The pH of TMP samples was measured as described by Wang et al. (21), using a pH meter (PHS-3E, INASE Scientific Instrument Co., Ltd., Shanghai, China).

The TMP particle size was analyzed using the method of Chen et al. (6). The average particle sizes of diluted TMP (1 mg/ml) samples were determined at 25°C, using a Zetasizer (Nano-ZS90, Malvern Instrument Co. Ltd., Malvern, UK). The refractive index of particles, refractive index of dispersant, equilibrium time, and absorption parameter were 1.46, 1.33, 2 min, and 0.01, respectively.

A recent study was followed for turbidity measurement with slight modifications (23). Briefly, the absorbance of TMP was performed at 660 nm after the diluted TMP samples (1 mg/ml) were placed at room temperature for 30 min, using the UV-5100 spectrophotometer. A₆₆₀ was used to indicate the turbidity of the TMP samples.

The protein solubility of TMP samples was measured according to a published method (24), using BSA as the standard protein, with slight modifications. Briefly, 8 ml of the TMP samples (1 mg/ml) were taken and centrifuged at 10,000 × g for 10 min. Then, 1 ml of supernatant and 1 ml of TMP samples without centrifugation were added to 4 ml of biuret reagent, respectively. After mixing well, the absorbance was measured at 540 nm after reaction for 30 min in a dark chamber at room temperature. The protein solubility was expressed as follows:

Where S, C_b, and C_a represent the protein solubility of TMP samples (%), total protein content before centrifugation and protein content of the supernatant, respectively.

The H₀ was analyzed as described by Jia et al. (25). Briefly, 1 ml of TMP sample (5 mg/ml) was added to 200 μL of BPB solution (1 mg/ml) and mixed evenly, using a mixture of 200 μL of BPB solution (1 mg/ml) and 1 ml of 0.6 mol/L NaCl buffer as control. After standing at room temperature for 2 h and centrifuging at 6,000 × g for 15 min, 0.5 ml of supernatant was diluted into 4.5 ml of 0.6 mol/L NaCl buffer, and the absorbance of the diluted supernatant was measured at 595 nm using the UV-5100 spectrophotometer. The amount of BPB bound to TMP was used to represent the surface hydrophobicity of TMP, which was calculated as follows:

Where A₁ and A₂ represent the absorbance value of control and TMP samples, respectively.

The reactive sulfhydryl (SH) group of TMP samples was examined according to a previously published method (26). Briefly, after 50 μL of 10 mmol/L DTNB solution (phosphate buffer, pH 8.0) was mixed with 4 ml of TMP sample (1 mg/ml) and then incubated at 25°C for 20 min, the absorbance of the mixture was recorded at 412 nm using the UV-5100 spectrophotometer. The reactive SH group content was expressed as follows:

Where A₄₁₂, D, E_M, and C represent the absorbance of the mixture at 412 nm, dilution factor, molar extinction coefficient (13,600 L⋅mol^–1⋅cm^–1) and TMP concentration (mg/ml), respectively.

The intrinsic fluorescence spectra were analyzed using a Cary Eclipse fluorescence spectrophotometer (G9800A, Agilent Technologies Ltd., Santa Clara, CA, USA) with slight modifications (11). Briefly, after the TMP samples were dissolved in 0.6 mol/L NaCl buffer and adjusted to 0.2 mg/ml, the intrinsic fluorescence spectra of TMP samples were recorded at excitation wavelength, emission wavelength, excitation slit width, and emission slit width of 280, 300–400, 5, and 5 nm, respectively.

The circular dichroism (CD) spectra were measured using a CD spectropolarimeter (Chirascan, Applied Photophysics Ltd., Surrey, UK) with slight modifications (27). Briefly, after the TMP samples were diluted to 0.067 mg/ml and then injected into a quartz cuvette, the CD spectra were scanned in the far-UV range (200–260 nm) at room temperature. The results were described by the average of three scans, and the circle two data were expressed by the average molar ellipticity (θ) in deg⋅cm²/dmol. The scan rate, response time, bandwidth, and sensitivity were 100 nm/min, 0.5 s, 1.0 nm, and 20 mdeg, respectively. The proportions of four secondary structures were analyzed by CDNN software.

The preparation of O/W emulsion followed a recent report (6), with slight modifications. Briefly, 28 ml of TMP samples (10 mg/ml) were added to 7 ml of soybean oil, followed by homogenization at 8,000 r/min for 1 min.

The EAI and ESI of TMP emulsions were examined as reported (11). Briefly, immediately (0 min) and 10 min later after homogenization, 50 μL of TMP emulsion of the bottom was diluted to 5 ml of 0.1% sodium dodecyl sulfate (SDS) (w/v) solution. After mixing well, the absorbance of the mixture was measured at 500 nm by the UV-5100 spectrophotometer, using SDS solution as blank. EAI and ESI were calculated as follows:

Where N, θ, and C represent the dilution factor (100), volume fraction of oil in emulsion (0.2) and initial concentration of TMP (g/ml), respectively, and A₀ and A₁₀ represent the absorbance at 500 nm after 0 and 10 min, respectively.

The droplet size measurement was performed using the Nano-ZS90 Zetasizer according to the published method (11). Briefly, about 1 ml of fresh emulsion diluted 500-fold with 0.6 mol/L NaCl buffer was loaded into a quartz cuvette to measure droplet size.

The microstructure of TMP emulsions was observed using an optical microscope (DM-2000 LED, Leica Microsystems Co. Ltd., Wetzlar, Germany) (6). Briefly, 10 μL of the homogenized emulsion was dropped in the center of the slide and slowly covered with a cover glass to ensure no bubbles formed. The image was obtained by a 20× objective lens.

The rheology of TMP emulsions was examined using a rotational rheometer (MCR72, Antompa, Graz, Austria) with slight modifications (28). Briefly, using a 50 mm plate test, 3 ml of TMP emulsion was evenly coated on the test platform, and a layer of silicone oil was applied around TMP emulsion to prevent evaporation of TMP emulsion during the heating process. The frequency, strain, initial temperature, heating rate, termination temperature, and crack spacing were 0.1 Hz, 1%, 20°C, 1°C/min, 80°C, 1.0 mm, respectively. Storage modulus (G′) and loss modulus (G″) were analyzed as test indexes.

The H₀ represents the exposure of hydrophobic groups in protein molecules (29). This work is to determine the H₀ by analyzing the binding of hydrophobic amino acids of proteins to BPB. Compared with control group, HC for 5 min significantly decreased the binding amount of BPB (P < 0.05; Figure 2A). The decrease in H₀ was based on an increase in protein solubility (Table 1) (33), since the hydrophobic amino acids of TMP might be distributed inside the molecule by HC, leading to enhancing protein-water interactions and exposing less hydrophobic groups (34). With the further increase of HC treatment time (5–20 min), the H₀ of TMP increased significantly (P < 0.05), and reached the highest value (73.11 ± 1.23 μg) after HC treatment for 20 min. This was because the cavitation effect unfurled TMP structure and exposed hydrophobic groups, resulting in increased H₀. Similar to other physical modified MP studies (35–37). In addition, the temperature effect of HC in the local region of the suspension may be another important factor that causes the conformational change of TMP. Similarly, Pan et al. (30) suggested that the temperature effect of ultrasonic might alter the tertiary structure of MP due to the susceptibility of fish MP to thermal denaturation. These results suggested that HC changed the H₀ due to its influence on protein-water and protein intermolecular interactions and the exposure of hydrophobic groups.

The reactive SH groups refer to the SH groups located on the surface of the protein network, which can reflect MP tertiary structure conformational changes (7). Compared with control group, the reactive SH content was decreased significantly after HC treatment for 5 min (P < 0.05; Figure 2B), indicating that the structure of TMP and disulfide bond formation were changed, possibly because the reactive SH on TMP surface was converted to disulfide bonds by the HC-induced cavitation effects. Similarly, Yang et al. (17) reported that HC could lead to the conversion of free SH into disulfide bonds in SPI. However, compared with the 5 min, the reactive SH content increased significantly with the increase of HC treatment time (P < 0.05), indicating that HC accelerated the expansion and structural changes of protein molecules, leading to exposing more SH group hidden inside the protein molecules (19).

EAI and ESI represent the capacity of a protein to be adsorbed by the interface between the water phase and oil sphere during the formation of protein emulsion and the ability of a protein to maintain at the oil-water interface of emulsion after storage period, respectively (6, 24). Compared with control group (0 min), all HC treatments significantly increased the EAI of TMP emulsion (P < 0.05), and EAI was increased significantly with the increase of HC treatment time (except 10 and 15 min) (P < 0.05; Figure 3A). Different from EAI, ESI showed a trend of increasing first and then decreasing, that is, increasing gradually within 0–10 min of HC treatment and decreasing gradually with further treatment (10–20 min). The increase in EAI and ESI of TMP might be due to the cavitation effect generated by HC that decreases the particle size of TMP (Table 1), making more TMP easily adsorbed on the oil-water interface, resulting in the improvement of emulsifying capacity (17). Furthermore, lower protein turbidity and higher solubility (Table 1) accelerated the diffusion of TMP in the emulsion (6, 23). Interestingly, excessive HC treatment (20 min) increased particle size and turbidity and decreased solubility (Table 1) compared with the other groups, leading to a decrease in ESI but not in EAI as expected. This phenomenon might be attributed to an increase in H₀ (Figure 2A), which led to the enhancement of the interaction between adjacent molecules at the oil-water interface (7). Some other studies have reported that MP (10, 12) and SPI (17, 19) showed a good positive correlation between EAI and H₀, and increasing H₀ was conducive to improve EAI of proteins. This study showed that HC for 10 min could improve the EAI and ESI of TMP significantly.

Droplet size determines the rheology and stability of emulsion, and generally smaller droplet size is associated with higher emulsifying capacity (23, 38). The droplet size of TMP emulsion decreased significantly from 2,754 ± 110 nm (0 min) to 2,138 ± 182 nm (10 min) (P < 0.05) with the increase in HC treatment time (0–10 min), but no significant difference was observed between 5 min and 10 min treatments (P > 0.05; Figure 3B). However, with the continuous extension of HC treatment time (10–20 min), the droplet size of TMP emulsion increased significantly (P < 0.05), and this opposite trend was attributed to the overtreatment of the protein (7). These results indicated that appropriate HC could be conducive to decrease the droplet size of TMP emulsion and improve its emulsifying properties.

The microstructure of TMP emulsions as affected by HC was enlarged 200 times under an optical microscope, as shown in Figure 3C. The soybean oil droplets in the control group (0 min) were relatively large and unevenly distributed, which was attributed to the high interfacial tension between water and soybean oil (6). Compared with the untreated group, HC for 10 min had fewer large oil droplets, suggesting that HC induced TMP expansion and conformational changes, thereby decreasing the interfacial tension between water and soybean oil. Similar results have been reported in ultrasonic modification of MP (6). However, it was observed that the oil droplets size in the fresh emulsion increased, and the distribution was more uneven with the further extension of HC treatment time (10–20 min). The results of optical microscopic images could intuitively show the oil droplet size, corresponding to the previous results of the droplet size (Figure 3B). These results showed that compared with control group, HC for 10 min decreased the oil droplet size and improved the uniformity of emulsion.

The rheological behavior of emulsions prepared with MP was analyzed to understand the viscoelastic properties of MP emulsions and the functionality of muscle protein in meat processing (39). The rheological properties of the TMP emulsions as affected by HC are shown in Figure 4. The G′ of TMP emulsion showed a decrease at the initial stage of heating (20–22°C), which might be attributed to the formation of weak and preliminary interactions of the protein aggregates prior to heating and destruction after initial heating (11). Compared with other groups, HC for 10 min had the highest G′ value at the initial stage and scanning end, indicating that HC for 10 min had the most robust gel structure of TMP emulsion (28). In addition, after HC treatment for 10 min, the particle size of TMP was decreased and an emulsion with more uniform and smaller oil droplets was formed. HC for 10 min decreased the particle size of TMP and formed smaller and more uniform oil droplets in the emulsion. Li et al. (11) showed that smaller oil droplets could fill effectively and evenly into the network of MP gel during heating, resulting in better elasticity of MP emulsion gel. Therefore, HC for 10 min improved the elastic properties of TMP emulsion and prepared emulsion with stronger rheological properties. The changes in G″ of TMP emulsions were similar to that of G′. The G″ value of the TMP emulsion treated by HC for 10 min during about 50∼70°C heating was higher than that of the control (0 min). For all emulsions, the G″ values were substantially lower than that of G′ values indicating the formation of a gel-like behavior (39). The values of G′ and G″ increased with the increase of HC treatment time, indicating that HC promoted the formation of firm and dense gel structure of proteins. However, with the further extension of HC treatment time, the viscoelastic properties of the emulsion decreased, possibly due to the formation of uneven and rough gel structure. Rheological measurements showed that appropriate HC (10 min) improved the rheological properties of TMP-based emulsion as well as the structure of TMP.

The tertiary and secondary structure changes of the HC-induced TMP were measured by intrinsic fluorescence spectra and CD spectra, which reflected the changes in the physicochemical structure and emulsifying properties of TMP to verify the proposed schematic model. In general, intrinsic fluorescence spectroscopy is used to assess the local environment of certain chromophores, especially tryptophan residues, and can monitor tertiary structural changes in proteins (7). The fluorescence intensity of all treatment groups was lower than that of the control group (Figure 6A), suggesting that HC destroyed hydrophobic interactions, allowing the structure of TMP molecules to unfold, which caused the movement of tryptophan residues to more polar environments. Similar results have been reported in the ultrasonic treatment of MP (30, 31). For example, Jiang et al. (31) speculated that cavitation effects generated by ultrasound exposed the tryptophan group to the solvent, leading to a decrease in fluorescence intensity of MP. Pan et al. (30) pointed out that ultrasound disrupted hydrophobic interactions between MP molecules and moved tryptophan residues to a more polar environment. Interestingly, the fluorescence intensity of TMP decreased when HC treatment time increased from 0 to 15 min but increased after 20 min. This might be due to protein aggregation (Table 1), increased H₀ (Figure 2A), and specific modification of tryptophan residues in TMP, which combinedly resulted in less exposure of tryptophan residues to water (12).

In addition, in terms of the maximum fluorescence emission wavelength (λ_max), the λ_max of the control group (334 nm) was higher than 330 nm, indicating that tryptophan was in a polar environment (41). The larger the redshift of λ_max, the larger the conformational change of the protein. With the extension of HC treatment time, λ_max of TMP redshifted from 334 to 338 nm, which might be due to the exposure of tryptophan and the microenvironment becoming more polar, causing the tertiary conformation of TMP to become looser (Figure 5) (12).

The CD spectra of TMP as affected by HC are shown in Figure 6B. In general, the α-helical structure is formed mainly by the hydrogen bond between the carbonyl oxygen (C=O) and amino hydrogen (NH^–) groups in the polypeptide chain (42). Two negative peaks of α-helical structures were detected at 210 and 221 nm, and HC decreased the intensity of these two peaks, indicating that HC destroyed the intramolecular hydrogen bond of TMP, thus promoting the unfolding of the protein (43). At the same time, Table 2 also shows that HC significantly decreased the α-helix content (P < 0.05). Additionally, compared with control group, the β-sheet, β-turn, and random coil contents of all HC treatment groups were increased significantly (P < 0.05), but no significant differences were observed between HC treatment groups (P > 0.05). The transformation of secondary structure of TMP might be caused by the hydrogen bond change induced by HC. Similar conclusions have been reported in using other physical modification techniques to change the secondary structure of MP, such as ultrasound (44), high-pressure homogenization (23). Moreover, Yang et al. (17) reported that swirling cavitation increased β-sheet content and decreased α-helix content, which might improve the emulsifying properties of SPI. Consistent with the experimental results, HC increased the β-sheet content and decreased the α-helix content of TMP significantly (P < 0.05) compared with control group, which might be another important reason for the enhancement of emulsifying properties of TMP (Figures 3, 4). However, the decrease in emulsifying properties after HC for 15 and 20 min might be attributed to the increase in particle size and turbidity as well as the decrease in solubility (Table 1).