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. 2026 May 5;36:103948. doi: 10.1016/j.fochx.2026.103948

pH-induced changes in hydrophobicity and key amino acid exposure regulate myoglobin digestibility

Hui Liu a,b, Yaxuan Li a,b,c, Kai Shan c, Chunbao Li c,⁎⁎, Xiaoyan Tang a,b,⁎
PMCID: PMC13197811  PMID: 42179951

Abstract

Meat processing can cause a wide range of pH variations, which in turn affect protein digestibility. The objective of this study was to elucidate the mechanism linking pH-induced structural alterations to the in vitro digestion of myoglobin. Spectroscopic techniques and molecular dynamics simulations were employed to evaluate physicochemical changes across a pH gradient. At low pH, myoglobin exhibited increased hydrophobicity, and molecular dynamics simulations indicated a higher affinity for protease binding. The protein adopted a flexible structure with increased hydrophobic amino acids exposure and weakened hydrogen bonds, collectively enhancing digestibility. These findings provide new insights into improving the digestibility of myoglobin within the gastrointestinal tract.

Keywords: Digestibility, Heme cavity, Hydrophobicity, In vitro digestion, Myoglobin

Highlights

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    The hydrophobic amino acid exposure is the key for myoglobin digestion.

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    Low pH conditions significantly improved in vitro protein digestibility.

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    Acidic conditions trigger heme cavity opening and aromatic residue exposure.

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    Reduced hydrogen bonding increases myoglobin flexibility for protease access.

1. Introduction

Meat products are rich in various proteins, including salt-soluble myofibrillar proteins, water-soluble sarcoplasmic proteins, and insoluble matrix proteins. These proteins provide essential nutrients to the body (Zhang et al., 2020). Myoglobin, for example, is an iron-rich metalloprotein composed of a heme prosthetic group and a globin polypeptide, and it adopts a relatively stable conformation (Reedy, Elvekrog, & Gibney, 2008).

The digestion and proteolysis rates of dietary proteins in the gastrointestinal tract serve as classical criteria for evaluating their nutritional quality (Remond, Savary-Auzeloux, Gatellier, & Sante-Lhoutellier, 2008). However, proteins have significantly different capacities of undergoing digestion (Li, Bassey, & Zhou, 2023; Zhao et al., 2022). A healthy organism depends on adequate digestion of dietary proteins and absorption of their breakdown products and low protein digestibility can impair growth and may even contribute to certain diseases (Joye, 2019; Kitada, Ogura, Monno, & Koya, 2019). Notably, the persistence of undigested proteins in the colon is linked to a higher risk of colorectal cancer (Kim et al., 2019). Protein digestibility is highly dependent on the source of the meat (Zhang et al., 2020). Concurrently, gastrointestinal dysfunction may delay proteolysis, thereby compromising the overall digestibility of meat products (Lee et al., 2021). Some studies have shown that although protein solubility is generally higher under normal gastric pH (1.5–3.5), intermolecular interactions, structural alterations, surface exposure of amino acids, and aggregate formation can reduce solubility and thereby affect protein digestibility (Dekkers, Kolodziejczyk, Acquistapace, Engmann, & Wooster, 2016; Liu, Wang, Zheng, Lu, & Arora, 2008). In vivo studies indicate that myoglobin exhibits inferior digestibility compared to other meat proteins, evidenced by the fact that incompletely digested myoglobin residues persist into the colon (Xie, Wang, Zhao, Zhou, & Li, 2020). However, little is currently known about myoglobin structural modifications during processing that are relevant to its digestibility.

In recent years, there has been growing attention to the conformational changes and molecular mechanisms of dietary proteins under different processing conditions (He et al., 2020; Jiang et al., 2017). Accurate characterization of these processes often relies on advanced analytical techniques and sample preparation methods (Azooz et al., 2024; Azooz, Al-Wani, Gburi, & Al-Muhanna, 2022; Brodkorb et al., 2019). Normally, the pH of meat and meat products range from 5.0 to 7.0. However, acid treatment or fermentation may reduce the pH of meat product. Protein conformational changes and the degree of aggregation can significantly alter protein degradation and the absorption of digested products, and a key feature contributing to these differences is how these proteins function in an acidic gastric environment (Dekkers et al., 2016). Extensive research indicates that varying environmental pH levels dictate protein structural characteristics and functional properties (Buchholz et al., 2020). For instance, pH shifts have been reported to alter hydrophobicity and aggregation in various food matrices, including plant and aquatic proteins, thereby altering protease susceptibility (Cui, Zhao, Yuan, Zhang, & Ren, 2013; He et al., 2020; Kristinsson & Hultin, 2003; Luo, Boom, & Janssen, 2015; Sanchez, Ortiz, Nino, Anon, & Patino, 2003). These structural modifications, such as the exposure of buried hydrophobic residues, may alter digestive-enzyme binding sites and expose additional cleavage sites, thereby improving in vitro protein digestibility. Consequently, current research extensively employs in vitro models to elucidate the mechanisms governing protein digestive dynamics.

Previous studies indicated that myoglobin is difficult to digest and degrade in vitro because the intermolecular forces stabilizing its structure are strong, resulting in poor binding affinity for pepsin (Li et al., 2020). The hydrophobic amino acids of myoglobin are typically buried within the protein interior; Liu et al. (2021) demonstrated that salt treatment can alter the conformational stability of these residues and thereby affect myoglobin digestibility. Digestion properties of pepsin and trypsin propose that changes in target protein hydrophilicity and hydrophobicity may be the key to overcome the issue of poor myoglobin digestibility in vivo. Therefore, exploring how changes in hydrophilicity and hydrophobicity affect myoglobin digestibility may lead to a breakthrough in improving the digestibility of myoglobin in the gastrointestinal tract.

In this study, in vitro digestion experiments were conducted to explore how different pH conditions, simulating fermentation or acid treatment, affect the structure and digestibility of myoglobin. Spectroscopic techniques combined with molecular docking and dynamics simulations were employed to analyze the physicochemical properties under varying pH models. The primary objective of this work was to elucidate the mechanism by which pH-induced structural modifications, specifically changes in hydrophobicity and microenvironment, influence the digestive stability of myoglobin, providing insights for improving its nutritional value.

2. Materials and methods

2.1. Materials

Myoglobin isolated from porcine skeletal muscle (CAS No. 100684–32-0, purity ≥95%), pepsin from porcine gastric mucosa (2500 U/mg) (CAS No. 9001-75–6), and trypsin from porcine pancreas (200 U/mg) (CAS No. 9002-07-7) were obtained from Promega (Madison, USA). The bicinchoninic acid (BCA) protein assay kit (Cat. No. 23225) was purchased from Thermo Scientific (Rockford, IL, USA). The other chemical reagents were purchased from local companies.

2.2. Sample preparation

Phosphate buffered solution (PBS, 0.2 mM) was prepared and the pH was adjusted to 3, 4, 5, 6, and 7 with concentrated hydrochloric acid. Myoglobin was dissolved in the different pH-adjusted phosphate buffered solutions at a concentration of 2 mg/mL and desalted using 3 kDa ultrafiltration tubes. The protein concentration in the filtrate was determined by the BCA method. Subsequently, samples were stored at 4 °C for further analysis.

2.3. UV–visible spectroscopy

To monitor changes in the heme environment and tertiary structure, UV–Vis spectroscopy was performed using a spectrophotometer (Thermo Scientific, Fremont, CA, USA). Myoglobin solutions were incubated at different pH levels for 0 min, 30 min, 60 min, 90 min, 120 min and diluted to 0.2 mg/mL. Spectra were recorded from 190 to 450 nm at 30 °C after calibrating with ultrapure water (n = 5).

2.4. Circular dichroism (CD) spectroscopy

Secondary structure alterations were assessed using a CD spectrometer (J-1500, Jasco, Tokyo, Japan). Myoglobin solutions (2 mg/mL) at different pH were incubated for 120 min and then diluted to 0.2 mg/mL. CD spectra (average of three scans) were recorded from 260 to 180 nm using a 0.1 cm pathlength quartz cuvette with the cell holder temperature at 30 °C.

2.5. Fluorescence spectroscopy

Tertiary structural changes and amino acid microenvironments were probed using a fluorescence spectrophotometer (RF-5301, Shimadzu, Kyoto, Japan) according to the procedure described by Shen, Huang, Zhao, and Sun (2019) with minor modifications. Fluorescence emitted by the diluted myoglobin samples (0.2 mg/mL) was recorded using the same spectrophotometer. To measure endogenous fluorescence, 1 mL of a 0.2 mg/mL myoglobin solution was transferred into a 1 cm quartz cuvette. Endogenous fluorescence was measured with excitation at 280 nm and emission scanning from 280 to 600 nm (slit widths: 5.0 nm). Synchronous fluorescence spectra were collected to characterize tyrosine (Δλ = 20 nm) and tryptophan (Δλ = 60 nm) residues with a scan speed of 1200 nm/min.

2.6. Surface hydrophobicity

Surface hydrophobicity, indicative of hydrophobic pocket exposure, was determined using the ANS fluorescent probe method (Chen et al., 2016; Li, Zhao, & Xu, 2022) with minor modifications. The myoglobin solutions at different pH were diluted to 1 mg/mL. Then, 20 μL ANS (15 mM, 0.1 M PBS, pH 7.0) was mixed thoroughly with 4 mL myoglobin solution and incubated in the dark for 20 min. Fluorescence was measured using an Infinite M200 PRO microplate reader (ThermoScientific, Fremont, CA, USA) at the excitation and emission wavelengths of 375 nm and 470 nm, respectively.

2.7. Particle sizing

A nanoparticle size and zeta potential analyzer (Zetasizer Nano ZS, USA) was used to evaluate the aggregation state of myoglobin based on the method of Ye, Hua, Zhang, Zhao, and Yang (2021). The relative refractive index was set to 1.33, and the temperature was set to 25 °C. Each sample (1 mL) was measured 3 times in parallel, equilibrated for 120 s before each measurement, and scanned 12 times. The square glass vessel was used to determine particle size.

2.8. Differential scanning fluorimetry (DSF)

Thermal stability and unfolding temperatures (Tm) were determined via DSF on a QuantStudio 5 instrument (ThermoFisher, USA) based on the method of Simeonov (2013) with minor modifications. Myoglobin (0.5 mg/mL) was mixed with SYPRO Orange dye (1500 final dilution). Fluorescence was monitored from 25 to 95 °C (Ex/Em: 470/587 nm). The data from the first derivative of fluorescence intensity obtained during heating were analyzed with the Origin software, and melt curves were generated by the same software.

2.9. In vitro digestion

Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared according to the INFOGEST protocol described by Brodkorb et al. (2019), and in vitro digestion was carried out following the procedure of Liu et al. (2022). Briefly, the myoglobin solutions incubated at different pH for 120 min were desalted through ultrafiltration tubes. Then, the samples were concentrated by a vacuum apparatus (Savant RVT5105, Thermo Fisher Scientific, USA), and diluted with SGF solution to a concentration of 10 mg/mL for further experiments. SGF digestion was initiated by mixing 800 μL of myoglobin in SGF buffer and 200 μL pepsin (2500 U/mL, dissolved in SGF) for a final volume of 1.0 mL. The samples were incubated at 37 °C for 2 h with shaking at 200 rpm. The pH of the mixture was then adjusted to 7.0 with 1 M NaOH to inactivate pepsin and ensure the optimal pH for trypsin activity. SIF digestion was prepared by mixing 100 μL trypsin (1000 U/mL, dissolved in SIF) with 1.0 mL of the pepsin-digested products and the final volume was adjusted to 2.0 mL with 900 μL SIF. The reaction mixtures were incubated at 37 °C for 2 h with shaking at 200 rpm. The reaction was terminated by heating the mixtures at 95 °C for 5 min. The samples were then chilled and collected as gastric and intestinal digestion products for subsequent digestibility determination.

2.10. Protein digestibility determination

The initial protein concentration of myoglobin was 10 mg/mL and designated as A0. After digestion, the undigested protein was precipitated by anhydrous alcohol and then resuspended in ultrapure water. The protein contents were quantified after gastric digestion and designated as A1. The protein contents were also quantified after intestinal digestion and designated as A2. The initial (A0) and remaining protein contents (A1) and (A2) were determined by the BCA assay kit and digestibility was calculated as follows:

Protein digestibility under gastric digestion%=A0−A1/A0×100 (1)
Protein digestibility under intestinal digestion%=A0−A2/A0×100 (2)

2.11. Homology modeling and molecular dynamics simulations

The monomeric structure of myoglobin (PDB ID: 1MNH) was obtained from the RCSB protein databank (https://www.rcsb.org/). The original structure was visualized and optimized using the Visual Molecular Dynamics (VMD) software. Molecular dynamics (MD) simulations were performed using the NAMD 2.14 software with the CHARMM27 force field (MacKerell et al., 1998), and simulations were carried out under different pH conditions. Protonation states for titratable residues at pH 3–7 were predicted using the H++ server (http://biophysics.cs.vt.edu/). Protonation conditions corresponding to pH 3–7 were introduced by protonating the amino acid residues and the TIP3P water molecule was used as the solvent model. Additionally, during the simulations, Cl− and Na+ were added to ensure sufficient amounts of neutralizing counterions. Molecular dynamics simulation was performed over three steps, i.e., 1 ns of system equilibration, 1000 steps of energy minimization and 1000 ps of heating, and 50 ns of classical molecular dynamics simulation.

2.12. Statistical analysis

All data are presented as mean ± standard deviation (SD). Data were subjected to one-way analysis of variance (ANOVA) to evaluate the effects of pH and time on the measured parameters. Mean differences were assessed using Duncan's multiple-range test in the SAS program (version 8.1, SAS Institute Inc., Cary, NC, USA). Figures were produced by GraphPad Prism8.0 (GraphPad Software, Inc., La Jolla, USA).

3. Results

3.1. UV–visible spectroscopy of myoglobin

The ultraviolet absorption spectrum revealed two characteristic peaks of myoglobin, with the first peak appearing at approximately 280 nm. This peak is generated by the π - π * transition of functional groups of the substrate and represents the microenvironmental change for hydrophobic amino acids, such as phenylalanine, tyrosine and other aromatic amino acids in a protein (Li et al., 2022). The second peak, observed at approximately 408 nm, is characteristic of the heme group and reflects structural changes in both the heme and the surrounding protein backbone (Li, Zhao, & Xu, 2022).

Figs. 1a–f shows the UV–Vis spectra of myoglobin under different pH conditions and absorption peaks of 280 nm and 408 nm. These peaks indicate that changes in pH, particularly at pH 3 (Figs. 2a–c), lead to alterations in the secondary structure of myoglobin and significant modifications in the heme microenvironment within the hydrophobic cavity. As shown in Figs. 1b-e, at pH 4 to 7, the position of myoglobin hydrophobic amino acids did not change significantly when the incubation time was extended, and heme was still buried in the hydrophobic cavity of the protein.

Fig. 1.

Fig. 1

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UV absorption spectra of myoglobin under different pH conditions. (a-e) pH 3 to 7, respectively, (f) 120 min for different pH.

Fig. 2.

Fig. 2

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Circular infrared spectra of myoglobin under different pH conditions. (a) infrared spectrum; (b) α-helix content; (c) random coil content.

The heme and myoglobin skeleton exhibited many interaction forces, and the binding between heme and globin is too stable to be easily altered (Figs. 3a-e). This stability may be explained by the high pH which does not affect the stability of the myoglobin helical chain in the context of heme (Kanai et al., 2017). At pH 3, the absorption peak intensity of heme at 409 nm decreased with prolongation incubation, but the peak intensity at 280 nm increased significantly (p < 0.05). This indicates that at low pH, hydrophobic amino acids are more exposed to the surroundings. However, the heme was buried deeper in the hydrophobic cavity of myoglobin due to the driving force of the salt and movement of the globin helix (Figs. 3f-g). In addition, limited interaction forces (Fig. 3a) contributed to this phenomenon (Kanai et al., 2017; Khan et al., 2019). Therefore, the structure of myoglobin was altered under extremely acidic conditions.

Fig. 3.

Fig. 3

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Interaction force statistics and three-dimensional position of heme microenvironment simulated by 50 ns molecular dynamics; (a) pH 3; (b) pH 4; (c) pH 5; (d) pH 6; (e) pH 7; (f) three-dimensional position of heme microenvironment at the condition of pH 3; (g) three-dimensional position of heme microenvironment at the condition of pH 7.

3.2. Circular dichroism spectroscopy of myoglobin reveals low pH affecting protein stability

CD spectroscopy was used to investigate the secondary structural changes of myoglobin under different pH conditions (Fig. 2, Fig. 4). Two characteristic peaks at 209 nm and 223 nm (Fig. 2a) represent alpha-helices and beta-sheets, respectively (Buchholz et al., 2020; Sun, Wang, Liu, Kong, & Chen, 2020). As the pH increased from 3 to 7, the intensity of the α-helical peaks gradually decreased (Figs. 2a–b). At all tested pH values, α-helices accounted for approximately 70% of the myoglobin secondary structure, suggesting that pH variation did not severely disrupt the overall structure of the protein. As shown in Fig. 2c, the random coil content of myoglobin increased significantly at pH 3, indicating that acidic conditions can alter the intramolecular interactions (Figs. 3a) within the protein and further modify the secondary structure content. Fig. 4 illustrates the changes in myoglobin secondary structure composition after a 50-ns molecular dynamics (MD) simulation performed under different pH conditions. At pH 3, the total number of alpha-helices in the secondary structure decreased over the period of incubation (Fig. 4a). Since the stability of α-helices is primarily maintained by hydrogen bonds among amino acid side chains (Liu et al., 2008), lower pH conditions may reduce protein stability and consequently affect protein digestibility. Additionally, molecular dynamics simulations performed on other groups did not yield similar results (Fig. 4b-e). These findings are consistent with those shown in Fig. 2. Furthermore, this experimental observation of reduced stability is corroborated by the Root Mean Square Deviation (RMSD) analysis from the MD simulations discussed, which confirms increased backbone fluctuations under acidic conditions.

Fig. 4.

Fig. 4

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Secondary structure content statistics of myoglobin by 50 ns molecular dynamics simulation; (a) pH 3; (b) pH 4; (c) pH 5; (d) pH 6; (e) pH 7.

3.3. Particle size and surface hydrophobicity of myoglobin

Solvent Accessible Surface Area (SASA) serves as a critical indicator of protein unfolding and the exposure of enzyme cleavage sites, which is central to understanding digestibility. In this section, SASA analysis derived from MD simulations was integrated with experimental measurements of particle size and surface hydrophobicity to comprehensively evaluate the structural stability of myoglobin (Fig. 5). Experimental results indicated that the distribution pattern of myoglobin particle sizes reflected the aggregation state under different pH conditions. As shown in Fig. 5a, the particle size did not significantly change with the extension of incubation time. After 120 min of incubation, myoglobin aggregation was obvious in the pH 4 group, while its hydrophobicity was lower than the pH 3 group but higher than the other groups (Fig. 5b). This suggests that protein aggregation at pH 4 is primarily driven by electrostatic interactions rather than hydrophobic forces (Tedeschi et al., 2017). As the pH approaches the isoelectric point region, the net charge repulsion on the protein surface decreases, thereby favoring intermolecular association and aggregation. The changes in surface hydrophobicity can affect the physical and chemical properties of proteins and change their structure (Shen, Zhao, & Sun, 2019). The hydrophobicity of proteins under low pH conditions was significantly higher than that for higher pH groups (Fig. 5b). Hydrophobic forces within protein molecules determine structural stability to a certain extent, and are an essential in supporting the stability of protein spatial structure (Kato & Nakai, 1980). Thus, more hydrophobic amino acids were exposed at pH 3. SASA is contingent on the primary and secondary structures of the protein and is a good indicator to estimate the degree of amino acid exposure to the solvent (Zhang et al., 2021). Fig. 5c and d present the solvent accessible surface area of myoglobin after 50 ns of molecular dynamics simulation. Under low pH conditions, the solvent accessible surface areas of myoglobin hydrophobic amino acids were significantly higher. The result is consistent with the data on hydrophobicity (Fig. 5b), implying that the low pH environment can change the structure of myoglobin, which in turn, affects its digestibility (Fig. 8). The microenvironmental changes of heme were also analyzed and the degree of exposure for the hydrophobic amino acids around heme (red area) is shown in Fig. 5e and f. It was observed that lower pH conditions promoted the exposure of the hydrophobic cavity housing the heme group, consistent with the hydrophobicity changes shown in Fig. 5c and d. Collectively, the data indicate that low pH conditions induce partial unfolding and aggregation of the myoglobin structure, characterized by increased surface hydrophobicity and enlarged particle size.

Fig. 5.

Fig. 5

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Changes of particle size and hydrophobicity of myoglobin under different pH conditions; (a) particle size; (b) hydrophobicity; (c-d) solvent accessible surface areas of global and hydrophobic amino acid for myoglobin, respectively; (e-f) expansion degree of myoglobin hydrophobic pockets at pH 3 and pH 7, respectively.

Fig. 8.

Fig. 8

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Digestibility of myoglobin treated by pepsin (a) and trypsin (b), respectively.

3.4. Fluorescence spectrometry of myoglobin aromatic amino acids

3.4.1. Intrinsic fluorescence spectra

The intrinsic fluorescence of a protein refers to the variation in fluorescence intensity of specific amino acid residues (e.g., tryptophan, tyrosine, and phenylalanine) when the protein is exposed to excitation light of different wavelengths. This change in fluorescence intensity can accurately reflect structural changes in the protein (Li, Zhao, & Xu, 2022). The peak of the intrinsic fluorescence spectra was observed at 330 nm and the fluorescence intensity reached 5360 A.U. at pH 3, which was significantly higher than the other pH groups (p < 0.05, Fig. 6a). At pH 5 and 6, the fluorescence intensity of myoglobin was the lowest, indicating that there was fluorescence quenching of tryptophan. The changes in the maximum emission peak shift can provide important information about alterations in the polarity of the microenvironment surrounding the fluorophore (Liu et al., 2022), which suggests that at pH 3 the tertiary structure of the protein has undergone certain changes, with the hydrophobic cavity of myoglobin opening up, exposing its aromatic amino acids residues to a more hydrophilic microenvironment. During incubation, the aggregation of proteins makes the unexposed amino acids stretch toward the polar environment, which enhances the intensity of its intrinsic fluorescence (Li, Liu, et al., 2022). In addition, at pH 3, the myoglobin structure was unfolded, the hydrophobic cavity of heme was open, and more aromatic amino acids were exposed (Figs. 5c-f). Under low pH conditions, the structure of myoglobin undergoes significant changes, exposing aromatic amino acids residues. This exposure may represent key sites for protease action on myoglobin, serving as a potential mechanism behind the altered digestibility of myoglobin.

Fig. 6.

Fig. 6

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Fluorescence spectroscopy and hydrophobic amino acid SASA analysis of myoglobin under different pH conditions; (a) endogenous fluorescence; (b) tryptophan synchronous fluorescence; (c) tyrosine synchronous fluorescence; (d) The SASA of the tyrosine; (e) The SASA of the tryptophan.

3.4.2. Synchronous fluorescence spectra

Synchronous fluorescence spectrometry enables good selectivity, high sensitivity and low interference, and can be used to determine hydrophobic amino acids in a protein microenvironment. Based on the difference between the emission wavelength and excitation wavelength (Δλ), the spectra of tyrosine and tryptophan can be characterized (Wang, Song, Kong, & Yu, 2005). Fig. 6b shows the synchronous fluorescence spectrum of tyrosine residues. The fluorescence intensity was found to increase gradually with a decline in pH, and the synchronous fluorescence spectra achieved the maximum value of 218 A.U. at pH 3 of incubating for 120 min. At this pH, the synchronous fluorescence spectra appeared as a red shift during the incubation, and the λmax increased significantly from 370 nm to 385 nm (p < 0.05). This result indicated that the microenvironment of tyrosine residues in myoglobin had shifted from a less polar to a more polar state, leading to structural modification of the protein (Pan et al., 2020). This phenomenon also corroborates the findings in Fig. 5, indicating that the hydrophobic cavity of myoglobin opens up, exposing more hydrophobic amino acids to the polar microenvironment. It has been demonstrated that other protein unfolding increases the exposure of hydrophobic groups—including aliphatic and aromatic residues—that were previously buried within the protein core (Pan et al., 2020).

The tryptophan fluorescence spectra were similar to that of tyrosine with the spectra gradually increasing with incubation time at the pH conditions tested (Fig. 6c). However, at pH 3, the synchronous fluorescence spectrum exhibits a blue shift, indicating that the microenvironments of tyrosine residues have become less polar and their exposure has decreased. Nonetheless, how lower pH conditions increase fluorescence intensity resulting in a blue shift of tryptophan emission suggests complex microenvironmental changes. While previous studies attribute similar shifts to disrupted quenching effects or altered energy transfer efficiency (Ajaj et al., 2009; Guzow et al., 2002), the current data primarily highlight a distinct structural transition under acidic conditions. Further studies are needed to understand the association between protein structure and amino acid residues. A similar finding was also reported in an experiment using a micelle suspension and whey protein treatment, and the transformation was attributed to the low-polarity environment (Gatti, Risso, & Pires, 1995). Molecular dynamics simulations were conducted to examine changes in the hydrophobicity of tyrosine and tryptophan residues, and the results are presented in Figs. 6d-e. After 50 ns of simulation, compared to the pH 7 group, the reduction to pH 3 significantly increased the SASA of hydrophobic tyrosine residues (p < 0.05), while the difference in SASA of hydrophobic tryptophan residues was not significant between the two pH conditions (p > 0.05). This indicates an increased exposure of tyrosine residues and a notable alteration in their microenvironment. The greater exposure of hydrophobic amino acids facilitates their binding with proteases. Therefore, it can be inferred that the change in pH modifies the microenvironment of hydrophobic amino acids—primarily tyrosine and phenylalanine—favoring protease catalysis and consequently altering the digestive properties of myoglobin.

3.5. Differential scanning fluorimetry

A fluorescent probe-based technology was used to monitor the structural stability of myoglobin and the extent of its conformational unfolding. SYPRO Orange is an environmentally sensitive hydrophobic dye. When the hydrophobic regions of a protein become exposed, the dye specifically binds to these regions, thereby enhancing fluorescence intensity. The maximum fluorescence intensity of the thermal denaturation curve represents the degree of protein exposure (Xue, Qian, Brad Kim, Xu, & Zhou, 2018). The fluorescence intensity of the pH 3 group was significantly higher than the other groups (Fig. 7a), which indicates that the hydrophobic amino acids in myoglobin were partly exposed after 120 min incubation at pH 3. This observation provides further evidence for the above spectral findings. Previous studies also have shown that fluorescence decreased with increasing temperature, which may be caused by the increase in hydrophobic forces leading to fluorescence quenching (Johnson, Savas, Kartje, & Hoops, 2014). The intensity of fluorescence decline was significantly higher than that in the higher-pH groups, indicating that the thermal stability of myoglobin was reduced at pH 3 (Fig. 7a). Fig. 7b and c illustrate the changes in the number of hydrogen bonds within the overall structure of myoglobin during simulation under high and low pH conditions. It can be observed that the number of hydrogen bonds in the high pH group remains in the range of 45–70, while that in the low pH group remains between 30 and 50, significantly lower than in the high pH samples (Fig. 7f). A reduction in the molecular forces maintaining the protein structure is an important factor leading to the weaker protein stability and hydrogen bonds play a crucial role in maintaining protein conformational stability. It is inferred that the reduced hydrogen bonding in the low pH group increases the structural flexibility of myoglobin, thereby facilitating its interaction with proteases. The interactions between hydrophobic amino acids, particularly phenylalanine, and the surrounding protein microenvironment during the simulation were analyzed (Fig. 7d and e). In the low-pH group, phenylalanine formed fewer hydrogen bonds with surrounding residues, rendering the local protein structure more flexible and more susceptible to enzymatic hydrolysis (Fig. 7g). Thus, low pH conditions resulted in decreasing protein stability and increasing hydrophobicity, thus activating amino acids at the favored target sites of digestive enzymes.

Fig. 7.

Fig. 7

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Stability analysis of myoglobin at different pH values; (a) The thermal denaturation of myoglobin during heating scanning fluorimetry; (b) The number of hydrogen bonds at pH 7; (c) The number of hydrogen bonds at pH 3; (d) The number of interaction forces of phenylalanine at pH 7; (e) The interaction force of phenylalanine at pH 3.

3.6. Digestibility of myoglobin

The nutritional value of dietary protein is related to its utilization rate, which is determined by its digestibility and absorption in the digestive tract (Yang et al., 2025). This can be investigated by simulating the physiological conditions of the digestive tract and by dynamically modeling the digestive process (Brodkorb et al., 2019). Fig. 8a and b show the digestibility of myoglobin during the gastric and intestinal phases, respectively. The gastric digestibility of myoglobin decreased from 52.68% at pH 3 to about 40% at pH 7 (p < 0.05, Fig. 5a). This significant enhancement in gastric digestibility at low pH is physiologically relevant, as it potentially improves the bioavailability of essential amino acids and heme iron absorption. This could be attributed to the change in myoglobin hydrophobicity. The exposed hydrophobic amino acids promote the interaction between substrate and enzyme to form a stable and precise catalytic conformation (Liu et al., 2022). Pepsin primarily recognizes and cleaves peptide bonds adjacent to hydrophobic, especially aromatic amino acid residues (e.g., phenylalanine, tryptophan, and tyrosine) (Tang et al., 2023; Yang et al., 2025). So, the increased flexibility of hydrophobic amino acid residues may also contribute to the improved digestibility observed at pH 3.

The change in the intestinal trypsin digestibility profile of myoglobin paralleled that of its gastric digestibility (Fig. 8b), with a decrease from about 60% at pH 3 to about 55% at pH 6. However, these changes in tryptic digestibility were not significantly different between the groups. This limited enhancement in digestibility likely originates from the unique specificity of the trypsin active center, which preferentially targets the smaller fragments produced by prior pepsin digestion of myoglobin (Liu et al., 2022).

3.7. Molecular docking and dynamics simulation

Based on the findings above, it can be concluded that the opening of the hydrophobic cavity in myoglobin, which subsequently destabilizes the protein structure and increases the flexibility of key amino acid residues, constitutes two critical factors influencing the digestibility of myoglobin. To provide a molecular-level validation of the experimental findings regarding structural unfolding and digestibility, data derived from 50 ns molecular dynamics simulation were further analyzed. As previously shown in Figs. 3a-e, the lower pH conditions significantly reduced the number of interaction bonds between heme and the surrounding hydrophobic cavity, exposing the hydrophobic groups and the embedding of heme (Figs. 3f-g). The RMSD values of main chain atoms were determined to assess the stability of myoglobin under different pH conditions (Fig. 9b). After 50 ns of simulation, the RMSD values under all pH conditions reached equilibrium. However, the RMSD values were lower at pH 7 than at pH 3, indicating that the myoglobin structure was less stable and underwent more conformational fluctuations under acidic conditions. The root mean square fluctuation (RMSF) value represents the freedom of movement of each atom in the molecule. As shown in Fig. 9a, there was no significant difference in the flexibility of myoglobin residues between different pH conditions. However, some differences existed around the amino acid residues 118–123, which are the preferred sites for pepsin and trypsin (Li, Huang, Dong, & Liu, 2018). Therefore, the representative phenylalanine at position 123 (Phe123) was selected for further analysis. It should be noted that while Phe123 serves as a primary example, other aromatic residues (e.g., Tyr and Trp) likely function collectively to provide accessible binding sites (Zhang et al., 2021). Under lower pH conditions, the number of hydrogen bonds between Phe123 and its surrounding microenvironment is significantly fewer than under higher pH conditions (Figs. 9c-d). At pH 3, Phe123 interacted with His119, whereas at pH 7, Phe123 interacted with both His119 and His116 (Figs. 9f-g). This indicates that at higher pH, the stability of key amino acids of myoglobin is improved and this affects the breakage of peptide bonds. In addition, the SASA values for Phe123 were higher at pH 3 than at pH 7 (Fig. 9e), which could promote the exposure of key digestive enzyme catalytic sites and improve myoglobin digestibility.

Fig. 9.

Fig. 9

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Molecular simulation and docking of myoglobin under different pH conditions; (a) RMSF; (b) RMSD; (c-d) change in the number of hydrogen bonds at pH 3 and pH 7; (e) The SASA of phenylalanine under different pH conditions; (f-g) bonding of phenylalanine under pH 3 and pH 7 conditions.

In summary, the simulation results confirm that acidic conditions facilitate the exposure of hydrophobic residues and increase SASA, thereby transforming the rigid myoglobin structure into a more flexible conformation conducive to digestion.

4. Conclusion

This study systematically investigated the variations in myoglobin digestibility under various pH conditions mimicking fermentation and processing environments. The results demonstrate that acidic environments (pH 3–4) significantly promote myoglobin digestibility by triggering a specific conformational transition. Crucially, low pH prompts the opening of the hydrophobic heme cavity, leading to the exposure of previously buried aromatic residues such as phenylalanine and tyrosine. This process, synergized with weakened hydrogen bonding, shifts the rigid myoglobin conformation toward a flexible state, thereby enhancing protease accessibility to cleavage sites. For example, the controlled acidification techniques, such as fermentation or marinating, can be strategically employed to modify the protein structure of meat products. This approach offers a practical pathway to improve the nutritional value and protein bioavailability of meat, particularly for specific populations with compromised digestive functions, such as the elderly.

CRediT authorship contribution statement

Hui Liu: Writing – original draft, Visualization, Methodology, Funding acquisition, Data curation. Yaxuan Li: Software, Methodology, Conceptualization. Kai Shan: Visualization, Investigation. Chunbao Li: Writing – review & editing. Xiaoyan Tang: Writing – review & editing, Supervision, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the grants from the National Science Foundation of the People's Republic of China (32402013), the Agriculture Research System of China (Grant No. CARS-35), the Agricultural Science and Technology Innovation Program of CAAS and National Key Research and Development Program of China (CAAS-ASTIP-IQSTAP-2024).

Contributor Information

Chunbao Li, Email: chunbao.li@njau.edu.cn.

Xiaoyan Tang, Email: meatstandard@126.com.

Data availability

Data will be made available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data will be made available on request.

Articles from Food Chemistry: X are provided here courtesy of Elsevier

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