Improvement of structural, physicochemical, and rheological properties of porcine myofibrillar proteins by high-intensity ultrasound treatment for application as Pickering stabilizers

Graphical abstract

Abstract

This study aimed to evaluate the potential of time-dependent (0, 15, 30, 60, 120 min) treatment of porcine-derived myofibrillar proteins (MPs) with high-intensity ultrasound (HIU) for utilizing them as a Pickering stabilizer and decipher the underlying mechanism by which HIU treatment increases the emulsification and dispersion stability of MPs. To accomplish this, we analyzed the structural, physicochemical, and rheological properties of the HIU-treated MPs. Myosin heavy chain and actin were observed to be denatured, and the particle size of MPs decreased from 3,342.7 nm for the control group to 153.9 nm for 120 min HIU-treated MPs. Fourier-transformed infrared spectroscopy and circular dichroism spectroscopy confirmed that as the HIU treatment time increased, α-helical content increased, and β-sheet decreased, indicating that the protein secondary/tertiary structure was modified. In addition, the turbidity, apparent viscosity, and viscoelastic properties of the HIU-treated MP solution were decreased compared to the control, while the surface hydrophobicity was significantly increased. Analyses of the emulsification properties of the Pickering emulsions prepared using time-dependent HIU-treated MPs revealed that the emulsion activity index and emulsion stability index of HIU-treated MP were improved. Confocal laser scanning microscopy images indicated that small spherical droplets adsorbed with MPs were formed by HIU treatment and that dispersion stabilities were improved because the Turbiscan stability index of the HIU-treated group was lower than that of the control group. These findings could be used as supporting data for the utilizing porcine-derived MPs, which have been treated with HIU for appropriate time periods, as Pickering stabilizers.

1. Introduction

An emulsion is a heterogeneous system of two immiscible liquids in which one liquid is dispersed in the form of droplets in the other liquid [1]. Generally, it is classified as water-in-oil (W/O) or oil-in-water (O/W) according to the arrangement of the two liquids. O/W emulsions are widely used in the food and pharmaceutical industries as they can be easily manufactured in a cost-effective manner and can capture the functional ingredients well [2]. Due to the current global challenges like population aging, there is a growing demand for clean-label products [3]. This has led to increasing interest in Pickering emulsions which uses small solid particles at the interface without using conventional surfactants [4], [5]. Many studies have shown that Pickering emulsions are less susceptible to the changes in the external environment because solid particles are irreversibly adsorbed to the droplet interface [4], [6]. It is known that starch nanoparticles, soybean protein isolate, and chitin can be used as stabilizers to produce Pickering emulsions [7], [8], [9]. However, the types of stabilizers for the Pickering emulsion are still limited, and therefore, research on the development of food material-based Pickering emulsion stabilizers is needed.

Myofibrillar proteins (MPs), which account for 55 to 60 % of muscle proteins, are salt-soluble proteins. This means that they are soluble in concentrated saline solutions (ionic strength above 0.6 M) as well as extremely low ionic strength; they are composed mainly of actin, myosin, and tropomyosin [10], [11]. MPs are the most important functional component of meat protein, playing an important role in its emulsification, gelation, and water retention properties [9]. Moreover, due to the amphiphilicity and low allergenicity of the proteins constituting MPs, it has the potential to be used as a Pickering emulsion stabilizer [5]. For this reason, studies on the physicochemical properties of MPs, including transglutaminase-treated MPs [12], gel preparations using MPs [5], and O/W emulsions stabilized with MPs and Tween 20 [13], have been reported. However, despite these advantages, when MPs are used alone as emulsifiers in the preparation of an emulsion, aggregation and flocculation are likely to occur, resulting in low physical stability [14].

Ultrasound (US) is an acoustic wave with a frequency exceeding 20 kHz (human hearing threshold) and is classified into high-intensity (20–100 kHz), medium-intensity (100–1,000 kHz), and low-intensity (1,000–10,000 kHz) US [15]. US treatment has received considerable attention in recent years as a safe and eco-friendly technology [16]. Low-frequency high-intensity ultrasound (HIU) is most widely used in the food industry. Several studies have demonstrated that HIU treatment affects the structural properties and improves the physicochemical properties of proteins. Zhu et al. [17] reported that the water solubility and emulsification ability of walnut protein isolates were improved by US treatment (600 W for 15 min). Amiri et al. [18] indicated that HIU treatment (100 and 300 W for 30 min) reduced the particle size of bovine-derived MPs and improved their rheological and functional properties. Lee et al. [19] reported that HIU treatment (35 kHz, 800 W, 60 min) could improve sensory characteristics through tenderizing without impairing the quality of the bovine semitendinosus muscle. Studies have reported the physicochemical properties of MPs after US treatment, but not many have identified the cause or confirmed its potential as an O/W emulsion stabilizer. To the best of our knowledge, studies reporting the dispersion stability of Pickering emulsions prepared with HIU-treated MPs are scarce.

In this study, HIU, which is a non-heat generating process, was applied for various time periods to MPs extracted from porcine muscle. The structural, physicochemical, and rheological properties of the HIU-treated MPs were determined. Furthermore, after preparing an O/W Pickering emulsion using HIU-treated MPs, the morphology and characteristics of the droplets were analyzed. In addition, the dispersion stability of the O/W emulsion was measured using a laser scanning turbidimetric method. Through these analytical methods, we tried to investigate the factors affecting the functional improvement of porcine-derived MPs under various HIU treatment time conditions and to confirm the potential of HIU-treated MPs as a Pickering emulsion stabilizer.

2. Materials and methods

2.1. Preparation of MP

Porcine ham muscles (Musculus semitendinosus, Musculus semimembranosus, and Musculus biceps femoris) were purchased from a local market. Muscles (48 h post-mortem) were prepared from three market-weighted crossbreeds (L × Y × D). After removing the excess connective tissues, muscle samples were vacuum-packed and stored at −20 °C [20]. MPs were extracted according to the method described by Jia et al. [21] with some modifications. In brief, an extraction buffer (0.01 M potassium phosphate with 0.1 M KCl, pH 7.4) equivalent to four times (w/v) the weight of the thawed muscle sample was added, homogenized for 1 min, and centrifuged at 12,000 × g for 15 min using a centrifuge (Continent 512R, Hanil Co., ltd, Incheon, Korea). Thereafter, the supernatant was removed, an extraction buffer equivalent to four times the weight of the residue was added again to homogenize it for 1 min. and centrifugation was performed under the same conditions as above. After repeating the same process twice, distilled water equivalent to four times the weight of the recovered residue was added and homogenized for 1 min. The supernatant was removed by centrifugation, and this process was also repeated twice. The final residue, MPs, was diluted with 0.58 M saline (0.49 M NaCl, 17.8 mM Na₅P₃O₁₀ and 1 mM NaN₃, pH 8.3), and the protein concentration was adjusted to 5 mg/mL before use.

2.2. HIU treatment

HIU treatment was performed as per the method described by Zhang et al. with slight modifications using an ultrasonic processor (VCX750; Sonic & Materials, Newtown, CI, USA) [22]. After adjusting the protein concentration to 5 mg/mL, 300 mL of the MP solution was transferred to a circulating cold water jacketed beaker. During US treatment, the probe (diameter 25 mm) was immersed into the sample to a depth of 2 cm from the bottom, and the sample temperature was maintained at 10 ± 0.2 °C. The treatment conditions were set to 20 kHz, 200 W (80 % amplitude), and a pulse mode of 5 s on and 5 s off was used. Samples were ultrasonicated for 0, 15, 30, 60, and 120 min, respectively, and after treatment, samples were stored at 4 °C until analysis.

2.3. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was performed to examine the protein molecular weight distribution of the HIU-treated MP solution. After adjusting the protein concentration of the sample to 1 mg/mL, it was mixed with 4x Laemmli sample buffer (312.5 mM Tris-HCl [pH 6.8], 50 % glycerol, 5 % SDS, 5 % β-mercaptoethanol, 0.05 % bromophenol blue, EBA-1052, Elpisbiotech, Daejeon, Korea) in a 3:1 ratio and heated at 100 °C for 5 min. The sample was then cooled to 4 °C for 5 min and analyzed by SDS-PAGE on a 12 % resolving gel containing a 4 % stacking gel. The operating voltage for the electrophoretic run was 80 mA. The gel was stained using a 0.25 % Coomassie Brilliant Blue R250 (B7920; Sigma-Aldrich, St. Louis, MI, USA) and molecular weight was estimated by comparing with standard protein markers (pre-stained DokDo-MARK, EBM-1032, Elpisbiotech, Daejeon, Korea).

2.4. Dynamic light scattering (DLS) analysis

The mean particle size, particle size distribution, polydispersity index (PDI), and zeta potential were measured using dynamic light scattering (DLS) analysis. Samples were diluted 10-fold using a buffer solution. Thereafter, the sample was injected into the sample-holder cell and measured at 25 °C using a Nano-ZS Nanosize analyzer (Malvern Instruments ltd., Worcestershire, UK) with a 633-nm laser and a detector angle of 90°.

2.5. Fourier transform infrared spectroscopy (FT-IR)

Fourier transform infrared spectroscopy (FT-IR) was used to analyze the molecular conformation of MPs treated with HIU at various times. After removing salt via dialysis, the samples were freeze-dried. The transmittance and reflectance of the protein in powder form were measured in the range of 4,000–400 cm⁻¹ using a Cary 630 FT-IR spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA) in the attenuated reflectance (ATR) mode.

2.6. Circular dichroism (CD) spectroscopy

The circular dichroism (CD) spectrum of the MP solutions treated with HIU was measured using a CD spectrometer (Chirascan, Applied Photophysics ltd., Surrey, UK). Samples were analyzed using a quartz cuvette with an optical path length of 1.0 mm in the wavelength range of 200–260 nm with a bandwidth of 1 nm. Contents of secondary protein structural elements were calculated using the CD Spectra Deconvolution (CDNN) software (Applied Photophysics ltd., Leatherhead, UK).

2.7. pH

The pH values of the HIU-treated MP solutions adjusted to a protein concentration of 5 mg/mL were measured using a pH meter (Orion Star A111, Thermo Fisher Scientific, Waltham, MA, USA) at room temperature.

2.8. Turbidity

The turbidity of the HIU-treated MP solution was determined by measuring the absorbance. Briefly, after adjusting the protein concentration of the MP solution to 5 mg/mL, 1 mL of the homogeneously mixed protein solution was placed in a cuvette, and the absorbance was measured at 600 nm using a UV/Vis-spectrophotometer (Spectra max Plus 384; Molecular Devices Inc., San Jose, CA, USA).

2.9. Surface hydrophobicity

Two hundred microliters of bound bromophenol blue (BPB 1 mg/mL in distilled water) were added to an MP sample (1 mL) diluted to a protein concentration of 1 mg/mL, vortexed and left at room temperature for 10 min. Subsequently, the sample was centrifuged at 2,000 × g for 15 min, the collected supernatant was diluted 10-fold, and the absorbance was measured at 595 nm using a UV/Vis-spectrophotometer. A phosphate buffer was used as a control. Hydrophobicity was calculated using the following equation:

$$\mathrm{B}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}\mathrm{B}\mathrm{P}\mathrm{B}(\mathrm{\mu }\mathrm{g})=\frac{200\mathrm{\mu }\mathrm{g}\times ({\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}}_{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}-{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}}_{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}})}{{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}}_{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}$$ (1)

2.10. Carbonyl content measurement

The carbonyl content of the HIU-treated MPs was measured using the 2,4-dinitrophenylhydrazine (2,4-DNPH) colorimetric method (Berardo, Claeys, Vossen, Leroy, & De Smet, 2015). Briefly, 0.4 mL of the sample was mixed with 2,4-DNPH dissolved in 2 M HCl. After incubating in the dark for 30 min, 0.5 mL of 40 % trichloroacetic acid was added to the sample, mixed, and centrifuged at 5,000 × g for 15 min at 4 °C. The supernatant was discarded, and the pellet was dissolved in guanidine hydrochloride (6 M). The carbonyl concentration was evaluated by measuring the absorbance at 370 nm.

2.11. Rheological characterization

The rheological properties of HIU-treated MP solutions were determined using a Physica MCR 102 rheometer (Anton paar GmbH, Graz, Austria). Measurements were performed after 5 min equilibration at 20 °C using a gap of 1 mm and a parallel plate with a diameter of 25 mm. Apparent viscosities were analyzed at shear rates from 0.1 to 100 s⁻¹. Strain sweeps test over the range of 0.1–100 % at a constant angular frequency of 10 rad/s was conducted to determine the linear viscoelastic region (LVE). Subsequently, a frequency sweep test was performed in the angular frequency range from 0.1 to 100 rad/s. Storage modulus (G'), loss modulus (G''), and loss factor (tan δ) were recorded using the RheoCompass v.1.30 software (Anton paar GmbH).

Determination of emulsification properties.

2.12. Preparation of Pickering emulsions

In order to prepare a Pickering emulsion, an HIU-treated MP solution with a protein concentration adjusted to 5 mg/mL was used as an aqueous solution. After accurately weighing 25 % (w/w) of medium chain triglyceride (MCT) oil, the MCT oil and protein solution were homogenized at 11,000 rpm for 2 min using a high-speed blender (Ultra-Turrax T25, IKA, Staufen, Germany). The prepared emulsions were immediately used in subsequent experiments.

2.12.1. Emulsion activity index (EAI) and emulsion stability index (ESI)

Twenty-five microliters of the sample were added to 5 mL 0.1 % sodium dodecyl sulfate (SDS) solution, stirred, and absorbance of the sample was measured at 500 nm, after 0 (A₀) and 30 (A₃₀) min, respectively. The emulsion activity index (EAI) and emulsion stability index (ESI) were calculated as follows:

$$\mathrm{E}\mathrm{A}\mathrm{I}(m^2/\mathrm{g})=\frac{2\times 2.303\times A_0\times 200}{c\times \mathrm{Ø}\times {10}^4}$$ (2)

$$\mathrm{E}\mathrm{S}\mathrm{I}\left(\mathrm{m}\mathrm{i}\mathrm{n}\right)=\frac{A_0}{A_0-A_{30}}\times 30$$ (3)

where 200 is the dilution factor, c is the protein concentration (g/mL), and Ø is the volume of the emulsion's oil fraction [23].

2.12.2. Droplet characterization

The mean particle size of the emulsion droplets was measured using a Cilas 1090 laser diffraction particle size analyzer (Cilas, Orléans, France). The volume mean diameter (d_4,3) and surface mean diameter (d_3,2) were obtained for the mean particle size of emulsion droplets [24]. Also, the zeta potential of the emulsion droplet was measured using DLS analysis, as described in Section 2.4.

2.12.3. Confocal laser scanning microscopy (CLSM)

The morphology of emulsion droplets was observed using a confocal laser scanning microscope (CLSM, FV3000, Olympus, Tokyo, Japan) with reference to the method of Liu and Lanier [25]. A 0.02 g of Nile Blue A in 10 mL of distilled water was used to stain emulsion droplets. Twenty microliters of Nile Blue A solution were added to 5 mL of a 10-fold diluted emulsion sample. From this, 20 μL of the stained sample was placed on a concave glass slide and covered with a glass coverslip (0.17 mm thickness). Samples were scanned at room temperature using a 20 × objective lens. The samples were excited using two wavelengths – at 488 nm with an argon laser (fluorescent dye excitation in the oil phase) and at 633 nm using a helium neon laser (fluorescent dye excitation in the protein phase). Emission spectra were collected at 500–620 nm for the oil phase and 650–750 nm for the protein phase, and the two resulting images were overlaid using the FluoView FV3000 software (Olympus, Tokyo, Japan).

2.12.4. Laser scanning turbidimetry

The turbidimetric properties of Pickering emulsions stabilized by ultrasonicated MPs were measured using a Turbiscan ASG (Formulaction, L'Union, France). After freshly prepared emulsions were placed in the measuring cell, it was scanned using an infrared light source at 880 nm every 2 min for 3 h. The dispersion stability was evaluated through the backscattering profile (ΔBS, %) and Turbiscan stability index (TSI). The TSI value was calculated with the following equation:

$$\mathrm{T}\mathrm{S}\mathrm{I}={\sum }_i\frac{{\sum }_h\left|{\mathit{scan}}_i\left(h\right)-{\mathit{scan}}_{i-1}(h)\right|}{H}$$ (4)

where H is the total height of the sample in the measuring cell, i is the time from 1 to k (k = total measuring time/measuring speed), and scan_i(h) is the backscattering value at a given height h, respectively [26].

2.13. Statistical analysis

All experiments were repeated three times, and the data were expressed as mean ± standard deviation. Statistical Package for the Social Sciences software (SPSS, Version 20.0; SPSS Inc., Chicago, IL, USA) was used for data analysis. The significance (P < 0.05) of the difference between means was determined through Duncan's multiple range test.

3. Results and discussion

3.1. Structural changes of HIU-treated MPs

3.1.1. Molecular weight distribution

SDS-PAGE of HIU-treated MPs under various time conditions is shown in Fig. 1a. Myosin heavy chain (MHC) and actin were observed as major bands at molecular weights of 210 and 41 kDa, respectively. In addition, tropomyosin, myosin light chain (MLC) 1, MLC 2, and MLC 3 bands were also observed. As the time of HIU treatment increased, MHC and actin degradation was evident, as reflected in the decrease in intensity of the bands. A previous study also demonstrated a decrease in MHC band intensity when analyzed using SDS-PAGE after US treatment (20 kHz, 240 W, 37 % amplitude, 6 min) of MPs extracted from pork [27] or US treatment (20 kHz) of thawed MPs extracted from small yellow croakers [11]. Bubbles are generated during US treatment in liquid foods, and the bubble size and implosion strength are determined by the frequency [28]. At high frequencies, the compression caused by sound waves is insufficient to separate the liquid molecules. On the other hand, the HIU at the low frequency used in this study (20 kHz) causes micro-streaming currents and cavitation [10], [28], [29]. High shear forces, temperatures, and pressures, as well as turbulence, occur when cavitation bubbles are rapidly created and vigorously grown and collapsed [27], [30]. As a result, cavitation has the effect of dispersing the aggregates and decomposing the polymer [29]. Temperature, pressure, viscosity, and ultrasonic intensity are important factors affecting the generation and maintenance of cavitation; it is also known that when the temperature, pressure, and viscosity increase, the cavitation intensity tends to decrease [28]. In contrast, the ultrasonic effect can be further promoted with the increase in ultrasonic intensity. However, since the temperature and ultrasonic intensity conditions were unified in all treatment groups, the results of this study suggest that as the HIU treatment (20 kHz, 200 W, 80 % amplitude) time increased from 0 min to 120 min, the MPs experienced structural change and deterioration, leading to an increase in their molecular weight distribution. On the other hand, Zhu et al. [17] reported no change in molecular weight after HIU treatment performed under similar conditions (200 W, 15–30 min) followed by SDS-PAGE analysis for walnut protein isolate. This discrepancy could be attributed to the variations between the animal species (pig, beef, or chicken), protein origin (plant or animal), and HIU treatment conditions (frequency, intensity, and time) [31].

Fig. 1.

Effect of high-intensity ultrasound (HIU) treatment time on structural changes in myofibrillar proteins (MPs): (a) SDS-PAGE, (b) mean particle size and polydispersity index (PDI), (c) particle size distribution, and (d) FT-IR.

3.1.2. Particle size distribution

Fig. 1b shows the mean particle size and PDI of HIU-treated MPs measured by DLS analysis. The mean particle size of MPs without HIU treatment was 3,342.7 ± 275.5 nm. It was observed that the particle size significantly decreased to ≤ 500 nm when MPs were treated with HIU for ≥ 15 min (P < 0.05). The sample treated with HIU for the longest time of 120 min had the smallest mean particle size of 153.9 ± 4.9 nm. The particle size reduction of MPs by HIU treatment can be explained by protein denaturation and non-covalent rupture, which is in line with the results of previous works of literature [18], [22], [29], [32]. Zhang et al. reported that the particle size of chicken-derived MPs was reduced from 2,084.57 to 271.27 nm after US treatment (20 kHz, 1,000 W) for 15 min compared to that in the control group [22]. Similarly, Li et al. showed that the particle size of chicken-derived MP was reduced from 1,095.0 to 391.7 nm by sonication (20 kHz, 450 W, 60 % amplitude) for 6 min [31]. PDI is an indicator of uniformity of the degree of distribution of particles [33]. The PDI value showed a tendency to decrease compared to the control group by HIU treatment, but it was not statistically significant (P > 0.05). With respect to particle size distribution (Fig. 1c), one peak with a large particle size and one small peak with a small particle size were observed in the control group. However, with the increase in HIU treatment time from 0 to 120 min, the center of the largest peak moved towards the left of the graph, confirming the decrease in MP particle size. The HIU-treated sample showed a bimodal distribution, with the first small peak attributed to monomers or oligomers and the second peak due to the contribution of the structure of the MPs [34]. The myofibril structure consists of thin filaments composed of actin and thick filaments composed of myosin [35]. The large particle size and well-ordered structure of myofibrils can be destroyed, polymerized, and dissociated by turbulence and mechanical forces generated during the HIU process, releasing myosin, actin, and thick filaments [36]. These results are consistent with the previous SDS-PAGE results in which MHC and actin were degraded (Fig. 1a). However, in the HIU-treated sample for 120 min, one small peak was observed in the large particle size, confirming that some MPs were aggregated by HIU for a long time. Similar results have also been presented earlier by Zhao et al. [37]. They reported that when porcine MPs were treated with US at 20 kHz at a power level of 0–600 W for 6 min, the particle size kept decreasing until the power reached 500 W but started increasing above 500 W due to protein aggregation. Thus, it is evident that excessive HIU processing power or time might cause aggregation of proteins, resulting in particle size increase.

3.1.3. Molecular conformation

To provide additional evidence for protein structural modification in HIU-treated MPs at various time periods, structural information was obtained by FT-IR spectroscopy (Fig. 1d). In the FT-IR spectrum, the main spectra are amide A, Amide Ⅰ and Amide Ⅱ. Amide A (3,200–3,400 cm⁻¹) reflects the asymmetric and symmetric stretching of the N—H bond of the amino group and the stretching vibration of intermolecular O—H, so that changes in hydrogen bonds between peptide chains can be estimated [1], [38]. Amide Ⅰ (1,600–1,700 cm⁻¹) is associated with stretching vibrations of C O and C—N in the polypeptide backbone, and Amide Ⅱ (1,480–1,580 cm⁻¹) is associated with N—H bending and C—N stretching vibrations [39]. HIU treatment did not significantly affect the overall FT-IR spectrum of MP, suggesting that no significant changes occurred in the polypeptide bac]. However, the Amide A peak position showed a slight blue shift (3,270 to 3,288 cm⁻¹) as the HIU treatment time increased from 0 to 60 min. This implies changes in intramolecular or intermolecular interactions, where strong cavitation and mechanical forces generated by HIU treatment are shown to induce dissociation of the myofibrils by destroying non-covalent interactions containing N—H or O—H in MP [1], [40]. The absorption of the Amide Ⅰ band is closely related to the stretching vibration of C—O derived from the vibration of water and the α-helix structure. As the HIU treatment time increased, the intensity of the Amide Ⅰ band tended to increase. In addition, compared to the control group, HIU-treated MPs showed a lower absorption band at 1,090 cm⁻¹. Thus, it can be inferred that C—H stretching vibrations can be promoted by HIU treatment and that HIU induces protein unfolding by disrupting hydrogen bonding and interfering with hydrophobic interactions [41], [42].

3.1.4. Secondary structure

The secondary structures of HIU-treated MPs were determined using CD spectroscopy, and the α-helix, β-sheet, β-turn, and random coil ratios are presented in Table 1. The proportion of α-helix tended to increase due to HIU treatment, but the β-sheet and random coil tended to decrease significantly compared to the control group (P < 0.05). Yu et al. [43] reported that the α-helix increased and the β-sheet decreased as the ultrasonic strength was increased when the mussel MPs were treated with HIU, which was similar to the results of this study. This secondary structural change of MPs could be attributed to cavitation, strong shear, and turbulence occurring during HIU treatment. β-sheets unfolding occurred as a result of such factors, which may have led to transformation to α-helix and β-turn [43]. However, some studies related to sonicated proteins reported a trend opposite to this study in that α-helix decreased and β-sheet increased, but this may be due to differences in sonication conditions and protein components analyzed [1], [11], [44].

Table 1.

Effect of high-intensity ultrasound (HIU) treatment time on secondary structural components of myofibrillar protein (MP) estimated from circular dichroism spectra.

Treatment time (min)	α-helix	β-sheet	β-turn	Random coil
0	18.59 ± 0.85c	43.10 ± 3.32a	18.90 ± 0.18c	19.41 ± 1.26 a
15	37.67 ± 1.92b	22.51 ± 2.13b	20.37 ± 0.31a	19.44 ± 0.52 a
30	46.88 ± 2.35a	16.33 ± 1.53c	20.04 ± 0.24ab	16.74 ± 1.26b
60	43.31 ± 3.19ab	18.32 ± 2.14c	19.85 ± 0.21ab	17.85 ± 1.09 ab
120	48.68 ± 3.90a	15.10 ± 2.37c	19.60 ± 0.33b	16.62 ± 1.20b

Mean values with different letters within the same samples are significantly different (P < 0.05) by Duncan’s multiple range test, n = 3.

3.2. Physicochemical properties of HIU-treated MPs

3.2.1. pH and turbidity

The pH of MPs treated with HIU under various time conditions is shown in Fig. 2a. No significant difference was observed between the samples treated with HIU for 0 min and 15 min (P > 0.05). However, when the HIU treatment time was 30 min or more, the pH was significantly reduced compared to the control (P < 0.05). It is presumed that the acidic residues of MP were exposed by HIU treatment, which causes strong cavitation, shear, and mechanical forces [37]. Turbidity is an index for measuring protein aggregation, and the higher the protein aggregation level, the higher the turbidity [43]. Fig. 2b presents the turbidity results of MPs treated with HIU under various time conditions. The control had the highest turbidity at 0.15 (A_600nm), which may be due to the larger aggregates and uneven distribution of protein molecules. As the HIU treatment time increased from 0 min to 60 min, the turbidity of the samples decreased significantly (P < 0.05), but there was no significant difference in the turbidity of the samples with HIU treatment for 60 and 120 min (P > 0.05). HIU treatment leads to protein particle size reduction and structural changes, which increases the specific surface area of the scattered light, ultimately reducing turbidity [29]. Numerous studies have also confirmed reduced protein turbidity as a result of HIU treatment [16], [22], [43]. However, although the results pertaining to particle size distribution indicated (Fig. 1b) that HIU treatment for ≥ 120 min induces MP re-aggregation and the formation of proteins with large particle sizes, a significantly increased level of turbidity was not observed compared to the 60-min HIU treatment group [16].

Fig. 2.

Effect of high-intensity ultrasound (HIU) treatment time on physicochemical properties of myofibrillar proteins (MPs): (a) pH, (b) turbidity, (c) hydrophobicity, and (d) protein oxidation.

3.2.2. Surface hydrophobicity

Protein surface hydrophobicity, which reflects changes in the number of surface hydrophobic amino acid residues, is related to protein solubility, emulsification, and stability [22], [43]. In addition, surface hydrophobicity is an important factor influencing protein’s functional properties and tertiary structure formation [31]. The surface hydrophobicity of the control group was the lowest, and the surface hydrophobicity increased significantly as the HIU treatment time increased (P < 0.05). Similarly, Zhang et al. [22] reported that the content of reactive sulfhydryl groups (SH) increased by sonication and that the SH groups embedded in the protein moved to the surface during the particle size reduction process by ultrasonication. These results indicate that the protein structure was modified as the protein unfolded during HIU treatment, increasing the exposure of the hydrophobic aromatic amino acid group surrounded by the non-polar environment located inside the MP [31]. However, there was no significant difference between the 60- and 120-min samples (P > 0.05). This phenomenon may be due to HIU treatment for an excessive time which protects the hydrophobic groups by re-aggregating the protein [43], and re-agglomeration of the protein was also confirmed in the previous particle size distribution results (Fig. 1c).

3.2.3. Protein oxidation

Carbonyl content is an important indicator of the degree of protein oxidation in food. The results of the evaluation of protein oxidation degree after HIU treatment for various time conditions indicate that the carbonyl content of native MPs not treated with HIU was the lowest at 4.02 ± 0.05 nmol/mg protein. However, it was observed that the carbonyl content significantly increased as the HIU treatment time increased (P < 0.05). In the case of the longest HIU-treatment time of 120 min, the carbonyl content was the highest at 8.01 ± 0.07 nmol/mg protein. From previous studies, it is known that the generation of reactive oxygen species (ROS) increased by US, and some amino acid residues, including cysteine and phenylalanine, are susceptible to protein oxidation by ROS [45], [46]. Such oxidative deformation may lead to structural changes in proteins, such as unfolding, crosslinking, and fragmentation, which expose hydrophobic groups to the surface [47]. In this study, it was also confirmed that as the HIU treatment time increased, the carbonyl group and surface hydrophobicity increased with a positive correlation.

3.3. Rheological characteristics of HIU-treated MPs

In order to understand the effect of HIU on the function of muscle protein in food processing including meat, the rheological characteristics, such as the viscoelasticity of HIU-treated MPs, were determined, which is the basis of emulsification properties for formulation and texture of the products [31]. Fig. 3a shows the results of apparent viscosity measured to investigate the fluidity of HIU-treated MP under various time conditions. As the HIU treatment time increased, the apparent viscosity tended to be lower, and all samples showed shear thinning behavior as the viscosity decreased with increasing shear rate. These results may be due to the formation of small particles by cavitation and strong shearing forces occurring during HIU treatment which breaks down protein chains, thereby preventing protein binding and aggregation [22].

Fig. 3.

Effect of high-intensity ultrasound (HIU) treatment time on rheological properties of myofibrillar proteins (MPs): (a) apparent viscosity, (b) strain sweep test, (c) storage and loss moduli (G′ and G′′), and (d) loss factor (tan δ) under frequency sweep test.

The storage modulus (G') as a function of strain for HIU-treated MP under various time conditions is shown in Fig. 3b. The control group exhibited an LVE at a strain of<5 %, followed by a non-linear region. On the other hand, HIU-treated samples showed LVE at a strain of<10 %, confirming that HIU treatment expanded the LVE of the MP solution. Earlier it was found that the G' and G'' values of the sonicated soybean isolate protein dispersion were lower than that of the unsonicated control, and the LVE was more expanded [48]. Also, in all samples, G' was dominant over G'' in LVE. In the case of samples treated with HIU for 0, 15, and 30 min, a crossover point, considered as a yield stress index, was confirmed, showing flow behavior due to structural failure at strains <100 %.

The frequency sweep test is an excellent method to characterize the viscoelastic properties of MP solutions; it was performed to better understand the structural changes of proteins by HIU treatment (Fig. 3c). All samples showed that G' was greater than G'' at frequencies above 1 rad/s, which could be attributed to the weak gel structure of the protein. It was confirmed that the G' and G'' values became smaller as HIU treatment time increased, which may be because HIU treatment induces protein unfolding and weakens non-covalent bonds, such as intermolecular hydrogen bonds. Yu et al. [43] reported that the β-sheet content decreased as the treatment intensity increased during HIU treatment (20 kHz, 600 W, 16 min) of Mytilus edulis proteins and G' decreased, which was similar to the results of this study. Low tan δ values and high G' indicate a strong gel network. It was confirmed that tan δ was higher and G' was lowered by HIU treatment (Fig. 3d). The above results suggest that HIU treatment altered the structure and rheological properties of MP.

3.4. Emulsification properties of HIU-treated MPs

3.4.1. ESI and EAI

EAI and ESI were measured to evaluate the emulsification properties of HIU-treated MP under various time conditions (Fig. 4a). EAI refers to the ability of a protein to be adsorbed on an emulsion droplet interface, and ESI refers to the ability of the protein to remain at the interface after storage time [43]. EAI and ESI were significantly increased as the HIU treatment time increased from 0 to 60 min (P < 0.05). In a previous study, the emulsification ability of beef MP and chicken muscle fiber protein were improved by HIU treatment [18], [31]. In particular, the apparent viscosity of the MP solution was decreased compared to the control group by the HIU treatment (Fig. 3a), which indicates increased mobility of MP and may have affected faster absorption at the interface of the Pickering emulsion. This phenomenon may be caused by the reduction of the particle size of MP (Fig. 1b) and the increase in surface hydrophobicity (Fig. 2b) by HIU treatment, which increases protein absorption rate at the water and oil interface [43]. Furthermore, α-helix has amphiphilic properties and plays an important role in adsorption to the droplet interface, and it is known that a protein containing a higher content of α -helix is adsorbed more to the droplet interface than a protein containing a higher content of β-sheet [49]. In this study, it was confirmed that the content of a-helix increased as the HIU treatment time increased. However, when treated with HIU for 120 min, EAI and ESI tended to decrease compared to 60 min, which may be due to excess protein re-aggregation through non-covalent and covalent interactions and reduced surface hydrophobicity, resulting in reduced emulsification capacity [31], [50]. Evidence of protein re-aggregation by excessive HIU treatment was also confirmed in the previous particle size distribution results (Fig. 1c).

Fig. 4.

Emulsification properties of Pickering emulsion stabilized by myofibrillar protein (MP) treated with high-intensity ultrasound (HIU) at various times: (a) emulsion activity index (EAI) and emulsion stability index (ESI), (b) mean particle size and polydispersity index (PDI), (c) zeta potential, and (d) confocal laser scanning microscopy images.

3.4.2. Droplet characterization and morphology

The mean particle size and zeta potential were analyzed to determine the effect of the HIU on MPs’ droplet properties of the Pickering emulsion. The mean particle size of the Pickering emulsion produced by MP was expressed as d_4,3 and d_3,2 (Fig. 4b), and it was found that the droplet size was significantly reduced from 51.18 to 19.55 μm for d_4,3 and from 41.35 to 17.62 μm for d_3,2 (P < 0.05). Based on the above results, it seems that as the HIU treatment time increased, denaturation of MHC and actin took place, and the MP particle size became smaller, resulting in the formation of smaller droplets. It was found that the absolute value of the zeta potential of the Pickering emulsion prepared with the MP treated with HIU significantly increased compared to the zeta potential of the emulsion prepared with the MP not treated with HIU (P < 0.05; Fig. 4c). The increased zeta potential can be explained by HIU-induced physical degradation and unfolding of protein aggregates, leading to exposure of internal polar sites [1]. Accordingly, it is expected that the electrostatic repulsive force between the droplets will increase due to the increase of the absolute zeta potential of the emulsion prepared with HIU-treated MP, and the resistance to the unstable dispersion state might increase.

The morphology of the Pickering emulsion stabilized by HIU-treated MP under various time conditions was observed by CLSM (Fig. 4d). In the image, the red spheres represent the oil phase, while the blue ones correspond to MP. MP was adsorbed at the droplet interface, forming a relatively dense layer on the oil droplet. In addition to the previous analysis of the mean particle size of the droplets (Fig. 4b), it was confirmed once again that small droplets were formed compared to the control group by the HIU treatment, which can be seen in the CLSM images. Moreover, in the Pickering emulsion prepared with MP that was not treated with HIU, a thicker blue protein interfacial layer was confirmed than in the Pickering emulsion prepared with MP treated for 60 or 120 min. Without HIU treatment, MP with a large particle size was adsorbed to the interface, resulting in a thicker interface. In addition, protein aggregates not adsorbed to the emulsion interface were also observed in the CLSM image of the emulsion prepared with MP with an HIU treatment time of 0 or 15 min.

3.4.3. Dispersion stability

The dispersion stability of Pickering emulsions prepared with HIU-treated MP at various times from 0 to 120 min was measured at 25 °C for 3 h, and the backscattering profiles (BS) are shown in Fig. 5a–e. The x-axis represents the height of the measurement cell, and the y-axis represents the percentage change of BS with respect to the initial state. In case of the control group prepared with MP without HIU treatment, three major destabilization phenomena occurred. The first is the sedimentation phenomenon at the bottom of the cell. MP, which was not adsorbed to the droplet interface, subsided and precipitated as the storage time elapsed, leading to an increase in BS [51]. Proteins not adsorbed to the droplet interface were also confirmed in the previous CLSM image (Fig. 4d). The second is flocculation or coalescence that occurs in the middle of the cell. When agglomeration occurs in colloids with a particle size>0.6 μm, the BS in the middle part is lowered [52]. Finally, the third is creaming and clarification that occurs at the top of the cell. Emulsion droplets composed of oil were less dense than water, so creaming occurred as they floated with increasing storage time. However, over time, droplet precipitation in the cream layer may have reduced BS again, and another possibility is that the BS at the top of the cell is lowered by the separation of water and oil due to the low emulsification capacity of naïve MPs. Pickering emulsions prepared with MP treated with HIU for 15 and 30 min showed similar destabilization. BS in the middle part of the cell tended to increase over storage time, which is a shape that occurs when agglomeration occurs in a colloidal solution containing particles smaller than 0.6 μm [52]. Also, at the top of the cell, a clarification phenomenon was observed, similar to the 0 min treatment group. On the other hand, emulsions prepared with HIU-treated MP for 60 and 120 min showed a stable emulsion dispersion phenomenon. Clarification occurred at the bottom of the cell, and creaming occurred at the top of the cell [33]. Thus, various phenomena such as sedimentation, flocculation, and creaming occurred, and TSI was calculated to compare the storage stability of the samples (Fig. 5f). The TSI of the Pickering emulsion prepared with MP without HIU treatment was the highest, and this confirms that the dispersion stability increased as the TSI is lowered by the HIU treatment.

Fig. 5.

(a-e) Backscattering profile and (f) turbiscan stability index (TSI) of Pickering emulsion stabilized by myofibrillar protein (MP) treated with high-intensity ultrasound (HIU) for various time periods: (a) 0 min, (b) 15 min, (c) 30 min, (d) 60 min, and (e) 120 min.

4. Conclusion

In this study, we have investigated the effect of HIU treatment time (0, 15, 30, 60, and 120 min) on the structural, physicochemical, and rheological properties of porcine-derived MPs; the potential of MPs as a Pickering stabilizer and the mechanism for increasing emulsification and dispersion stability of the HIU-treated MP. It was confirmed through SDS-PAGE, DLS, FT-IR, and CD spectroscopic analyses that MHC and actin were denatured by HIU treatment and that the secondary/tertiary structure of the protein was modified. Accordingly, as the HIU treatment time increased, the turbidity of MP decreased compared to the control, and the surface hydrophobicity significantly increased. Also, rheological properties such as apparent viscosity G' and G'' tended to decrease by HIU treatment. Since the EAI and ESI of HIU-treated MP were enhanced, the possibility of using HIU-treated MP as a Pickering stabilizer looks promising. The small particle size, increase in surface hydrophobicity, and decrease in apparent viscosity due to strong cavitation and shear force generated during HIU treatment contributed to the rapid and stable adsorption of HIU-treated MP at the Pickering emulsion interface and the formation of smaller droplets. In addition, it was confirmed that the TSI of the HIU-treated group was lower than that of the control group, showing good dispersion stability. In the context of an aging population and increasing demand for clean-label foods, these findings could be used as supporting data for the use of porcine-derived MPs, which have been treated with HIU for appropriate time periods, as a Pickering stabilizer.

CRediT authorship contribution statement

Yun Jeong Kim: Methodology, Formal analysis, Data curation, Writing – original draft, Writing – review & editing. Min Hyeock Lee: Data curation, Writing – original draft, Writing – review & editing. Se-Myung Kim: Conceptualization, Data curation, Writing – review & editing. Bum-Keun Kim: Conceptualization, Writing – review & editing, Supervision. Hae In Yong: Conceptualization, Writing – review & editing, Supervision. Yun-Sang Choi: Conceptualization, Data curation, Visualization, Writing – original draft, Writing – review & editing, Supervision.

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.