Introduction
Retail lighting within display cases influences consumer appeal in countries where meat is marketed in chilled storage. However, retail light can enhance photooxidation of both protein and lipids. Studies have shown that meat stored under retail display conditions is more prone to photooxidation (Özyürek et al., 2021) than meat stored in dark conditions. Further, research has characterized the wavelength dependence of oxymyoglobin (OxyMb) oxidation (Bertelsen and Skibsted, 1987). Myoglobin is a water-soluble heme protein primarily responsible for meat color (Cornforth, 1994; King et al., 2023). Although meat color is not a reliable indicator of microbial levels (Smith et al., 2024), consumers often use meat color as a primary determinant to assess freshness during purchasing (Mancini et al., 2022; Lybarger et al., 2023). The United States beef industry incurs an annual loss of approximately $3.73 billion due to deviations from a bright cherry-red color (Ramanathan et al., 2022). Therefore, elucidating the roles of factors involved in meat discoloration is important to maximize the benefits of beef production.
Myoglobin oxidation is species specific. For example, studies have shown that myoglobin from species with more histidine residues in the primary structure oxidizes faster than those with fewer histidines (Yin et al., 2011). Hence, bovine myoglobin, which has 13 histidines, oxidizes faster than pork myoglobin, which has 9 histidines (Suman et al., 2007). 4-Hydroxy-2-nonenal (HNE) is a secondary lipid oxidation product formed from the oxidation of n-6 polyunsaturated fatty acids. Proximal histidine-93 and distal histidine-64 are important for stabilizing OxyMb. HNE adduction of histidine residues affects the redox stability of myoglobin by modifying its tertiary structure, which makes heme iron more prone to oxidation (Suman et al., 2007; Tatiyaborworntham et al., 2012). Although species specificity in OxyMb oxidation and HNE-induced oxidation is well characterized, limited knowledge is available on the species-specific effects on photooxidation.
Similar to light, oxygen levels within meat packages can increase oxidative reactions and discoloration. Several in vitro studies have used purified myoglobin to understand OxyMb oxidation properties. More specifically, in vitro myoglobin studies have focused on the role of pH, lipid oxidation products (Alderton et al., 2003; Suman et al., 2007; Yin et al., 2011; Tatiyaborworntham et al., 2012), oxygen partial pressure (Kiyimba et al., 2017), meat processing ingredients such as lactate and phosphate (Mancini et al., 2010), and fatty acids (Hoa et al., 2021) on myoglobin oxidation. However, limited studies have considered species-specific and HNE effects of oxygen diffusion from the atmosphere on OxyMb oxidation. The hypothesis of the study was that energy rays from display light or oxygen would increase HNE-induced species-specific OxyMb oxidation. In the current research, the impact of 4 different factors (species, HNE, light, and storage time in experiments 1–2) was determined to gain an understanding of OxyMb oxidation. Therefore, the objective of this study was to examine (1) effects of light and dark storage conditions on HNE-induced beef and pork OxyMb oxidation at 4°C at pH 5.6 and pH 7.4 in vitro (experiment 1) and (2) effects of aerobic and anaerobic systems on HNE-induced beef and pork OxyMb oxidation at 4°C at pH 5.6 in vitro (experiment 2).
Materials and Methods
Reagents used
Potassium phosphate monobasic (KH2PO4), potassium phosphate dibasic (K2HPO4), sodium dithionite, urea, and tris-hydroxymethyl amino-methane hydrochloride (Tris-HCl) were purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO). HNE (Cayman Chemical Co., Ann Arbor, MI) and PD-10 desalting columns packed with Sephadex G-25 resin columns were purchased from GE Healthcare (Piscataway, NJ). All chemicals used were of high purity, meeting the standards of reagent grade or higher.
Myoglobin isolation and purification
Beef and pork myoglobins were purified by ammonium sulfate precipitation and gel filtration chromatography following the protocol described by Faustman and Phillips (2001). For both species, cardiac tissue was ground and homogenized with 10 mM Tris-HCI, 1 mM EDTA (pH 8.0) buffer at 4°C. The homogenates were centrifuged at 5,000 × g for 10 min, and the supernatant was subjected to 70% ammonium sulfate saturation. The solution was then centrifuged at 18,000 × g for 20 min. The resultant supernatant was fully saturated with ammonium sulfate (100%) and centrifuged at 20,000 × g for 1 h. The solid pellet was mixed with homogenization buffer and dialyzed 3 times using a solution containing 10 mM Tris-HCI and 1 mM EDTA (pH 8.0) for 24 h. Myoglobin and hemoglobin were separated using a Sephacryl 200HR gel filtration column with 2.5 × 100 cm dimensions. The elution buffer consisted of 5 mM Tris-HCI and 1 mM EDTA at pH 8.0 and 4°C. The flow rate was set at 60 mL/h. Isolated beef and pork myoglobins were stored in −80°C freezer until use.
Oxymyoglobin preparation
Both beef and pork myoglobins were thawed at 4°C. OxyMb was prepared by hydrosulfite-mediated reduction of purified myoglobin (0.1 mg sodium hydrosulfite to 1 mg myoglobin). Residual hydrosulfite was removed using a PD-10 desalting column. Reduced myoglobin was converted to OxyMb by bubbling with oxygen. Myoglobin concentration was confirmed using absorbance at 525 nm (assuming an excitation coefficient of 7.6 mM−1 cm−1 at 525 nm; Broumand, 1958). The pH of the reaction mixture [either at pH 5.6 (represents approximate postmortem muscle pH) or 7.4 (represents physiological pH)] was achieved by passing myoglobin solutions through PD-10 columns pre-calibrated with respective phosphate buffers (50 mM) at pH 5.6 or pH 7.4.
Oxymyoglobin reaction with 4-hydroxy-2-nonenal
OxyMb was incubated with HNE (0.085 mM porcine OxyMb + 0.6 mM HNE; 0.085 mM bovine OxyMb + 0.6 mM HNE) at 4°C and pH 5.6 and pH 7.4. A molar ratio of myoglobin to HNE was maintained at 1:7 for both species (Yin et al., 2011). Controls consisted of OxyMb plus a volume of ethanol equivalent to that used to deliver the aldehyde to treatment mixtures.
Treatment allocation for light and dark storage and open and closed systems
A schematic representation of experiments 1 (light and dark) and 2 (aerobic and anaerobic systems) is included in Figure 1.
Schematic representation of treatment allocation. MetMb, metmyoglobin; OxyMb, oxymyoglobin.
Experiment 1: Light and dark storage study at pH 5.6 and pH 7.4
In experiment 1, light and dark conditions were created in a sliding door cooler (BSI, Stafford, TX) set at 4°C. Fluorescent light (GE Electric, Boston, MA) in the cooler was the primary source of light. The light intensity within the sliding door cooler was measured using a handheld light intensity meter (Amprobe LM-200, Amprobe, China). The light intensity was approximately 1,583 lx. The dark condition was created by wrapping the cuvette in aluminum foil. The myoglobin mixture containing HNE was incubated in the cuvette, and the myoglobin redox state was determined by scanning absorption from 400–700 nm using a Shimadzu UV-2600 UV-VIS spectrophotometer (Shimadzu Co, Kyoto, Japan). For both species, myoglobin and HNE concentrations were 0.085 mM and 0.6 mM, respectively. The cuvettes were repeatedly measured on 0, 2, 4, and 6 d of storage for pH 5.6 and 0, 2, 4, 6, and 8 d of storage for pH 7.4. Myoglobin is stable at greater pH; hence, a longer storage period was used at pH 7.4 to achieve similar metmyoglobin (MetMb) levels as at pH 5.6. Percent MetMb was determined by measuring the wavelength maxima at 503, 557, and 582 nm (Tang et al., 2004). A similar approach was used for studies at pH 5.6 and pH 7.4. A blank contained only phosphate buffer at pH 5.6 and pH 7.4 for respective experiment conditions. Experiment 1 was replicated 3 times.
Experiment 2: aerobic and anaerobic system study at pH 5.6
In experiment 2, aerobic and anaerobic system represents oxygen diffusion from the atmosphere to the OxyMb and HNE mixture. A similar approach utilized in experiment 1 was followed to prepare OxyMb and HNE addition. The samples were incubated in a sliding door cooler at 4°C and pH 5.6. In the aerobic system, the OxyMb-HNE mixture was added into a cuvette without a screw cap (allows atmospheric air to come in contact with myoglobin-HNE mixture; FireflySci Type 46 semi-micro spectrophotometer cuvette, Northport, NY), while in the anaerobic system the OxyMb-HNE mixture was added to a FireflySci Type 46 semi-micro spectrophotometer cuvette (with a volume of 700 μL with SEPTA closed screw caps to prevent diffusion of oxygen from outside). The aerobic system cuvettes were wrapped with an aerobic polyvinyl chloride film to minimize water vapor evaporation. MetMb levels were repeatedly measured on 0, 2, 4, and 6 d of storage, according to Tang et al. (2004). Experiment 2 was also replicated 3 times.
Statistical analysis
The data for experiments 1 and 2 were analyzed separately. Both experiments were replicated 3 times. It is well known that the reactivity of HNE is greater at pH 7.4 than at pH 5.6 (Alderton et al., 2003; Elroy et al., 2015). Hence, pH 5.6 and pH 7.4 results were analyzed separately. A completely randomized design with factorial arrangement was used to evaluate the combined effects of HNE on light and dark conditions as well as aerobic and anaerobic systems on species-specific myoglobin oxidation. The fixed effects in experiment 1 included lipid oxidation product (HNE), light and dark conditions, species, storage time, and their interactions. The fixed effects in experiment 2 included lipid oxidation product (HNE), aerobic and anaerobic system, species, storage time, and their interactions. Type-3 tests of fixed effects for changes in MetMb redox state during storage time were evaluated using the PROC GLIMMIX procedure of SAS (SAS 9.4; SAS Inst.; Cary, NC). Repeated option in Mixed Procedure was used to assess the covariance–variance structure resulting from repeated measurements on the same sample during incubation. Least-squares means were generated for significant t tests (P < 0.05) and separated using least significant differences.
Results
Summary of statistical interactions
Analysis of variance (ANOVA) table containing various interactions in experiments 1 and 2 are summarized in Table 1. In the current research, the highest-order interactions were reported. It is well documented that pH has significant effects on HNE-induced oxidation. Hence, the pH results were analyzed separately.
ANOVA table summarizing P-values of various interactions in experiments 1 and 2
| Experiment 1 | Experiment 2 | ||
|---|---|---|---|
| Effect | pH 7.4 | pH 5.6 | pH 5.6 |
| time | <0.0001 | <0.0001 | <0.0001 |
| species | <0.0001 | <0.0001 | <0.0001 |
| time × species | <0.0001 | 0.008 | <0.0001 |
| source | <0.0001 | <0.0001 | <0.0001 |
| time × source | <0.0001 | 0.0003 | <0.0001 |
| species × source | 0.92 | 0.62 | <0.0001 |
| time × species × source | 0.11 | 0.50 | <0.0001 |
| HNE | <0.0001 | <0.0001 | <0.0001 |
| time × HNE | <0.0001 | <0.0001 | <0.0001 |
| species × HNE | 0.94 | 0.01 | <0.0001 |
| time × species × HNE | <0.0001 | 0.64 | <0.0001 |
| source × HNE | 0.0001 | <0.0001 | 0.14 |
| time × source × HNE | 0.002 | 0.71 | 0.01 |
| species × source × HNE | 0.20 | 0.29 | 0.02 |
| time × species × source × HNE | 0.02 | 0.56 | 0.23 |
Experiment 1: effects of light and dark. Experiment 2: effects of aerobic and anaerobic systems (whether atmospheric air/oxygen is allowed to come in contact with myoglobin or not). Source in experiment 1: light and dark. Source in experiment 2: aerobic and anaerobic system. ANOVA, analysis of variance; HNE, 4-hydroxy-2-nonenal.
Experiment 1a: Effects of light and dark storage on species-specific effects on 4-hydroxy-2-nonenal at pH 5.6
Four different types of 2-way interactions were significant for the effects of light and dark storage conditions on HNE-induced beef and pork OxyMb oxidation at 4°C and pH 5.6 (Figures 2–5).
Effects of storage time and light and dark conditions on HNE-induced beef and pork oxymyoglobin oxidation at pH 5.6 and 4°C. Standard error of storage time and light/dark conditions = 0.56. Least-squares means within each interaction type with different letters (a–h) are significantly different (P < 0.05). Light: oxymyoglobin samples were exposed to light; dark: oxymyoglobin samples were wrapped in aluminum foil. HNE, 4-hydroxy-2-nonenal.
Effects of storage time on HNE-induced beef and pork oxymyoglobin oxidation at pH 5.6 and 4°C. Standard error of storage time and HNE = 0.80. Least-squares means within each interaction type with different letters (a–k) are significantly different (P < 0.05). Control: no HNE was delivered to oxymyoglobin solution. However, ethanol used to dissolve HNE was added to control oxymyoglobin solution. HNE, 4-hydroxy-2-nonenal.
Effects of species and HNE on oxymyoglobin oxidation in light and dark storage at pH 5.6 and 4°C. Standard error of species and HNE = 0.43. Least-squares means within different letters (a–c) are significantly different (P < 0.05). Control: no HNE was delivered to oxymyoglobin solution. However, ethanol used to dissolve HNE was added to control oxymyoglobin solution. HNE, 4-hydroxy-2-nonenal.
Effects of light and dark storage on HNE-induced beef and pork oxymyoglobin oxidation at pH 5.6 and 4°C. Standard error of HNE and light/dark conditions = 0.43. Least-squares means within each interaction type with different letters (a–d) are significantly different (P < 0.05). Light: oxymyoglobin samples were exposed to light; dark: oxymyoglobin samples were wrapped in aluminum foil. Control: no HNE was delivered to oxymyoglobin solution. However, ethanol used to dissolve HNE was added to control oxymyoglobin solution. HNE, 4-hydroxy-2-nonenal.
As expected, there were time × source (light/dark) and time × HNE interactions that resulted in MetMb levels (Figures 2–3). There were no differences (P > 0.05) in MetMb levels on days 0 and 1 between light and dark samples. However, MetMb levels were greater (P <0.05) in light samples from days 3 to 6 than for samples stored in the dark. There were no differences (P > 0.05) in MetMb levels between control and HNE on day 0 of storage. However, from day 1 of storage to day 6, there were differences (P < 0.05) between control and HNE treatments.
There were species × HNE and source (light/dark) × HNE interactions that resulted in MetMb formation (Figures 4–5). In both species, HNE increased MetMb formation compared with control samples. However, beef HNE samples had greater (P < 0.05) MetMb than pork HNE samples. Light condition increased (P < 0.05) MetMb in presence of HNE compared with control samples without HNE. In both light and dark conditions, HNE increased MetMb formation. However, the change in MetMb between HNE and control (HNElight – controllight) was greater (P < 0.05) in light conditions compared to HNE and control (HNEdark – controldark) in the dark.
Experiment 1b: Effects of light and dark storage on species-specific effects on 4-hydroxy-2-nonenal at pH 7.4
There was a time × species × light × HNE interaction (P < 0.05) that resulted in MetMb formation at pH 7.4 (Figure 6). On day 0, there were no differences (P > 0.05) between species × light × HNE. On day 8, bovine control samples in light had greater (P < 0.05) MetMb than bovine control samples kept in the dark. On day 8, bovine HNE samples in light had greater (P < 0.05) MetMb than bovine control samples kept in light. Interestingly, pork myoglobin on day 8 had lower oxidation (P < 0.05) than beef samples on day 8 for both light and dark. However, HNE increased (P < 0.05) pork metmyoglobin formation compared with control pork samples both in light and dark conditions.
Four-way interaction of time × species × source × HNE on oxymyoglobin oxidation at pH 7.4 and 4°C. Least-squares means with different letters (a–x) in both panels are significantly different (P < 0.05). Standard error for time × species × source × HNE interaction = 0.52. Light: oxymyoglobin samples were exposed to light; dark: oxymyoglobin samples were wrapped in aluminum foil. Control: no HNE was delivered to oxymyoglobin solution. However, ethanol used to dissolve HNE was added to control oxymyoglobin solution. HNE, 4-hydroxy-2-nonenal.
Experiment 2: Effects of aerobic and anaerobic systems on species-specific effects on 4-hydroxy-2-nonenal at pH 5.6
There was a source (aerobic and anaerobic) × HNE × storage time interaction that resulted in MetMb formation (Figure 7). On day 0 of storage, HNE-treated samples had greater MetMb than control samples both in aerobic and anaerobic conditions. However, on day 2 of storage, MetMb levels in control myoglobin samples in an aerobic system were greater (P < 0.05) than in anaerobic myoglobin samples. HNE addition increased MetMb formation both in aerobic and anaerobic systems more than control myoglobin without HNE on day 6 of storage.
Effects of aerobic and anaerobic systems on HNE-induced beef and pork oxymyoglobin oxidation at pH 5.6 and 4°C. Least-squares means with different letters (a–m) are significantly different (P < 0.05). Standard error for time × source × HNE interaction = 0.80. Control: no HNE was delivered to oxymyoglobin solution. However, ethanol used to dissolve HNE was added to control oxymyoglobin solution. The aerobic or anaerobic system indicates either allowing or not allowing atmospheric oxygen in contact with myoglobin solution. HNE, 4-hydroxy-2-nonenal.
A species × source × HNE interaction revealed that bovine myoglobin samples had greater (P < 0.05) MetMb levels than their porcine counterparts (Figure 8). For example, beef myoglobin samples with and without HNE had greater (P < 0.05) MetMb than pork myoglobin samples with and without HNE when kept in an open system. However, anaerobic system had lower (P < 0.05) MetMb levels for species × HNE treatment than in aerobic system.
Effects of aerobic and anaerobic systems on HNE-induced beef and pork oxymyoglobin oxidation at pH 5.6 and 4°C. Least-squares means with different letters (a–h) are significantly different (P < 0.05). Standard error for species × source × HNE interaction = 0.28. Control: no HNE was delivered to oxymyoglobin solution. However, ethanol used to dissolve HNE was added to control oxymyoglobin solution. The aerobic or anaerobic system indicates either allowing or not allowing atmospheric oxygen in contact with myoglobin solution. HNE, 4-hydroxy-2-nonenal.
Discussion
Various biochemical analyses have characterized the pre- and post-harvest factors that influence meat color (Tang et al., 2004; Seyfert et al., 2007; King et al., 2011; English et al., 2016; McKeith et al., 2016; Ke et al., 2017; Nair et al., 2018; Mitacek et al., 2019). Furthermore, in vitro studies have also determined the role of pH, lipid oxidation products, and light on myoglobin oxidation and reduction (Mancini and Ramanathan, 2008; Naveena et al., 2010; Yin et al., 2011; Scott et al., 2023). However, limited studies have examined the combined effects of the species specificity of HNE-induced OxyMb oxidation in light or dark conditions. Similarly, limited studies have considered the role of oxygen diffusion from the outside on OxyMb oxidation.
Previous studies have characterized the prooxidant nature of light and oxygen toward myoglobin (George and Stratmann, 1952; Brantley et al., 1993). In support, the current study also noted greater myoglobin oxidation with light or oxygen. An elevated oxygen concentration can promote free radical generation in the presence of light. In this research, myoglobin samples were exposed to continuous fluorescent lighting to simulate retail store display conditions. Thus, the presence of ultraviolet (UV) radiation in display light can serve as a prooxidant and trigger the oxidation of OxyMb. The UV rays in light can generate superoxide anion radicals (O2-), which can produce hydrogen peroxide (Lind et al., 1990). In addition, heme-containing porphyrin rings act as a photosensitizer (Chakraborti, 2003). Thus, heme can transition from a ground state to an excited state, transferring an electron to molecular oxygen and producing reactive oxygen species. Therefore, a greater level of MetMb was observed in light conditions compared to dark conditions.
For the first time, we noted a species-specific effect of light and oxygen on HNE-induced OxyMb oxidation in vitro. Previous research showed that histidine residues were the primary locations for HNE adduction in bovine OxyMb (Alderton et al., 2003; Suman et al., 2007). Pork myoglobin has 9 histidine residues, while bovine myoglobin contains 13 histidine residues. Greater number of ionizable histidine residues in beef myoglobin favors more oxidation. More specifically, lower pH favors the protonation of proximal histidine, weakens iron and oxygen bonds, reduces oxygen affinity, and results in more oxidation (Capece et al., 2006). In addition, fewer histidine residues in pork myoglobin would decrease the number of sites available for HNE adduction. Consequently, pork myoglobin may have become less vulnerable to nucleophilic attack by HNE. Thus, this study noted that bovine myoglobin had greater oxidation than pork myoglobin (without HNE). However, HNE increased light-induced oxidation in beef myoglobin more than in pork myoglobin.
HNE-induced OxyMb oxidation was greater when equine OxyMb was exposed to 80% oxygen compared to atmospheric oxygen levels (Kiyimba et al., 2017). In support, the current research also observed that when oxygen was allowed to diffuse from the outside, greater oxidation was noticed in bovine samples than in pork myoglobin samples (without adding HNE). Similar to light, greater oxygen levels increased HNE-induced OxyMb oxidation in bovine samples more than in pork samples. A previous study did not observe differences in HNE binding with greater oxygen levels compared to normal atmospheric oxygen level (Kiyimba et al., 2017). In the current research, the impact of HNE under light/dark or oxygen diffusion on covalent binding of myoglobin was not evaluated. Determining the HNE adduction to myoglobin will provide a better understanding of parameters such as light or oxygen levels on myoglobin redox stability.
Effects of pH on species-specific metmyoglobin formation
Although the focus of this study was not to compare pH, a separate analysis was also conducted using control myoglobin samples (not including HNE-treated myoglobins) to know the effects on MetMb formation (Figure 9). In order to compare the same time periods, MetMb levels at pH 5.6 and pH 7.4 for beef and pork were analyzed at 0, 2, 4, and 6 d of storage. For beef, pH 5.6 had greater MetMb levels than pH 7.4 on days 2 and 4. However, day 6 had greater MetMb levels at pH 7.4 than at pH 5.6. For pork, pH 5.6 had greater MetMb levels at respective time periods than at pH 7.4. These results support the initial phase of previously published studies (Alderton et al., 2003; Suman et al., 2007), and the observed increase in MetMb levels on day 6 is unclear. Greater MetMb levels on day 6 in beef might be due to increased storage time (6 d) in this research compared with shorter storage (4 d; Alderton et al. 2003).
Effects of pH × species × time on control beef and pork oxymyoglobin oxidation in dark storage at 4°C (HNE is not included in this analysis). To make the storage time similar, only 6 days of storage were considered for analysis for pH 7.4. Least-squares means with different letters (a–n) are significant (P < 0.05); standard error for pH × species × time = 0.4. HNE, 4-hydroxy-2-nonenal.
Conclusion
The present study evaluated the impacts of light and oxygen diffusion on HNE-induced beef and pork OxyMb oxidation. The results indicate species-specific effects on HNE-induced OxyMb oxidation under light. Bovine OxyMb oxidized more than pork myoglobin when incubated under light conditions. A greater number of histidine residues might have favored more ionizable groups and increased oxidation in samples without added HNE. HNE and light increased oxidation in beef compared to the samples without HNE. Similar to the impact of light, diffusion of oxygen from outside increased beef and pork OxyMb. The current study indicates beef myoglobin is more prone to oxidation in light and oxygen than pork myoglobin.
Conflict Of Interest
The authors declare no conflicts of interest regarding the content of this manuscript.
Acknowledgements
This research was supported, in part, by Ranjith Ramanathan’s Leo and Kathy Noltensmeyer Endowed Research Chair funds.
Author Contributions
Rishav Kumar conducted the study, collected data, and wrote the draft manuscript; Surendranath Suman edited the manuscript, Runnan Li was involved with the myoglobin extraction, Gretchen Mafi edited the manuscript; Morgan Pfeiffer edited the manuscript; Ranjith Ramanathan analyzed data, edited the manuscript, secured funding, and provided supervision.
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