Dual enhancement of pork color stability: Antioxidant activity and myoglobin structural compactness mediated by L-histidine/L-arginine

Abstract

This study aimed to investigate the effect of L-histidine (His) and L-arginine (Arg) on the color of pork and reveal the possible mechanism. The a* value increased by 2.36 %, 4.78 % and 8.65 % with 0.10–0.30 % His, and by 3.45 %, 8.40 % and 13.06 % with 0.10–0.30 % Arg, respectively (P < 0.05). His/Arg increased oxymyoglobin levels while reducing metmyoglobin levels in dose-dependent patterns. Concomitantly, His/Arg increased sulfhydryl content and decreased reactive oxygen species, TBARS, and carbonyl levels (P < 0.05). Multi-spectral analyses revealed that His/Arg increased the α-helix content and inhibited the unfolding of tertiary structure of myoglobin mainly by interactions with tyrosine residues. Molecular docking simulations suggested that His/Arg could form stable complexes with myoglobin through hydrogen bonding and hydrophobic interactions. These changes might create a protective barrier to limit myoglobin oxidation, thus enhancing color stability. Overall, His/Arg enhanced pork color stability by exerting antioxidant activity and strengthening structural compactness of myoglobin, with Arg demonstrating greater efficacy than His.

Graphical abstract

Highlights

• •His/Arg increased the L* value, a* value, and decreased the b* value of pork.
• •The antioxidancy of His/Arg altered the content of different types of Mb.
• •His/Arg increased α-helix content and stabilized Mb's tertiary structure.
• •His/Arg could bind with Mb through hydrogen bonds and hydrophobic interactions.
• •Arg demonstrated superior antioxidancy and enhanced Mb structurability to His.

1. Introduction

Color is the consumer's first impression of meat and plays a crucial role in its sensory acceptance (Ragucci et al., 2024). The oxidation of proteins and lipids in meat is a major factor contributing to color deterioration in meat and meat products (Faustman, Sun, Mancini, & Suman, 2010). Myoglobin (Mb), a pigment protein, is primarily responsible for determining meat color (Mancini & Hunt, 2005). After slaughter, the oxygenated myoglobin (Mb (Fe²⁺) O₂, bright red) is inevitably oxidized to metmyoglobin (MetMb (Fe³⁺)), leading to a brown discoloration that significantly reduces the market value of the meat (Su et al., 2024). Therefore, maintaining the reduced state of Mb and stabilizing meat color retain critical and challenging issues for the meat industry.

Many physical and biochemical methods have been studied to stabilize meat color. High pressure processing (HPP) can improve meat color by inhibiting microbial growth and reducing lipid/Mb oxidation, but improper treatment may induce MetMb formation via heme iron oxidation, causing meat to turn brown (Bak et al., 2017; Bolumar et al., 2021; Chun et al., 2014). Modified atmosphere packaging (MAP) with optimized oxygen atmospheres can enhance meat color stability by stabilizing OxyMb via suppressing microbial growth and enzymatic oxidation, whereas suboptimal gas ratios promote pigment autoxidation through heme iron destabilization (Li, Guo, et al., 2022; Yan et al., 2024; Gokoglu, 2019; Amaral et al., 2021). Nitrite is widely used to enhance meat color, but it raises safety concerns due to potential toxicity, respiratory depression, and carcinogenic N-nitrosamine formation. (Huang et al., 2020; Ma et al., 2024; Rodrigues et al., 2023; Shakil et al., 2022). Plant extracts can preserve color through their antibacterial and antioxidant properties, but they also suffer from drawbacks such as strong odors, instability and susceptibility to degradation (Xie et al., 2023; Zhu et al., 2022; Zhu et al., 2024). Hence, it's urgent to develop safer, more economical and effective methods to maintain meat color stability.

As basic amino acids, L-histidine (His) and L-arginine (Arg) have been broadly implemented in meat processing due to multifunctional benefits, such as enhancing meat tenderness, and water-holding capacity (Fan et al., 2024; Gunasekaran et al., 2023; Hayakawa et al., 2023; Wu, Jiang, Gao, Yu, Yang, et al., 2023; Zhang et al., 2020; Zhang et al., 2021). It has also been shown that His can enhance the a* values of cured loins (Zhang et al., 2018), while Arg can improve beef color stability (Tuell et al., 2021). These effects may arise from their antioxidant activity through free radical scavenging and iron-chelating capacity (He et al., 2025; Xu et al., 2024; Zhou et al., 2014; Zhou et al., 2015). In addition, studies also demonstrate that His results in higher total pigmentation in cured pork sausages (Bae & Jeong, 2024), while Arg incorporation increases a* values concomitant with MetMb reduction of cured sausage (Ning et al., 2019). Therefore, it can be hypothesized that His/Arg might enhance pork color through their antioxidant activity and maintaining structural compactness of Mb. Nevertheless, the mechanism underlying the interaction between His/Arg and Mb remains unclear.

This study aimed to investigate the effect of His and Arg on pork color and reveal the possible mechanism through a multi-scale approach. We firstly examined the effect of His/Arg on pork color and and Mb relative content. Secondly, the levels of reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS), as well as sulfhydryl content were determined to assess the antioxidant effects of His/Arg. Thirdly, we evaluated the effects of His/Arg on structural changes of Mb by circular dichroism (CD), ultraviolet–visible (UV–Vis) spectroscopy and endogenous fluorescence emission spectroscopy. Then, we focused on exploring interaction between His/Arg and Mb through synchronous fluorescence spectroscopy, molecular docking and molecular dynamics simulation. Finally, a His/Arg-mediated color protection mechanism was proposed, as illustrated in Fig. 1. These findings may offer a theoretical basis for the use of basic amino acids to improve meat quality.

Fig. 1.

Mechanism of interaction of L-histidine and L-arginine with myoglobin.

2. Materials and methods

2.1. Materials

All the analytical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The longissimus dorsi muscles of six-month-old ternary crossbred pigs (Duroc × Long White × Large White, 100 kg ± 10 kg body weight) were taken from local slaughterhouse (Wenshi Livestock Co., Ltd. Zhenjiang, Jiangsu, China). After slaughter, the LD muscles were immediately removed from carcasses and rapidly transported to the laboratory within 1 h (4 °C).

2.2. Sample preparation

The meat was sliced longitudinally along the fiber orientation into evenly sized strips (1.5 × 5 × 1.5 cm³, 10.0 ± 0.1 g). The prepared samples were randomly allocated into seven experimental groups. His and Arg solutions at concentrations of 0.10 %, 0.20 %, and 0.30 % (w/v) were prepared using deionized water (pH 7.0) and maintained at 4 °C. Pork samples were placed in His/Arg solution (pH adjusted to 7.0 with 1 M hydrochloric acid) at 4 °C, using deionized water as the control group. The ratio of meat samples to marinade was 1:4 (w/v). After treatment, the samples were dried on filter paper to remove surface moisture within 30 min, and then immediately subjected to quality assessment and Mb extraction.

2.3. Color

The lightness (L* value), redness (a* value), and yellowness (b* value) of the pork was ascertained by the method of Guo et al. (2024) with an automated colorimeter (Physical Optical Instruments Co., Ltd., Shanghai, China).

2.4. Mb sample preparation

The Mb was extracted and purified using a method based on our previous study (Guo et al., 2024) with slight modifications. Meat samples were mixed with a pre-chilled extraction solution (10 mM Tris-HCl, pH 8.0, 4 °C, containing 25 g/L Triton X-100 and 1 mM EDTA) at a ratio of 1:3 (w/v). The mixture was homogenized using a homogenizer (Model FSH-2 A, Yinen Experimental Instrument Factory, Changzhou, China) at 12,000 rpm for 15 s, followed by another 15 s of homogenization after a 15 s interval. The homogenate was then centrifuged at 4 °C for 10 min at 9600 ×g. The supernatant collected after filtration through double-layer filter paper was the crude Mb extract. Solid ammonium sulfate was added to the crude extract to achieve a 50 % saturation of ammonium sulfate.

The mixture was sealed and kept at 4 °C in the dark for 3 h. And then，the mixture was centrifuged at 10,000 ×g for 15 min at 4 °C to remove the precipitate. Ammonium sulfate was further added to the supernatant to reach a saturation of 90 %, and the mixture was allowed to stand and centrifuged under the same conditions as above. The precipitate was dissolved in 10 mM Tris-HCl (pH 8.0, 4 °C) and dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 7000 Da. The dialysis solution was refreshed every two hours throughout the process. After dialysis, the solution was centrifuged at 5000 ×g for 10 min at 4 °C to obtain the purified Mb solution, which was stored at −20 °C. The absorbance of the Mb solution was measured using a UV spectrophotometer (model P1, MAPADA Instrument Co., Ltd., Shanghai, China). The concentration of Mb was calculated according to the Beer-Lambert law as described by Trout (1989):

Mb (mg/mL) = (A₅₂₅ - A₇₀₀) × 2.303.

where A525 and A700 are the absorbance of Mb extract at 525 nm,700 nm.

2.5. Mb relative percentage determination

The absorbance of Mb (0.1 mg/mL) was measured at 525 nm, 545 nm, 565 nm, 572 nm. Based on the absorbance, W₁ (A₅₇₂/A₅₂₅), W₂ (A₅₆₅/A₅₂₅), and W₃ (A_545/A₅₂₅) were calculated. The respective proportions of DeoMb, MetMb and OxyMb were computed based on the method of Qi et al. (2024).

DeoMb% = 100 × (0.369 × W₁ + 1.140 × W₂–0.941 × W₃ + 0.015).

OxyMb% = 100 × (0.882 × W₁₋ 1.267 × W₂ + 0.809 × W₃₋0.361).

MetMb% = 100 × (− 2.514 × W₁ + 0.777 × W₂ + 0.8 × W₃ + 1.098).

2.6. ROS levels

ROS levels in meat were measured using a commercial assay kit (Biosharp Biotechnology Co., Ltd., Hefei, China). The procedure was summarized as follows: 0.1 g of meat sample was homogenized in PBS (4 °C) to obtain a cell suspension. The cell suspension was centrifuged (1000 ×g, 4 °C, 10 min), and 10 μM of 2,7-dichlorodihydrofluorescein diacetate solution was added to the cell pellet. The mixture was then incubated at 37 °C in the dark for 30 min before centrifugation. The pellet was resuspended in PBS, and fluorescence detection was performed using a fluorescence spectrophotometer (model F-7000, Hitachi Scientific Instruments Ltd., Beijing, China), with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The level of ROS in meat samples was represented by the magnitude of the fluorescence value.

2.7. TBARS

Meat samples (5 g ± 0.01 g) were mixed with 50 mL trichloroacetic acid solution and incubated at 50 °C for 30 min using a thermostatic shaker. After cooling to room temperature, the mixture was filtered through double-layer quantitative filter paper. 5 mL of collected filtrate was mixed with 5 mL aqueous thiobarbituric acid solution and sealed and then heated in a 90 °C water bath for 30 min in dark. Absorbance at 532 nm was measured.

2.8. Protein oxidation

Carbonyl content was detected using the 2,4-dinitrophenylhydrazine (DNPH) as the measuring substance, with a slight modification based on our previous method (Guo et al., 2023). Record the absorbance values of the samples and the control group at 370 nm. Carbonyl content was calculated accordingly:

$$\text{Carbonyl content}\left(\text{nmol}/\mathrm{mg}\text{protein}\right)=125\times {10}^7\times \left(A_{\mathit{sample}}-A_{\mathit{control}}\right)/\mathrm{22,000}$$

where A_sample and A_control mean the of samples and the control group at 370 nm.

The R-SH and T-SH contents were measured by combining 1 mL of Mb (0.1 mg/mL) solution with 2 mL of Tris-glycine-EDTA buffer (6.9 mg/mL glycine, 10.4 mg/mL Tris, 1.2 mg/mL EDTA, pH 8.0). For the T-SH assay, 480 mg/mL urea was included in the buffer. After the mixture had been maintained at 25 °C for 5 min under light-protected conditions, its absorbance at 412 nm was documented. The sulfhydryl content was computed according to the following formula (Guo et al., 2024):

$$\text{Sulfhydryl content}\left(\text{μmol}/\mathrm{mg}\right)=\mathrm{10,000}\times A_{\mathit{412}}/13600$$

where A₄₁₂ means the absorbance of samples at 412 nm.

2.9. CD spectra

The secondary structure of Mb uses products of circular dichroism (CD, Chirascan type, Applied Physics Inc., UK). Mb (0.01 mg/mL) was transferred to 1 mm quartz glass, and CD spectra were collected in the range of 195–260 nm. The spectra were averaged over three scans. The percentages of the four secondary structures were determined using the protein secondary structure estimation program (CDNN method) provided by the Christcan spectrometer.

2.10. UV–vis absorption spectra

UV–vis absorption spectroscopy was determined using quartz cuvettes with a pathlength of 1 cm, to which 2 mL of Mb (0.1 mg/mL) was added. Measurements were performed using a UV spectrophotometer (model P1, MAPADA Instrument Co., Ltd., Shanghai, China) with absorption wavelengths ranging from 250 to 450 nm. The buffer solution containing 5 mM Tris-HCl was set as blank.

2.11. Endogenous fluorescence spectra

The endogenous fluorescence spectra were measured according to the method of Qi et al. (2024) with slight adjustments. The density of Mb solution used was 0.1 mg/mL. The fluorescence spectral range was 290–400 nm, and the excitation wavelength was 280 nm. The samples were measured at room temperature and stored at 4 °C.

2.12. Synchronous fluorescence spectra

The synchronous fluorescence spectra were measured based on our previous study with minor modifications (Guo et al., 2024). The concentration of Mb used was 0.1 mg/mL. Δλ was set to 15 nm and 60 nm, respectively, where Δλ = λex-λem (λex is the excitation wavelength and λem is the emission wavelength). The samples were stored at 4 °C before testing at room temperature.

2.13. Molecular docking

Molecular docking was carried out to model the interaction between Mb and His as well as Arg. The crystal structure of Mb, with the Protein Data Bank code 1MNK, was retrieved from the Protein Data Bank (https://www.rcsb.org/pdb). The molecular structures of His (CAS: 71–00 - 1) and Arg (CAS: 74–79 - 3) were gotten from the PubChem molecular database (https://pubchem.ncbi.nlm.nih.gov/). The AmberTools22 suite was employed to preprocess Arg and His, while hydrogenation and RESP charge calculations were performed using Gaussian 16 W. The resulting potential parameters were incorporated into the topology files for molecular dynamics simulations. AutoDock Vina was utilized for molecular docking, and the binding pocket prediction was carried out with Fpocket (Le Guilloux, Schmidtke, & Tuffery, 2009; Macari et al., 2020). A total of 10 docking conformations were generated, with the one exhibiting the lowest absolute binding free energy selected as the optimal result. The docking interactions were further visualized in three-dimensional and two-dimensional formats using Pymol (DeLano, 2002; Guo et al., 2024) and LigPlot (Laskowski & Swindells, 2011), respectively.

2.14. Molecular dynamics simulation

MD was accomplished by GROMACS 2022, with learning conditions referenced from our previous article (Abraham et al., 2015; Der Spoel et al., 2005). Trajectories were analyzed for solvent-accessible surface area (SASA), Root Mean Square Deviation (RMSD), radius of gyration (Rg), and Root Mean Square Fluctuation (RMSF) using GROMACS utilities. Binding free energies were calculated via the MM/PBSA method (Wang et al., 2022).

2.15. Statistical analysis

All experimental procedures were carried out in triplicate (n = 3). The obtained results were shown as the mean value ± standard deviation (SD). Statistical analyses, specifically Duncan's multiple range test and one-way analysis of variance (ANOVA), were carried out using IBM SPSS Statistics 26 software (SPSS Inc., located in Chicago, USA). The significance level was set at P < 0.05. Graphs were plotted using Origin Pro 2021 software (Origin Lab Corporation, Northampton, USA).

3. Results and discussion

3.1. Color

Color is a primary determinant in meat products evaluation, directly influencing consumer purchasing behaviors (Su et al., 2024). The colors of various treatments were presented in Table 1 and Fig. 2a. Compared with control, the L* value of His and Arg treatments was significantly increased (P < 0.05), which might be due to the enhanced water-holding capacity that increased surface moisture and light scattering (Guo et al., 2024). The a* value showed a dose-dependent increase and b* value showed a dose-dependent decrease in the presence of His and Arg (P < 0.05), which might be ascribed to the antioxidant activities of His/Arg via ferrous ions chelating and free radicals scavenging abilities (Guo et al., 2022; Zhang et al., 2021; Zhou et al., 2014; Zhou et al., 2015; Zhu et al., 2023). The result agreed with the study of Zhang et al. (2018), who found that partial substitution of NaCl with His improved the a* value and decreased b* value of cured cooked loin (P < 0.05). Notably, Arg treatments exhibited significantly higher a* value than His treatments at equivalent concentrations (P < 0.05). This might be attributed to Arg might have higher antioxidant activity than His, as demonstrated in subsequent studies. Therefore, the present result suggested that the presence of His and Arg could enhance the meat color.

Table 1.

Effect of different concentrations of His and Arg on the color of pork.

Samples	L*	a*	b*
CT	51.37 ± 0.32f	16.54 ± 0.24e	7.36 ± 0.55a
0.10-His	55.55 ± 0.14d	16.93 ± 0.08d	5.72 ± 0.43b
0.20-His	57.46 ± 0.24c	17.33 ± 0.24c	6.10 ± 0.12b
0.30-His	60.28 ± 0.09a	17.97 ± 0.03b	5.63 ± 0.06b
0.10-Arg	52.37 ± 0.31e	17.11 ± 0.24cd	7.06 ± 0.18a
0.20-Arg	56.99 ± 0.66c	17.93 ± 0.05b	6.26 ± 0.24b
0.30-Arg	58.79 ± 1.07b	18.70 ± 0.18a	5.83 ± 0.14b

Note: The values are the means ± SD. Different letters indicate significant differences (P < 0.05) (n = 3).

Fig. 2.

Surface color of different treatments (a). Effect of different concentrations of His and Arg on the relative content of Mb (b). Values are means ± SD. “a-d" indicated significant differences in DeoMb of different treatments. “A-D" indicated significant differences in OxyMb of different treatments. “v-z” indicated significant differences in MetMb of different treatments (P < 0.05) (n = 3).

3.2. Mb relative content

Pork color reflects proportions of DeoMb, OxyMb, and MetMb, indicating Mb redox status (Su et al., 2024). As shown in Fig. 2b, compared with control, His/Arg treatments significantly increased DeoMb and OxyMb levels, whlie decreasing MetMb levels compared with control (P < 0.05). This was in accordance with the research of Zhou, Ye, Nishiumi, Qin and Chen, 2014, Zhou, Ye, Wang, Qin and Li, 2015, who found that both His and Arg could reduce the MetMb levels accompanied by the increase of OxyMb levels. As mentioned above, both His and Arg had an antioxidant activity, which effectively inhibited the oxidation of ferrous iron to ferric iron (Guo et al., 2022; Xu et al., 2018; Zhang et al., 2021). In addition, it might facilitate the elevation of NADH content, thereby enhancing the electron supply for the decrease of MetMb (Kim et al., 2009). Remarkably, both His and Arg induced a dose-dependent increase in OxyMb levels, with the increase reaching up to 23.21 % for His treatment and 24.85 % for Arg treatment. Concurrently, there was a corresponding decrease in MetMb levels, which dropped to 33.30 % for His treatment and 32.29 % for Arg treatment. These results were aligned with changes of meat color.

3.3. ROS

Meat discoloration arises from interdependent oxidative systems in which Mb-derived ROS initiate lipid and protein oxidation cascades (Krasulya et al., 2021; Wang et al., 2021). As shown in Table 2, His/Arg treatments exhibited a dose-dependent suppression of ROS levels (P < 0.05), with Arg demonstrating significantly superior efficacy to His at equivalent concentrations. This observation aligned with previous studies, which showed that His and Arg had high free radicals scavenging abilities (Guo et al., 2022; Xu et al., 2018). This phenomenon might be attributed to two primary reasons: Firstly, both His and Arg possessed the ability to chelate iron, forming tridentate complexes through their amino (−NH₂) and carboxyl (-COOH) groups (Campen, 1972; Xu et al., 2018; Vera-Aviles et al., 2018; Ha et al., 2023). This interaction effectively inhibited the Fenton reaction, thereby reducing the generation of ROS. Secondly, His could directly neutralize ROS to form 2-oxo-histidine via single π-electron donation and single proton transfer through its imidazole ring. Notably, Arg could also directly neutralize ROS to form Glutamic-semialdehyde via dual π-electron donation and dual proton transfer through its α-guanidino group (Lass et al., 2002; Stadtman & Levine, 2003). This enhanced electron/proton transfer capacity likely accounted for why Arg exhibited superior efficacy than His in ROS suppression. In summary, the results indicated that His and Arg could protect Mb from ROS attack and subsequently enhance meat color.

Table 2.

Effects of different concentrations of His and Arg on ROS, TBARS, carbonyl and sulfhydryl content in pork.

Samples	Carbonyl μmol/mL	T-SH μmol/mg	R-SH μmol/mg	ROS/mg	TBARS mg/mL
CT	0.73 ± 0.01a	231.90 ± 0.20d	82.00 ± 4.80f	25.34 ± 0.14a	0.62 ± 0.02a
0.10-His	0.65 ± 0.02b	272.80 ± 2.69b	101.87 ± 0.99d	20.25 ± 0.18c	0.36 ± 0.01d
0.20-His	0.50 ± 0.01c	225.73 ± 3.36d	84.57 ± 1.20f	18.85 ± 0.09d	0.28 ± 0.01e
0.30-His	0.47 ± 0.01c	246.80 ± 9.41c	92.30 ± 3.45e	16.31 ± 0.48f	0.13 ± 0.02f
0.10-Arg	0.66 ± 0.03b	510.90 ± 2.26a	170.27 ± 6.14a	20.87 ± 0.29b	0.46 ± 0.01b
0.20-Arg	0.54 ± 0.06c	514.60 ± 1.10a	154.60 ± 3.94b	17.99 ± 0.63e	0.41 ± 0.01c
0.30-Arg	0.22 ± 0.05d	511.73 ± 1.12a	141.00 ± 1.39c	14.49 ± 0.15j	0.37 ± 0.01c

Note: The values are the means ± SD. Different letters indicate significant differences (P < 0.05) (n = 3).

3.4. TBARS

Lipid oxidation and its secondary oxidation products promote the oxidation of Mb and concomitant meat discoloration during processing and storage of meat products (Zhu et al., 2022).

As the most reliable direct biomarker for evaluating lipid oxidation in meat products (Xu et al., 2018), TBARS values were determined (Table 2). As shown, TBARS values significantly diminished with the increasing addition of His and Arg (P < 0.05), with His demonstrating superior efficacy over Arg at equivalent concentrations, indicating that His and Arg could reduce the lipid oxidation. The results were supported by the previous studies demonstrating that Arg could primarily mitigate lipid oxidation of Litopenaeus vannamei during freeze-thaw cycles (He et al., 2025), while His could reduce TBARS levels during dry-cured loin processing (Zhang et al., 2015). For Arg treatment, Arg exhibits scavenging capacity against lipid-derived radicals (LOO·) via π-electron donation and proton transfer through its α-guanidino group, thus inhibiting the accumulation of lipid oxidation and its secondary oxidation products (Vera-Aviles et al., 2018; Zhang et al., 2023). In addition, Arg had high ferrous ions chelating activity, which could inhibit lipid oxidation triggered by free iron ions (He et al., 2025; Xu et al., 2018). Similarly, His also had high chelating activity of ferrous ions and scavenging capacity against hydroperoxides via π-electron donation and proton transfer through its imidazole ring (Guo et al., 2022; Stadtman & Levine, 2003). Moreover, His might inhibit lipoxygenase activity so as to attenuate the rate of lipid oxidation (Zhang et al., 2015), potentially explaining its greater efficacy than Arg in suppressing lipid oxidation. As is known, the inhibition of lipid oxidation concomitantly suppressed Mb oxidation, resulting in enhanced color stability (Sun et al., 2024). The results were in accordance with the changes of color and Mb relative content.

3.5. Protein oxidation

Carbonyl content serves as a widely accepted indicator for evaluating the oxidation of Mb (Li et al., 2023). As shown in Table 2, the carbonyl content was significantly decreased by 10.96 %, 31.51 % and 35.62 % in the presence of 0.1–0.3 % His, and by 9.59 %, 26.03 % and 69.86 % in the presence of 0.1–0.3 % Arg, respectively (P < 0.05). The results aligned with the previous research wherein Arg decreased the carbonyl content of MPs in the Antarctic krill (Zhang et al., 2025), while His exhibited analogous attenuation of oxidative carbonylation of porcine MPs (Guo et al., 2022).

Sulfhydryl groups are subject to aggression from hydroxyl radicals mediate the synthesis of inter- and intramolecular disulfide bonds, making them as important indicators for evaluating the oxidation levels of protein (Xu et al., 2024). Compared with the control, the content of T-SH and R-SH were significantly increased with the addition of His and Arg (P < 0.05), indicating the prevention of the formation of cross-linking of SH groups. This corroborated earlier findings, showing that the T-SH and R-SH content of porcine MPs was increased in the presence of His (Guo et al., 2022), while that of Antarctic krill MPs was increased in the existence of Arg (Zhang et al., 2025). Notably, Arg displayed markedly greater efficacy compared to His at equivalent concentrations. As previously mentioned, Arg demonstrated a more effective capacity than His in neutralizing ROS through dual π-electron donation and dual proton transfer by its α-guanidino group (Lass et al., 2002; Stadtman & Levine, 2003), thus potentially reducing the ROS attack on sulfhydryl groups.

Overall, the addition of His and Arg decreased carbonyl and rose SH content of Mb, indicating the prevention of Mb oxidation. These observations might result from the ferrous ion chelating capacity and hydroxyl radical-scavenging activity of His and Arg (Guo et al., 2022; Xu et al., 2018), which effectively prevented the synthesis of carbonyl derivatives and disulfide bonds from amino acid residues in Mb.

3.6. Secondary structure

Secondary structural alterations of Mb were analyzed by CD spectra (Fig. 1a and Table 3). The CD spectra presented two negative peaks at 210 nm and 223 nm, which are consistent with the α-helix structure of Mb (Su et al., 2024). The α-helical structure was the dominant secondary structure of Mb regardless of His/Arg addition, followed by random coil. This was consistent with the results of Su et al. (2024). Compared with control, the α-helix content was increased by 16.67 %, 17.67 % and 20.70 %, and by 54.67 %, 55.67 % and 58.70 % in the presence of 0.1–0.3 % Arg, respectively (P < 0.05). Concurrently, the β-sheet content was correspondingly decreased, indicating the structure of Mb transitioned to a more ordered state. On one hand, His/Arg could prevent the unfolding of Mb induced by oxidation through their antioxidant activities (Zhou et al., 2014; Zhou et al., 2015). On the other hand, His/Arg might directly interact with the amino acid residues of Mb to form more stable His/Arg-Mb complex, which was further investigated in the subsequent sections.

Table 3.

Effect of different concentrations of His and Arg on the content of secondary structure in Mb.

Samples	α-helix (%)	β-sheet (%)	β-turn (%)	Random coil (%)
CT	38.00 ± 0.90c	26.40 ± 1.45a	8.30 ± 0.90bc	27.30 ± 1.45c
0.10-His	41.85 ± 1.25b	19.15 ± 1.55d	10.20 ± 3.20b	28.80 ± 0.40bc
0.20-His	46.50 ± 1.25b	10.53 ± 1.63b	9.30 ± 2.52b	33.67 ± 2.68a
0.30-His	46.55 ± 1.25b	13.38 ± 1.33c	12.24 ± 2.53b	27.83 ± 0.03c
0.10-Arg	54.67 ± 1.17a	0.59 ± 0.51f	13.27 ± 1.08ab	31.47 ± 1.12ab
0.20-Arg	55.67 ± 2.01a	2.10 ± 1.08e	16.30 ± 3.26a	25.93 ± 0.55c
0.30-Arg	58.70 ± 4.60a	4.10 ± 0.20e	7.95 ± 3.30bc	29.35 ± 1.55bc

Note: The values are the means ± SD. Different letters indicate significant differences (P < 0.05) (n = 3).

3.7. UV–Vis

Mb, as a heme-containing globular protein, exhibits high intense UV–vis absorption characteristics arising from conjugated π-system of its prosthetic heme group, where the oxygenation state of the iron-porphyrin complex directly affects the structure of Mb (Su et al., 2024). Therefore, UV–vis absorption variation was determined to reflect the structural changes of Mb. As shown in Fig. 3b, two primary peaks were observed at 280 nm and 410 nm. The absorption peak at 280 nm correspondsed to the π–π* transitions of aromatic amino acid residues (tyrosine, tryptophan, and phenylalanine) and served as a sensitive indicator of changes in microenvironmental polarity. The peak observed at 410 nm originated from the conjugated π-system within the heme–globin complex, reflecting the electronic interaction between the porphyrin ring and the apoprotein (Li, Liu, et al., 2022). Upon adding His and Arg, the intensity of the 280 nm peak decreased and exhibited a red shift in a concentration-dependent manner, indicating alterations in the microenvironment surrounding tryptophan and tyrosine residues. These variations might be due to the interactions that occur between His/Arg and the aromatic residues within Mb (Guo et al., 2022; Liu et al., 2021; Qi et al., 2024). Likewise, the intensity of the 410 nm peak was reduced in the presence of His and Arg, suggesting a decrease in the proportion of highly oxidized MetMb (Su et al., 2024). This trend corresponded to the observed variations in relative Mb content described earlier. The implications of these structural modifications in Mb stability were investigated through following MD.

Fig. 3.

Circular dichroism spectra (a). The UV–vis absorption spectra (b) and the fluorescence spectra (c, d, e) of Mb treated with different concentrations of His and Arg.

3.8. Endogenous fluorescence spectra

The aromatic residues tryptophan, phenylalanine and tyrosine of proteins are widely utilized as sensitive probes for detecting protein changes due to their micro-environment-dependent spectral properties (Wu, Xu, Ruan, Chen, Li, et al., 2023). Therefore, the changes of endogenous fluorescence were determined. As illustrated in Fig. 3c, the fluorescence intensity of Mb was increased with the increasing of His and Arg, indicating an enhanced affinity between His/Arg and Mb (Xu et al., 2024). Besides, it could be inferred that His/Arg further enhanced the folding of Mb structure and burial of tryptophan residues in Mb, aligned with the above changes of Mb secondary structure. In addition, a red shift of fluorescence spectrum was observed in the presence of His and Arg, which might be resulted from the extension vibration peak of the C C bond is enhanced by additional conjugated units (Qi et al., 2024).

3.9. Synchronous fluorescence spectra

Synchronized fluorescence was further determined to investigate conformational changes of proteins, with wavelength interval (Δλ) of 15 nm and 60 nm specifically tracking microenvironmental shifts surrounding tyrosine and tryptophan residues, respectively (Tian et al., 2022). As illustrated in Fig. 3 (d, e), all samples exhibited maximum fluorescence emission peaks at around 300 nm. When Δλ = 15 or 60 nm, His/Arg induced a dose-dependent enhancement in fluorescence intensity of tyrosine and tryptophan residues, with Arg treatment displaying higher fluorescence intensity, respectively (P < 0.05). The results indicated increased hydrophobicity in the micro-environment surrounding tyrosine and tryptophan residues (Yin et al., 2022). In other words, His/Arg might enhance the folding of Mb structure and burial of tyrosine and tryptophan residues in Mb. This was in accordance with the results of endogenous fluorescence spectra. Concomitantly, a red-shifted fluorescence was observed in the presence of Arg. This phenomenon may be attributed to several factors: First, it had been established that an increase in conjugated units could enhance the stretching vibration peaks of C C and lead to a red shift of the absorption peaks (Huang et al., 2015). As demonstrated above, Arg caused the folding of Mb, which might reduce the distance between the porphyrin ring and either tryptophan or tyrosine, thus enhancing the stretching vibration peaks of C C and leading to a red shift of the absorption peaks. Second, the guanidinium group of Arg might partially interact with the aromatic residues of Mb by cation-π interaction, thus altering the local polar environment and resulting in a red-shift (Shukla & Trout, 2010). Same changes were observed in the presence of His at the wavelength interval of 15 nm. This might also be due to the enhanced stretching vibration peaks of C C caused by the increased conjugation effect and the interaction between the imidazole ring of His and aromatic residues of Mb by cation-π interaction (Chen et al., 2016). Notably, a slight, blue-shifted fluorescence was observed in the presence of His at the wavelength interval of 60 nm. This suggested that the folding of Mb induced by His might position the tyrosine residues closer to the porphyrin ring rather than tryptophan. Besides, His was likely to interact more significantly with the tyrosine residues of Mb than with tryptophan. Moreover, the fluorescence intensity of tyrosine residues was higher than that of tryptophan residues, indicating that tyrosine residues dominated the intrinsic fluorescence emission of Mb (Yin et al., 2022). Therefore, His/Arg might modify the conformation of Mb via interaction with tyrosine residues, with binding sites likely positioned adjacent to tyrosine residues (Xu et al., 2024).

3.10. Molecular docking

Molecular docking elucidates the conceivable engagement processes between Mb and His/Arg residues (Xu et al., 2023). Ten independent docking simulations of Mb with His/Arg were conducted, and the spatial arrangement with the minimum binding free energy was selected as the representative structure to identify the interaction site. The calculated binding free energies for the His-Mb and Arg-Mb complexes were − 6.712 kcal/mol and − 6.276 kcal/mol, respectively, indicating that Arg exhibits a stronger binding affinity toward Mb than His (Wu, Xu, Ruan, Chen, Li, et al., 2023). As shown in Fig. 4, the docking results of His/Arg with Mb showed strong binding and multiple binding sites. Specifically, the interactions between His and Mb were mainly hydrophobic interactions, hydrogen bonding, π-stacking, and salt bridges, and the interaction sites were Phe 43, Ile 99, Leu 104, Val 64, Lys 42, Thr 68, Tyr 103, and His 93, with Phe 43, Ile 99, Leu 104, and Val 64 being hydrophobic residues. Notably, His could interact with the terminal histidine residue His93A in Mb via salt bridges and π–π stacking interactions, which may influence the local environment around the heme group and help protect it from oxidative damage (Ma et al., 2024). For Arg, the interaction forces with Mb were mainly hydrogen bonding and hydrophobic interactions, and the interaction points were Phe 33, Phe 43, Ile 99, Leu 32, Ile 107, Lys 42, Thr 36, Thr 68, and Tyr 103, of which the hydrophobic residues were Phe 33, Phe 43, Ile 99, Leu 32, and Ile 107.Hydrogen bonding served as the primary interaction between His/Arg residues and Mb, playing a crucial role in maintaining its structural stability (Zeng et al., 2014). The formation of these hydrogen bonds is likely responsible for stabilizing the α-helical structure of Mb (Guo et al., 2021), which was in accordance with the above results of CD spectra. Interestingly, the interactions between His/Arg with Mb involved numerous hydrophobic amino acids, suggesting that His/Arg could interact with the hydrophobic regions of Mb. This interaction might lead to the exposure of hydrophobic groups or the formation of hydrophobic molecular clusters, thereby facilitating a tighter folding of Mb (He et al., 2025). Similar phenomena were also observed in the multispectral analyses (3.8, 3.9, 3.10). Such folding behavior may contribute to enhanced protection of the heme group within Mb. In summary, both His and Arg were capable of forming stable complexes with Mb and inducing conformational changes through interactions such as hydrogen bonding and hydrophobic forces. These structural alterations are likely beneficial for maintaining the stability of the Mb molecule.

Fig. 4.

Molecular docking of myoglobin to Arg (a,b) and His(c,d).

3.11. MD

3.11.1. RMSD

RMSD is a critical parameter that reflects conformational changes within a simulation system and is essential for evaluating its stability (Guo et al., 2024). As shown in Fig. 5a, in the first 20 ns of the modeling, the RMSD values for the Mb complex with His and Arg increased rapidly, which is typical during the initial equilibration phase as the system adjusts to solvent effects (Qi et al., 2024). Between 20 and 40 ns, the RMSD value for the Arg-Mb complex remained slightly lower than that of the His-Mb complex, eventually stabilizing within the range of 0.15 to 0.2 nm. Furthermore, when compared to Mb alone, the RMSD values for the His-Mb and Arg-Mb complexes were lower, suggesting that the presence of Arg and His contributes to the enhanced stability of Mb (Gui et al., 2023).

Fig. 5.

The root mean square deviation (RMSD) values (a), root mean square fluctuation (RMSF) values (b), radius of rotation (Rg, c), solvent accessible surface area (SASA, d), and number of hydrogen bonds (e) of Mb binding to His and Arg.

3.11.2. RMSF

RMSF serves as an indicator of the conformational flexibility of proteins. Elevated fluctuations in RMSF values suggest that proteins exhibit greater flexibility and possess relatively less stable structural conformations (Wu, Xu, Ruan, Chen, Li, et al., 2023). As shown in Fig. 5b, RMSF values in the His-Mb complex were lower than those in the uncomplexed Mb system, implying that the His-Mb complex exhibits greater conformational stability (Qi et al., 2024). Additionally, compared to the single Mb system, the Arg-Mb complex displayed decreased fluctuations in the amino acid residue regions of 20–40, 50–70, and 85–100. This observation implied that Arg mitigates significant fluctuations in these flexible regions, thereby resulting in a more compact conformation in these areas (Wu et al., 2025).

3.11.3. Rg, SASA and hydrogen bonds

Using Rg, protein structural activity can be assessed in order to estimate the distribution of atoms around the protein axis (Wu, Xu, Ruan, Chen, Li, et al., 2023). As shown in Fig. 5c, the Rg values of both His-Mb and Arg-Mb complexes showed a gradual increase before the 60 msec threshold, suggesting that His/Arg may promote Mb unfolding at the beginning. After the 60-microsecond threshold, the Rg values of both Arg-Mb and His-Mb complexes decreased gradually, which indicated that both Arg and His were effective in inhibiting the rapid unfolding of Mb structures (Qi et al., 2024).

With SASA it is possible to evaluate the surface area of proteins to determine how they interact with other substances (Qi et al., 2024). As illustrated in Fig. 5 d, after 20 ns, the SASA value of the His-Mb complex did not exhibit a significant difference compared to that of Mb alone. In contrast, the SASA value of the Arg-Mb complex progressively surpassed that of Mb over time, suggesting that Arg exerts a more substantial influence on the Mb structure than His, thereby enhancing the reactivity with the active site structure and improving the surface activity of Mb (Baildya et al., 2020). Similar findings were corroborated by multispectral measurements and surface hydrophobicity assays. Fig. 5e presented the calculated hydrogen bond contacts between His/Arg and Mb. The results indicated a relatively low number of hydrogen bonds, aligning with the docking results. A hydrogen bond was defined as the interaction between a hydrogen donor and a hydrogen acceptor. The role of hydrogen bonds in protein stability had garnered considerable attention, as they were not only fundamental to the chemical structure and reactivity of proteins but also essential for comprehending their structure and properties (Li, Liu, et al., 2022).

3.11.4. Binding free energy

The interaction between protein values and small molecules can be better assessed by binding free energy, which encompasses a variety of forces including van der Waals forces, polar solvation energy, and non-polar solvation energy, electrostatic forces, (Farhadian et al., 2019). As presented in Fig. 6a, the total binding free energy of the His-Mb complex was −16.12 Kcal/mol, while that of the Arg-Mb complex was −17.19 Kcal/mol, indicating notable interactions between His, Arg, and Mb. The His-Mb complex exhibited negative values for van der Waals forces (−45.09 Kcal/mol), electrostatic energy (−14.94 Kcal/mol), non-polar solvation energy (−3.01 Kcal/mol), and molecular mechanics term energy (−33.72 Kcal/mol), alongside a positive polar solvation energy (20.62 Kcal/mol). A similar trend was observed in the Arg-Mb complex. These findings suggested that polar solvation energy might hinder binding (Sun et al., 2024).

Fig. 6.

Schematic representation of the binding free energy between His-Mb complex (a,b) and Arg-Mb (c,d) complex. The free energy landscape map of Mb (e) and His-Mb complex (f) and Arg-Mb complex (g). Note: VDWAALS: van der Waals energy, EEl: Electrostatic energy, EGB: Polar solvation energy, ESURF: Non-polar solvation energy, GGAS: Total gas phase free energy, GSOLV: Total solvation free energy, TOTAL: GSOLV + GGAS.

The amino acid residues involved in the interactions that contribute to the energy of the His-Mb complex are depicted in Fig. 6b. In particular, the residues THR 39, HIS 93, PHE 43, VAL 64, THR 67, LYS 42, ILE 99, TYR 103, LEU 104, and PHE 138 presented negative energy values, indicating their role in the association between His and Mb. In contrast, the Arg-Mb complex included a greater number of amino acids contributing to energy interaction, specifically LEU 32, THR 68, LEU 89, ILE 99, PHE 106, and ILE 107. This suggested that Arg exhibited a stronger interaction with Mb than His.

The relative Gibbs free energy was computed using Rg and RMSD values, and a 2D free-energy landscape was generated to visualize the lowest-energy conformations sampled during MD simulations. Free-energy landscapes are valuable for characterizing the stability of protein-ligand interactions. The presence of numerous small blue-violet clusters in the free-energy profile indicates weak or unstable interactions, whereas strong and stable interactions yield a virtually unified, uniform energy allocation (Wu et al., 2025). In the free-energy landscape of Mb (Fig. 6c), a rough, uneven minimum-energy cluster was detected. For the His-Mb complex (Fig. 6d), the minimum energy cluster for the His-Mb complex was characterized by a relatively rough but more pronounced structure. In contrast, the free energy landscape of the Arg-Mb complex, as depicted in Fig. 6e, exhibited a smoother and more singular minimum energy cluster. These findings suggest that the Arg-Mb complex exhibits greater stability than the His-Mb complex (Feng et al., 2024; Guo et al., 2024).

3.12. Put forth meat color protection mechanism

Based on these findings, we proposed a possible mechanism to reveal how His/Arg enhanced pork color stability (Fig. 1). Postmortem accumulation of free radicals drove Mb auto-oxidation through direct radical-mediated protein damage and lipid oxidation cascades, inducing heme iron oxidation (Fe²⁺ → Fe³⁺) and subsequent meat discoloration (Su et al., 2024). As basic amino acids, His and Arg had demonstrated potent free radical scavenging capacity and ferrous ions chelating activity (Zhang et al., 2021). The findings from the analysis of ROS, TBARS, carbonyl, and sulfhydryl content indicated that His and Arg could shield Mb from free radical assault and enhance the conversion of MetMb via hydrogen donation, thereby promoting meat color stability. In addition, multi-spectral analyses revealed the capacity of His and Arg to inhibit the unfolding of secondary and tertiary structures of Mb, potentially through interactions with tyrosine residues, thereby contributing to the stabilization of free-moving rings within the amino acid residues of Mb (Xu et al., 2024). This structural compaction mechanism likely enhanced the oxidative resistance of Mb's heme pocket. Molecular docking simulations further demonstrated that both His and Arg could form stable complexes with Mb through hydrogen bonding and hydrophobic interactions. These interactions create a steric barrier that limits oxygen penetration and oxidant accessibility to the heme center, thereby effectively suppressing Mb oxidation and improving pork color stability.

4. Conclusions

This study elucidated a dual mechanism for enhancing pork color, induced by His and Arg. The results indicated that His/Arg elevated the a* values and the proportion of OxyMb. In addition, His/Arg decreased ROS, TABARS, carbonyl content, while increasing sulfhydryl content. These results suggested that His/Arg might protect Mb structure from free radical damage and facilitate the reduction of MetMb, thereby improving pork color. Furthermore, multi-spectral analyses indicated that His/Arg inhibited the unfolding of secondary and tertiary structures of Mb through interactions with tyrosine residues, thus enhancing the structural stability of Mb and subsequently improving the oxidative resistance of Mb's heme pocket. Notably, Arg demonstrated greater efficacy than His in exerting antioxidant effects and strengthening the structural compactness of Mb. The results might provide a reference for the use of His/Arg to improve meat quality.

Nonetheless, given the limited range of His/Arg concentrations selected for this study, further investigation is warranted to explore the effects of varying His/Arg concentrations on meat color stability. Moreover, the antioxidant roles of His/Arg needs to further clarification. Considering that Mb is susceptible to browning during storage due to continuous oxidation, a more comprehensive study is necessary to assess the effects of His/Arg on the color and Mb characteristics of pork during storage, as well as to elucidate their antioxidant roles.

CRediT authorship contribution statement

Xiuyun Guo: Funding acquisition. Chao Fu: Writing – original draft, Resources. Shuangyi Xu: Software, Resources. Jiangpeng Yao: Visualization, Validation, Conceptualization. Kaixian Zhu: Conceptualization. Xiangren Meng: Conceptualization.

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.

Data availability

Data will be made available on request.