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Ultrason Sonochem
Ultrason Sonochem
Ultrasonics Sonochemistry
1350-4177
1873-2828
Elsevier

S1350-4177(24)00307-9
10.1016/j.ultsonch.2024.107059
107059
Original Research Article
Insight into the effects of ultrasound-assisted intermittent tumbling on the gelation properties of myofibrillar proteins: Conformational modifications, intermolecular interactions, rheological properties and microstructure
Zhang Ruyu
Zhou Lei
Zhang Wangang wangang.zhang@yahoo.com
⁎
State Key Laboratory of Meat Quality Control and Cultured Meat Development, Key Laboratory of Meat Products Processing, Ministry of Agriculture, Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
⁎ Corresponding author at: College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. wangang.zhang@yahoo.com
05 9 2024
11 2024
05 9 2024
110 10705925 7 2024
28 8 2024
4 9 2024
© 2024 The Authors. Published by Elsevier B.V.
2024

https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Graphical abstract

The aim of the present study was to evaluate the effects of ultrasound-assisted intermittent tumbling (UT) at 300 W, 20 kHz and 40 min on the conformation, intermolecular interactions and aggregation of myofibrillar proteins (MPs) and its induced gelation properties at various tumbling times (4 and 6 h). Raman results showed that all tumbling treatments led the helical structure of MPs to unfold. In comparison to the single intermittent tumbling treatment (ST), UT treatment exerted more pronounced effects on strengthening the intermolecular hydrogen bonds and facilitating the formation of an ordered β-sheet structure. When the tumbling time was the same, UT treatment caused higher surface hydrophobicity, fluorescence intensity and disulfide bond content in the MPs, inducing the occurrence of hydrophobic interaction and disulfide cross-linking between MPs molecules, thus forming the MPs aggregates. Additionally, results from the solubility, particle size, atomic force microscopy and SDS-PAGE further indicated that, relative to the ST treatment, UT treatment was more potent in promoting the polymerization of myosin heavy chain. The MPs aggregates in the UT group were more uniform than those in the ST group. During the gelation process, the pre-formed MPs aggregates in the UT treatment increased the thermal stability of myosin, rendering it more resistant to heat-induced unfolding of the myosin rod region. Furthermore, they improved the protein tail–tail interaction, resulting in the formation of a well-structured gel network with higher gel strength and cooking yield compared to the ST treatment.

Keywords

Intermittent tumbling
High-intensity ultrasound
Myofibrillar proteins
Intermolecular interactions
Protein aggregation
Gelation properties
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pmc1 Introduction

Tumbling technology is well known for curing, tenderizing, and improving the slice coherency of meat products. During the tumbling process, meat pieces rotate with the rotation of the tumbler or the inner paddle, inducing them to fall, rub and experience physical shock. These mechanical actions could destroy the muscle structure, weaken the mechanical strength of myofibers, and expand the intracellular space. Consequently, the meat pieces become more tender, and the external marinade solution penetrates the meat matrix more quickly and distributes more evenly [1], [2]. Additionally, meat pieces become more manageable for subsequent filling and shaping treatments. Recognizing its advantages, tumbling treatment is extensively applied in the procedure of restructured meat products (e.g., cooked ham, restructured pork steak, sausages, and surimi) and prepared meat products (e.g., prepared pork chops and marinated chicken breast). Furthermore, it has been increasingly employed in the development of health-conscious and green-label foods, including meat products with reduced phosphate content [3]. Notably, for restructured meat products, tumbling treatment exerts a crucial role in improving the adhesion between individual meat chunks to mimic the original intact muscle food [4], [5]. Following the entry of the marinade solution into the meat, there is an increase in electrostatic repulsion and ion strength in the muscle. This, coupled with the disruption of muscle structure, facilitates the extraction of myofibrillar proteins (MPs). Through sustained compression, the extracted MPs accumulate on the meat surface, forming a cohesive creamy exudate that tightly adheres to meat chunks. During the subsequent heating process, these proteins could form a protein gel “layer” that covers the meat chunks, preventing water loss [6]. In practical processing, motivated by the need for increased cooking yield, the majority of tumbling procedures are performed using a single intermittent tumbling (ST) mode. This mode involves alternating work and rest sessions, allowing the tumbled meat chunks to have sufficient time for the extraction and swelling of MPs. Nevertheless, it presents a clear drawback of being time-consuming: the entire tumbling process may take a considerable amount of time (10–16 h), potentially falling short of the desired efficiency for the modern food industry [2]. Consequently, there is an urgent need to explore innovative technologies to enhance the efficiency of ST treatment.

High-intensity ultrasound (HIU, 20–100 kHz, 10–1,000 W/cm2) is well known for inducing cavitation effects, where bubbles instantaneously collapse within the liquid medium [7]. This consequently causes the shearing force, turbulence, and microstreaming [8]. Numerous studies have applied HIU as an auxiliary method to promote curing efficiency of meat products [9], [10], [11], [12]. However, there is limited research on combining HIU with tumbling technology for meat curing, and the existing studies mainly adopt the “segmented” approach, where samples are processed separately in an ultrasonic device either before or after tumbling treatment. This method could lead to issues such as high equipment separation, low operational efficiency, complex procedures, significant raw material losses, and increased safety risks.

Therefore, to address the aforementioned challenges, we have innovatively redesigned the tumbling devices, that fulfilled the HIU synchronized assistance with the ST treatment (UT) [13]. Furthermore, our previous research on pork curing and cooked ham production proved that the HIU could promote the ST treatment by further disrupting the integrity structure of myofilaments, which subsequently accelerated the brine entry into the inner muscle and facilitated the extraction of MPs. As a result, the UT treatment could increase the tumbling efficiency and improve the quality of cooked ham [1], [6]. Considering that MPs constitute the predominant component of meat protein (50–60 %), their physicochemical and functional properties substantially impact the quality of meat products. Particularly, MPs possess gelation capacity. During the heating process, MPs could interact with each other and form a gel network that traps water and oil within a three-dimensional structure. The quality of the gel plays a crucial role in determining the texture, juiciness, slice-ability, and product yield of meat products. It is essential to pay close attention to the physicochemical and gelation properties to regulate the quality of meat products.

However, there is still a gap in understanding the influence and mechanisms of UT treatment on the gelation properties of MPs. Therefore, this study implemented the ST and UT treatments with 4 and 6 h of tumbling time. The effects of ST and UT on the physicochemical properties and conformation of MPs were examined using Raman analysis, surface hydrophobicity, intrinsic fluorescence intensity, and sulfhydryl-disulfide bond content. Subsequently, the protein interaction pattern and the morphology were evaluated at multiple scales, including solubility, particle size, atomic force microscope (AFM) and SDS-PAGE. The heat-induced protein–protein interactions were analyzed based on the thermal sweep rheological properties of MPs. The final gel quality was assessed by considering the gel strength, cooking yield and microstructure. This study could advance a comprehensive understanding of the effects of UT treatment on the conformational changes, protein interaction patterns, and induced gelation properties of MPs.

2 Materials and methods

2.1 Materials and reagents

The experimental meat sample chosen for this study was pork thigh (mesoglutaeus, Sushi Meat Co., Ltd in Huai'an, Jiangsu province, China) within 24 h of slaughter. The meat samples were trimmed into lean meat pieces (100 × 40 × 40 mm3) and were stored at −20 °C under vacuum packaging. All other chemicals were analytical reagent grade.

2.2 Tumbling treatment

Before the start of the experiment, the meat samples were completely thawed at 4 °C and injected with 25 % (w/w) brine, following the procedures described in our previous study [6]. Subsequently, the meat samples underwent tumbling treatment, with the ST treatment being designated as the control group. The tumbling conditions were set as follows: the work/rest time of one cycle was 20 min/10 min, the vacuum level was −0.08 MPa, the rotation speed was 10 rpm and the tumbling temperature was 4 °C. For the UT treatment, HIU was synchronized with the ST treatment, operating at a frequency of 20 kHz and a power of 300 W during the working phase of the ST treatment. Conversely, the HIU ceased operation when the ST treatment was at a standstill. In addition, the HIU was designed to assist the ST during the first two cycles, until the total work time of HIU reached 40 min, while the total tumbling times were established at 4 and 6 h, respectively. It should be noted that the parameters of HIU were selected based on our previous pork curing research, which demonstrated that these conditions could accelerate curing efficiency and improve pork quality [1]. Additionally, the total tumbling time was determined according to the production of cooked ham. Under both 4 and 6 h of UT treatment, the UT treatment was found to be more effective in improving the quality of cooked ham than the ST treatment [6].

2.3 Myofibrillar proteins extraction

The extraction of MPs followed the methods of Zhou et al. [14] with a slight change. Approximately 50 g of meat samples from each replication were blended with 200 mL of buffer A (0.1 M NaCl, 2 mM MgCl2, 1 mM EGTA, 10 mM Na2HPO4, pH 7.0) and homogenized at 10,000 g for 1 min (PD 500-TP, Prima Technology Group Co., Ltd., UK). Subsequently, the homogenate was centrifuged for 15 min at 2,000 g to collect the residue. This step was repeated three times. The resulting residue was washed twice with 200 mL of buffer B (0.1 M NaCl, pH 6.0). Before the final centrifugation, the homogenous liquid was filtered through gauze. Ultimately, the MPs pellet was collected and stored at 4 °C.

2.4 Conformation of MPs

2.4.1 Secondary structure and microenvironment

The MPs pellet was placed on a silicon slice and detected within the 400 to 3,600 cm−1 range using a Raman spectrometer (Horiba/Jobin Yvon, Longjumeau, France) with three scans and a 30-second exposure time. Protein chemical groups and their Raman spectral assignments were referenced from Herrero et al. [15].

2.4.2 Tertiary structure

The tertiary structure of MPs was ascertained by adopting our previous methods [13] with a few modest adjustments. For the intrinsic fluorescence intensity, the MPs solution was diluted to 0.2 mg/mL and scanned in the fluorescence mode with the excitation at 280 nm and the emission in the range of 325–400 nm (Spectramax M2e Microplate Reader, Molecular Devices Inc., San Francisco, CA, USA). For determining the surface hydrophobicity of MPs, the excitation was set at 370 nm, and the emission was recorded in the range of 415–570 nm.

2.4.3 Sulfhydryl content and disulfide bond content

The total and free sulfhydryl contents were measured according to Fu et al. [16] with appropriate changes. A volume of 0.5 mL of MPs solution (1 mg/mL) was mixed with 5 mL of total sulfhydryl buffer (8 M urea, 10 mM EDTA-Na2, pH 6.0) and 100 μL of Ellman's reagent. Then, the mixture was left to stand for 25 min at 40 °C. For the determination of free sulfhydryl content, a total of 5.5 mL of MPs solution (1 mg/mL) was mixed with 100 μL of Ellman's reagent and reacted for 1 h at 4 °C in the dark. The disulfide bond content was measured using the 2-nitro-5-thiosulfo benzoate (NTSB) method [17]. Two milliliters of MPs solutions (2 mg/mL) were mixed with 3 mL of fresh and diluted NTSB solution. Subsequently, the mixture was incubated for 25 min in the dark at room temperature. After the reaction, all the mixtures were read at 412 nm.

2.5 Aggregation behavior of MPs

2.5.1 Protein solubility

The MPs solution was diluted to a concentration of 2 mg/mL. Five milliliters of the MPs solutions were transferred to a plastic tube and then centrifuged at 10,000 g for 10 min at 4 °C (Beckman Coulter, Brea, CA). The protein content was assessed using the Biuret method. MPs solubility was determined by calculating the proportion of protein concentration in the supernatant relative to the original protein concentration [18].

2.5.2 Particle size and zeta potential

The MPs solution was diluted to 0.5 mg/mL using buffer C and ultrapure water in preparation for the assessment of particle size and Zeta potential. The dispersant was set as water with a refractive index of 1.330, and the equilibration time was established at 120 s (Malvern Zetasizer, Malvern Nano ZS90, Malvern, UK).

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

Protein aggregation pattern was detected using the reduced and non-reduced SDS-PAGE method. The MPs solution (1 mg/mL) was mixed with 4 × sample buffer with or without 10 % β-mercaptoethanol (GenScript, Nanjing, China). Subsequently, the blend was heated at 95 °C for 5 min. Precisely, 10 μL of the prepared sample solutions were loaded into the 8 % precast gel (GenScript, Nanjing, China) and run at 100 V until the bromophenol blue reached the bottom of the gel. After staining the bands, the image of the gel was scanned and analyzed on the program of Molecular Imager GelDoc TM XR+imaging system (Bio-Rad Laboratories, Hercules, CA). In addition, the polymer band located in the upper gel was cut for the determination of Nano liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry (Nano LC-ESI-MS/MS) technology, according to Kang et al. [19].

2.5.4 Atomic force microscope (AFM)

The AFM topography of MPs was investigated using a scanning probe microscope (SPM-9700HT, Shimadzu, Kyoto, Japan). After diluting MPs sol with ultrapure water to 0.1 mg/mL, 10 μL of the MPs solutions were applied to the freshly cleaved mica substrate and dried at 25 °C until the solvent evaporated. The AFM image was then captured in an area of 5 × 5 μm2. The data were analyzed and visualized using the open-source software Gwyddion 2.43 (https://gwyddion.net/).

2.6 Rheological properties

The MPs sol (30 mg/mL) was painted evenly on the platform of the Rheometer (MCR 301, Anton Paar, Graz, Austria) with the 50 mm parallel probe. The temperature sweep test was performed within the 1 % stain, and the gap was 1 mm. The temperature was increased from 20 to 80 °C at a rate of 2 °C/min and then decreased from 80 to 20 °C at the rate of 5 °C/min [20].

2.7 Gel properties

2.7.1 Preparation of MPs gel

The MPs solution (30 mg/mL) in the 50 mL plastic centrifuge tube was gently centrifuged at 300 g for 5 min to eliminate bubbles. After that, all tubes were heated from 4 to 71 °C at a rate of 1°C/min and held at 71 °C for 30 min. Note that the final temperature was chosen in accordance with low-temperature meat products, which are conventionally heated until the center temperature reaches 71 °C in practical production [6]. After heating, the gel samples were cooled to 25 °C and equilibrated overnight at 4 °C before further determination.

2.7.2 Gel strength

The MPs gel was cut into a cylinder with 2 cm height and equilibrated at 25 °C for 20 min. The gel strength was determined using the texture analysis with the P/5 probe (TA XT Plus, Stable Micro Systems, Surrey, UK). The speeds for pre-testing, testing, and post-testing were determined to be 2.0, 1.0, and 2.0 mm/s, respectively. The force that compressed the gel with a distance of 5 mm was regarded as the gel strength (g).

2.7.3 Cooking yield of MPs gel

The cooking yield of the MPs gel was calculated as the weight percentage before and after heating [21].

2.7.4 Water distribution of MPs gel

The MPs gel was sectioned into a uniform cube with a height of 2 cm and detected by the low-field nuclear magnetic resonance (LF-NMR). The gel cube was positioned within the chamber of the Niumag pulsed NMR analyzer (NMI20-040H-I, Suzhou Niumag Analytical Instrument Corporation, Jiangsu, China). The program was configured as CPMG with a sampling bandwidth of 100 and a scan repetition of 4 [6].

2.7.5 Microstructure of MPs gel

Atomic Force Microscopy (AFM) was employed to examine protein aggregation during the gelation process. Following the method outlined in section 2.5.3, the 0.1 mg/mL MPs solution underwent heating using the steps detailed in section 2.8.1. The microstructure of the MPs gel was analyzed through scanning electron microscopy (SEM). The MPs gel was cut into an intact cube (0.5 × 0.5 × 0.5 cm3) and fixed in the 2.5 % glutaraldehyde. The following steps were adopted by our previously established method [6].

2.8 Statistical analysis

Each treatment was repeated four times. The data were analyzed using the factorial ANOVA with Fisher's LSD (Least Significant Difference), and it can be identified as significant when the P<0.05. The results were presented as mean ± standard error.

3 Results and discussion

3.1 Raman spectroscopy analysis

3.1.1 Secondary structure of MPs

The primary constituent of MPs is myosin, known for its “bean sprout” shape, featuring a double-globular head region and an α-helical rod-shaped tail. The secondary structure of MPs is depicted in Fig. 1A. Untreated MPs exhibited a typical composition, which predominantly consisted of α-helix (43.09 %), followed by β-sheet (23.30 %), β-turn (18.92 %), and random coil (14.70 %). After the tumbling treatments, irrespective of tumbling time and methods, there was a substantial decrease in α-helix content (P<0.05), accompanied by a significant increase in β-sheet content (P<0.05). It indicates that all the tumbling treatments modified the overall secondary structure of MPs. Considering that the α-helix stability relies on the intramolecular hydrogen bonds formed by carbonyl oxygen (C=O) and amino hydrogen (N–H) from the same peptide chain, this alteration in the secondary structure further illustrates that both the ST and UT treatments could disrupt the intramolecular hydrogen bonds and unfold the double-stranded tail region [18], thereby exposing the active groups in the tail region. Moreover, it was evident that at the same tumbling time, UT treatment imparted higher β-sheet content to the MPs compared to the ST treatment, with the maximum β-sheet content being observed in the UT-6 h treated MPs (P<0.05). In contrast, the ST treatment induced the formation of a more random coil structure in MPs (P<0.05). It further reveals that UT treatment facilitated the formation of intermolecular hydrogen bonds between two adjacent peptide segments, leading to the conversion of α-helix to β-sheet, endowing a more ordered structure in the MPs. Conversely, ST treatment tended to induce the conversion of the α-helix to the random coil, wherein the exposed active groups underwent irregular rearrangement, forming more unordered structures.Fig. 1 Raman spectroscopy analysis of myofibrillar proteins after single intermittent tumbling (ST) and ultrasound-assisted intermittent tumbling (UT) treatments. (A) Secondary structure content; (B) Normalized intensity of tryptophan residues band (I760) and tyrosyl doublet bands (I850/830); (C) Disulfide bonds conformation in 450–600 cm−1 region; (D) Normalized intensity of trans-gauche-trans conformation of disulfide bond (I545). UN represents the untreated myofibrillar proteins. Different letters (a-d) indicate significant differences (P<0.05).

3.1.2 Changes in the microenvironment of MPs

The Raman spectrum also provides the microenvironment information about proteins. The I760 refers to the normalized intensity of tryptophan residues and reflects their microenvironment polarity as the I760 increases along with the increase of hydrophobicity [22]. As depicted in Fig. 1B, when the tumbling time was the same, either at 4  or 6 h, the UT treatment exerted a more pronounced effect to enhance the I760 than the ST treatment (P<0.05), which confirmed that, in comparison to the ST treatment, UT treatment was more efficient in relocating the tryptophan residues to the hydrophobic microenvironment [23].

The I850/830 vibrations are assigned to the para-substituted benzene ring of the tyrosine residues. It is regarded as an indicator of hydrogen bonds involving the phenolic hydroxyl group [15]. When the value is in the range of 0.7–1.0 or lower than 0.3, it implies that the tyrosine residues are encased in the hydrophobic environment and serve as hydrogen bonds donors to form strong internal hydrogen bonds with protein molecules. Conversely, a higher value (>1.0) means that the tryptophan residues are exposed to the aqueous environment and engage in forming moderate or weak hydrogen bonds with water molecules [21]. As shown in Fig. 1B compared to the untreated MPs, all tumbling treatments obviously reduced the I850/830, reaching its minimum value in the UT-6 h group (P<0.05). It indicates that the tumbling treatments, particularly the UT-6 h treatment, could force the tryptophan residues to move to the hydrophobic microenvironment and facilitate the formation of strong hydrogen bonds between phenolic hydroxyl groups with neighboring protein molecules [21]. It agrees with the findings of the secondary structure, indicating that the UT treatment promoted the involvement of MPs in intermolecular interactions, resulting in stronger and more ordered hydrogen bonds compared to the ST treatment.

Disulfide bonds are crucial for preserving the conformation of MPs [24]. Its conformation could be identified within the 500–550 cm−1 wavelength region of the Raman spectrum. The peaks at 510, 516–530 and 535–545 cm−1 are corresponding assigned to the gauche-gauche-gauche (g-g-g), gauche-gauche-trans (g-g-t) and trans-gauche-trans (t-g-t) conformations of the disulfide bond. The g-g-g and g-g-t conformations are indicative of the intramolecular disulfide bond, whereas the t-g-t conformation corresponds to the intermolecular disulfide bond [24]. The untreated MPs exhibited a distinct peak located at 502 and 521 cm−1 and a subtle peak at 540 cm−1 (Fig. 1C). This indicates that the disulfide bond in the untreated MPs predominantly formed via intramolecular head-head linkage, involving the g-g-g and g-g-t conformations. The ST-4 h treatment was not sufficient to cause a significant change in the conformation of the disulfide bond. In contrast, the ST-6 h treatment resulted in the disappearance of the peak at 502 cm−1, accompanied by the enhancement of peaks at 524 and 540 cm−1. It demonstrates that the ST-6 h treatment facilitated the transformation of the intramolecular disulfide bond g-g-g to g-g-t and induced the formation of intermolecular disulfide bond with the t-g-t conformation. It might be related to the fact that the ST-6 h treatment could input the energy, causing the g-g-g with low potential energy to be transferred to high potential energy conformation [25]. Additionally, the ST-6 h treatment could destroy the α-helix structure and unfold the coiled rod region (as discussed in the secondary structure section). Thus, the active sulfhydryl groups initially buried in the rod region could interact with each other and form the intermolecular disulfide bond. Notably, the UT-4 h treatment amplified the peak at 546 cm−1 compared to the ST-6 treatment. Also, Fig. 1D shows that the normalized intensity of the 545 cm−1 band (I545) in the UT-4 h group increased to the same level as in the ST-6 h group (P>0.05), meaning that the UT-4 h treatment was efficient in enhancing the building of intermolecular disulfide bond. Furthermore, extending the tumbling time to 6 h for the UT treatment resulted in the disappearance of the peak near 510 cm−1. Only two peaks at 521 and 545 cm−1 were observed in the UT-6 h group. In particularly, the peak at 521 cm−1 was more intense than that in the UT-4 h and ST-6 h groups. It indicates that the UT-6 h treatment could introduce more energy, predisposing the intramolecular disulfide bond to a higher potential energy state and enabling the coexistence of intra- and intermolecular disulfide bond in the MPs.

3.2 Tertiary structure of MPs

For the surface hydrophobicity (So), ST treatment exhibited only a slight enhancement in So compared to the untreated MPs (Fig. 2A). In contrast, UT treatment had a more pronounced effect on So, elevating it to a significantly higher level than both untreated MPs and ST treatment (P<0.05). It is consistent with our previous research that the ultrasonic cavitation effect could further disrupt the spatial structure of the MPs, resulting in greater exposure of hydrophobic groups [13]. The hydrophobic interaction between the hydrophobic groups is the dominant driving force for protein aggregation [26]. The increased exposure of hydrophobic groups in the UT-treated MPs increased the potential for protein collision and interaction, potentially leading to protein reaggregation.Fig. 2 The tertiary structure of myofibrillar proteins after single intermittent tumbling (ST) and ultrasound-assisted intermittent tumbling (UT) treatments. (A) Surface hydrophobicity (SO); (C) Fluorescence emission spectrum; (D) Fluorescence intensity; (E) Total and sulfhydryl contents and disulfide bond content. UN represents the untreated myofibrillar proteins. Different letters (a-d) indicate significant differences (P<0.05).

On the other hand, most of the hydrophobic groups on MPs carry negatively charged residues (e.g., tyrosine and phenylalanine). As these hydrophobic groups become exposed on the surface of MPs, the negative charge on the protein surface could be increased. Fig. 2B shows the zeta potential of MPs after different tumbling treatments. Compared to the untreated MPs, all the tumbling treatments caused a considerable increase in the absolute zeta potential value (P<0.05). In particular, the UT treatment could slightly enhance the absolute zeta potential value compared to the ST treatment, thereby increasing the intermolecular electrostatic repulsion more significantly than the ST treatment [18]. Consequently, the MPs in the UT groups exhibited greater resistance against associative forces and regulated the ordered reaggregation of the MPs [27].

The exposure degree of tryptophan residues was assessed using intrinsic fluorescence based on their endogenous chromogenic groups, which were sensitive to the microenvironment [13]. No shift in the maximum excitation wavelength was observed after the tumbling treatments (Fig. 2C). However, the fluorescence intensity gradually increased compared to the untreated MPs (Fig. 2D), indicating that the tumbling treatments unfolded and stretched the MPs structure, consequently exposing the initially embedded tryptophan residues to the surface of the MPs [28]. Furthermore, when subjected to the same tumbling time, UT treatment led to a more significant increase in fluorescence intensity than ST treatment (P<0.05). It suggests that the UT treatment had a more effective impact on the unfolding of the MPs structure. Changes in fluorescence intensity are consistent with the results of I760 in section 3.1.2. Both findings illuminate that the microenvironment of tryptophan residues became more hydrophobic, especially in the UT-treated MPs samples. This might be attributed to the rearrangement and aggregation of MPs, as more hydrophobic groups were exposed and accumulated on the MPs surface. This enhanced the intermolecular attraction driven by hydrophobic forces, resulting in more hydrophobic interactions and thus forming the hydrophobic pockets surrounding the tryptophan residues. Therefore, the hydrophobicity of the microenvironment was increased.

The total, free sulfhydryl contents and disulfide bond content were employed to monitor the transformation of sulfhydryl-disulfide interchange and evaluate the tertiary structures of MPs. As depicted in Fig. 2E, in comparison to untreated MPs, tumbling treatment led to a reduction in the total sulfhydryl content (P<0.05). Specifically, UT treatment substantially decreased the total sulfhydryl content compared to ST treatment at the same tumbling time (P<0.05). It could be attributed to the ultrasonic cavitation effect, which discomposes water molecules and generates free radicals, subsequently oxidizing the susceptible sulfhydryl groups and significantly reducing the total sulfhydryl content [29]. Conversely, the increase in free sulfhydryl content in the ST-6 h, UT-4 h, and UT-6 h treatment groups suggests a simultaneous unfolding of the protein structure, exposing initially shielded sulfhydryl groups to the molecular surface. Particularly in the UT-6 h treatment group, where the free sulfhydryl content was approximately equal to the total sulfhydryl content, demonstrating that most sulfhydryl groups were exposed to the MPs surface [30]. Consequently, this increased the accessibility of highly active sulfhydryl groups for forming new disulfide bonds [31], as evidenced by the significantly higher content of disulfide bonds in the UT groups compared to the ST groups treated for an equivalent tumbling duration (P<0.05). Considering the conformational changes in the disulfide bond outlined in section 3.1.2, it can be inferred that the UT treatment outperformed the ST treatment in modifying the structure of MPs. This resulted in reorientation and facilitated both head-head and tail–tail cross-linking in MPs.

3.3 Protein aggregation behavior

3.3.1 Solubility

The conformational results indicate that the unfolding and aggregation occurred simultaneously in MPs molecules after the tumbling treatments. The original hydrophilic-hydrophobic balance of MPs was disturbed by the unwinding of the MPs structure, causing it to expose the inner hydrophobic groups, sulfhydryl groups and the negatively charged amino acid residues. The UT treatment enhanced this change in the MPs more than the ST treatment. It, in turn, stimulated the protein interaction and cross-linking, resulting in the aggregation of the MPs.

The solubility could be used for indicating the extent of protein aggregation [27], as depicted in Fig. 3A. After tumbling treatment, the solubility of MPs was significantly improved compared to the untreated MPs, except for the ST-4 h treatment (P<0.05). It might be related to the fact that the extent of structure unfolding was more vigorous than the MPs aggregation, making relatively more sites available for protein-water interaction, thus improving the solubility of MPs. Additionally, tumbling treatment induced myofibril fragmentation and depolymerization, resulting in smaller protein particles, contributing to increased solubility by expanding the specific surface area in contact with water molecules [27]. Extending the tumbling time to 6 h for the UT treatment resulted in improved solubility compared to the UT-4 h treatment (P<0.05). This enhancement might be attributed to the restoration of the hydrophilic-hydrophobic equilibrium and stabilization of smaller particles in the UT-6 h group.Fig. 3 Protein aggregation behavior of myofibrillar proteins after single intermittent tumbling (ST) and ultrasound-assisted intermittent tumbling (UT) treatments. (A) Solubility; (B) Particle size; (C) Atomic force microscopy (AFM) images of protein aggregates; (D) SDS-PAGE. M: standard marker; 1: ST treatment with 4 h; 2: ST treatment with 6 h; 3: UT treatment with 4 h; 4: UT treatment with 6 h. UN represents the untreated myofibrillar proteins. Different letters (a-d) indicate significant differences (P<0.05).

3.3.2 Particle size distribution

Fig. 3B exhibits the average particle size and distribution of MPs under different tumbling treatments. The untreated MPs displayed the trimodal distribution with peaks at 193, 863 and 5,558 nm, respectively. Although the particle size cannot directly reflect the morphology and structure of MPs, it was generally reckoned that the first peak could be assigned as the monomers and smaller oligomers with 8–20 myosin molecules [32]. The second peak was related to the larger oligomer of MPs and the myofilament structure, and the largest peak was associated with other polymers [33]. After all the tumbling treatments, it was evident that the largest size peak had completely disappeared, indicating that the largest polymers had been destroyed. Following the ST-4 h treatment, the smallest size peak at 193 nm was shifted to 105 nm. The second peak was shifted from 825 to 712 nm, suggesting that the ST-4 h treatment could dissociate the native smaller oligomer of MPs and disaggregate the initial myofilament structure, thereby reducing their particle size. In the ST-6 h treatment, the two peaks distributed below 1,000 nm tended to merge into one peak, indicating that the MPs molecules tended to aggregate [33]. Under the UT treatment, the particle size peak completely merged into a single narrow peak, suggesting that the homogeneous MPs aggregates formed. Particularly, in the UT-6 h treatment, the average particle size was smaller than that in the UT-4 h treatment (P<0.05). This might be because the protein aggregates in the UT-6 h group were aggregated from smaller particles. Extending the tumbling time for the UT treatment could strengthen the disruptive effect on the original MPs oligomer and myofilament structure, releasing smaller particles in the MPs system. Meanwhile, under the continuous action of molecular attraction and molecular repulsion, smaller protein particles could interact and form uniform protein aggregates with a smaller size.

3.3.3 AFM

The AFM results (Fig. 3C) are consistent with the particle size. The untreated MPs exhibited various sizes of aggregates in the 2D image. The height of the largest aggregates could reach 76 nm in the 3D image, confirming that the untreated MPs had an intact myofilament structure and different sizes of protein aggregates. After the ST-4 h treatment, the protein aggregates tended to become more uniform as the smallest and largest aggregates were eliminated, and the height of the cross-section was more homogeneously distributed. This change was more pronounced after the ST-6 h treatment. Additionally, the roughness (Rq) was significantly reduced in the ST-4 h and ST-6 h treatments compared to the untreated MPs (P<0.05). It indicates that the ST treatment focused on breaking down the original larger protein aggregates. In contrast to the ST treatment, the 2D AFM image of the UT treatment groups showed larger and spherical MPs aggregates. Fan et al. [34] demonstrated that this observation indicated the formation of new protein aggregates. The height of the cross-section revealed distinctive peaks with similar heights. The Rq was also increased compared to the ST treatment (P<0.05), indicating that the UT treatment facilitated the MPs debris released from the original MPs aggregates to be further reaggregated, forming new protein aggregates.

3.3.4 SDS-PAGE

The reduced and non-reduced SDS-PAGE was operated to examine the pattern of protein–protein interactions and cross-linking. As shown in Fig. 3D, all the groups exhibited the typical bands of the myosin heavy chain (MHC) and actin. Particularly in the non-reduced condition, both ST and UT treatments intensified the oligomer band at the top of the gel, accompanied by weakened bands of MHC. This tendency was most distinctive in the UT-6 h treatment. Quantifying the relative density of the oligomer and MHC bands (Table 1) aligned with the visual results. With the application of tumbling treatments, the relative density of the oligomer significantly increased, reaching the highest value in the UT-6 h treatment (P<0.05). In contrast, the relative density of the MHC band significantly decreased (P<0.05), suggesting that the MHC might be the primary source of the oligomer. Therefore, we identified the protein composition of the oligomer, revealing in Table 2 that the relative abundance of MHC in the oligomer could reach a high of 56.4 %-77.1 %. Also, the UT treatment could enhance the ratio of MHC compared to the untreated MPs and ST-treated MPs. It indicates that the UT treatment was inclined to polymerize MHC to form the oligomer. Liu et al. [18] reported similar effects of HIU and demonstrated that this phenomenon was attributed to the disruption of the myosin filament structure, which subsequently stimulated MHC rearrangement. Under the reduced SDS-PAGE, the oligomer can be partially reduced, and the bands of MHC were partially recovered. It suggests that the disulfide bond played a major role in the formation of the protein aggregates. Nonetheless, some oligomers resisted reduction, suggesting the involvement of other intermolecular interactions, such as the hydrophobic interactions and intermolecular hydrogen bonds, as mentioned in the secondary and tertiary structure.Table 1 Relative density of oligomer, myosin heavy chain (MHC) and actin bands under non-reducing (− βME) and reducing conditions (+βME).

	Bands	UN	ST-4 h	ST-6 h	UT-4 h	UT-6 h	
− βME	Oligomer	1.46 ± 0.038c	1.62 ± 0.11b	1.69 ± 0.098ab	1.74 ± 0.067ab	1.81 ± 0.027a	
MHC	1.03 ± 0.068a	0.75 ± 0.10b	0.74 ± 0.13b	0.66 ± 0.032b	0.67 ± 0.074b	
+ βME	Oligomer	0.64 ± 0.0071a	0.51 ± 0.039c	0.50 ± 0.018c	0.58 ± 0.047b	0.56 ± 0.044bc	
MHC	4.45 ± 0.056a	4.17 ± 0.040b	4.07 ± 0.065b	4.32 ± 0.042a	4.41 ± 0.14a	
UN: untreated myofibrillar proteins; ST: single intermittent tumbling; UT: ultrasound-assisted intermittent tumbling. Different letters (a-d) indicate significant differences (P<0.05). The standard marker at 250 kDa was used as a reference.

Table 2 Identified protein composition and relative abundance (%) of the oligomer in the non-reducing condition based on LC-ESI-MS/MS sequencing and analysis.

group	Sequence Header 1	Protein Mass (D) 2	Relative abundance (%)	
UN3	>sp|Q9TV62|MYH4_PIG Myosin-4 OS=Sus scrofa OX=9823 GN=MYH4 PE=2 SV=1	224008.64	60.4 %	
ST3-4 h	>sp|Q9TV62|MYH4_PIG Myosin-4 OS=Sus scrofa OX=9823 GN=MYH4 PE=2 SV=1	224008.64	60.4 %	
ST3-6 h	>sp|Q9TV62|MYH4_PIG Myosin-4 OS=Sus scrofa OX=9823 GN=MYH4 PE=2 SV=1	224008.64	56.4 %	
UT3-4 h	>sp|Q9TV62|MYH4_PIG Myosin-4 OS=Sus scrofa OX=9823 GN=MYH4 PE=2 SV=1	224008.64	65.6 %	
UT3-6 h	>sp|Q9TV62|MYH4_PIG Myosin-4 OS=Sus scrofa OX=9823 GN=MYH4 PE=2 SV=1	224008.64	77.1 %	
1 The header of an identified protein present in the NR database, which is limited to 300 characters.

2 The calculated molecular weight of each identified protein based on its amino acid sequence in the current NR database.

3 UN: untreated myofibrillar proteins; ST: single intermittent tumbling; UT: ultrasound-assisted intermittent tumbling.

Overall, the above results indicate that the tumbling treatment, especially the UT treatment, could induce the MPs to unfold and aggregate, forming homogeneous and smaller protein aggregates. The pre-aggregates are a prerequisite for the MPs gel formation. Particularly, uniform aggregates with the appropriate size are conducive to forming a well-organized gel [23]. Therefore, the UT treatment enhanced the potential to fabricate a better MPs gel than the ST treatment.

3.4 Rheological properties of MPs

The storage modulus (G') serves as a descriptor for protein–protein interactions during the heating process and correlates with the elasticity of the MPs gel. The G' curve shows three typical phases in this study (Fig. 4A). In the case of untreated MPs, G' exhibited a slight increase at the onset of the temperature rise from 20 to 42.5 °C, followed by a sharp increase, reaching its peak at 47.6 °C. This elevation can be attributed to the unfolding and weak cross-linking of the myosin head region, forming a loosely structured myosin gel. The second phase was denoted as the gel weakening phase, in which G' sharply decreased due to the dissociation of the myosin rod sub-fragment. Consequently, the gel network was disrupted, leading to increased filament fluidity. As the temperature continued to rise, G' increased again owing to the tail–tail interactions, strengthening the protein gel and forming an irreversible gel network [35]. Following the tumbling treatments, the overall G' profile remained unaltered during heating. Conversely, during cooling, the UT treatment exhibited a greater increase in G' than the ST treatment for the same tumbling time. Consequently, the UT-treated MPs resulted in a higher final G' (Fig. 4B). This implies that the UT treatment improved the cross-linking and interaction between protein molecules, forming a more stable and elastic MPs gel, potentially enhancing the quality of the protein gel. Notably, the UT-6 h treatment achieved the highest G' level during the heating and cooling process, possibly due to the smaller and more uniform particle size of the UT-6 h groups, which increased the formation of irreversible interactions [36]. The UT-6 h treatment also delayed the gel weakening transition temperature to 51.6 °C, which might be attributed to the conformational change. The UT-6 h treatment strengthened the tail–tail hydrophobic interaction and cross-linking before heating, providing resistance against heat-induced denaturation. In addition, a higher number of solubilized proteins in the UT-6 h groups contributed to a denser protein matrix, thereby promoting the thermal aggregation of MPs and creating steric hindrance to inhibit the unwinding of the tail region [35].Fig. 4 Rheological properties of myofibrillar proteins after single intermittent tumbling (ST) and ultrasound-assisted intermittent tumbling (UT) treatments. (A) Storage modulus (G') during heating and cooling; (B) Storage modulus (G') at the overall gelation process. UN represents the untreated myofibrillar proteins.

3.5 Physicochemical properties of MPs gel

3.5.1 Gel strength

Gel strength is closely related to the internal structure of the MPs gel and is commonly used to evaluate the quality of MPs gels [37], [38]. Fig. 5A shows the change trends of gel strength under different tumbling treatments. Following the ST-6 h, UT-4 h and UT-6 h treatments, there was a notable improvement in gel strength from 27.50 g (untreated MPs gel) to 34.20, 33.97, and 36.90 g, respectively (P<0.05). This improvement indicates a substantial reinforcement of protein interactions, fortifying the protein network. However, no significant differences were observed among the ST-6 h, UT-4 h and UT-6 h treatment groups, possibly indicating that the gel strength had reached its maximum level with no further room for improvement. The UT treatment expedited the increase in gel strength, reaching its peak in only 4 h, whereas the ST treatment required 6 h for a similar effect. This acceleration can be attributed to the UT treatment strengthening the active groups exposed on the surface of the MPs, leading to increased intermolecular interactions and facilitating the formation of a compact network in the MPs gel.Fig. 5 Physicochemical properties of myofibrillar proteins gel after single intermittent tumbling (ST) and ultrasound-assisted intermittent tumbling (UT) treatments. (A) Gel strength and cooking yield; (B) Water distribution curve; (C) T2 relaxation time; (D) Water proportion. T2b: bound water; T21: immobilized water; T22: Free water. P2b: the proportion of bound water; P21: the proportion of immobilized water; P22: the proportion of Free water. UN represents the untreated myofibrillar proteins. Different letters (a-d) indicate significant differences (P<0.05).

3.5.2 Cooking yield of MPs gel

The cooking yield of the MPs gel is indicative of water-holding capacity during the gelling process and directly correlates with the product yield of meat products. As depicted in Fig. 5A, all tumbling treatments significantly increased the cooking yield compared to the untreated MPs gel (P<0.05). It signifies that tumbling treatments enhanced gel formation and minimized water channels, rendering it challenging for water molecules to escape from the gel network [37]. Notably, the UT treatment achieved the same cooking yield level as the ST-6 h treatment in just 4 h (P>0.05), and the cooking yield reached its maximum under the UT-6 h treatment (P<0.05). It demonstrates the efficacy of the UT treatment in enhancing the gelation properties of MPs, which could be attributed to the higher solubility in the UT group, allowing more protein molecules to participate in gel formation. Furthermore, the pre-formed aggregates in the UT treatment exposed a greater number of active groups on the MPs surface, providing greater accessibility. This facilitated aggregation during the heating process, resulting in a compact gel with reinforced capillary forces for more effective water binding [23].

3.5.3 Water distribution in MPs gel

The internal water composition and distribution in the MPs gel were assessed using the LF-NMR. The T2 relaxation time reflects the mobility of the water molecules, with the shorter relaxation times indicating tighter binding and lower mobility. All the MPs gels prepared in this study showed three peaks, representing the three states of water: bound water (T2b) in the range of 1–10 ms, immobile water (T21) in the range of 10–100 ms, and free water (T22) in the range of 100–1000 ms (Fig. 5B) [22]. After the ST-6 h, UT-4 h, and UT-6 h treatments, both the T2b and its corresponding proportion (P2b) increased significantly compared to the untreated MPs gel (P<0.05) (Fig. 5C and D). Conversely, the T21 and P21 declined (P<0.05) and reached a lower value in the UT groups. This might be because the UT treatment regulated protein aggregation more efficiently, resulting in ordered and smaller protein aggregates. Consequently, this increased the number of binding sites for water, facilitating the conversion of immobile water into bound water. The T22 values showed a downward trend after the tumbling treatments, reaching the lowest value in the UT-6 h group (P<0.05), indicating that the UT-6 h treatment caused the free water to be more tightly bound. Meanwhile, the P22 increased in the UT groups compared to the ST treatment at the same tumbling time (P<0.05), suggesting that the UT treatment retained more free water in the MPs gel than the ST treatment (P<0.05). It might be stemmed from the fact that the UT treatment facilitated the formation of a well-structured protein gel, effectively averting free water loss during the heating process. As a result, there was an augmentation in the amount of free water [39], thus leading to a higher cooking yield in the UT groups, as evidenced in section 3.5.2. Therefore, by combining the physicochemical properties of MPs gel under the different tumbling treatments, it was found that the UT-4 h treatment proved to be more efficient, achieving comparable results in a shorter tumbling time compared to both the ST-6 h and UT-6 h treatments. Consequently, when efficiency is the primary goal, the UT-4 h treatment is recommended. However, when specifically aiming to optimize the gelation properties of MPs, the UT-6 h treatment demonstrated superior potential, indicating that extending the tumbling time in the UT treatment could further enhance the quality of meat products.

3.5.4 Microstructure of MPs gel

Fig. 6A illustrates the microstructure of the MPs gel as observed by SEM. The untreated MPs gel exhibited larger cavities, and the surface of the gel network appeared rough. After the ST treatment, the gel network showed continuous visibility, with smaller pores, and the water channel was blocked [40], explaining the improved cooking yield in the tumbling groups. The UT treatment significantly enhanced this change. The gel surface became smoother, and uniform pores were embedded in the dense protein network, trapping water in the porous structure and making it difficult to separate. Also, it was observed that the gel network of UT groups transformed from spherical to fibrous aggregation. This phenomenon may be attributed to the UT treatment enhancing myosin tail–tail interactions, promoting the formation of structurally homogeneous aggregates of MPs molecules before heating. Subsequently, during the heating process, the rod-like tails of MPs molecules further aggregated in a regular manner, resulting in the formation of fibrillar aggregates, which were eventually interwoven into a dense gel network with the application of thermal energy.Fig. 6 Microstructure of myofibrillar proteins during the heating process after single intermittent tumbling (ST) and ultrasound-assisted intermittent tumbling (UT) treatments. (A) Scanning electron microscopy (SEM) observation of protein gel; (B) Atomic force microscopy (AFM) images of protein thermal aggregates. UN represents the untreated myofibrillar proteins.

To verify this speculation, the morphology of the protein aggregates formed during the heating process was observed using AFM (Fig. 6B). The untreated MPs exhibited irregular aggregation, featuring different shapes of aggregates, such as a string of beads and a daisy wheel shape. After the ST-4 h treatment, the majority of the protein aggregation transformed into the daisy wheel shape. This phenomenon, as noted by Yamamoto et al. [41], stemmed from myosin head-head interactions forming clumps, with myosin tails extending radially from the clump. When extending the tumbling time to 6 h for the ST treatment, the MPs aggregation displayed a distinct difference from that of the ST-4 h and untreated MPs, forming an ordered string of beads. This was due to the fact that the pre-formed aggregation tended to be converted into tail–tail interaction during the gelation process, thus facilitating the regular arrangement of MPs molecules. The UT-4 h treatment improved MPs interaction similarly to the ST-6 h treatment, with more MPs participating in gel formation, establishing the primary fibrillar gel network. Following the UT-6 h treatment, protein aggregation exhibited a thicker fibrillar shape and parallel arrangement. It proved that the UT treatment, especially the UT-6 h treatment could strengthen the tail–tail associations, forming an ordered orientation of protein aggregation and potentially promoting the formation of a more ordered and denser protein gel.

4 Conclusion

This research marks the first investigation into how the UT treatment modifies the protein interaction behavior and gelation properties of MPs. The conformational results indicated that, compared to the ST treatment, the UT treatment significantly disrupted the myofibril structure and released the smaller MPs particles. With more active groups being exposed on the surface of MPs, the UT treatment induced the ordered aggregation of MPs by the head-head crosslinks and tail–tail interactions resulting in smaller and more ordered MPs aggregates than those formed by the ST treatment. During the subsequent heating process, the UT treatment significantly increased the final G', particularly the UT-6 h treatment enhanced the thermostability of the MPs, making it more resistant to denaturation at the gel weakening stage. In contrast to the daisy wheel shape aggregates formed in the ST-4 h treatment, the UT treatment tended to induce thick fibrillar-like aggregation and further increase tail–tail interactions to strengthen the gel network. Consequently, the gel network in the UT groups was denser and stronger than in the ST groups, contributing to higher gel strength and cooking yield in the UT groups. Furthermore, the UT-6 h treatment induced more significant conformational modification in MPs compared to the UT-4 h treatment, thereby amplifying the protein interactions during gel formation and resulting in better gelation properties of MPs. This study provides a comprehensive understanding of the UT treatment that influences the conformation and gelation properties of MPs, further providing a highly efficient method to regulate the quality of meat products.

CRediT authorship contribution statement

Ruyu Zhang: Writing – review & editing, Writing – original draft, Software, Methodology, Formal analysis, Data curation, Conceptualization. Lei Zhou: Investigation. Wangang Zhang: Writing – review & editing, Supervision, Resources, Funding acquisition.

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.

Acknowledgment

This work was financially supported by the 10.13039/501100001809 National Natural Science Foundation of China (32372358 ), the 10.13039/501100010038 Earmarked Fund for China Agriculture Research System (CARS-35 ).
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