A. Compared to the MP samples, the sulfhydryl content of the UMP samples slightly increased, while UMP-TA, UMP-QR and UMP-RSV all showed significant decrease. This might be attributed to the effect that the ultrasonic pretreatment loosened the protein structure and sulfhydryl groups buried inside the molecule were gradually exposed to the surface, and polyphenol compounds can interact with the free sulfhydryl groups in the proteins, resulting in a decrease in the total sulfhydryl content of MP. Bian et al found that ultrasonic cavitation may lead to the unfolding of the MP structure resulting in the exposure of reactive sulfhydryl groups to the surface of the proteins [12]. Han et al. found that the addition of polyphenols such as catechin, epigallocatechin, and (−)-epigallocatechin gallate to scallop gonadal protein isolates resulted in a decrease in the total sulfhydryl group content, which might due to the interaction of hydroxyl groups in the polyphenols with the sulfhydryl groups in the scallop gonadal proteins, resulting in a decrease in the sulfhydryl group content [14].

Fig. 2.

Total sulfhydryl content (A); UV spectrum (B).

3.5. UV spectra analysis

The ultraviolet absorption spectra of proteins are mainly due to the absorption of ultraviolet light by the side-chain groups of tryptophan and tyrosine residues, and the differences in the absorption of the ultraviolet spectra can reflect the conformation change of proteins [26]. As can be seen from Fig. 2B, the characteristic peaks of the MP samples appeared near the wavelength of 270 nm. The UV absorption spectra of myofibrillar proteins increased slightly after sonication and the addition of polyphenols, suggesting that ultrasonic-assisted polyphenol binding can promote MP structure unfolding and exposure of chromophores in the hydrophobic nonpolar region to polar solvents. In addition, the absorption peak at 280 nm was slightly red-shifted, indicating that the binding of polyphenols with MP reduced the microenvironmental polarity of the aromatic residues, suggesting that the addition of polyphenols induced the exposure of the MP hydrophobic moiety, which altered the conformation of the protein. Ye et al provided information on the structure of the rutin-SPI complex using UV–visible spectroscopy, which indicated the presence of interactions between rutin and SPI, possibly involving tyrosine and tryptophan moieties and attributed to polyphenol and protein interaction [27].

3.6. Fourier infrared spectroscopy (FT-IR)

FT-IR can reflect changes in side-chain motifs of protein structure [28]. The IR spectra of the different samples are shown in Fig. 3A. The broad peak between 3200–3500 cm⁻¹ in the MP exhibited the characteristic absorption of –OH stretching of hydrogen bonding. While, after ultrasonic-assisted polyphenol binding the maximum absorption peak of MP changed from 3303.45 cm⁻¹ to 3295.67 cm⁻¹, indicating that the hydrogen bonding interaction between the proteins-polyphenols was strengthened.

Fig. 3.

Fourier Infrared Spectroscopy (A); Circular dichroism (B); Protein Secondary Structure content (C).

The typical absorption peaks occurred at 1700–1600 cm⁻¹ and 1600–1500 cm⁻¹ indicating the amide I group, and amide II group of protein, respectively. The absorption peak at 1658.62 cm⁻¹ indicates the amide I band, which corresponds to C = O stretching or COO– binding with hydrogen bonding [29]. After ultrasonic-assisted polyphenol binding, the peak of the amide I band of MP shifted to 1656.55 cm⁻¹, indicating that the secondary structure of MP was changed and the change might be caused by hydrogen bonding or electrostatic attraction through non-covalent binding with the polyphenols. The absorption peak between the amide II region of MP at 1536.91 cm⁻¹ was attributed to the bending vibration of the N-H bond and the stretching vibration of the C-N bond. After ultrasonic-assisted polyphenol binding, the maximum peak between amide II of MP shifted from 1536.91 cm⁻¹ to 1538.95 cm⁻¹, which might due to hydrogen bonding and hydrophobic interactions formed between MP and the polyphenols.

3.7. Far-UV circular dichroism (CD) spectroscopy

Far-ultraviolet (190–260 nm) circular dichroism spectra can reflect the secondary structure change of proteins after polyphenol binding [26]. The secondary structure of proteins can be characterized by the ratio of α-helices, β-folds, β-turns, and irregular coils. The α-helices are ordered arrangements of protein molecules maintained by intramolecular hydrogen bonds, while the β-folds are ordered arrangements of protein compartments maintained by intermolecular hydrogen bonds the α-helix, β-fold, β-turn, and irregular curl contents of MP under different treatment are shown in Fig. 3B and Fig. 3C. Compared with MP, ultrasonic pretreatment combined with polyphenols addition reduced the α-helix content in secondary structure. Among them, there was a decrease in the α-helix content of the UMP-TA (from 78 % to 35 %) and an increase in the β-fold content (from 3 % to 15 %) in comparison with MP. Both ultrasonic pretreatment and non-covalent binding of polyphenols promoted the structure change from α-helix to β-fold. The change of secondary structure by ultrasound might be partly due to the cavitating and mechanical benefits, which could break the hydrogen bond and lead to the transformation of α-helix to β-folding. In addition, the change by polyphenols incorporation could be attributed to the interaction of polyphenols with amino acid residues of MP. Stathopulos et al. reported that ultrasound treatment could cause proteins to aggregate and form β-structures [30]. This effect is particularly noticeable in proteins that naturally contain a lot of α-helical structures, which tend to decrease in the aggregates as the β-structures become more prominent.

3.8. Fluorescence spectroscopy analysis

Endogenous fluorescence is caused by aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) [7]. Especially, since the fluorescence emission of Trp is very sensitive to the environment, it is always applied to verify modifications of protein tertiary structure [31]. Compared with the untreated MP sample, the structure of UMP after ultrasonic pretreatment was extended and more hydrophobic groups inside were exposed to the protein surface, and the fluorescence intensity showed and enhanced phenomenon [32]. As can be seen in Fig. 4A, after further binding with polyphenols (TA, QR and RSV), the fluorescence intensity was all decreased in comparison with UMP, indicating that in the presence of polyphenols, proteins interacted with polyphenols causing a fluorescence burst phenomenon. Joye et al also found that the fluorescence intensity of maize alcohol-soluble proteins decreased with an increasing concentration of resveratrol added [33]. The calculation of the number rate of sample bursts caused by the addition of polyphenols (TA, QR and RSV) is shown in Table 1. The rate constant (K_q) of different polyphenol types binding with proteins were all greater than 2.0 × 10¹⁰ L mol⁻¹ s⁻¹, indicating that the fluorescence quenching of MP and polyphenols (TA, QR and RSV) is a static quenching caused by the non-covalent interaction between them.

Fig. 4.

Endogenous fluorescence (A); Synchronous fluorescence at Δλ = 60 (B); Synchronous fluorescence at Δλ = 15 (C); surface hydrophobicity (D).

Table 1.

Fluorescence quenching parameters of UMP-Polyphenols complex.

UMP-P	Q295	F295	F0/F295	Kq295
UMP	0	490	1	−
UMP-TA	0.05	233	2.20	2.4 × 1011
UMP-QR	0.05	393	1.24	4.8 × 108
UMP-RSV	0.05	379	1.29	5.8 × 108

Synchronized fluorescence spectroscopy could provide molecular microenvironment information around the aromatic amino acid residues by measuring the emission wavelength shift. The peak shift at the position of the λ_max (maximum emission wavelength) corresponds to the change of polarity around the chromophore molecules. When Δλ between excitation and emission wavelength is fixed at 15 nm and 60 nm, synchronized fluorescence can reflect the microenvironment information around tyrosine or tryptophan residues, respectively [34]. As can be seen in Fig. 4B and Fig. 4C, the λ_max of the Trp residues are slightly red-shifted, suggesting that the hydrophobic environment of Trp residues is reduced, and thus more exposed to the solution. The results indicated that the combination of polyphenols (TA, QR and RSV) and MP could alter the microenvironment around the hydrophobic aromatic amino acid residues of MP. TA, QR and RSV all contained a high number of hydroxyl groups, which can introduce a large number of hydrophilic groups when they are combined with MP, thus leading to a decrease in the hydrophobicity of the microenvironment around the amino acid residues. This might be due to the combination effect of the unfolding of protein structure by ultrasonic pretreatment, which exposes more hydrophobic groups to the structural surface, and the introduction of more hydrophilic groups (hydroxyl groups) through the addition of polyphenols (TA, QR and RSV).

3.9. Surface hydrophobicity (H₀) index

The surface hydrophobicity (H₀) index can reflect the distribution of hydrophobic groups on the surface of proteins, which can further reflect the affinity balance of the protein [35]. As can be seen in Fig. 4D, the hydrophobicity of the sample a decreased after ultrasonic pretreatment, and the hydrophobicity of the sample further decreased after the addition of polyphenols. Since polyphenols have one or more hydroxyl and carboxyl groups, The addition of polyphenols will bring more hydroxyl groups to the protein surface, thus increasing the hydrophilicity of the sample, resulting in a decrease in its H₀ index, which is also consistent with the protein solubility increased with the polyphenols addition shown in Fig. 1A. Yang et al proposed that when non-covalent interactions between polyphenols and proteins occur, a large number of hydroxyl groups attached to the polyphenol aromatic ring are introduced to the surface of the protein molecule, thus reducing the protein surface hydrophobicity [36].

3.10. Dynamic interfacial tension analysis

Dynamic interfacial tension is a fundamental quantity associated with the assembly properties of proteins on the oil–water interface and plays an important role in the formation and stabilization of emulsion. From Fig. 5(A-E), the interfacial tension of the samples decreased with time, indicating that protein adsorption on the oil–water interface is a dynamic process [37]. All proteins showed similar interfacial behavior, after the onset of adsorption, the pressure increased rapidly until saturation. Therefore the interfacial tension gradually decreases until it reaches equilibrium after 12000 s. Compared to the interfacial tension stabilized value of MP (21.22 mN/m), the interfacial tension of the samples pretreated with ultrasound as well as polyphenol addition was significantly reduced(15.25, 8.66, 13.50, and 9.14 mN/m for UMP, UMP-TA, UMP-QR and UMP-RSV, respectively). This might be attributed to the expansion of protein structure by ultrasonic pretreatment and the improvement of hydrophilic/hydrophobic balance of protein by polyphenols addition. Feng et al also found that ultrasonic treatment could significantly reduce the oil/water interfacial tension of soybean protein [38].

Fig. 5.

Dynamic interfacial tension of different samples: MP (A); UMP (B); UMP-TA (C); UMP-QR (D); UMP-RSV (E).

3.11. Analysis of emulsification and foaming properties

The emulsification activity index (EAI) can be applied to characterize the protein emulsification capacity by the oil–water interfacial area adsorbed per unit mass. The emulsion stability index (ES) can represent the ability of the protein to maintain the stability of the emulsion for a certain period of time [10]. The EAI and ES results of samples are shown in Fig. 6(A). In comparison with the MP group, after ultrasonic treatment and polyphenols addition, the EAI was significantly increased (from 21.57 m²/g of MP to 30.13, 28.79, 31.81 and 30 m²/g of UMP, UMP-TA, UMP-QR and UMP-RSV, respectively). The ES result also showed a significant increase compared to the MP sample group (from 84.76 min of MP to 116.30, 124.25, 106.47, and 106.48 min of UMP, UMP-TA, UMP-QR and UMP-RSV, respectively). The development of emulsifying activity and stability of MP by ultrasonic preconditioning assisted polyphenol addition might be attributed to different factors: (1) the expansion of protein structure, the increase of conformational flexibility, and the decrease of surface tension caused by ultrasonic pretreatment [2]. (2) the interaction of polyphenols helped to improve the hydrophilic/hydrophobic balance and promote the adsorption and stability of protein on the oil–water interface [39].

Fig. 6.

Emulsifying properties of different samples (A); Foaming properties (B).

As an important indicator of protein property, the foaming properties can reflect the interface properties between air and water for proteins [40]. As can be seen from Fig. 6B, the foaming capacity (FC) in all treatment groups was significantly increased compared to the MP group (from 39.25 % of MP to 39.87, 50.13, 51.05 and 52.10 % of UMP, UMP-TA, UMP-QR and UMP-RSV, respectively). and foaming stability (FS). The improvement of the foaming properties of protein was also related to the modification of protein structure through ultrasonic pretreatment combined with polyphenol addition. Ultrasonic preprocessing can stretch the structure of proteins, making it easier to unfold and adsorb at the air–water interface. The addition of polyphenols can enhance the hydrophilic balance of protein and make the adsorption of protein at the air–water interface more stable. Li et al also reported that the addition of TA could improve the foam stabilization of SPI, and analyzed that it was mainly due to the non-covalent interaction between SPI and TA (hydrogen bonding and van der Waals forces) [41].

3.12. Analysis of antioxidant properties

The scavenging capacity of DPPH and ABTS free radicals as well as the FRAP reduction capacity could reflect the antioxidant capacity of the protein samples. As shown in Fig. 7A, after ultrasonic preconditioning assisted polyphenol addition, the DPPH radical scavenging activity all represented a significant increase in comparison with the MP group (from 28.55 % of MP to 28.56 %, 76.5 %, 71.25 % and 69.20 % of UMP, UMP-TA, UMP-QR and UMP-RSV, respectively). As shown in Fig. 7B, the ABTS radical scavenging activity also showed the same increase in contrast to the MP group after ultrasonic preprocessing combined with polyphenol addition (from 28.88 % of MP to 29.55 %, 84 %, 71.25 % and 74.70 % of UMP, UMP-TA, UMP-QR and UMP-RSV, respectively). FRAP assay was used to measure the conversion of Fe3⁺ to Fe2⁺. Fig. 7C shows that after ultrasonic pretreatment combined with polyphenol addition, the FRAP were all significantly higher than that of MP, which indicated that the polyphenols could provide electronic 3⁺ ions to Fe and reduce them to Fe2⁺. Therefore, the non-covalent interaction of polyphenols with MP could provide a novel way to confer protein reduction properties. The above three sets of antioxidant performance test results all indicated that polyphenol addition could endow MP with strong antioxidant properties, which might due to interaction with polyphenol introduce strong antioxidant groups (such as phenolic hydroxyl groups) into the protein structure surface, thereby improving its antioxidant capacity. Ultrasonic preprocessing allowed the protein structure to stretch and expose more hydrophilic amino acid residues, and make it easier for non-covalent interaction with polyphenols. In addition, the cavitation force generated by the sonication reaction promotes a partial unfolding of the protein structure, exposing more aromatic amino acids (tryptophan) exhibiting high antioxidant activity due to their involvement in direct electron transfer [42], [43], [44]. In addition, the ultrasound-assisted protein–polyphenol coupling has a high content of polyphenols [11].

Fig. 7.

Antioxidant capacities of different samples: DPPH radical scavenging capability(A); ABTS radical scavenging capability(B); FRAP Reducing power (C).

4. Conclusions

In this study, ultrasound-assisted polyphenol incorporation was used to investigate the structural and functional properties of MP. It was found that ultrasonication and the addition of polyphenols (TA, QR and RSV) resulted in the solubility increase, H₀ and dynamic interfacial tension decrease, and EAI, ES, and FS increase of MP. Ultrasound assisted polyphenols addition led to a significant decrease of the oil/water interfacial tension (from 21.22 mN/m of MP to 8.66 mN/m of UMP-TA sample) and a significant increase of the EAI (from 21.57 m²/g of MP to 28.79 m²/g of UMP-TA sample) and ES(from 84.76 min of MP to 124.25 min of UMP-TA). In addition, ultrasound-assisted polyphenol incorporation could enhance the antioxidant properties of MP, with the DPPH and ABTS radical scavenging rate of UMP-TA increase of 47.7 % and 55.2 % in comparison with MP, respectively. This might be due to the effect that ultrasonic preconditioning can stretch the MP structure and improve the interaction between polyphenols and proteins. In addition, the effect of polyphenols on MP depends on the structure and type of polyphenols. Among the three polyphenols, tannic acid has the most hydroxyl groups and the strongest affinity with methyl parathion. The multispectral analysis represented that the interaction between MP and polyphenols (TA, QR and RSV) was mainly driven by hydrogen bonding and hydrophobicity. The non-covalent interactions with polyphenols could alter the conformational flexibility, surface charge properties and hydrophilic balance of protein, which effectively improved the emulsification, foaming as well as antioxidant properties of MP. The present study demonstrated that ultrasonic pretreatment combined with polyphenol noncovalent binding as a useful physical treatment could provide an effective reference path to improve the processing value of myofibrillar protein from golden threadfin in the healthy food industry.

CRediT authorship contribution statement

Xianglian Wei: Writing – original draft, Methodology, Data curation, Conceptualization. Chunxia Zhou: Writing – original draft, Methodology, Data curation, Conceptualization. Donghui Luo: Formal analysis, Conceptualization. Guili Jiang: Writing – review & editing. Zilong Zhao: Visualization. Wenduo Wang: Writing – review & editing, Supervision, Project administration, Funding acquisition. Pengzhi Hong: Writing – review & editing, Supervision, Project administration, Funding acquisition. Zuman Dou: Writing – review & editing.

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.