Elucidating the mechanism of accelerated ripening and enhanced flavor in Hunan bacon under temperature cycling with liquid smoke application: Insights from myofibrillar protein-2,3-butanedione interactions

Abstract

This study elucidates the molecular mechanism underlying the enhanced flavor formation in Hunan bacon processed under temperature cycling (4 °C – 55 °C) with liquid smoke. Focusing on the interaction between myofibrillar proteins (MPs) and the key smoke flavor compound 2,3-butanedione, we employed multi-technique approaches including HS-SPME-GC-MS, spectroscopy, and molecular dynamics simulation. Results showed that temperature cycling induced a partially unfolded yet dynamic MP structure, characterized by increased surface hydrophobicity and accessible binding sites, which significantly enhanced the binding capacity and stability for 2,3-butanedione. Thermodynamic analysis revealed the interaction was enthalpy-driven under cycling conditions, facilitating efficient flavor retention. These findings provide a protein-flavor interaction perspective to explain the accelerated ripening and superior sensory quality of bacon processed by cyclic temperature treatment, offering insights for optimizing smoked meat production.

Graphical abstract

Highlights

• •Temperature cycling induces a partially unfolded, dynamic structure in myofibrillar proteins.
• •Surface hydrophobicity and accessible binding sites increase under cycling conditions.
• •Binding capacity and stability for 2,3-butanedione are highest with temperature cycling.
• •The interaction is enthalpy-driven, enhancing flavor retention.
• •Molecular simulations confirm structural changes and stronger binding via hydrogen bonds.

1. Introduction

The formation of flavor in smoked meat products is an extremely complex process (M. Zhang et al., 2021), involving protein thermal degradation, lipid oxidation, Maillard reactions (Wu et al., 2023), and their interactions with the food matrix (Fu et al., 2022). Hunan bacon, a traditional dry-cured and smoked meat product (Ai-Nong and Bao-Guo, 2005), is highly prized for its distinctive sensory attributes. However, its conventional smoking process often requires prolonged ripening periods and high-temperature treatments (Bai et al., 2025), which can lead to inconsistent quality and loss of volatile flavor compounds. In recent years, liquid smoke application has emerged as a controllable alternative to traditional smoking (D. Zhang et al., 2026). Notably, 2,3-butanedione (diacetyl) is a key volatile compound in liquid smoke (Vazquez et al., 2025), is an occurring natural α-diketone and is volatil (Y. Wang et al., 2024),characterized by a high odor activity value and a pronounced buttery aroma (Farsalinos et al., 2015), playing a significant role in enhancing the overall flavor profile of smoked products.

To mitigate the drawbacks inherent in traditional processing, our team introduced an oscillating temperature approach (oscillating between 4 °C and 55 °C) coupled with the application of liquid smoke (Zou et al., 2024). Preliminary studies have demonstrated that this process significantly shortens the ripening time of Hunan bacon while improving its flavor and texture compared to constant high-temperature treatments. It is hypothesized that these improvements are closely linked to modifications in muscle proteins, which serve as primary flavor carriers through covalent and non-covalent interactions with volatile compounds. The dynamic thermal changes induced by temperature cycling likely alter protein conformation, thereby affecting their binding capacity for key flavor molecules. However, the underlying molecular mechanisms—specifically, how the structural dynamics of myofibrillar proteins (MPs) under temperature cycling modulate their interactions with critical smoke flavorants like 2,3-butanedione—remain poorly understood.

Proteins act as important flavor carriers in food systems, capable of adsorbing flavor substances through covalent or non-covalent interactions (Qian et al., 2024). Changes in protein conformation critically influence their binding capacity and patterns with flavor compounds (Z. Wang et al., 2025). Under dynamic heat treatments such as temperature cycling, proteins undergo frequent temperature fluctuations, leading to particularly intense and unique structural dynamics (Peng et al., 2024). This inevitably plays a key role in determining their flavor adsorption behavior. Nevertheless, systematic research on the structural changes of myofibrillar proteins during temperature cycling and their adsorption mechanisms for key flavor compounds remains scarce, hindering further optimization and application of this process for flavor control.

Therefore, this study aims to elucidate the interaction mechanism between myofibrillar proteins and 2,3-butanedione under simulated temperature cycling conditions. The specific objectives are to: (1) systematically characterize the effects of constant low temperature (4 °C), constant high temperature (55 °C), and temperature cycling treatments on the physicochemical properties and structure of myofibrillar proteins; (2) quantitatively evaluate the binding capacity of myofibrillar proteins for 2,3-butanedione and related thermodynamic parameters; (3) investigate structural modifications of the proteins upon binding using spectroscopic techniques; and (4) reveal the interaction sites and dynamic processes through molecular docking and molecular dynamics simulations. By integrating multiple technical approaches, this research seeks to unravel the protein-flavor interaction mechanisms at the molecular level during temperature cycling, providing a theoretical foundation for flavor optimization and process control in Hunan bacon and other smoked meat products.

2. Materials and methods

2.1. Materials and reagents

Pork used in the experiments was purchased from the local market. 2,3-Butanedione was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Other chemical reagents used were of analytical grade.

2.2. Myofibrillar proteins (MPs) extraction

Myofibrillar proteins were extracted following the method of (Park et al., 2006) with slight modifications. Briefly, Fresh pork longissimus dorsi was thoroughly minced, and mixed with 10 mM PBS (0.1 M NaCl, 2 mM MgCl₂, 1 mM EGTA, pH 7.0) at a ratio of 1:4 (w/v). The mixture was homogenized twice on ice (11000 rpm, 40 s each) and centrifuged (2500×g, 15 min, 4 °C). The supernatant was discarded, the precipitate collected, and this procedure repeated three times. The precipitate was resuspended in 0.1 M NaCl at 1:4 (w/v), and washed four times under the same homogenization and centrifugation conditions. Before the final centrifugation, the precipitate was filtered through four layers of gauze to remove connective tissues, and its pH was adjusted to 6.25 with 0.1 M HCl. Myofibrillar protein was the main component of the resulting precipitate. The protein concentration was determined via a microplate reader, strictly following the protocols of the Solarbio BCA Protein Assay Kit.

2.3. Preparation of protein samples with different treatments

The extracted proteins were dispersed in 20 mM phosphate buffered saline (PBS) containing 0.6 M NaCl (pH 6.25) and adjusted to a concentration of 20 mg/mL (Gan et al., 2019). The samples were subjected to three different temperature treatments: 4 °C (4 °C), constant 55 °C (55 °C), and variable temperature cycling (VT). For each temperature condition, 2,3-butanedione was added at final concentrations of 0, 2, 4, 6, 8, and 10 mM, respectively.

2.4. Determination of total sulfhydryl content

The total sulfhydryl content was determined using the Ellman's method (DTNB method) (Ellman, 2022). One gram of the sample was mixed with 10-fold volume of 0.6 M KCl, homogenized for 30 s and filtered through two layers of gauze. Then 0.5 mL of the filtrate was pipetted and thoroughly mixed with 4 mL of 50 mM PBS (8 M urea, 0.6 M NaCl, 10 mM EDTA, pH 7.4), followed by centrifugation at 12000×g for 10 min. A 3 mL of the supernatant was mixed with 0.4 mL of Ellman's reagent (0.1% 5,5′-dithiobis (2-nitrobenzoic acid)), and the mixture was incubated for 10 min at room temperature in the dark. The absorbance was measured at 412 nm. A standard curve was constructed with reduced glutathione (GSH) as the standard and phosphate buffer as the blank, and the total sulfhydryl content in the protein was calculated according to the absorbance values.

2.5. Determination of carbonyl content

The carbonyl content was determined using the DNPH method (Oliver et al., 1987). Briefly, 1.6 mL of 2 mol/L hydrochloric acid containing 1% DNPH was added to 0.8 mL of the MP solution (5 mg/mL), and the mixture was incubated for 1 h at room temperature in the dark. Subsequently, 2 mL of 40% (w/v) trichloroacetic acid (TCA) was added to precipitate proteins. After standing for 20 min, the mixture was centrifuged at 8000 r/min for 15 min at 4 °C. The precipitate was washed with 2 mL of ethanol:ethyl acetate (1:1, v/v) repeatedly until the supernatant turned colorless and transparent. Finally, the precipitate was dissolved in a phosphate buffer (pH 6.5) containing 6 mol/L guanidine hydrochloride, and incubated at 37 °C until complete dissolution. The absorbance was measured at 370 nm using a microplate reader. The carbonyl content was calculated with a molar extinction coefficient of 22000 L/(mol·cm) for carbonyl groups, and the results were expressed as nmol DNPH per mg protein.

2.6. Determination of protein surface hydrophobicity

Surface hydrophobicity was determined using the bromophenol blue (BPB) binding method (Chelh et al., 2006). The protein concentration was adjusted to 5 mg/mL using 20 mM PBS (containing 0.6 M NaCl, pH 6.25). Then, 1.0 mL of protein solution was mixed with 200 μL of 1.0 mg/mL BPB solution. The mixture was centrifuged at 4 °C, 3000 g for 15 min. The supernatant was diluted 10-fold, and the absorbance was measured at 595 nm against a blank prepared by mixing BPB solution with PBS.

2.7. Determination of protein solubility

The protein concentration was adjusted to 10 mg/mL using 20 mM PBS (containing 0.6 M NaCl, pH 6.25). Then, 5 mL of the protein solution was placed in a 15 mL centrifuge tube and kept at 4 °C for 2 h. This was followed by centrifugation at 4 °C, 10000 g for 20 min. The protein concentration in 1 mL of the supernatant was determined using the biuret method or BCA method to calculate solubility (Z. Zhang et al., 2017).

2.8. Determination of protein turbidity

The protein concentration was adjusted to 1 mg/mL using 20 mM PBS (containing 0.6 M NaCl, pH 6.25). The absorbance at 370 nm was measured using a UV spectrophotometer to characterize the turbidity of the solution (You et al., 2023).

2.9. Adsorption properties analysis

Different volumes of 2,3-butanedione methanol stock solution were added to a series of protein sample solutions to achieve final concentrations of 0, 2, 4, 6, 8, and 10 mM. The sample vials were sealed, vortexed, and equilibrated at room temperature for 24 h to ensure binding equilibrium.

Headspace-gas chromatography-mass spectrometry (HS-GC-MS) was used for analysis (Wei et al., 2022). HS conditions: sample equilibration at 37 °C for 30 min, pressurization time 2 min, injection time 1 min, pressurization gas: high-purity helium. GC conditions: HP-5 column (30 m × 0.25 mm, 0.25 μm); splitless injection mode; temperature program: initial temperature 60 °C held for 3 min, then ramped at 10 °C/min to 180 °C; carrier gas: helium, flow rate 1.0 mL/min. MS conditions: electron impact (EI) ion source, ionization energy 70 eV, detector voltage 350 V, ion source temperature 240 °C; mass scan range m/z 30∼450, scan rate 3.00 scans/s (Guo et al., 2019). The release rate of the flavor compound was expressed as the percentage of the peak area of the flavor compound in the headspace of the protein sample vial (S ∼ MPs∼) relative to the peak area of the same flavor compound in the headspace of the blank control vial (without protein, S∼0∼):

$$\text{Release}\text{Rate}(\%)=\frac{{\mathrm{S}}_{\text{MPs}}}{{\mathrm{S}}_0}\times 100\%$$

The adsorption capacity of the protein for the flavor compound, i.e., the flavor retention rate, was calculated as:

$$\text{Flavor}\text{Retention}\text{Rate}(\%)=100\%-\text{Release}\text{Rate}(\%)$$

The Klotz model was used to qualitatively and quantitatively analyze the binding interaction between the protein and ligand (Wongprasert et al., 2024):

$$\nu =\frac{n\left[L\right]}{K+\left[L\right]}$$

where:

$$\nu =\frac{\frac{\left[HS\right]c-{\left[HS\right]}_P}{\left[HS\right]c}\times O}{C_P}$$

$$\left[L\right]=\frac{{\left[HS\right]}_P}{{\left[HS\right]}_C}\times O$$

O is the total flavor compound concentration (M), Cp is the protein concentration (M), [HS]p is the headspace flavor compound concentration in the protein-containing sample, and [HS]c is the headspace flavor compound concentration in the protein-free sample.

2.10. Secondary structure analysis

Sample preparation for CD spectroscopy was consistent with that for flavor retention capacity measurement. After mixing the protein with 2,3-butanedione and reacting at room temperature for 30 min, measurements were taken. A CD spectropolarimeter equipped with a 0.1 cm path length quartz cell was used. A 20 mM PBS buffer (containing 0.6 M NaCl, pH 6.25) was used for background subtraction. Each sample was scanned four times, averaged after background subtraction. Scanning was performed at room temperature under a nitrogen atmosphere, over a wavelength range of 190-250 nm with a 1 nm interval. The average molecular mass per amino acid residue was taken as 110 g/mol for secondary structure calculation (Estévez, 2011).

2.11. Fluorescence quenching spectroscopy

Sample preparation for fluorescence spectroscopy was consistent with that for flavor retention capacity measurement. After thorough mixing, each sample was reacted at the corresponding temperature (293 K, 303 K, 310 K) for 30 min and then measured immediately. Fluorescence spectra were recorded under the following conditions: excitation and emission slit widths both 10 nm, excitation voltage 500 V, excitation wavelength 280 nm, emission scan range 300-500 nm, scan speed 500 nm/min.

Binding thermodynamic parameters were analyzed using the modified Stern-Volmer equation and the Van't Hoff equation:

$$\frac{F_0}{F_0-F}=\frac{1}{f_a{K}_a}\frac{1}{\left[L\right]}+\frac{1}{f_a}$$

$$\Delta G=-RT\ln K=\Delta H-T\Delta S$$

2.12. Molecular docking and molecular dynamics simulation

Molecular dynamics (MD) simulations were performed using Gromacs software (version 2019.6)(Abraham et al., 2015). Myosin crystal structure (PDB ID: 6ysy) was retrieved from the Protein Data Bank, preprocessed by removing crystallographic water and heteroions and supplementing missing atoms, and simulated with the GROMOS54a7 force field. The processed protein was centered in a cubic box, solvated with the SPC/E water model (1.0 nm solvent buffer), and the system was supplemented with 0.6 M NaCl to mimic muscle physiological ionic environment and neutralized. System energy was minimized via the steepest descent method, followed by 400 ps equilibration under NVT and NPT ensembles sequentially, bringing the final equilibrium temperature of the system to 298.15 K (Nosé and Klein, 1983) and the equilibrium pressure to 100 kPa.

Production MD simulations were run for 150 ns with trajectory sampling at the specified temperature. Bond vibrations were constrained by LINCS, long-range electrostatics calculated via PME, and van der Waals interactions cut off at 1.0 nm. Pressure and temperature were coupled using Parrinello-Rahman and V-rescale methods, respectively. The system temperature was ramped to 328.15 K (55 °C) at 10 °C/ns and held constant thereafter. The integration time step was 2 fs, and trajectory data were saved every 1 ps for analysis.

Ligand 2,3-butanedione was sketched in ChemDraw Professional 17.1, and its stable 3D structure was obtained via molecular mechanics optimization and protonation in Chem3D 17.1. AutoDock Tools software (Morris et al., 2009) was used to prepare the myosin receptor and the 2,3-butanedione ligand, generating standard PDBQT topology files. For blind docking, a 40 × 40 × 40 A grid box (1 A resolution) was constructed, with the 55 °C (328.15 K) MD-derived final myosin conformation as receptor. Flexible docking was performed via AutoDock Vina (energy_range = 5, exhaustiveness = 20). The top 20 poses with the most favorable binding free energy were selected for average binding energy calculation and binding mode analysis.

2.13. Data processing and statistical analysis

All experimental analyses were independently repeated three times. Results are expressed as mean ± standard deviation. SPSS statistical software was used for statistical analysis and significance testing (significance level set at P < 0.05). Origin 24 software was used for data plotting and image processing. The MetaboAnalyst 6.0 online platform was used for principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA).

3. Results

3.1. Changes in myofibrillar protein properties under different temperature conditions

The physicochemical properties of myofibrillar proteins (MPs) were significantly influenced by the different temperature regimes, as summarized in Fig. 1. Compared to constant 4 °C incubation, both constant 55 °C and variable temperature (VT, 4–55 °C cycling) treatments induced pronounced alterations in sulfhydryl content, carbonyl formation, surface hydrophobicity, solubility, and turbidity, indicating substantial protein oxidation and structural modification.

Fig. 1.

Total thiol (A),carbonyl (B),Hydrophobicity (C), solubility (D), and turbidity (E) in different treatment groups.

Total sulfhydryl content decreased markedly from 95.34 nmol/mg protein at 4 °C to 11.16 nmol/mg protein at 55 °C, reflecting extensive oxidation of free thiol groups. Under VT conditions, the sulfhydryl content (37.31 nmol/mg protein) was significantly higher than at constant 55 °C but still considerably lower than at 4 °C. This suggests that temperature cycling induced moderate oxidation, possibly due to repeated exposure to high temperature promoting thiol-disulfide interchange or other oxidative modifications (Gatellier et al., 2010), while the low-temperature phases may have partially alleviated further oxidation. Carbonyl content, a well-established marker of protein oxidation, increased sharply from 1.03 nmol/mg protein at 4 °C to 5.45 nmol/mg protein at 55 °C. The VT sample showed a similarly high carbonyl level (5.09 nmol/mg protein), indicating that protein oxidation occurred extensively under both elevated and cycled temperature conditions. This implies that even intermittent heating can induce substantial oxidative damage to MPs, likely through side-chain modification of amino acids such as lysine, arginine, and proline. Surface hydrophobicity increased with rising temperature (Estévez, 2011), from 53.6 μg at 4 °C to 152.9 μg at 55 °C, demonstrating significant protein unfolding and exposure of hydrophobic residues (Sante-Lhoutellier et al., 2007). The VT sample exhibited an intermediate yet elevated hydrophobicity (132.2 μg), which still indicates considerable structural unfolding (Cao et al., 2019). This change suggests that thermal treatment, whether constant or cyclic, disrupts the native conformation of MPs, facilitating the interaction of hydrophobic patches with other molecules or promoting aggregation (Zhu et al., 2025). Protein solubility dropped dramatically from 85.69% at 4 °C to 17.09% at 55 °C, consistent with heat-induced denaturation and aggregation. Under VT conditions, solubility remained low (20.15%), slightly higher than at constant 55 °C but markedly lower than at 4 °C. This supports the observation that thermal cycles still drive substantial protein aggregation, though the repeated cooling phases may slightly mitigate complete insolubility. Turbidity followed a similar trend, increasing from 0.398 at 4 °C to 1.053 at 55 °C, with VT showing an intermediate value of 0.765. Higher turbidity indicates the formation of larger protein aggregates or precipitated complexes under thermal stress. The moderate turbidity under VT implies that aggregation occurred but to a lesser extent than under constant high temperature, potentially resulting in a different aggregate size or morphology.

These results clearly demonstrate that temperature plays a critical role in modifying the structural and oxidative state of myofibrillar proteins. Constant 55 °C treatment induced severe oxidation, denaturation, and aggregation, leading to drastically reduced solubility and increased turbidity. The VT conditions produced changes that were, in many respects, intermediate between the two constant temperatures but notably closer to the high-temperature profile—especially in carbonyl content and hydrophobicity. This indicates that the repeated heating phases during cycling are sufficient to drive significant protein oxidation and unfolding, while the cooling phases may slow but not fully reverse these processes. Importantly, the moderate sulfhydryl retention and slightly lower turbidity under VT compared to constant 55 °C suggest that the cycling regime may allow for partial structural rearrangement rather than irreversible precipitation. Such a state—where proteins are partially unfolded, oxidized, yet still somewhat soluble—could create a more dynamic interface for interaction with small flavor molecules from liquid smoke. Enhanced hydrophobicity and accessible reactive sites (e.g., carbonyls and exposed side chains) may facilitate covalent or non-covalent binding with smoke-derived compounds, thereby accelerating flavor incorporation and maturation.

3.2. Binding capacity and thermodynamic parameters of myofibrillar proteins for 2,3-butanedione

The binding interaction between myofibrillar proteins (MPs) and 2,3-butanedione was quantitatively evaluated by determining the flavor retention rate using GC-MS and further modeled with the Klotz equation to derive thermodynamic parameters. The results, including the flavor retention rates at varying initial 2,3-butanedione concentrations and the calculated binding parameters, are presented in Table 2. The flavor retention rate, representing the percentage of 2,3-butanedione bound to the protein matrix, exhibited a clear dependence on both the incubation temperature and the initial flavorant concentration. A general trend across all groups was a decrease in retention rate with increasing 2,3-butanedione concentration (from 2 mM to 10 mM) in Fig. 2, indicative of the progressive saturation of available binding sites on the proteins. Notably, the VT treatment consistently resulted in the highest flavor retention rates across all concentrations tested, with the most pronounced advantage observed at lower concentrations. At 2 mM, the retention rate under VT conditions (19.25%) was significantly higher than those under constant 4 °C (14.75%) and constant 55 °C (13.11%) conditions. This superior capacity to sequester the flavor compound, especially at lower, more physiologically relevant concentrations, directly correlates with the enhanced initial flavor uptake observed in the VT-processed bacon.

Table 2.

Binding parameters of Klotz models in different treatment groups.

Treatment group	K	n	R2
4 °C	103 ± 13c	9.61 ± 1.81a	0.9584
55 °C	110 ± 21bc	7.33 ± 2.23a	0.8363
VT	131 ± 14bc	10.37 ± 3.80a	0.9349

Fig. 2.

The flavor retention rates under different treatments, the concentrations of 2,3-butanedione were 0, 2, 4, 6, 8, and 10 mM, respectively.

The binding parameters obtained from the Klotz model provide deeper insight into the nature of the interaction. The equilibrium binding constant (K), which reflects the binding affinity or strength, was highest for the VT-treated MPs (131 ± 14 M⁻¹), followed by the 55 °C (110 ± 21 M⁻¹) and 4 °C (103 ± 13 M⁻¹) groups. Although the differences in K among groups did not reach extreme significance, the numerically highest value for VT suggests a moderately stronger binding interaction between 2,3-butanedione and the protein matrix formed under thermal cycling. More strikingly, the number of binding sites per protein molecule showed a clear trend. The VT condition yielded the highest value (10.37 ± 3.80), which was substantially greater than that of the 55 °C group (7.33 ± 2.23) and slightly higher than the 4 °C group (9.61 ± 1.81). This indicates that proteins subjected to temperature cycling present a greater number of accessible sites for binding 2,3-butanedione.

The integrated analysis of flavor retention data and Klotz model parameters robustly demonstrates that the VT conditioning of myofibrillar proteins creates a uniquely favorable matrix for the binding of key smoke flavor compounds. The significantly higher flavor retention rates, particularly at lower concentrations, are of paramount practical importance. In the initial stages of bacon processing or liquid smoke application, the concentration of volatiles interacting with the meat surface is relatively low. The superior binding efficiency of VT-conditioned proteins at this stage would lead to more effective capture and retention of delicate flavor molecules, reducing their loss and initiating flavor development earlier.

3.3. Secondary structural changes of myofibrillar proteins induced by 2,3-butanedione under different temperature conditions

The secondary structure of myofibrillar proteins (MPs), as determined by circular dichroism (CD) spectroscopy, was significantly altered by both incubation temperature and the presence of 2,3-butanedione (Fig. 3). A prominent observation was the vastly different baseline (0 mM 2,3-butanedione) secondary structure among the three temperature conditions. At constant 4 °C, MPs maintained a relatively ordered structure with a high α-helix content (38.6%) and moderate random coil (39.4%). In stark contrast, both constant 55 °C and VT treatments drastically reduced the native α-helix content to 19.1% and 17.8%, respectively, while concurrently increasing the random coil fraction to 54.5% and 48.7%. This confirms that thermal treatment, whether constant or cyclic, induces substantial unfolding and loss of ordered secondary structure. The effect of increasing 2,3-butanedione concentration on protein structure was highly temperature-dependent. Under constant 4 °C, the addition of 2,3-butanedione led to a notable increase in α-helix content (from 38.6% to 45% at 10 mM) and a concomitant decrease in random coil (from 39.4% to 27.5%). This suggests that the flavor molecule may interact with and stabilize the partially unfolded protein structure at low temperature, potentially promoting refolding or inducing a more ordered conformational state. Conversely, under constant 55 °C, the secondary structure was less responsive to 2,3-butanedione. Although a slight increase in α-helix (from 19.1% to 24%) and a decrease in random coil were observed at the highest concentration (10 mM), the structure remained predominantly disordered (random coil >46.8% at all concentrations). This indicates that the severe, heat-induced denaturation is largely irreversible, limiting the ability of 2,3-butanedione to meaningfully reorganize the protein scaffold. The VT condition displayed a unique and intermediate behavior. Similar to the 55 °C treatment, the baseline structure was highly unfolded. However, upon addition of 2,3-butanedione, the VT samples showed a clear trend of increasing α-helix (from 17.8% to 24.6%) and a significant reduction in β-sheet content (from 22.6% to 13.7%). The random coil content remained high but stable (∼48-52%). This pattern implies that 2,3-butanedione interaction under VT conditions facilitates a specific structural rearrangement—partially recovering helical order while disassembling some aggregated β-sheet structures that likely formed during thermal cycling (Ishigaki et al., 2023).

Fig. 3.

Circular dichroism spectra in different treatment groups. The concentrations of 2,3- butanedione in the system were 0, 2, 4, 6, 8, and 10 mM, respectively.4 °C treatment group (A), 55 °C treatment group (B), and variable temperature treatment group (C),.Secondary structure content of myofibrillar proteins in different treatment groups. The concentrations of 2,3-butanedione in the system were 0, 2, 4, 6, 8, and 10 mM, respectively. 4 °C treatment group (D), 55 °C treatment group (E), variable temperature treatment group (F), after adding different concentrations of 2,3-butanedione.

The CD spectroscopy results provide crucial molecular-level insight into the state of MPs that governs their flavor-binding capacity, as observed in Section 3.2. The high adsorption capacity observed under VT conditions can be attributed to this specific, dynamic structural state. Firstly, the initial unfolded state (high random coil) induced by VT cycling creates a flexible protein matrix with abundant exposed binding sites, such as hydrophobic patches and reactive amino acid side chains. This is consistent with the high surface hydrophobicity measured in Section 3.1. Secondly, the structural rearrangement triggered by 2,3-butanedione in VT samples—increased α-helix and decreased β-sheet—is mechanistically informative (Acharya et al., 2020). The rise in α-helix content implies that the flavor molecule may function as a structure-inducing ligand, binding to and stabilizing select polypeptide segments into helical conformations (Hussein, 2011). More importantly, the decrease in β-sheet content is particularly significant. In protein aggregation, intermolecular β-sheets are often associated with insoluble, non-functional aggregates. The reduction of β-sheet upon 2,3-butanedione addition in VT samples indicates that the flavor compound may interfere with or reverse some aggregation pathways, potentially "loosening" the protein network. This action would increase the accessibility of internal sites and create a more open yet partially re-ordered structure ideal for entrapping flavor molecules.

3.4. Fluorescence quenching analysis of the interaction between myofibrillar proteins and 2,3-butanedione

To elucidate the interaction mechanism between myofibrillar proteins (MPs) and 2,3-butanedione, fluorescence spectroscopy was conducted (Fig. 4). Myofibrillar proteins have a maximum fluorescence emission at 330-340 nm. After high-temperature heating and oxidation treatment, the maximum fluorescence intensity decreased and a red shift occurred, indicating changes in protein structure, consistent with the findings of (Xu et al., 2019). 2,3-Butanedione itself has no fluorescence but quenched myofibrillar protein fluorescence in a concentration-dependent manner, indicating its action through static quenching or an electron transfer mechanism.

Fig. 4.

Fluorescence quenching spectra of myofibrillar proteins were obtained by adding different concentrations of 2,3-butanedione to different treatment groups (293K). The concentrations of 2,3-butanedione in the system were 0, 2, 4, 6, 8, and 10 mM, respectively. The 4 °C group (A), 55 °C group (B),and variable temperature group (C). Klotz fitting models of different treatment groups after adding 2,3-butanedione. 4 °C group (D), 55 °C group (E), and variable temperature group (F).

Moreover, the quenching mechanism of myofibrillar proteins by 2,3-butanedione was analyzed using the Stern-Volmer equation. The derived quenching constants (Ksv) and bimolecular quenching rate constants (Kq) for MPs subjected to different pretreatment conditions and subsequently titrated with 2,3-butanedione at varying measurement temperatures (293, 303, and 310 K) are summarized in Table 1. The Ksv values exhibited distinct patterns depending on both the pretreatment condition and the measurement temperature (Tang et al., 2020). A key observation is the significant influence of the pretreatment on the protein's binding affinity. At the lowest measurement temperature (293 K), MPs from the 4 °C and VT pretreatments showed the highest and statistically similar Ksv values (72.74 and 72.98 M^-1, respectively), which were significantly greater than that of the 55 °C pretreatment (63.70 M^-1). This indicates that at 293 K, proteins from the 4 °C and VT conditions possessed a higher apparent binding affinity or accessibility for 2,3-butanedione. The response of Ksv to increasing measurement temperature revealed crucial mechanistic differences. For the 4 °C pretreatment, Ksv decreased dramatically from 72.74 M⁻¹ at 293 K to 40.68 M⁻¹ at 310 K. This strong inverse temperature dependence is characteristic of static quenching, where a ground-state complex is formed, and its stability often decreases with rising temperature.

Table 1.

Stern-Volmer quenching constant Ksv in different treatment groups.

Treatment group	T	Ksv	R2	Kq∗108
4 °C	293K	72.74 ± 1.62b	0.9268	72.74 ± 1.62b
4 °C	303K	56.57 ± 1.51e	0.9428	56.57 ± 1.51e
4 °C	310K	40.68 ± 2.43hi	0.9926	40.68 ± 2.43hi
55 °C	293K	63.70 ± 0.94c	0.9810	63.70 ± 0.94c
55 °C	303K	42.05 ± 0.71hi	0.9822	42.05 ± 0.71hi
55 °C	310K	42.35 ± 1.34hi	0.9197	42.35 ± 1.34hi
VT	293K	72.98 ± 0.09b	0.9623	72.98 ± 0.09b
VT	303K	59.23 ± 0.87d	0.9825	59.23 ± 0.87d
VT	310K	49.68 ± 0.84g	0.9969	49.68 ± 0.84g
VT	293K	57.42 ± 2.81de	0.7445	57.42 ± 2.81de

In contrast, for the 55 °C pretreatment, the Ksv values at 303 K and 310 K (42.05 and 42.35 M^-1) were significantly lower than at 293 K (63.70 M^-1) and showed little variation between the two higher temperatures. This suggests that the binding sites or protein conformation after severe heat treatment became less sensitive to further thermal changes, possibly due to irreversible aggregation. Most notably, the VT pretreatment displayed an intermediate yet distinct pattern. While Ksv also decreased with rising measurement temperature (from 72.98 M⁻¹ at 293 K to 49.68 M⁻¹ at 310 K), the values at 303 K and 310 K were consistently and significantly higher than those for the 4 °C and 55 °C pretreatments at the same measurement temperatures. For instance, at 310 K, the Ksv for VT (49.68 M^-1) was about 22% and 17% higher than for the 4 °C and 55 °C pretreatments, respectively. All calculated Kq values were on the order of 10⁸ M⁻¹s⁻¹, which is two orders of magnitude greater than the maximum diffusion-controlled quenching rate constant (∼2.0 × 10¹⁰ M⁻¹s⁻¹). This confirms that the quenching process for all samples is primarily static quenching, arising from the formation of a non-fluorescent complex between MPs and 2,3-butanedione, rather than dynamic collisions.

This enhanced binding can be interpreted in the context of the protein's structural state. As established in Sections 3.1, 3.3, VT pretreatment induces a partially unfolded, dynamic conformation with exposed hydrophobic clusters and reactive sites, while potentially mitigating the formation of large, impervious aggregates seen in the 55 °C pretreatment. This "optimally denatured" state appears to create a more favorable and thermally resilient landscape for forming stable complexes with 2,3-butanedione. The complex formed with VT-pretreated protein exhibits greater stability (higher Ksv) at physiologically relevant higher temperatures compared to complexes formed with either the native-like (4 °C) or the over-aggregated (55 °C) proteins.

3.5. Binding kinetics and thermodynamic analysis of the myofibrillar protein-2,3-butanedione interaction

To gain deeper insight into the binding mechanism, the interaction between myofibrillar proteins (MPs) and 2,3-butanedione was further characterized by determining the binding constant (K) at multiple temperatures and calculating the corresponding thermodynamic parameters via the Van't Hoff equation. The results are summarized in Table 3, The negative Gibbs free energy change (ΔG) across all conditions (4 °C, 55 °C, and VT) confirms the spontaneity of the interaction. However, the distinct enthalpy (ΔH) and entropy (ΔS) contributions reveal fundamentally different binding mechanisms. The binding for both 4 °C- and VT-conditioned MPs was characterized by large negative values of ΔH and ΔS, indicating an enthalpy-driven process accompanied by a significant decrease in system disorder. Notably, the VT condition exhibited the most pronounced values (ΔH = −75.55 kJ/mol; ΔS = −187.30 J/mol·K), suggesting that the interaction here involves the strongest exothermic release of energy and the greatest loss of conformational freedom upon complex formation. In contrast, the interaction for 55 °C-conditioned MPs displayed a less negative ΔH (−49.06 kJ/mol) and a less negative ΔS (−97.67 J/mol·K), implying a more balanced contribution between enthalpy and entropy to the overall binding spontaneity.

Table 3.

Binding kinetic constants in different treatment groups.

Treatment group	T	K (∗103)	ΔH (kJ/mol)	ΔS (J/mol•K)	ΔG ((kJ mol-1)
4 °C	293K	4.40	−67.96	−160.22	−20.44
4 °C	303K	1.93			−18.87
4 °C	310K	1.03			−17.74
55 °C	293K	4.41	−49.06	−97.67	−20.44
55 °C	303K	2.38			−19.47
55 °C	310K	1.55			−18.78
VT	293K	4.83	−75.55	−187.30	−20.67
VT	303K	1.73			−18.80
VT	310K	1.04			−17.89

The exceptionally strong enthalpy-driven binding observed under VT conditions can be directly attributed to the unique structural state of the MPs, as elucidated in prior sections. The partially unfolded yet dynamic conformation induced by thermal cycling, featuring maximally exposed hydrophobic interfaces and reactive groups, appears to create an optimally complementary surface for 2,3-butanedione. This structural milieu facilitates the formation of a multitude of strong, specific non-covalent interactions—such as hydrogen bonds and van der Waals forces—accounting for the highly favorable ΔH. Concurrently, the large negative ΔS likely results from the substantial confinement and ordering of both the flexible protein chains and the flavorant molecule within a stable, defined complex. This stands in contrast to the 55 °C condition, where excessive aggregation buries potential interaction sites, weakening enthalpic gains, and to the 4 °C condition, where the more rigid native structure may limit the optimal spatial alignment for such extensive interactions.

These thermodynamic distinctions have direct and significant implications for the accelerated flavor formation in Hunan bacon processed under temperature cycling. The formation of a highly stable, enthalpy-driven complex between VT-conditioned MPs and 2,3-butanedione ensures efficient sequestration and retention of this key volatile compound from the liquid smoke, preventing its loss during processing. More importantly, this strong binding likely serves a dual function: it acts as a stable reservoir that protects the flavorant, while the tight association may also position it favorably to participate in or catalyze subsequent flavor-development reactions, such as further interactions with lipid oxidation products or integration into the Maillard reaction network during ripening. Consequently, this molecular-scale efficiency in initial capture and stabilization translates macroscopically to a more rapid intensification and complexification of the overall flavor profile, effectively shortening the required maturation time and yielding a superior final product compared to traditional constant-temperature processes.

3.6. Molecular simulation

MD simulation of myosin at 55 °C revealed dynamic changes in its structural stability (Fig. 5). RMSD analysis showed intense conformational changes within the first 30 ns, after which it equilibrated (RMSD ∼1.0 nm), indicating system stabilization. The radius of gyration (Rg) increased briefly at 10 ns and then continued to decrease, suggesting the protein structure became more compact after initial relaxation. RMSF analysis showed that the local flexibility of amino acid residues, especially at the tail ends, increased significantly after heating, suggesting irreversible conformational disruption. Solvent accessible surface area (SASA) analysis further revealed structural rearrangements: SASA decreased within 0-30 ns, indicating structural relaxation and disorder in flexible regions; SASA increased significantly within 30-45 ns, indicating that high temperature continuously disrupted hydrogen bonds and electrostatic interactions, leading to dissociation of secondary structure and exposure of internal hydrophobic residues. Evidence from secondary structure changes confirmed that high temperatures induce substantial structural rearrangement and instability. In summary, MD simulations demonstrate that heating compromises myosin structural stability, eliciting conformational alterations, secondary structure disruption, and residue disordering.

Fig. 5.

Molecular dynamics simulation of myosin conformation. RMSD (A), Rg (B), RMSF (C), solvent accessible surface area (D), binding energy (E), secondary structure (F): total amount (1), α helix (2), β fold (3), and irregular curl (4). Protein binding sites (1), binding site microenvironment (2), and binding forces (3) in 4 °C (A) and 55 °C treatments (B).

This study based on the three-dimensional structure and chemical features of the protein, the binding pockets of myosin were predicted using DOGsite Scorer (Fig. 5). The results showed that heating caused partial unfolding and refolding of myosin. The dissociation of α-helix and β-sheet structures led to a significant loss of backbone hydrogen bonds, consequently causing changes in the location and size of the binding pockets (Laganowsky et al., 2022). Affinity was assessed by calculating the average binding energy from 10 simulations. It was found that after heating, the binding energy between 2,3-butanedione and myosin increased from −3.62 ± 1.07 kJ/mol to −3.72 ± 1.02 kJ/mol. In terms of binding mode, 2,3-butanedione primarily binds to myosin through hydrogen bonds, and the increase in binding energy after heating might be related to the formation of new hydrogen bonds.

4. Conclusion

This study demonstrates that temperature cycling (4 °C – 55 °C) during liquid smoke application promotes a unique structural state of myofibrillar proteins—partially unfolded yet dynamically stable—which significantly enhances their binding capacity for the key flavor compound 2,3-butanedione. Compared to constant low- or high-temperature treatments, cycling conditions increased protein surface hydrophobicity, exposed more binding sites, and improved flavor retention through an enthalpy-driven interaction mechanism. Molecular dynamics simulations further confirmed that thermal cycling alters myosin conformation, facilitating stronger binding with 2,3-butanedione via hydrogen bonds and hydrophobic interactions. These findings provide a mechanistic explanation for the accelerated ripening and enhanced flavor quality of Hunan bacon under cyclic temperature processing, highlighting the potential of targeted protein-flavor interaction strategies to optimize smoked meat production and flavor precision control.

CRediT authorship contribution statement

Zhi Huang and Enqi He: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Writing-original draft; Lei Zhou and Aihua Lou: Data curation; Investigation; Bo Liu: Methodology; Yan Liu and Zhizhong Zhang: Supervision; Visualization; Haohua Fu: Conceptualization, Resources; Wei Quan and Qingwu Shen: Funding acquisition; Project administration; Supervision; Writing - review & edition.

Funding sources

This work has been supported by the Key Research and Development Plan Project of Hunan Province (2024JK2146, 2024JK2155), the key research project of changsha city (kq2503007), The science and technology innovation Program of Hunan Province (2024RC3185).

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.