CC BY
0EFFECT OF MYOGLOBIN CONTENT ON THE BEEF MEAT COLOR DURING STORAGE: NEW TECHNOLOGY FOR ACCURATE EVALUATION OF MEAT COLOR
1Dissanayake K.D.K.K., 2Samadiy M.A. 3Rifky A.L.M., 4Nurmukhamedov K.S.
1,2Karshi Engineering-Economics Institute, Karshi, 180100, Uzbekistan 3Eastern University, Sri Lanka, Chenkalady, Sri Lanka 4Tashkent Chemical-Technological Institute, Tashkent, Uzbekistan https://doi.org/10.5281/zenodo.13831860
Abstract. This study examines the correlation between changing myoglobin levels and variations in meat color after a 15-day storage period. It studies extensively how sodium nitrate affects the consistency of meat color. Myoglobin levels were measured using spectrophotometry techniques, which revealed variations in the amounts of deoxymyoglobin, oxymyoglobin, and metmyoglobin over the course of the observation period. Results show a clear correlation between meat's redness and myoglobin concentration, with higher myoglobin concentrations corresponding to deeper red colors. Sodium nitrate was added, which enhanced the color's initial redness and maintained it for the duration of storage. This indicates sodium nitrate functions as a color stabilizer by possibly preventing oxidation processes or interacting with myoglobin. These results were supported by sensory assessments carried out with a panel of consumers, which showed the significance of sodium nitrate in preserving a pleasing appearance and possibly extending the shelf life by preventing color deterioration.
Statistical analysis demonstrates significant differences in myoglobin levels and meat color parameters over time, emphasizing the need for continuous monitoring and optimization of processing conditions. These results underscore the importance of sodium nitrate as a food additive in meat products and highlight avenues for further research to balance color stability with potential health considerations. This study contributes valuable insights into understanding the interplay between myoglobin, sodium nitrate, and meat color, crucial for ensuring consumer satisfaction and food quality assurance.
Keywords: meat color, myoglobin level, spectrophotometer, sodium nitrate.
INTRODUCTION
Myoglobin, the primary compound in meat responsible for its inherent color, is more preference of consumers and is a key quality indicator of meat [1]. The meat red color is determined by myoglobin, and its color may change due to oxidation and browning during storage and processing [2]. Variations in the spacing among myofilament structure, length of sarcomeres, as well as the arrangement of sarcoplasmic peptide influence to differences in meat lightness [3]. The oxidation of myoglobin results in the formation of metmyoglobin, which can be quantified using color parameters like a*, C*, AE*, and the reflectance ratio R630/R580 [4]. The achromatic aspects of meat color are caused by light scattering from the microstructure of muscle cells, influenced by factors such as myofilament spacing, protein solubility, and cytoskeleton integrity [5]. Understanding the role of myoglobin and its interaction with meat microstructure is essential for assessing meat color and ensuring consumer satisfaction.
The amount of sodium nitrate in meat is linked to myoglobin levels and color. Studies have shown that adding d-sodium erythorbate to pork sausage can reduce myoglobin level and lipid oxidation speed while increasing amount of heme iron, leading to slower discoloration. Additionally, the oxygenation level of myoglobin before freezing and the duration of frozen storage can influence the color of beef. Steaks with higher myoglobin oxygenation tend to have increased oxygen penetration and higher a* values, indicating a brighter red hue. Furthermore, the presence of NO-synthase and arginine in meat can impact its color when heated. For NO-synthase activity, adjusting the pH value, duration, and temperature conditions may result in desired color effects, presenting NO-synthase as a possible alternative beneficial component [8].
2. Methods
Different techniques can be used to evaluate the relationship between the concentration of sodium nitrite and myoglobin content and color in processed meat. Technique is to measure the myoglobin redox forms, such as metmyoglobin (Met-MB), deoxymyoglobin (Deoxy-MB), and oxymyoglobin (Oxy-Mb), using spectrophotometric Technology [10]. Additionally, spectrophotometers can be used to determine the color parameters of processed meat, including redness (a*), yellowness (b*), lightness (L*), facilitate to evaluate relationship between processed meat's myoglobin concentration and color and sodium nitrite levels [6].
2.1 Measurement of Meat Myoglobin level of meat muscle.
As a preservative, add 120 mg/kg of beef sample. For processed meat products and dry meat, the recommended maximum amount of sodium nitrate is 500 mg/kg [11]. Muscle meat 0.5 grams were blended thoroughly and then subjected to centrifugation at 10,000 times gravity for 10 minutes. A spectrophotometer (Shimadzu make- uv-visible spectrophotometer model no: uv-1800, Japan) was used to determine the supernatant absorbance at 576 nm, using a blank as the reference. Myoglobin extinction coefficient millimolar (mM) at 576 nm is used for determining the outcome, which are presented as (mg)milligrams of total myoglobin per (g) gram of meat [12].
About sample one gram underwent homogenization (HG-200 Homogenizer 270 x 310 x 560mm 0 - 20000rpm) with 8 milliliters of sodium phosphate reagent (0.04 mol/L, pH 6.8). Following this, Whatman filter was used to filter the supernatant. Using a spectrophotometer (1800 uv spectrophotometer, Japan), the absorbance of the final extract was measured at wavelengths of 555 nm, 577 nm, and 630 nm against a blank [13].
To calculate the total myoglobin content in meat, The amounts of (Deoxy-MB) deoxymyoglobin, (Oxy-Mb) oxymyoglobin, and (Met-MB) metmyoglobin, along with their corresponding extinction coefficients, must be taken into consideration [14,15].
2.1.1 Measure Absorbance
Measure the absorbance of the meat sample at specific wavelengths. Myoglobin has characteristic absorption peaks at different wavelengths. Deoxymyoglobin absorbs strongly at around 555 nm, Oxymyoglobin absorbs strongly at around 577 nm, and Metmyoglobin absorbs strongly at around 630 nm [14,15].
Every type of myoglobin has a specific extinction coefficient, which determines the amount of light at a certain wavelength it is able to absorb. The extinction coefficients for myoglobin forms are typically provided in scientific literature or can be experimentally determined [16].
2.1.2 Calculate Concentrations of myoglobin in beef meat
The Beer-Lambert Law is used to calculate the concentration of each form of myoglobin based on their absorbance and extinction coefficients [17,18].
Table 1. Formula for calculating myoglobin concentration in meat by using
spectrophotometer absorbance
Beer-Lambert Law Rearranging for concentration
A = 8 X c X l A C = exl
The terms A stands for absorbance, s for molar absorptivity/extinction coefficient, c for myoglobin content, and l for the spectrophotometer cuvette's path length [19-22].
2.2 Measurement of Meat Color of meat muscle.
The brightness, yellowness and redness of the beef meat were measured after it had been opened and allowed to fully bloom for 30 minutes. These CIE L*, a*, and b* values used to quantify, respectively [23], utilizing a spectrophotometer (CM-5, Konica Minolta Sensing Inc., Osaka, Japan). The spectrophotometer was calibrated using a standard white and black plate following every test. [24,25].
2.3 Statistical evaluation.
The analysis was carried out in three replicates and the results were got average value (n=3) [26]. Samples of beef meat stored for varying durations (0, 5, 10, and 15 days) were analyzed in each trial [27,28]. With SAS 9.4 (SAS Institute Inc., USA), a common linear model was utilized to data analysis. The findings are illustrated as mean values with (SEM) standard error of the mean. Specific distinctions through the mean value data were assessed using Tukey's multiple comparison test at a specific level of p<0.05 [28-30].
3. Results and Discussion
3.1 Color determination of Meat.
Myoglobin concentration, sensory analysis, and redness value of beef meat during 15 days of keep at freezing conditions (4°C). The research findings indicate a significant variation in the myoglobin content of meat over a period of 15 days of storage. Myoglobin is a protein responsible for the color of meat and undergoes chemical changes during storage, affecting its color and freshness. The three main forms of myoglobin observed in the study are deoxymyoglobin, oxymyoglobin, and metmyoglobin, each representing different stages of oxidation.
Table 2. Myoglobin percentage of deoxymyoglobin, oxymyoglobin, and metmyoglobin during
storage days.
Storage Days
Myoglobin % 0 5 10 15
DeoxyMyoglobin (%) 3.45 6.01 9.56 5.54
OxyMyoglobin (%) 58.05 51.96 85.88 37.33
MetMyoglobin (%) 38.50 42.03 4.56 57.13
Fresh meat (day 0), typically exhibits a high proportion of oxymyoglobin, giving it a bright red color. The presence of deoxymyoglobin suggests that the meat was adequately oxygenated during processing and packaging. Metmyoglobin, while present, is at a relatively low level, indicating minimal oxidation at this stage.
Over the first 5 days of storage, there is a noticeable increase in deoxymyoglobin and metmyoglobin levels, accompanied by a decrease in oxymyoglobin. This indicates that oxidation
processes are occurring, leading to a shift in the myoglobin forms. The decrease in oxymyoglobin suggests a loss of oxygen availability, likely due to packaging or environmental conditions. By day 10, there is a significant increase in oxymyoglobin levels, indicating a potential reoxygenation of the meat.
100,00%
80,00%
£ '.n 60,00%
O
M o 40,00%
^ 2E
20,00%
0,00%
3,45% 0
DeoxyMyoglobin (%)
6,01%
5 10
Storage Days OxyMyoglobin (%)
5,54% 15
MetMyoglobin (%)
Figure 1. Deoxymyoglobin, oxymyoglobin and metmyoglobin percentage variation of beef
meat during storage days This could be due to factors such as exposure to air or changes in packaging. The proportion of deoxymyoglobin continues to increase, suggesting ongoing oxidation processes, although at a slower rate. Metmyoglobin levels decrease substantially, indicating a partial reversal of oxidation. When the storage duration is completed, there is a notable decrease in oxymyoglobin levels, accompanied by a significant increase in metmyoglobin. This indicates that the meat has undergone extensive oxidation, resulting in a brownish coloration characteristic of metmyoglobin. The decrease in deoxymyoglobin suggests a reduction in the availability of reducing agents, further contributing to oxidation.
The results demonstrate that the myoglobin content of meat undergoes dynamic changes during storage, influenced by impacts such as presence of oxygen, packaging, and environmental conditions. Myoglobin forms are continuously monitored to provide useful information on the quality and freshness of processed beef products. This information guides handling and storage procedures to preserve the finest possible color and flavor.
3.2 Myoglobin levels in beef meat sample during 15 days of storage at freezing conditions
(4°C)
Table 3. the redness value of the meat changed during storage according to the total
myoglobin content
Storage Days 0 5 10 15
Myoglobin level 1 mg/g. 0.8 mg/g. 0.6 mg/g. 0.5 mg/g.
Redness value CIE a* 13.09 11.90 10.56 10.02
The myoglobin level reaches its highest point on the first day at 1 mg/g, and the redness value is relatively high at 13.09. This indicates elevated beef meat redness is associated with higher myoglobin concentration. After five days, the redness value had dropped to 11.90 and the myoglobin level had dropped by 0.2 mg/g. Still, it is evident that redness and myoglobin levels are correlated—redness declines while myoglobin levels fall.
The trend continues on day 10, when the myoglobin level is 0.6 mg/g and the redness value is 10.56, where both myoglobin level and redness have decreased further. The correlation between myoglobin level and redness persists. On the final day of observation, both myoglobin level and redness have decreased slightly compared to day 10. The myoglobin content was reduced to 0.1 mg/g, and the redness value is 10.02.
14
12
CÖ
W 10
myoglobin level vs redness value CIE a*
13,09
11,9
10,56
10,02
g 8
"cd
ë T3
<D
Pi
Myoglobin level 1 mg/g Storage Days o
0.8 mg/g. 5
0.6 mg/g. 10
0.5 mg/g. 15
4
2
0
Figure 2. Myoglobin content and beef meat redness value change during storage
The correlation between myoglobin level and redness remains evident, albeit with a diminished effect due to the decrease in myoglobin level.
The trend suggests that there is a positive correlation between myoglobin level and meat color redness. As myoglobin levels decrease over the storage period, the redness of the meat also decreases. This relationship is expected, as myoglobin is key factor for the red color of meat, and its degradation leads to a decrease in redness.
3.3 Effect of Sodium Nitrate to Meat color.
Without sodium nitrate, the initial redness value of the meat (2.31 ± 0.41) is lower compared to when sodium nitrate is added (3.59 ± 0.32). This suggests that sodium nitrate enhances the initial redness of the meat, likely due to its interaction with myoglobin, the protein responsible for meat color.
Table 4. Sodium nitrate added and without adding sodium nitrate for beef meat color variation
during storage days
Parameter Days
0 5 10 15
CIE L0* 76.46±2.22 77.21±1.82 76.52±1.20 76.18± 1.11
CIE L*sn 75.96±2.12 76.94 ± 1.54 75.01 ± 2.71 76.62 ± 2.71
CIE a*0 2.31 ± 0.41 3.54 ± 0.31 3.95 ± 0.36 3.25 ± 1.13
CIE a*sN 3.59 ± 0.32 3.69 ± 0.39 3.94 ± 0.33 3.98 ± 0.60
CIE b*0 12.64 ± 0.70 12.75 ±0.23 12.30 ± 0.60 12.50 ± 0.28
CIE b*sN 12.81 ± 1.34 12.15 ± 0.24 12.42 ± 0.45 12.66 ± 0.79
CIE L* a* b*sN with 120 mg/kg of sodium nitrate added to beef meat sample
CIE L* a* b*o without sodium nitrate in beef meat sample
After 5 days, the redness value increases significantly (3.54 ± 0.31), indicating a progression towards a redder color. By day 10, the redness value further increases (3.95 ± 0.36), indicating continued color development. However, by day 15, there's a slight decrease in redness value (3.25 ± 1.13), possibly indicating the onset of discoloration or degradation processes.
Redness value change with storage days and sodium nitrate 5 -
0 -
0 5 10 15
Storage Days
Redness value Without Sodium Nitrate Redness value With Sodium Nitrate
Figure 3. Redness value change with adding sodium nitrate during storage period
Similarly, the redness value increases over time, but the initial redness (3.59 ± 0.32) is higher due to the addition of sodium nitrate. The redness values at days 5 (3.69 ± 0.39), 10 (3.94 ± 0.33), and 15 (3.98 ± 0.60) show a consistent trend of maintaining or slightly increasing redness over time. Importantly, the redness values with sodium nitrate remain higher than those without sodium nitrate throughout the storage period, indicating better color stability.
4 Conclusion
The presence of sodium nitrate appears to enhance the initial redness and improve meat color stability over the storage days. This suggests that sodium nitrate acts as a color stabilizer, potentially by inhibiting oxidation processes or interacting with myoglobin to maintain a desirable red color. The results highlight the importance of sodium nitrate as a food additive in meat products for maintaining visual appeal and potentially extending shelf life by delaying color deterioration. While sodium nitrate shows benefits in preserving meat color, further research may explore its effects on other quality attributes such as flavor, texture, and safety. Additionally, investigating optimal concentrations of sodium nitrate to balance color stability with potential health concerns is essential for ensuring food safety and consumer acceptance.
REFERENCES
1. Fernández-Barroso MÁ, García-Casco JM, Núñez Y, Ramírez-Hidalgo L, Matos G, Muñoz M. Understanding the role of myoglobin content in Iberian pigs fattened in an extensive system through analysis of the transcriptome profile. Animal genetics. 2022 Jun;53(3):352-67.
2. Zhu W, Han M, Bu Y, Li X, Yi S, Xu Y, Li J. Plant polyphenols regulating myoglobin oxidation and color stability in red meat and certain fish: A review. Critical Reviews in Food Science and Nutrition. 2022 Sep 8:1-3.
3. Purslow PP, Warner RD, Clarke FM, Hughes JM. Variations in meat colour due to factors other than myoglobin chemistry; a synthesis of recent findings (invited review). Meat Science. 2020 Jan 1; 159:107941.
4. Salueña BH, Gamasa CS, Rubial JM, Odriozola CA. CIELAB color paths during meat shelf life. Meat science. 2019 Nov 1; 157:107889.
5. Hughes JM, Clarke FM, Purslow PP, Warner RD. Meat color is determined not only by chromatic heme pigments but also by the physical structure and achromatic light scattering properties of the muscle. Comprehensive Reviews in Food Science and Food Safety. 2020 Jan;19(1):44-63.
6. Kim, S., Lee, H.J., Kim, M., Yoon, J.W., Shin, D.J. and Jo, C., 2019. Storage stability of vacuum-packaged dry-aged beef during refrigeration at 4 C. Food Science of Animal Resources, 39(2), p.266.
7. Henriott ML, Herrera NJ, Ribeiro FA, Hart KB, Bland NA, Calkins CR. Impact of myoglobin oxygenation level on color stability of frozen beef steaks. Journal of Animal Science. 2020 Jul;98(7): skaa193.
8. Zaj^c M, Zaj^c K, Dybas J. The effect of nitric oxide synthase and arginine on the color of cooked meat. Food Chemistry. 2022 Mar 30; 373:131503.
9. Azeem SM, Madbouly MD, El-Shahat MF. Determination of nitrite in processed meat using digital image method and powdered reagent. Journal of Food Composition and Analysis. 2019 Aug 1;81:28-36.
10. Karwowska M, Kononiuk A, Wójciak KM. Impact of sodium nitrite reduction on lipid oxidation and antioxidant properties of cooked meat products. Antioxidants. 2019 Dec 21;9(1):9.
11. Ferysiuk, K., & Wójciak, K. M. (2020). Reduction of nitrite in meat products through the application of various plant-based ingredients. Antioxidants, 9(8), 711.
12. Li YS, Zhu NH, Niu PP, Shi FX, Hughes CL, Tian GX, Huang RH. Effects of dietary chromium methionine on growth performance, carcass composition, meat colour and expression of the colour-related gene myoglobin of growing-finishing pigs. Asian-Australasian Journal of Animal Sciences. 2013 Jul;26(7):1021.
13. Yu QP, Feng DY, Xiao J, Wu F, He XJ, Xia MH, Dong T, Liu YH, Tan HZ, Zou SG, Zheng T. Studies on meat color, myoglobin content, enzyme activities, and genes associated with oxidative potential of pigs slaughtered at different growth stages. Asian-Australasian journal of animal sciences. 2017 Dec;30(12):1739.
14. Nguyen, T., & Kim, J. G. (2019). A simple but quantitative method for non-destructive monitoring of myoglobin redox forms inside the meat. Journal of food science and technology, 56, 5354-5361.
15. Li, H., Haruna, S. A., Geng, W., Wei, W., Zhang, M., Ouyang, Q., & Chen, Q. (2021). Fabricating a novel colorimetric-bionic sensor coupled multivariate calibration for simultaneous determination of myoglobin proportions in pork. Sensors and Actuators B: Chemical, 343, 130181.
16. Bittante, G., Savoia, S., Cecchinato, A., Pegolo, S., & Albera, A. (2021). Phenotypic and genetic variation of ultraviolet-visible-infrared spectral wavelengths of bovine meat. Scientific Reports, 11(1), 13946.
17. Olivé, M., Engvall, M., Ravenscroft, G., Cabrera-Serrano, M., Jiao, H., Bortolotti, C. A., ... & Laing, N. G. (2019). Myoglobinopathy is an adult-onset autosomal dominant myopathy with characteristic sarcoplasmic inclusions. Nature communications, 10(1), 1396.
18. Yeh, Y. C., Haasdonk, B., Schmid-Staiger, U., Stier, M., & Tovar, G. E. (2023). A novel model extended from the Bouguer-Lambert-Beer law can describe the non-linear absorbance of potassium dichromate solutions and microalgae suspensions. Frontiers in Bioengineering and Biotechnology, 11, 1116735.
19. Casasanta, G., Falcini, F., & Garra, R. (2022). Beer-Lambert law in photochemistry: A new approach. Journal of Photochemistry and Photobiology A: Chemistry, 432, 114086.
20. Oshina, I., & Spigulis, J. (2021). Beer-Lambert law for optical tissue diagnostics: current state of the art and the main limitations. Journal of biomedical optics, 26(10), 100901-100901.
21. Shepherd, A. P., Sutherland, J. C., & Wilson, A. F. (1975). Continuous spectrophotometry measurements of arteriovenous oxygen difference. Journal of applied physiology, 39(1), 152155.
22. Lee, Y. F., & Kirchhoff, J. R. (1993). Design and characterization of a spectroelectrochemistry cell for absorption and luminescence measurements. Analytical Chemistry, 65(23), 34303434.
23. Jilcott Pitts, S., Moran, N. E., Laska, M. N., Wu, Q., Harnack, L., Moe, S., ... & Zaidi, Y. (2023). Reflection spectroscopy-assessed skin carotenoids are sensitive to change in carotenoid intake in a six-week randomized controlled feeding trial in a racially/ethnically diverse sample. J. Nutr.
24. Stafford, C. D., Taylor, M. J., Dang, D. S., England, E. M., Cornforth, D. P., Dai, X., & Matarneh, S. K. (2022). Spectro 1—A Potential Spectrophotometer for Measuring Color and Myoglobin Forms in Beef. Foods, 11(14), 2091.
25. Mancini, R. A., Ramanathan, R., Hunt, M. C., Kropf, D. H., & Mafi, G. G. (2022). Interrelationships between visual and instrumental measures of ground beef color. Meat and Muscle Biology, 6(1).
26. Sun, Y., Zhai, X., Zou, X., Shi, J., Huang, X., & Li, Z. (2023). A Ratiometric Fluorescent Sensor Based on Silicon Quantum Dots and Silver Nanoclusters for Beef Freshness Monitoring. Foods, 12(7), 1464.
27. Nerin, C., Tovar, L., Djenane, D., Camo, J., Salafranca, J., Beltran, J. A., & Roncales, P. (2006). Stabilization of beef meat by a new active packaging containing natural antioxidants. Journal of Agricultural and Food Chemistry, 54(20), 7840-7846.
28. Bagdatli, A., & Kayaardi, S. (2015). Influence of storage period and packaging methods on quality attributes of fresh beef steaks. CyTA-Journal of Food, 13(1), 124-133.
29. Rovira, P., Brugnini, G., Rodriguez, J., Cabrera, M. C., Saadoun, A., de Souza, G., ... & Rufo, C. (2023). Microbiological changes during long-storage of beef meat under different temperature and vacuum-packaging conditions. Foods, 12(4), 694.
30. Kim, S., Kim, G., Moon, C., Ko, K., Choi, Y., Choe, J., & Ryu, Y. (2022). Effects of aging methods and periods on quality characteristics of beef. Food science of animal resources, 42(6), 953.
31. Hernandez, M. S., Woerner, D. R., Brooks, J. C., Wheeler, T. L., Legako, J. F., & Brooks, J. C. (2022). Influence of aging temperature and duration on spoilage organism growth, proteolytic activity, and related chemical changes in vacuum-packaged beef longissimus. Meat and Muscle Biology, 6(1).
