A comprehensive study on the color preservation mechanism of isoquercitrin in meat-based food systems

Abstract

Meat color is the most intuitive indicator for consumers to judge meat freshness, which mainly depends on myoglobin (Mb). In order to explore the effect of isoquercetin on meat color preservation and its mechanism, this study mainly carried out systematic research through in vitro experiments, multi-spectral technology, molecular docking and molecular dynamics simulation. This study found that isoquercetin has a good protective effect on meat color. And isoquercetin has a static quenching effect on Mb. In addition, after forming a stable complex through hydrogen bonds and van der Waals forces, isoquercetin changed the secondary structure of Mb, resulting in a decrease in surface hydrophobicity by 9.67 % and a decrease in solubility by 27 %. Molecular dynamics and molecular docking experiments further confirmed the interaction between the two. This study confirmed that isoquercitrin can stabilize meat color through a variety of mechanisms, providing a theoretical basis for its application in meat processing.

Highlights

• •Isoquercitrin exhibits a protective effect on meat color stability.
• •Isoquercitrin bound myoglobin, altering its secondary structure conformation.
• •Hydrogen bonds and van der Waals forces were primary intermolecular interactions.
• •Isoquercetin–myoglobin binding enhanced the thermodynamic stability of myoglobin.

1. Introduction

Red meat has long been considered to be an important dietary source of essential nutrients such as protein, and vitamin B (Gagaoua & Picard, 2020). It is considered to be a healthy lifestyle food with high nutritional and biological value (Zhang et al., 2010). During the entire processing or storage process, red meat is prone to protein and lipid oxidation, resulting in discoloration, which leads to consumer rejection and ultimately causes economic losses to the meat processing industry (Singh et al., 2022). Color is the basic feature to evaluate the freshness, quality and acceptance of meat. Brightly coloured cherry red meat has always been the first choice to buy meat or its products (Anusha Siddiqui et al., 2023; Ramanathan et al., 2021).

Myoglobin (Mb) and hemoglobin (Hb) are the main pigments that cause red meat. Changes in these pigments can lead to the formation of different colors (Singh et al., 2022). Among them, Mb plays a leading role in the color of red meat during storage after slaughter (Singh et al., 2021). However, throughout postmortem processing and storage, some chemical changes, such as the oxidation of lipids and proteins, can lead to the oxidation of Mb, resulting in poor color and reduced acceptability (Singh et al., 2022). The ideal color of fresh meat is mainly affected by natural pigments. For example, bright cherry red is due to the action of oxymyoglobin (OxyMb), while purple is associated with deoxymyoglobin (DeoxyMb) (Singh et al., 2021). OxyMb undergoes a variety of biochemical changes during processing and storage, and eventually transforms into brown metmyoglobin (MetMb). Therefore, it is of great significance to find natural compounds that can protect myoglobin in the field of food science.

Isoquercitrin (Fig. 1) is a flavonoid widely found in fruits, vegetables and medicinal plants. As a glucoside derivative of quercetin, its strong antioxidant (Jung et al., 2010), anti-inflammatory (Han, Ma, et al., 2024) and anti-tumor activities (Fan et al., 2025) have attracted much attention. Studies have shown that isoquercetin can play a protective role by scavenging free radicals, inhibiting lipid peroxidation and regulating redox-related signaling pathways (Li et al., 2016; Shen et al., 2020). This highlights its important application potential in the field of food preservation and disease treatment.

Fig. 1.

Structural formula of isoquercitrin.

The research on the interaction between small molecules and proteins is of great significance in the food field. There have been some studies on the interaction between flavonoids and Mb. For example, Peng et al. studied the inhibitory effect of quercetin combined with Mb on lipid peroxidation (Peng et al., 2025). However, the solubility and bioavailability of quercetin are low, which greatly limits its application, and the research on the molecular mechanism of protecting meat color is not sufficient (Alizadeh & Ebrahimzadeh, 2022). Isoquercitrin, as a glucoside derivative of quercetin, has greatly improved solubility and bioavailability in aqueous solution (Hobbs et al., 2018). Therefore, studying the interaction mechanism between isoquercitrin and Mb can develop a more efficient and more suitable natural meat color protectant for food systems. Unfortunately, there is no literature report on the interaction mechanism between isoquercitrin and Mb. Therefore, it is necessary to explore the detailed molecular mechanism of isoquercitrin and Mb.

In order to explore the color preservation effect and mechanism of isoquercitrin in meat-based food systems, this study first conducted a preliminary in vitro study on the protective effect of isoquercitrin on meat color. Subsequently, Fourier transform infrared spectroscopy, fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy and other multi-spectral techniques, combined with molecular docking, molecular dynamics simulation and other research methods, systematically revealed the molecular mechanism of the combination between isoquercitrin and Mb. The research results not only clarify the molecular mechanism of isoquercitrin and Mb, but also provide a solid theoretical basis for its application in food preservation and muscle-related fields.

2. Materials and methods

2.1. Materials

Isoquercitrin (purity≥98 %) was purchased from Beijing Innochem Co., Ltd. (Beijing, China). Myoglobin (from horse heart muscle) was obtained from Dingzhou Baikesaisi Biotechnology Co., Ltd. (Jiangsu, China). Bromophenol blue (BPB) indicator was purchased from Aladdin Biochemical Reagent Co., Ltd. (Shanghai, China). BCA Protein Assay Kit from Jianglai Biotechnology Co., Ltd. (Shanghai, China). Other reagents were analytically pure, and the water used in the experiment was ultrapure water.

2.2. Methods

2.2.1. Sample preparation

2.2.1.1. Isoquercitrin-myoglobin complex freeze-dried powder

Firstly, 288 mg Mb was accurately weighed and dissolved in 144 mL PBS (pH = 6.8) to prepare a Mb solution with a concentration of 2 mg/mL. Subsequently, the isoquercitrin powder was pre-dissolved with 70 % ethanol and diluted with PBS to a concentration of 0–100 μM. The isoquercitrin solution of each concentration was mixed with Mb solution at a ratio of 1:1, and reacted at 4 °C for 1 h. After the reaction was sufficient, the freeze-dried powder with a final concentration of Mb of 1 mg/mL was prepared by freeze-drying.

2.2.1.2. Isoquercitrin-myoglobin complex solution

The 72 mg Mb was dissolved in 72 mL PBS buffer (pH = 6.8) to prepare 1 mg/mL Mb mother liquor. The quantitative isoquercitrin powder was accurately weighed, pre-dissolved with 70 % ethanol, diluted with PBS and prepared into 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 0 μM solution. And mixed with myoglobin solution 1:1, reacted at 4 °C for one hour, standby.

2.2.2. Study on the protective effect of isoquercitrin on meat color

Firstly, isoquercitrin solutions with concentrations of 0.05 % and 0.1 % were prepared with deionized water. Subsequently, fresh beef was cut into 5 cm × 5 cm × 1 cm meat pieces and divided into three groups. The two groups were added 20 mL 0.05 % and 0.1 % isoquercitrin solution, respectively, and the control group was added 20 mL deionized water. Both groups were soaked at 4 °C for 1 h, and then dried meat blocks were wiped and stored at 4 °C. Three groups of meat were photographed at the same time. In addition, the brightness (L*), redness (a*) and yellowness (b*) values of base pork were measured using a CR-400 Minolta colorimeter (Konica Minolta Sensing Americas Inc., Ramsey, NJ, USA) under D65 light source.

2.2.3. Fourier transform infrared spectroscopy

Isoquercitrin-myoglobin complex lyophilized powder 1–2 mg and KBr 200 mg were weighed by potassium bromide (KBr) tabletting method. The agate mortar was used to fully grind it clockwise. After tableting, it was placed in a Fourier transform infrared spectrometer (Nicolet IS50, Thermo Fisher Scientific, USA) for full-band scanning, with a scanning range of 400–4000 cm⁻¹ and 32 scans.

2.2.4. Ultraviolet-visible absorption spectrometry

The Ultraviolet-visible absorption spectra of each concentration sample solution in the range of 200–750 nm were measured by UV-2550 series Ultraviolet-visible spectrometer (Shimadzu, Japan), and each group was repeated three times.

2.2.5. Determination of solubility

Each sample was centrifuged twice (10,963×g, 15 min), and the supernatant was taken to determine the corresponding absorbance value, and the protein concentration was calculated according to the following formula (formula (1)):

$$\text{Solubility}\%=\text{supernatant protein concentration}/\text{initial protein concentration}\times 100\%$$ (1)

2.2.6. Determination of surface hydrophobicity

1 mL myoglobin-isoquercitrin complex (containing 1 mg/mL Mb solution) was taken, 200 μL 0.1 mg/mL BPB solution was added, vortexed and mixed, centrifuged twice (2000×g, 10 min). The supernatant was taken to determine the absorbance at 595 nm, and the content of protein-binding BPB was calculated as a hydrophobic index. The binding amount with BPB is mainly calculated quantitatively according to the following formula (formula (2)) (Han, Ma, et al., 2024).

$$\mathit{BPB}\text{bound}\left(\mu \mathrm{g}\right)=200\mu \mathrm{g}\times \frac{A_{\mathit{control}}-A_{\mathit{sample}}}{A_{\mathit{control}}}$$ (2)

2.2.7. Determination of the relative content of metmyoglobin

The absorbance values at 525 nm, 545 nm, 565 nm and 572 nm of the complex solutions with different concentrations were determined by ultraviolet spectrophotometer. And the relative content of Mb in the corresponding samples was calculated according to the following formula (formula (3)) (Han, Sun, et al., 2024):

$$\mathit{MetMb}\%=\left(-2.514R_1+0.777R_2+0.800R_3+1.098\right)\times 100\%$$ (3)

R₁ = A572/A525, R₂ = A565/A525, R₃ = A545/A525, where A525, A545, A565, A572 are the absorbance at 525 nm, 545 nm, 565 nm, 572 nm, respectively.

2.2.8. Synchronous fluorescence spectrum detection

Under the conditions of excitation wavelength of 200 nm and scanning rate of 2000 nm/min, the emission wavelength ranges of 215–565 nm and 260–610 nm were set respectively. The isoquercitrin-myoglobin complex solution was scanned by synchronous fluorescence spectroscopy to obtain its concentration-dependent fluorescence response signal.

2.2.9. Three-dimensional fluorescence spectrum detection

Using the same type of fluorescence spectrophotometer, the excitation wavelength range was set to 200–400 nm, the emission wavelength range was set to 270–600 nm, and the scanning rate was 12,000 nm/min. The three-dimensional fluorescence spectrum of isoquercitrin-myoglobin complex solution was determined, and the law of the three-dimensional fluorescence characteristics changing with the concentration was analyzed.

2.2.10. Endogenous fluorescent spectrum detection

The intrinsic fluorescence intensity of different concentrations of isoquercitrin-myoglobin complex solution was determined by RF-6000 fluorescence spectrophotometer at 293 K, 299 K and 305 K. The intrinsic fluorescence intensity of different concentrations of isoquercitrin-myoglobin complex solution was determined by RF-6000 fluorescence spectrophotometer. The excitation wavelength was 283 nm, the emission wavelength range was 290–550 nm, and the scanning rate was 600 nm/min. Based on the experimental data, the fluorescence quenching constant was calculated by Stern-Volmer equation (formula (4)), the binding constant and the number of binding sites were determined by double logarithmic equation (formula (5)), and the thermodynamic parameters of the system were deduced by Van ‘t-Hoff equation (formula (6)) and thermodynamic equation (formula (7)).

The formulas are as follows:

$$\frac{F_0}{F}=1+K_{\mathrm{q}}{\tau }_0\left[Q\right]=1+K_{\mathit{SV}}\left[Q\right]$$ (4)

$$\log \frac{F_0-F}{F}=\log K_{\mathrm{a}}+\mathrm{n}\log \left[Q\right]$$ (5)

$$\ln K_{\mathrm{a}}=‐\frac{\mathrm{\Delta }H}{\mathit{RT}}+\frac{\mathrm{\Delta }S}{R}$$ (6)

$$\mathit{\Delta G}=\mathit{\Delta H}-\mathit{T\Delta S}$$ (7)

Among them, F₀ and F are the fluorescence intensity when the quencher is not added and added, respectively; K_q is the quenching rate constant; K_sv is the Stern-Volmer quenching constant; [Q] is the quencher concentration; K_a is the binding constant; T is the absolute temperature; R is the ideal gas constant; ΔG, ΔH, and ΔS represent Gibbs free energy change, enthalpy change, and entropy change, respectively.

2.2.11. Differential scanning calorimetry to assess thermal stability

Each concentration of 3–5 mg of myoglobin-isoquercitrin complex freeze-dried powder was weighed and placed in a crucible for determination using a differential scanning calorimeter HS-DSC-101A-Pro (Shanghai Hesheng). The heating rate was 5 °C/min, and the temperature range was 20–100 °C. The initial temperature (T₀), peak temperature (T_max) and gelatinization enthalpy (ΔH) were recorded.

2.2.12. Molecular docking

The SDF format of the compound was downloaded from Pubchem database (https://pubchem.ncbi.nlm.nih.gov/). The three-dimensional structure of the protein was downloaded from the RCSB PDB database (www.rcsb.org). Docking with Discovery Studio 2019 Client.

2.2.13. Molecular dynamics simulation

MD simulation of Isoquercitrin and charmm36 force field was performed on Mb using GROMACS 2021.6 software. During the whole MD simulation process, the TIP3P water model provides a hydrated environment for 1 ns to reach water balance. Then, the energy is minimized by using the steepest descent method in 1000 steps to minimize the system heating from 0 K to 300 K deviation (RMSD), root mean square fluctuation (RMSF) and radius in 1000 ps. After 50 ns MD simulation, the rotation (Rg) is calculated to analyze the stability of the interaction between Mb and isoquercitrin.

2.2.14. Atomic force microscope detection

Different concentrations of myoglobin-isoquercitrin complex solution were diluted 100 times with PBS and placed on the surface of mica sheet for drying. The surface structures in the XY direction of 125 μm and the Z direction of 5 μm were scanned using an atomic force microscope (MultiMode 8-HR, Bruker, USA).

2.2.15. Statistical analysis

The experimental data were calculated by Excel, and all data were expressed as mean ± standard deviation (S.D.) (n = 3). Origin 2024 software and Graph Pad Prism 8 software were used to draw the relevant maps. One-way analysis of variance (ANOVA) and Tukey multiple comparison test and significant difference analysis were performed using IBM SPSS Statistics 26 software. Statistical significance was defined as p < 0.05, p < 0.005, p < 0.001 or p < 0.0001.

3. Results and discussion

3.1. Analysis of the protective effect of isoquercitrin on meat color

As shown in Fig. 2, it is the result of meat color change within seven days. From the picture, it can be seen that from the first day to the seventh day, the color of the meat in the control group changed significantly, and the color eventually changed to purple brown. The meat color of the group added with 0.05 % isoquercitrin solution showed local browning, and the degree of discoloration was lighter than that of the control group. In addition, the color change of meat in the 0.1 % isoquercitrin group was lighter than that in the above two groups. It shows that isoquercitrin can protect meat color.

Fig. 2.

The effect of different concentrations of isoquercitrin on the color of beef samples during storage.

In addition, it can be found from Table 1 that at the same time point, with the increase of isoquercitrin concentration, the redness value (a*) of beef was significantly increased, and the yellowness value (b*) was reduced, but there was no significant effect on the brightness value (L*). In addition, with the passage of storage time, it can be found that the a* and b* values of the control group decreased significantly, while the decrease of these values decreased significantly after the addition of isoquercitrin. It shows that isoquercitrin can protect meat color and delay the browning of meat color. Among them, the effect of 0.1 % isoquercitrin is relatively better.

Table 1.

Effects of different concentrations of isoquercitrin on beef color.

		Sample ID
Attribute	Display time (Day)	control	0.05 % Isoquercitrin	0.1 % Isoquercitrin
L*	1	45.18 ± 0.25aA	45.19 ± 0.57aA	45.12 ± 0.34aA
	3	44.96 ± 0.62aAB	44.98 ± 0.50aAB	45.28 ± 0.28aA
	5	43.82 ± 0.58aBC	44.28 ± 0.72aB	44.42 ± 0.42aB
	7	42.29 ± 0.65aC	43.24 ± 0.53aC	43.67 ± 0.39aC
a*	1	15.29 ± 0.26aA	15.81 ± 0.18abA	16.05 ± 0.18bA
	3	14.11 ± 0.13aB	15.49 ± 0.32bA	15.82 ± 0.17bA
	5	11.27 ± 0.66aC	14.34 ± 0.68bB	14.65 ± 0.39bB
	7	8.52 ± 0.29aD	10.08 ± 0.08bC	11.03 ± 0.17bC
b*	1	8.92 ± 0.46aA	8.92 ± 0.43aA	8.43 ± 0.43aA
	3	9.21 ± 0.71aA	8.76 ± 0.41bA	8.75 ± 0.24bA
	5	9.23 ± 0.65aA	9.09 ± 0.49abA	8.98 ± 0.50bA
	7	9.37 ± 0.43aA	9.12 ± 0.17abA	9.03 ± 0.14bA

Different lowercase letters on the same line indicate significant differences, and different uppercase letters on the same column in the same group indicate significant differences (p < 0.05).

3.2. Fourier transform infrared spectroscopy analysis

Modern protein Fourier transform infrared (FT-IR) spectroscopy has proven to be a versatile and sensitive technique, applicable to many aspects of protein characterization (Zuber et al., 1992). In the research process of this article, this method was used to explore the interaction process between isoquercitrin and Mb. The relevant results are shown in Fig. 3a. The absorption peak in the infrared spectrum corresponds to the vibration mode of the specific chemical bond in the molecule. It can reveal the interaction mechanism between isoquercitrin and Mb. It can be observed that in the range of 3250 cm⁻¹ to 3450 cm⁻¹, the complexes of different concentrations had characteristic absorption peaks. These peaks may be related to the hydroxyl group (—OH) in isoquercitrin and the amide group (—NH) in Mb protein, because they usually correspond to O—H and N—H stretching vibrations. It can be seen that with the increase of the concentration of isoquercitrin added, the peak position here also shifted to different degrees, indicating that isoquercitrin may form a hydrogen bond interaction with Mb. The absorption peaks at 1658.21 cm⁻¹, 1661.19 cm⁻¹, 1666.42 cm⁻¹, 1669.40 cm⁻¹, 1658.21 cm⁻¹, 1661.19 cm⁻¹, 1658.96 cm⁻¹ usually correspond to the amide I band (C O stretching vibration), which was the characteristic peak of protein secondary structure (such as α-helix, β-sheet). It can also be seen that after adding different concentrations of isoquercitrin, the position of the absorption peak shifted, suggesting that the presence of isoquercitrin changed the secondary structure of Mb protein. In addition, the peaks at 1541.04 cm⁻¹, 1538.81 cm⁻¹, 1532.84 cm⁻¹, 1535.82 cm⁻¹, 1541.04 cm⁻¹, 1541.04 cm⁻¹, 1544.03 cm⁻¹ usually corresponded to the amide II band (N—H bending vibration and C—N stretching vibration), which further supported the potential interaction between isoquercetin and Mb protein.

Fig. 3.

Fourier transform infrared spectroscopy of different concentrations of isoquercitrin-myoglobin complex (a), UV–visible absorption spectra of different concentrations of isoquercitrin-myoglobin complex (b), UV–visible absorption spectra of different concentrations of isoquercitrin (c), solubility (d), surface hydrophobicity (e) and relative content of methemoglobin (f) (*p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001 vs no isoquercitrin group).

In addition to the relevant information in the above infrared spectrum, we calculated the specific changes of each part of the secondary structure of Mb after the addition of isoquercitrin. The specific values are shown in Table 2. When isoquercitrin was not added, the proportions of β-sheet, Random coil, α-helix and β-turn were 16.36 %, 17.23 %, 34.07 % and 32.35 %, respectively. After adding isoquercitrin, the proportions of each part changed to varying degrees. Among them, α-helix is one of the most stable forms of protein secondary structure. With the increase of isoquercitrin concentration, the α-helix showed a trend of decreasing first and then increasing. When isoquercitrin was 100 μM, the value was 33.28 %, which was close to the original value. It indicated that Mb was temporarily unwound in order to adapt to isoquercitrin in the early stage, and formed a stable complex through structural rearrangement in the later stage, thus revealing the dynamic regulation process of the interaction between the two.

Table 2.

The relative content of each component of protein secondary structure of isoquercitrin-myoglobin complexes at different concentrations.

Isoquercitrin addition(μM)	β-Sheet (%)	Random coil (%)	α-Helix (%)	β-Turn (%)
0	16.36 ± 0.02a	17.23 ± 0.01a	34.07 ± 0.01a	32.35 ± 0.01a
3.125	27.82 ± 0.00b	13.21 ± 0.01b	17.33 ± 0.02b	41.73 ± 0.02b
6.25	20.66 ± 0.01c	0.03 ± 0.02c	6.87 ± 0.02c	72.44 ± 0.01c
12.5	19.02 ± 0.02d	10.57 ± 0.00d	5.58 ± 0.01d	64.83 ± 0.01d
25	18.94 ± 0.03e	10.47 ± 0.02e	13.25 ± 0.01e	57.34 ± 0.03e
50	22.09 ± 0.01f	22.54 ± 0.01f	18.25 ± 0.01f	37.13 ± 0.01f
100	22.12 ± 0.03f	21.47 ± 0.02g	33.28 ± 0.02g	23.12 ± 0.03g

Different letters in the superscript of the same column indicate significant differences (p < 0.05).

3.3. Ultraviolet–visible absorption spectroscopy

Ultraviolet–visible spectroscopy (UV–Vis) is a powerful tool for studying the steady-state and time-resolved studies of protein–ligand interactions, which can be used to study the electronic transitions and binding properties between molecules (Nienhaus & Nienhaus, 2005). In this study, the binding mode of isoquercetin and myoglobin was analyzed by this technique, and the results were shown in Fig. 3b and Fig. 3c. Fig. 3b showed the UV absorption spectra of isoquercitrin-myoglobin complexes at different concentrations. It can be seen that the maximum absorption peak of the complex appeared near 400 nm, corresponding to the Soret band of myoglobin, which can reveal the change of the microenvironment of heme prosthetic group. However, the change of the absorbance value here was almost negligible, indicating that the effect of isoquercitrin on heme prosthetic group was relatively small. The absorption peak around 280 nm was the characteristic absorption peak of aromatic amino acids in Mb, which can reflect the structural changes of Mb. The absorption peak in the range of 400–450 nm was the characteristic absorption peak of heme prosthetic group in Mb, which reflected the environmental changes of heme. It can be found that with the increase of isoquercitrin concentration (from 0 μM to 100 μM), the absorption peak intensity near 280 nm gradually increased, and different degrees of red shift occurred. This suggested that isoquercitrin may affect the surrounding environment of aromatic amino acids in Mb. The position and intensity of the absorption peak near 400–450 nm changed little, indicating that isoquercitrin had a relatively small effect on the heme prosthetic group. And the absorption peak near 280 nm, which was the characteristic absorption peak of aromatic amino acids in Mb, can reflect the degree of structural change of Mb. With the increase of isoquercetin concentration from 0 μM to 100 μM, the intensity of the absorption peak near 280 nm gradually increased and showed different degrees of red shift. In order to further determine whether this change was due to the influence of isoquercitrin's own absorption, we measured the UV absorption spectrum of isoquercitrin at the corresponding concentration separately, and the results were shown in Fig. 3c. It can be seen from the graph that the 280 nm position was close to the peak valley position, and with the increase of concentration, the absorbance curve almost showed a parallel increase state, which conforms to Lambert-Beer's law. However, the absorbance of the complex in Fig. 3b increases while a continuous red shift occurs. It shows that the amino acid residues of myoglobin interact with isoquercitrin to form hydrogen bonds, which led to the occurrence of red shift.

3.4. Solubility determination

The solubility of protein determines its stable state in solution, which is particularly important for protein structure and function (Asherie, 2019). The effect of isoquercitrin on the solubility of myoglobin is shown in Fig. 3d. As the concentration of isoquercitrin increases, the solubility of myoglobin gradually decreases from 88.10 % to 61.27 %, with a decrease of about 27 %. The reason for the decrease in solubility may be that, on the one hand, isoquercitrin binds to myoglobin, resulting in intermolecular aggregation. On the other hand, binding may lead to local conformational adjustment of myoglobin, exposing internal hydrophobic residues and further reducing solubility. With the increase of isoquercitrin concentration, the solubility decreased continuously, indicating that the binding process was concentration-dependent and conformed to the typical ligand-protein binding model.

3.5. Hydrophobicity

The hydrophobicity of protein surface can directly reflect the distribution of hydrophobic residues on the protein surface, which is closely related to the change of its surface properties. Therefore, changes in surface hydrophobicity caused by changes in molecular structure can be an ideal key indicator for predicting and evaluating changes in protein surface properties (Tang et al., 2021). In the course of this study, the surface hydrophobicity of Mb was also detected. It can be seen from Fig. 3e that with the increase of isoquercitrin concentration, the hydrophobicity of Mb decreased from 18.88 % to 9.21 %, a decrease of 9.67 %. This may result from isoquercitrin-Mb binding through hydrogen bonding/electrostatic interactions, which shielded hydrophobic residues and induced a more compact hydrophobic core conformation, thereby lowering surface hydrophobicity. On the other hand, the polar groups of isoquercitrin may increase the hydrophilicity of the complex. In addition, at 25 μM, the hydrophobicity of Mb decreased most significantly, which may be related to the binding saturation or conformational adjustment of isoquercitrin.

3.6. Relative content of metmyoglobin

The oxidation of Mb to MetMb is an important reason for the darkening of meat color (Bendall & Swatland, 1988). Therefore, it is very important to detect the relative content of MetMb. As shown in Fig. 3f, with the increase of isoquercetin concentration, the percentage of MetMb decreased from 99.1 % to 96.9 %, a decrease of 2.2 %. This indicates that isoquercetin has a weak inhibitory effect on the oxidation of myoglobin. This may be because isoquercetin has a certain antioxidant capacity, can scavenge reactive oxygen species (ROS), prevent oxidative chain reaction, and inhibit ions (Fe²⁺/Fe³⁺) to participate in the oxidation reaction, thereby reducing the formation of MetMb. In addition, after isoquercetin binds to myoglobin, its structure is stabilized, and the exposure and oxidative sensitivity of heme prosthetic group are reduced.

In short, isoquercetin reduced the degree of myoglobin oxidation (MetMb% reduction) to a certain extent in a concentration-dependent manner, and its mechanism may involve free radical scavenging, metal ion chelation and conformational stability.

3.7. Synchronous fluorescence spectroscopy

When the difference between excitation wavelength and emission wavelength (∆λ) was 15 nm, it mainly reflected the fluorescence characteristics of tryptophan residues. It can be seen from Fig. 4a that the fluorescence intensity of Mb gradually decreased with the increase of isoquercitrin concentration. The decrease in fluorescence intensity indicated that isoquercitrin bound to Mb, resulting in changes in the microenvironment of tryptophan residues.

Fig. 4.

The synchronous fluorescence spectra (a, b) of isoquercitrin-myoglobin complexes were obtained at the excitation wavelength and emission wavelength difference of 15 nm and 60 nm at different isoquercitrin concentrations. The three-dimensional fluorescence spectra showed the changes of the complexes at different isoquercitrin concentrations (c–i).

When the difference between the excitation wavelength and the emission wavelength (∆λ) was 60 nm, it mainly reflected the fluorescence characteristics of tyrosine residues. It can be seen from Fig. 4b that with the increase of isoquercitrin concentration, the fluorescence intensity of Mb also showed a downward trend. The decrease of fluorescence intensity indicated that the binding of isoquercitrin to Mb affected the microenvironment of tyrosine residues.

Combining the above two synchronous fluorescence spectra, it can be inferred that the binding of isoquercitrin to Mb may affect the local environment of tryptophan and tyrosine residues at the same time, indicating that the binding sites may be located near these residues.

3.8. 3D fluorescence spectroscopy

The results of 3D fluorescence spectra are shown in Fig. 4c–i. In the 3D fluorescence spectrum of blank Mb, the fluorescence peaks of tryptophan residues were located at Ex≈280 nm and Em ≈ 340 nm, and the fluorescence peaks of tyrosine residues were located at Ex≈275 nm and Em ≈ 300 nm (Em range 280–320 nm), indicating the typical fluorescence characteristics of Trp and Tyr residues in the unbound state. With the increase of isoquercitrin concentration from 3.125 μM to 100 μM, the Em peak of Trp residue gradually blue-shifted from 340 nm to 335 nm, indicating that the binding of isoquercitrin led to the enhancement of hydrophobicity of the microenvironment around Trp. The Em peak position (300 nm) of Tyr residue was not significantly shifted, but the fluorescence intensity continued to decrease with the increase of concentration, indicating that the binding mainly affected the local conformation of Trp residue.

In all treatment groups, it was observed that the fluorescence intensity decreased significantly with the increase of isoquercitrin concentration, and the blue shift of Trp peak was accompanied, which was consistent with the static quenching characteristics and formed a stable complex. Concentration-dependent fluorescence quenching further supported the interaction between isoquercitrin and myoglobin in a 1:1 binding mode. The blank group showed obvious Trp peak at Ex/Em = 280/340 nm, and the contour distribution was concentrated. In the treatment group, the intensity of the contour line in this area was weakened and diffused to the short wavelength direction, indicating that the microenvironment of Trp residues was hydrophobically adjusted after Isoquercitrin binding. In the high concentration group of 50 μM, a new peak appeared at Ex/Em = 280/335 nm, which further verified the binding-induced conformational changes.

The blue shift of Trp peak (Em = 340 → 335 nm) indicated that isoquercitrin binds to the hydrophobic region of Mb and stabilized the complex through hydrophobic interaction and hydrogen bond. The decrease of fluorescence intensity was consistent with the decrease of solubility (by 27 %) and hydrophobicity (by 51.2 %), indicating that the hydrophobic residues on the surface of Mb were masked and the local conformation tended to be compact after binding. 3D fluorescence spectroscopy data confirmed that isoquercitrin binds to Mb through a static quenching mechanism, mainly targeting the hydrophobic region where the Trp residue is located, inducing microenvironment hydrophobicity enhancement and conformational stabilization.

3.9. Endogenous fluorescence spectroscopy

In addition to the above two fluorescence spectra, the endogenous fluorescence spectra of the complexes at different temperatures were also detected in this study. The results are shown in Fig. 5a–e. It can be seen from Fig. 5a–c that with the increase of isoquercitrin concentration, the fluorescence intensity at different temperatures also showed a decreasing trend. Subsequently, we calculated the thermodynamic parameters of the complex at different concentrations based on the fluorescence spectra at three temperatures. The results are shown in Fig. 5d–e and Table 3. Among them, at three temperatures, the K_q values of the complexes were 8.1 × 10¹⁰ M⁻¹·s⁻¹, 6.3 × 10¹⁰ M⁻¹·s⁻¹, 5.7 × 10¹⁰ M⁻¹·s⁻¹, respectively, which were greater than the maximum value of the protein dynamic collision rate constant. It can also be explained that isoquercitrin-induced myoglobin fluorescence quenching is mainly achieved through a static quenching process. In addition, ΔG < 0, indicating that the process of star lake interaction between the two is a spontaneous process at the conditional temperature.

Fig. 5.

The fluorescence spectra (a–c) of isoquercitrin-myoglobin complexes at 293 K, 299 K and 305 K, and the corresponding quenching constants (d) and binding constants (e) at different isoquercitrin concentrations were obtained.

Table 3.

The quenching constants, bind constants and thermodynamic parameters for myoglobin-isoquercitrin complexes at 293 K, 299 K and 305 K.

T (K)	Ksv (103L/mol)	Kq (1011 M−1·s−1)	Ra2	Ka (102L/mol)	n	Rb2	ΔG (kJ/mol)	ΔH (kJ/mol)	ΔS (J/mol·K)
293	8.1 ± 0.76a	8.1 ± 0.76a	0.99 ± 0.02a	7.09 ± 0.23a	0.68 ± 0.03a	0.99 ± 0.03a	−135.03 ± 1.21a	−73.72 ± 1.62	216.07 ± 3.23
299	6.3 ± 0.34b	6.3 ± 0.34b	0.96 ± 0.01b	3.19 ± 0.45b	0.51 ± 0.01b	0.97 ± 0.01b	−138.33 ± 2.13b
305	5.7 ± 0.12c	5.7 ± 0.12c	0.95 ± 0.03b	2.16 ± 0.21c	0.36 ± 0.02c	0.98 ± 0.02ab	−142.62 ± 2.26c

Different letters in the superscript of the same column indicate significant differences (p < 0.05).

3.10. Thermal stability analysis

Differential scanning calorimetry is an important method to study the thermal stability of proteins. For proteins, differential scanning calorimetry can not only be used to determine the equilibrium thermodynamic stability and folding mechanism of proteins, but also qualitatively screen the thermal stability of proteins (Johnson, 2013). It can be seen from Fig. 6a that the denaturation temperature of Mb increased significantly with the increase of concentration. Specifically, the denaturation temperature of Mb increased from 85 °C in the control group to 130.65 °C at 100 μM, with an increase of 53.7 %. It can be seen that isoquercitrin significantly enhanced the thermal stability of Mb in a concentration-dependent manner. In addition, between 6.25 μM and 12.5 μM, T_m increased the most, indicating that the binding of isoquercitrin at low concentrations rapidly increased thermal stability.

Fig. 6.

Differential scanning calorimetry (a), root mean square deviation (b), root mean square fluctuation (c), radius of gyration (d), hydrogen bond dynamic change (e) and molecular docking (f) in molecular dynamics simulations at different isoquercitrin concentrations were performed.

This may be due to the fact that isoquercitrin binded to Mb through hydrogen bonding, hydrophobic interaction or electrostatic interaction, thereby reducing structural disorder and improving the ability to resist thermal denaturation. It may also result from that the synergistic effect of isoquercitrin-myoglobin complex inhibited protein unfolding and improved overall thermodynamic stability. While the thermal stability of Mb was improved, the surface hydrophobicity was reduced, which reduced the thermal aggregation or denaturation caused by masking the hydrophobic region. At the same time, the stable structure may reduce the exposure of oxidation sensitive sites, which was consistent with the decreasing trend of MetMb%.

3.11. Molecular dynamics simulation

Molecular dynamics simulation can better understand the structural changes of food proteins and small molecules at the molecular level, and is increasingly used in the food field (Hu et al., 2023). In order to further explore the effect of isoquercitrin on myoglobin, molecular dynamics simulation was used in this study. The results are shown in Fig. 6b–e. The RMSD (Fig. 6b) reflected the deviation between the protein structure and the initial structure during the simulation process. During the 150 ns simulation time, the RMSD value in the complex fluctuated between 0.20 nm and 0.32 nm, which was significantly higher than the RMSD value of myoglobin alone. This indicated that the overall conformation of myoglobin had undergone a more obvious adjustment. In addition, the RMSD values of the complex and myoglobin fluctuated and tended to be stable after 60 ns, indicating that the two systems had reached a state of conformational dynamic equilibrium. RMSF (Fig. 6c) reflected the flexibility of each amino acid residue in the protein. It can be found that in the simulation time of 140 ns, the RMSF fluctuation of isoquercitrin-myoglobin complex was similar to the RMSF fluctuation of myoglobin, and between 140 and 150 ns, the RMSF fluctuation of the two was quite different. It indicated that isoquercitrin did not significantly change the flexibility of the core structure of the protein after binding to myoglobin, but the specificity limited the fluctuation of the C-terminal disordered region. Rg (Fig. 6d) reflected the overall compactness of the protein. During the simulation time of 150 ns, the Rg values of the two were dynamically changed between 1.46 and 1.56 nm, and the baselines of the two were similar, but the Rg values of the complex were slightly lower than that of myoglobin. This showed that although the overall compactness of myoglobin was not changed after the formation of the complex, the dynamic fluctuation of the conformation was significantly reduced and the stability of the structure is enhanced. Fig. 6e showed the dynamic change of the number of hydrogen bonds during the formation of isoquercitrin-myoglobin complexes. It reflected that the binding process of isoquercitrin and myoglobin is dynamically adjusted. In addition, the formation of hydrogen bonds was also conducive to increasing the stability of the complex structure.

3.12. Molecular docking

Molecular docking can be used to predict the binding mode of small compounds or macromolecules in contact with receptors, and to predict the interaction between the two (Santos et al., 2019). In this study, the docking analysis was carried out by Discovery Studio 2019 Client to visualize the 2D and 3D molecular structures in protein complexes (Hussain et al., 2023; Khan, Hussain, et al., 2024; Khan, Maalik, et al., 2024). Fig. 6f showed the molecular docking results of isoquercitrin and Mb. It was found that the binding energy of isoquercitrin with Mb was −9.7 kcal/mol, and the binding energy was lower than −7 kcal/mol, indicating that the compound could form a strong binding with the protein active pocket, and the small molecule could enter the protein active pocket with a good configuration matching. Subsequently, the interaction analysis showed that the interaction between isoquercitrin and Mb was mainly caused by van der Waals force and hydrogen bond interaction (Arif et al., 2024; Iqbal et al., 2025; Khan et al., 2025). Among them, isoquercitrin formed van der Waals force with amino acids PHE98, ARG70, VAL63, PHE74, LEU71, PHE28, PHE105, VAL108, ASN107, SER39 and PHE42. In addition, three-dimensional structure analysis showed that isoquercitrin formed hydrogen bond interactions with amino acids ARG66, GLU94 and GLN104. These interactions enable the compounds to effectively bind to the protein pocket, thereby exerting their functions.

3.13. Atomic force microscopic analysis

The atomic force microscope can generate a spatial resolution map of the sample surface, which can show its surface characteristics (Allison et al., 2002). In this study, atomic force microscopy was used to study the surface microscopic effects of isoquercitrin on Mb. Fig. 7 shows the three-dimensional image of Mb by atomic force microscopy. In the control group, the size of Mb was smaller. However, with the increasing concentration of isoquercitrin, the aggregation of Mb increased significantly, and its size also increased accordingly. It showed that the addition of isoquercitrin increased the crosslinking phenomenon of Mb and increased its molecular size, which explained the reason why the surface hydrophobicity and solubility are reduced.

Fig. 7.

Atomic force microscopy results of different concentrations of isoquercitrin-myoglobin complexes (a–g).

4. Conclusion

In summary, this study systematically explored the protective effect of isoquercetin on meat color, and analyzed its interaction mechanism with Mb by multi-spectral technology and molecular docking (Fig. 8). Studies have found that isoquercitrin has a good protective effect on meat color, and it can bind to Mb through non-covalent interactions dominated by hydrogen bonds and van der Waals forces. After binding, the secondary structure of Mb was adjusted, the molecules were aggregated and the size was increased, resulting in a decrease in water solubility and surface hydrophobicity by about 27 % and 9.67 %, respectively, and a slight decrease in methemoglobin content. At the same time, its thermal stability increased to 130.65 °C. These changes in structure and properties provide a theoretical basis for the application of isoquercetin as a natural meat color protective agent in the food field and the development of high-quality meat products with stable color and excellent quality.

Fig. 8.

Summary of interaction between isoquercitrin and myoglobin.

CRediT authorship contribution statement

Xiangyang Zhang: Writing – original draft, Software, Methodology, Investigation. Yupei Sun: Writing – original draft, Software, Methodology, Investigation. Zongwei Yu: Software. Guoqiang Cai: Visualization. Jianzeng Xin: Writing – review & editing, Visualization, Methodology, Investigation. Sheng Liu: Writing – review & editing, Visualization, Resources, Funding acquisition.

Funding sources

This work was financially supported by the Natural Science Foundation Project of Shandong Province (ZR2023QH088).

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.

Appendix A. Supplementary data

Supplementary material.

Data availability

The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.