Discoloration Characteristics of 3 Major Muscles From Cattle During Cold Storage

Authors


Abstract

ABSTRACT:  Discoloration characteristics of 3 major muscles (LD, Longissimus dorsi; PM, Psoas major; SM, Semimemebranosus) from Korean native cattle (Hanwoo) were monitored during 7 d of cold storage at 4 °C. The muscles were obtained from 12 Hanwoo carcasses at 24 h postmortem. Meat color (CIE L*, a*, b*), myoglobin (Mb) concentration, chemical form, metmyoglobin (MetMb) reducing ability (MRA), mitochondria concentration, and thiobarbituric acid reactive substance (TBARS) were measured at 1, 3, 5, and 7 d of storage. Although there were no significant differences in CIE a* and b*-values between the 3 muscles at day 1, the values of PM muscle were significantly (P < 0.05) lower than those of LD and SM muscles at day 5 and 7. PM muscle showed a rapid decrease in the oxymyoglobin (OxyMb) and an increase in MetMb, which resulted in a significantly (P < 0.05) higher percentage of MetMb in PM muscle compared to LD and SM muscles. Also, the Mb and mitochondria concentration of PM muscle was significantly (P < 0.05) higher than those of LD and SM muscles. However, there were no significant differences in MRA, pH, or TBARS between the 3 muscles during 7 d of cold storage. It was concluded that rapid discoloration (that is, MetMb accumulation) in PM muscle of Hanwoo could be due to its higher contents of Mb and mitochondria.

Introduction

The color of fresh meat is considered to be the most important quality attribute for consumers. It is considered an indicator of freshness especially in red meat (beef, lamb, and so on). According to one report, surface discoloration in retail beef leads to revenue losses to the tune of around 15% in the U.S. meat industry (Smith and others 2000). Therefore, various research trials were carried out to identify the number of biochemical and physical factors affecting color and color stability. Meat color is influenced by many factors such as the concentration of haem pigments, particularly myoglobin (Mb), the chemical state of Mb, and the physical characteristics of the meat. On exposure to air, Mb combines with oxygen to form ferrous oxymyoglobin (OxyMb), which is bright red in color and presumed by consumers to indicate freshness. The extended contact of Mb with oxygen leads to the formation of the oxidized form, ferric metmyoglobin (MetMb), which is brown and unattractive. During cold storage, the rate of MetMb accumulation on the surface of meat is governed by many intrinsic factors (for example, pH, muscle metabolic type, animal age, breed, sex, and diet, and so on), extrinsic factors (for example, temperature, oxygen availability, type of lighting, surface microbial growth, and type of packing, etc) or by a combination of these factors (Renerre 1990). It is well established that beef muscles exhibit a wide range of color stability during cold storage (Renerre and Labas 1987). However, the mechanism of MetMb accumulation has not been well established. There are differences in Mb oxidation of bovine muscles (Trout and Gutzke 1995; Hwang and others 2007). Differences in Mb oxidation rate may vary depending on the type of muscle fiber and differences in the concentrations of endogenous antioxidants and pro-oxidants. MetMb reducing ability (MRA) is thought to prolong the color stability of muscles by reducing MetMb to Mb. Oxygen consumption rate is also identified as another major color determining characteristic (Madhavi and Carpenter 1993). According to Faustman and Cassens (1990), lipid and Mb oxidation are closely related in meat with an increase in one resulting in a similar increase for the other. Oxidation is thought to be related to direct Mb oxidation or destruction of Mb reducing systems by free radicals during lipid oxidation. Also, the color stability of muscles could be affected by the pH of muscles because MRA, oxygen consumption rate, and lipid oxidation are all influenced by changes in pH.

Recently, the beef industry in many countries is showing changes in trends towards marketing individual muscle cuts. The color characteristics of individual muscles and the factors responsible for discoloration are not well known, although the factors associated with MetMb accumulation on the surface of beef have been identified (McKenna and others 2005). Therefore, there is a need to characterize the color characteristics of individual bovine muscles. The objective of this study was to investigate the factors responsible for discoloration of 3 major muscles of Korean Hanwoo cattle meat during cold storage.

Materials and Methods

Sample preparation

Twelve Hanwoo carcasses were selected randomly from a commercial slaughterhouse at 24 h postmortem and 3 muscles including M. Longissimus dorsi, M. Psoas major, and M. Semimemebranosus were used to make steaks (3 cm thickness). Steaks from each muscle were placed in an oxygen permeable polyvinylchloride film, and stored at 4 °C for 7 d. Meat color (CIE L*, a*, b*), Mb, mitochondria concentration, percentage of Mb chemistry forms, thiobarbituric acid reactive substance (TBARS), and MetMb reducing ability (MRA) were measured at 1, 3, 5, and 7 d of storage. Three replicate measurements were taken for each of 12 replicates.

Color measurements

The meat color was measured on a surface of muscles using a Minolta Chromameter CR-300 (Minolta Co., Japan) that was standardized with a white plate (Y = 93.5, X = 0.3132, y = 0.3198).

Mb chemistry forms

The concentration of Mb forms was determined as described by Krzywicki (1982). Mb was extracted from meat samples with phosphate buffer of pH 6.8 and ionic strength of 0.04 (Warriss 1979). The final ratio of buffer to meat in extracts was 5:1. The absorbance of the extract was measured with a spectrophotometer (8453, UV-visible, Agilent Co., Palo Alto, Calif., U.S.A.) at 572, 565, 545, and 525 nm. A linear relationship between the absorbance and the concentration of Mb was checked for all wavelengths used. The concentration of Mb forms was calculated using the following equation:

image

where R1, R2, and R3 were absorbance ratio A572/A525, A565/A525, and A545/A525, respectively.

Mb concentration

Mb concentration was measured by the method of Warriss (1979) with modifications. Four grams of meat sample was homogenized with phosphate buffer of pH 6.8 and ionic strength of 0.04. After centrifuging the slurry at 5000 ×g for 30 min, the supernatant was filtered with Whatman No. 1 filter paper and the extract retained. One milliliter of Mb extract was oxidized with 100 μℓ of 60 mM K3F(III)(CN)6 and treated with 80 mM NaCN to measure the final volume cyanides to the addition of the cyanides. After the change in color from yellow to red with the addition of potassium cyanide, the absorbance was measured at 540 nm. Molar extinction coefficient of 11300 for cyanmetmyoglobin (CNMMb) was used to determine total molar concentration of Mb (Drabkin 1950). Molar concentrations were converted to milligrams of Mb by the following formulae:

image

where A540= absorbance at 540 nm, 11300 = molar extinction coefficient of CNMMb at 540 nm, 16800 = equivalent weight of the bovine Mb, 0.01 = volume of the Mb extract, 0.001 = volume of the potassium ferricyanide, and d= volume increase due to the addition of potassium cyanide.

Mitochondria concentration

Muscle mitochondria were isolated according to the Bhattacharya and others (1991) with modifications. Ten grams of muscle were finely minced in an initial isolation medium (100 mM sucrose, 10 mM EDTA, 100 mM Tris-HCl, pH 7.4). Prior to homogenization, the minced muscle was incubated in 20 mL of initial isolation medium. Twenty milligrams of nagarase solution were added into it and stirred in an ice blanket for 5 min. The nagarase solution was removed by rinsing in the initial isolation medium. The minced muscle was homogenized, and then the resulting homogenate was centrifuged at 1000 ×g for 10 min. The pellet was discarded and the supernatant was centrifuged at 10000 ×g for 15 min to obtain the mitochondrial pellet. The mitochondrial pellet was washed in 10 mL of initial muscle isolation medium containing 0.5% (w/v) (0.005 mg/mL) fatty acid free Bovine Serum Albumin. Finally, mitochondria were reharvested by centrifugation at 8000 ×g for 10 min in a 2nd muscle isolation medium (230 mM mannitol, 70 mM sucrose, 20 mM Tris-HCl, 5 mM KH2PO4, pH 7.4). The final pellet was resuspended in 1 mL of the 2nd isolation medium and protein content was determined.

Intramuscular fat content

The crude fat content was extracted from 3 g of the homogenized meat sample using the procedure of Folch and others (1957) with modification. Three grams of sample was combined with 30 mL of Folch solution I (chloroform: methanol, 2:1 [v/v]) and homogenized with a Polytron homogenizer (IKA Labortechnik T25-B, Selangor, Malaysia) for 30 s. The homogenate was filtered with Whatman No. 1 filter paper in a 100 mL measuring cylinder after stirring for 2 h at 4 °C. The filtered solution was stirred with 0.88% of NaCl (25% volume of the filtered solution), and then allowed to separate into 2 layers for 1 h at room temperature. After washing the wall of measuring cylinder with 10 mL of Folch solution II (chloroform: methanol: H2O = 3: 47: 50), the final volume of the lower layer was recorded. The upper layer (methanol and water layer) was removed using an aspirator, and 10 mL of the lower layer (chloroform containing lipid extracts) was taken in a dish to dry at 50 °C. The weight of dish was measured before and after drying, and the intramuscular fat content was calculated using the following equation:

image

where a= the final volume of the lower layer, b= weight of dish before drying, and c= weight of dish after drying.

Thiobarbituric acid reactive substances (TBARS)

TBARS was measured by the method of Buege and Aust (1978) with modifications. Five grams of meat were weighed into a 50 mL test tube and butylated hydroxyanisole (BHT; 50μℓ, 10%) added. The sample homogenized with 15 mL of deionized distilled water using polytron homogenizer for 15 s at the highest speed. Homogenated sample meat (2 mL) was transferred to a disposable test tube (3 × 100 mm), and thiobarbituric acid/tricholoroacetic acid (TBA/TCA) (4 mL) was added. The mixture was vortexed and then incubated in boiling water bath at 90 °C for 20 min to develop color. The sample was cooled in cold water for 10 min, vortexed again, and centrifuged for 15 min at 2000 ×g. The absorbance of the resulting supernatant solution was determined at 532nm against a bland containing 1 mL of distill water and 2 mL of TBA/TCA solution. The amounts of TBARS were expressed as milligrams of malondealdehyde (MA) per kilogram of meat.

MetMb reducing ability (MRA)

MRA was measured by the method of Lee and others (1981). Two grams of sample was homogenized with 10 mL of 25 mM PIPES buffer at pH 5.8. Five milliliters of homogenate were transferred to a 10 mL-volumetric flask and then 2 mL of 5 mM K2Fe (CN)6 were added. The mixture was incubated with occasional stirring for 1 h at 2–4 °C. After incubation, 0.1 mL of 5% ammonium sulfamated and 0.2 mL of 0.5 M lead acetate were added to the mixture, and then the mixture was allowed to stand for 5 min at room temperature. After 5 min, 2.5 mL of 20% TCA were added and brought up to volume with distilled water. After another 5 min, the mixture was filtered through Whatman filter paper No. 42. The absorbance of the filtrate was measured at 420 nm after 30 min from the end of the incubation at 2–4 °C. MRA was expressed as absorbance of 1 mM of K2Fe (CN)6 at 420 nm minus absorbance of the sample.

Statistical analysis

The data of this experiment was analyzed by the analysis of variance (ANOVA) procedure of statistical analysis systems (SAS 1999), and the Duncan's multiple range test was used to determine the significant differences among means at 5% level of significance (SAS 1999).

Results and Discussion

Changes in meat color measurements on the surface of Hanwoo muscles during cold storage are presented in Table 1. Lightness (L*-value) of M. Longissimus dorsi (LD) was significantly (P < 0.05) higher than that of M. Psoas major (PM) and M. Semimembranosus (SM). The L*-values of the 3 muscles did not significantly change during 7 d of cold storage. However, redness (a*-value) and yellowness (b*-value) of PM muscle were significantly (P < 0.05) decreased during cold storage. Although there were no significant differences in a* and b*-values among the 3 muscles at day 1, the values of PM muscle were significantly (P < 0.05) lower than those of LD and SM muscles at day 5 and 7. This suggests that color stability of PM muscle is less stable compared to LD and SM muscles. The lower a* and b* values of PM muscle are probably due to more accumulation of MetMb during cold storage. Although the CIE values (L*, a*, b*) have been found to be most correlated to visual determination of muscle surface color (Joo and others 1999). MetMb content has also been found to be slightly correlated with L*-value (Lindahl and others 2001). McKenna and others (2005) indicated that changes in L*-value during cold storage were very subtle and lightness appeared to play a minimal role in color stability of meat. In this study, it was also confirmed that the L*-values of 3 muscles were inconsistent during storage. Decreasing of a* and b* values in PM muscle was expected because the composition of muscle fiber type I, red muscle fiber, in PM muscle was higher than LD and SM muscles (Hwang and others 2007). Generally, muscles of low color stability have more oxidative muscle fibers, which are red fibers, whereas muscles of high color stability have more glycolytic muscle fibers, which are white fibers. Oxygen storage by Mb is consistent with the high proportion of enzymes involved in oxidative metabolism and the low levels of glycolytic enzymes found in red fibers (Aberle and others 2001). The mitochondria consisting of oxidative enzymes are more numerous and large in size in red muscle fibers than white muscle fibers. It was also observed that the Mb and mitochondria concentration of PM muscle was significantly (P < 0.05) higher than those of LD and SM muscles (Table 2). Therefore it could be possible that the rapid decreasing of a* and b* values in PM muscle is due to more active oxidative metabolism with higher concentrations of Mb and mitochondria (Hwang and others 2007).

Table 1—.  Changes in surface meat color of cattle muscles during cold storage.
TreatmentStorage (d)
1357
  1. Values are the means ± SD.

  2. 1LD: longissimus dorsi, PM: Psoas major, SM: Semimemebanosus.

  3. a,b,cMeans in the same row with different letters are not significantly different (P < 0.05).

  4. A,B,CMeans in the same column with different letters are not significantly different (P < 0.05).

L*LD142.8 ± 2.8A44.1 ± 3.4A42.1 ± 2.544.6 ± 3.1A
PM 41.5 ± 1.8AB40.3 ± 1.5B42.3 ± 3.141.3 ± 1.6B
SM39.9 ± 2.7B39.7 ± 2.4B41.3 ± 2.540.6 ± 2.3B
a*LD26.7 ± 2.4 26.2 ± 2.4  25.7 ± 2.0A24.9 ± 1.6A
PM 27.3 ± 2.4a24.0 ± 2.4b  21.7 ± 1.6Bc 21.4 ± 1.5Bc
SM25.2 ± 3.2 24.4 ± 2.9  24.5 ± 2.6A24.6 ± 2.3A
b*LD13.7 ± 1.3 13.6 ± 1.1A 13.3 ± 0.8A13.2 ± 1.2A
PM13.1 ± 1.0a 11.5 ± 0.7Bb  10.9 ± 0.6Bbc 10.5 ± 0.8Bc
SM12.7 ± 1.6 11.8 ± 1.7B 12.6 ± 1.4A12.4 ± 1.6A
Table 2—.  The contents of intramuscular fat, myoglobin, and mitochondria in 3 major muscles of cattle.
MusclesIntramuscular fat (%)Myoglobin (mg/g)Mitochondria (mg/kg)
  1. Values are the means ± SD.

  2. 1LD: Longissimus dorsi, PM: Psoas major, SM: Semimemebranosus.

  3. A,B,C Means in the same column with different letters are not significantly different (P < 0.05).

LD113.9 ± 2.4A7.33 ± 0.77B15.1 ± 3.0B
PM10.8 ± 2.6B8.87 ± 0.41A18.8 ± 2.6A
SM 7.3 ± 2.2C7.89 ± 0.72B 17.1 ± 1.2AB

Changes in the percentage of OxyMb (OxyMb%) and MetMb (MetMb%) among Hanwoo muscles were observed during 7 d of cold storage (Figure 1). OxyMb percent of PM muscle decreased rapidly compared to LD and SM muscles (P < 0.05), which substantiates our previous observations. Although an increase in MetMb percent was observed in all 3 muscles during cold storage, MetMb percent of PM muscle was significantly (P < 0.05) higher than that of LD muscle. Also, there were significant (P < 0.05) differences in OxyMb percent between PM muscle and other muscles during the entire length of cold storage. These results were consistent with those of McKenna and others (2005) who reported discoloration patterns of 19 bovine muscles. According to their discoloration categories for bovine muscles, LD, SM, and PM muscles exhibited high, intermediate, and very low color stability, respectively.

Figure 1—.

Changes in OxyMb percent (A) and MetMb percent (B) of cattle muscles for 7 d of cold storage.

The low color stability of PM muscle compared with other muscles has also been reported by other researchers (Bendall and Taylor 1972; O'Keefe and Hood 1982; Renerre and Labas 1987; Echevarne and others 1990). O'Keefe and Hood (1982) reported that PM muscle had higher enzymic activity than LD muscle This resulted in relatively higher oxygen consumption rate (OCR) with consequently low oxygen penetration depth (OPD) and rapid conversion of OxyMb to Mb, predisposing PM to rapid. Research findings have suggested that OPD values for PM muscle are lower than those reported for LD muscle (Bendall and Taylor 1972; MacDougall and Taylor 1975). Lanari and Cassens (1991) also reported that differences in the OCR of the muscle mitochondria could account in part for the effect of muscle type and breed on beef discoloration rate.

MRA of Hanwoo muscles was significantly (P < 0.05) decreased with increasing storage time, and irrespective of muscle types. During the first 3 d, there was a slight decrease in MRA value, however MRA value decreased significantly (P < 0.05) at 5 and 7 d of cold storage for all the 3 muscles (Table 3). Possible enzymatic pathways of MetMb reduction have been studied by many researchers (O'Keefe and Hood 1982; Renerre and Labas 1987; Faustman and Cassens 1990; Arihara and others 1995; Sammel and others 2002) and it is now accepted that the reduction process in meat is primarily enzymatic in nature with Nicotinamide adenine dinucleotide (NADH) as co-enzyme present in mitochondrial intermembrane space involved in MRA with the conversion of ferric Mb to its ferrous form. However, observation in this study suggests that NADH would not be involved with MRA during postmortem 3 d in relation with time to loss of structural integrity and functional properties of mitochondria. Our results are not in agreement with Bekhit and others (2001) who reported that MRA remained stable during postmortem storage and was not the principal determinant of color stability.

Table 3—.  Changes in metmyoglobin reducing ability of cattle muscles during cold storage.
MusclesStorage (d)
1357
  1. Values are the means ± SD.

  2. 1LD: Longissimus dorsi, PM: Psoas major, SM: Semimemebranosus.

  3. a,b,cMeans in the same row with different letters are not significantly different (P < 0.05).

LD10.46 ± 0.04a0.43 ± 0.05ab0.41 ± 0.04bc0.38 ± 0.01c
PM0.45 ± 0.05a0.47 ± 0.05a0.43 ± 0.05ab0.37 ± 0.01b
SM0.47 ± 0.03a0.44 ± 0.05ab0.43 ± 0.07b0.37 ± 0.03c

Renerre (1990) suggested that muscles with elevated mitochondrial concentrations will be highly oxidative and therefore will have low color stability. Muscle with low color stability had the highest MRA, whereas more color stable muscle had lower MRA (McKenna and others 2005). However, our results showed that all 3 muscles of Hanwoo had similar MRA during postmortem period. This implied that differences in color stability between Hanwoo muscles might not be affected by the MRA of muscles although decreasing MRA was related to accumulation of MetMb during cold storage.

On the other hand, the color stability of muscles could be affected by pH and lipid oxidation of muscle. MRA, MetMb accumulation, and lipid oxidation could be influenced by changes in muscle pH. However, in this study, there were no significant changes in pH between all muscles during 7 d of cold storage (data not shown). Moreover, no significant difference in TBARS values between all muscles was observed (Table 4) although intramuscular fat contents were significantly different (Table 2). According to Faustman and Cassens (1990), lipid oxidation and Mb oxidation were closely related in meat with an increase in one resulting in a similar increase for the other. This was thought to be related to direct Mb oxidation or destruction of Mb reducing systems by free radicals during lipid oxidation. McKenna and others (2005) reported that PM muscle was susceptible to autoxidation at earlier days during retail display whereas LD and SM muscles showed longer lag phases before oxidative rancidity by-products began accumulating. However, our results did not agree with their findings, and suggest that the differentiation of Mb oxidation on Hanwoo muscles was related more to Mb and mitochondria contents than to MRA, pH, or lipid oxidation of muscles.

Table 4—.  Changes in thiobarbituric acid reactive substances (mg MA/kg) of cattle muscles during cold storage.
MusclesStorage (d)
1357
  1. Values are the means ± SD.

  2. 1LD: Longissimus dorsi, PM: Psoas major, SM: Semimemebranosus.

  3. a,b,cMeans in the same row with different letters are not significantly different (P < 0.05).

LD1 0.16 ± 0.03c0.35 ± 0.12b0.43 ± 0.04ab0.54 ± 0.18a
PM0.14 ± 0.02c0.37 ± 0.08b0.49 ± 0.07ab0.57 ± 0.14a
SM0.12 ± 0.03c 0.33 ± 0.09b0.45 ± 0.12b0.52 ± 0.16a

Conclusions

PM muscle showed rapid decreases in redness and yellowness values during 7 d of cold storage. Also, among the 3 major muscles of Hanwoo, PM muscle showed the most rapid increase in percentage of MetMb and had higher concentration of Mb and mitochondria compared to LD and SM muscles. MRA of PM muscle was not different from LD and SM muscles, although the values of MRA of all the 3 muscles were decreased during cold storage. There were no differences in pH and TBARS values between muscles during cold storage. Therefore, it is concluded that rapid discoloration (that is MetMb accumulation) of PM muscle could be due to its higher contents of Mb and mitochondria among 3 Hanwoo muscles.

Acknowledgments

The authors acknowledge a graduate fellowship provided by the Ministry of Education and Human Resources Development, Korea. Jin-Yeon Jeong, Young-Hwa Hwang, and Gab-Don Kim were supported by a scholarship from the BK21 Program.

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