J. Anim. Sci. 2003. 81:1885-1894
© 2003 American Society of Animal Science
The effect of vitamin E supplementation on discoloration of injection-site lesions in retail cuts and the greening reaction observed in injection-site lesions in muscles of the chuck1
D. L. Roeber*,2,
K. E. Belk*,3,
T. E. Engle*,
T. G. Field*,
S. R. Koontz
,
J. A. Scanga*,
J. D. Tatum*,
G. L. Mason
,
D. Van Metre
,
F. B. Garry and
G. C. Smith*
* Department of Animal Sciences;
and
Department of Agricultural and Resource Economics;
and
Department of Microbiology, Immunology, and Pathology; and
and
Department of Clinical Sciences, Colorado State University, Fort Collins 80523-1171
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Abstract
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Concern has been raised about green discoloration of injection-site lesions in chuck muscles in modified-atmosphere packages. Objectives were: 1) to recreate green lesions, 2) to compare the severity of discoloration of injection-site lesions in chucks from carcasses of control or vitamin E-supplemented steers, and 3) to identify pigment(s) responsible for discoloration via in vitro color reactions. In Exp. 1, 23 steers (BW = 415 kg; 37 d before harvest) were injected with one of 12 pharmaceuticals, following label directions for route and dose, with the exception of a 5-mL maximum dose, to identify a product that could result in discoloration. Two vaccines (Products A and B) resulted in greening. In Exp. 2, 50 steers were injected (i.m.) with Product A and assigned to the control or vitamin E (1,000 IU/steer daily for 60 d) group. After retail display, 80 and 72% of steaks from the control and treatment groups, respectively, were discolored. Although vitamin E did not reduce (P = 0.53) greening, there was a trend (P = 0.10) toward delay discoloration of lesions from the treatment group. In Phase I of Exp. 3, pigments extracted from green lesions obtained from Exp. 2 were compared with solutions, exposed to a high partial pressure of oxygen (ppO), of myoglobin (Mb), copper sulfate, hydrogen peroxide (H2O2), vaccine, and aluminum hydroxide either alone or in combination. In Phase II of Exp. 3, solutions of two or more of Mb, Cu, sodium sulfide, sodium sulfite, sodium sulfate (Na2SO4), and H2O2 were made at pH 7.2 or 5.5 and exposed to low or high ppO. Normal muscle tissue displayed a 3.2 and 56.7% decrease in absorbance/µg of protein as wavelength changed from 654 to 656 nm and 656 to 658 nm, respectively. Pigments from control and treatment group green tissue displayed a 164.5 and 621.3% increase, respectively, in absorbance/µg of protein as wavelength changed from 654 to 656 nm. As wavelength changed from 656 to 658 nm, the absorbance/µg of protein for control and treatment group lesions decreased by 75 and 109%, respectively. The Mb+Cu+Na2SO4 solution, at pH 5.5 and high ppO, exhibited similar absorbance trends as green lesions indicating that greening may result from a Mb, Cu, and Na2SO4 interaction. Results indicated that greening varies with pharmaceuticals and oxidation of tissue cannot be controlled with vitamin E supplementation. Research on the causative agents of green discoloration, with an emphasis on compounds containing sulfate or Cu, is needed.
Key Words: Discoloration • Intramuscular Injection • Muscle Tissue • Subcutaneous Injection • Vitamin E
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Introduction
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Incidence of injection-site lesions in top sirloin butts of fed steers and heifers declined from a high of 21.3% in July 1991 (Dexter et al., 1994
) to 2.1% in July 2000 (Roeber et al., 2001). However, during the 2000 National Beef Quality Audit, concern was raised by a packer about green discoloration that occurs in injection-site lesions in muscles of the chuck in high-oxygen, modified-atmosphere packages (Smith et al., 2001).
Most green discoloration in muscles of fresh meat is associated with an alteration (saturation of a double bond) in heme structure (Lawrie, 1998) and is normally attributed to hydroperoxymetmyoglobin, sulfmyoglobin, or choleglobin pigments (Price and Schweigert, 1987).
Vitamin E has been used as a dietary supplement for steers because of its antioxidant properties, which help to delay muscle discoloration (Faustman et al., 1989a; Faustman and Wang, 2000). Some studies have used high levels of vitamin E supplementation with conventional feeding times (2,000 to 3,000 IU/d for 30 to 100 d preharvest), whereas others have explored the use of lower supplementation levels (300 to 500 IU/d) with extended time on feed (210 to 310 d) (Faustman et al., 1989b; Arnold et al., 1992; Smith et al., 1996). Delmore et al. (1998) reported that supplementation of cows with 50,400 IU of 
-tocopheryl acetate, even in short-term feeding (900 IU/d for 14 d), resulted in an improvement in retail caselife of beef from those cows.
The objectives of this study were: 1) identify a pharmaceutical product that could be used to recreate green discoloration in injection-site lesions, 2) compare severity of green discoloration of injection-site lesions in chucks from carcasses of steers that were or were not supplemented with vitamin E, and 3) determine the muscle pigment (hydroperoxymetmyoglobin, sulfmyoglobin, or choleglobin) potentially responsible for the green discoloration occurring in injection-site lesions by studying color reactions in vitro.
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Materials and Methods
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The protocols for Exp. 1 and 2 described below were conducted according to the guidelines stated in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 1999).
Experiment 1. Determination of the Pharmaceutical Product
Pharmaceutical companies and veterinarians were interviewed to estimate the market share for various pharmaceutical products used in the feedlot industry. Twelve products were chosen for evaluation based on market share, the class of product, and, if applicable, type of adjuvant used in the product. Eleven commercially available products (various vaccines, parasiticides, and antimicrobials) were designated as Products A through K. The twelfth product was an aluminum hydroxide gel adjuvant (Alyhydrogel 1.3%; Accurate Chemical and Scientific Corp., Westbury, NJ).
Twenty-three steers (average BW = 415 kg) were vaccinated 37 d before harvest with one of the 12 products (two animals per product, with the exception of only one animal being vaccinated with Alyhydrogel) in front of the point of the shoulder to identify a product that could be used to recreate the green discoloration in muscles of the chuck after being stored in a high-oxygen, modified-atmosphere package. Products labeled only for s.c. injection were administered as such with a 1.9-cm, 16-gauge needle; those labeled only for i.m. injection were administered as such with a 2.5-cm, 16-gauge needle; and those with a i.m./s.c. joint label were administered i.m. with a 2.5-cm, 16-gauge needle. Product administration followed label directions with the following exceptions: 1) Product A did not clear withdrawal before harvest, 2) Product B did not clear withdrawal before harvest, 3) only 5 mL of a 9-mL dosage of Product C was administered, 4) only 5 mL of a 13-mL dosage of Product F was administered, 5) only 5 mL of a 27-mL dosage of Product I was administered, and 6) all s.c. injections were administered using the non-tented technique by a licensed veterinarian.
Steers were harvested at the Colorado State University Meat Laboratory, chilled for 48 h, fabricated to generate chuck steaks, and then all cuts and byproducts were condemned. Chuck steaks (2.5-cm thick) were inspected for an injection-site lesion. Two steaks per chuck containing an injection-site lesion were placed on a styrofoam tray, overwrapped, and placed in a high-oxygen master pack at King Soopers (Denver, CO). When a lesion was not visible in the chuck, one steak was removed from the chuck approximately 15 cm posterior to the neck end (approximately the same location as the location of the injection on the live animal) and placed in a high-oxygen master pack. Following packaging, master packs were held in boxed storage for 5 d and then placed on tables in a cooler maintained at 2 ± 1°C under 24-h/d lighting conditions (Phillips delux warm white fluorescent lamps; Phillips, Somerset, NJ); the surface of the meat was exposed to 900 to 1365 lx), as recommended by AMSA (1991) as appropriate for simulation of retail display for 3 d.
Color Evaluation.
Injection-site lesions were subjectively monitored (0 = no discoloration; 1 = green discoloration) for greening by a trained individual at the time of packaging, at 6, 12, 24, 48, 72, and 96 h after packaging during boxed storage, and at 120 h, which represented the completion of boxed storage and the beginning of simulated retail display. Steaks also were evaluated every 12 h during simulated retail display.
Experiment 2. Effect of Vitamin E Supplementation on Green Discoloration
Cattle Selection and Administration of Injections.
Fifty yearling steers with known treatment histories (no steers previously receiving injections in the chuck and/or all previous injections administered on the left side of animals; right side was used for experimentation) were selected at a cooperating feedyard. Each of the 50 steers received a 5-mL intramuscular injection of Product A, administered by a collaborating veterinarian, on the right side in the serratus ventralis muscle immediately anterior to the shoulder blade and as close to the spinal column as was practical. The intramuscular vaccination was administered just anterior to the shoulder blade, the location in which the discolored lesions, when packaged in a high-oxygen, modified-atmosphere environment, have been identified. After injection, steers were randomly allocated to either the control (n = 25) or treated (vitamin E-supplemented, n = 25) group. Steers were fed in separate group pens and followed through the feeding phase to ensure that all other feedlot processing vaccinations were administered to the animal in the neck region on the opposite (left) side.
Vitamin E Supplementation.
Steers (n = 25) were supplemented with 1,000 IU/animal daily of vitamin E, which was top dressed on daily rations for 60 d (total of 60,000 IU/animal during finishing) before harvest.
Chuck Selection.
Steers from each group (control and treated) were shipped to harvest, and tag transfer was completed by Colorado State University personnel on the harvest floor. Chucks were tagged, identified, and tracked through fabrication. Chucks (NAMP 113; NAMP, 1997) were boxed, shipped to Colorado State University, and stored (2°C) in vacuum packages until 4 d postmortem. At 4 d postmortem, chuck steaks (2.5 cm thick) were inspected for an injection-site lesion. Two steaks from each of the chucks in which a lesion was visually identified were placed in high-oxygen, modified-atmosphere packages using the Reiser portioner and white barrier stryofoam trays (Sealed Air Corp., Duncan, SC) and placed into boxed storage. When an injection-site lesion was not visible in the chuck, one steak was removed from the chuck approximately 15 cm posterior to the neck end (approximate location of where the injection was given on the live animal) and placed in a high-oxygen, modified-atmosphere package for boxed storage.
Boxed Storage and Retail Case Display.
Packaged chuck steaks were placed in boxed storage for 3 d to simulate the typical amount of time that product is in transit from packaging until being placed in a retail display case. After 3 d of boxed storage, each chuck steak was placed on a table, in a cooler maintained at 2 ± 1°C, under lighting 24-h/d conditions (Phillips delux warm white florescent lamps; the surface of the meat was exposed to 900 to 1,365 lx) as recommended by AMSA (1991) to simulate retail display conditions.
Color Evaluation.
The color of each steak prior to packaging was measured using a HunterLab MiniScan XE handheld spectrophotometer equipped with a 6-mm aperture (HunterLab Associates, Inc., Reston, VA) to determine values for CIE L* (brightness; 0 = black, 100 = white), a* (redness/greenness; positive values = red, negative values = green), and b* (yellowness/blueness; positive values = yellow, negative values = blue) following procedures of the Commission Internationale del’Eclairage (CIE, 1976). Three readings for each of L*, a*, and b* were averaged for each injection-site lesion in the steak and from a comparable area in steaks with no visible lesion before high-oxygen packaging and after retail display. In addition, L*, a*, and b* readings were obtained in all steaks from muscle tissue that did not have a lesion and did not exhibit the greening reaction.
Color was subjectively evaluated (0 = no discoloration, 1 = green discoloration) by a trained individual at the time of packaging, at 6, 12, 24, and 48 h after packaging during boxed storage, and at 72 h, which represented the completion of boxed storage and the beginning of simulated retail display. Steaks also were subjectively evaluated for color every 12 h during simulated retail display.
Thiobarbituric and Collagen Analysis.
When chucks contained three steaks with visible injection-site lesions (n = 27; n = 15 from the vitamin E treatment group, n = 12 from the control group), the third steak was packaged in a high-oxygen, modified-atmosphere package and shipped to Food Safety Net Services (San Antonio, TX) for thiobarbituric (TBA) analysis (Tarladgis et al., 1960). Thiobarbituric acid analysis was conducted using samples from the location of the "greening" and from samples obtained 6 cm away from the green area to determine if differences in TBA values were associated with the extent/occurrence of discoloration. A 10-g sample for TBA analysis was taken from each steak at the location of the greening; the sample included surface discoloration and the center of the steak. Additionally, 9 of the 27 steaks (n = 5 from the vitamin E treatment group, n = 4 from the control group) were selected for hydroxyproline determination to assist in confirming the presence of injection-site lesions (Procedure AOAC #990.26; AOAC, 2000). Steaks for hydroxyproline determination were selected based on the location of the lesions. Hydroxyproline analysis was conducted using samples obtained at two locations from each of the nine steaks; locations were at the center of the lesion and at a point approximately 6 cm away from the center of the lesion in the same muscle.
Histopathologic Examination.
Following retail display, five chuck steaks per control and treatment group were evaluated by light microscopy at the injection-site lesion. Sections of chuck steak containing green injection-site lesions and grossly normal sections of the same muscle were fixed in 10% neutral buffered formalin for more than 24 h, routinely processed, paraffin embedded, cut at 5 µm, and stained with hematoxylin and eosin.
Statistical Analysis.
Chi squared analysis was used to test for differences in the frequency of green discoloration between cuts from steers that were and were not supplemented with vitamin E. Analysis of variance was used to test for differences in the amount of time necessary for green discoloration of injection-site lesions to be observed in the serratus ventralis muscle between steers that were or were not provided supplemental vitamin E. The ANOVA model included dietary vitamin E supplementation level (0 or 1,000) as a fixed main effect, and retail display time as a repeated measure. Data were analyzed using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC); a pairwise t-test was used to identify differences in mean values when the model demonstrated a treatment effect ( = 0.05).
Experiment 3. Determination of the Greening Reaction Observed in Injection-Site Lesions
Materials.
Product A was chosen for use during the in vitro determination to create the chemical reaction resulting in the green discoloration of injection-site lesions based on Exp. 1. All chemicals used were reagent grade or better and were purchased from Fisher Scientific (Houston, TX). Double-deionized water was used throughout the study. Myoglobin was from horse skeletal muscle (ICN Biomedical, Aurora, OH) and was used without further purification. Fresh 1 and 3% H2O2 was prepared for each phase of the experiment by diluting stock solution with deionized water. All experiments were conducted at room temperature.
Phase I. Preparation of "Green" Injection-Site Lesions.
Twenty (n = 10 lesions each from chuck steaks from steers in the control and Vitamin E treatment groups) green injection-site lesions from Exp. 2, as well as 18 green lesions supplied by a cooperating packing plant, were extracted from affected steaks using a sterile knife. Myoglobin pigments were extracted from the muscle using centrifugation as described by Smith and Carpenter (1970).
In Vitro Reactions.
Solubilized myoglobin was prepared according to Morey et al. (1973). In vitro solutions of muscle (MSC) or myoglobin (Mb), 3% hydrogen peroxide (H2O2), 1.3% aluminum hydroxide (AlOH), 1 ppm of copper sulfate (CuSO4), vaccine (VAC), and combinations of two or more of the solutions were made and are outlined in Table 1. Each solution was replicated eight times. Solutions were subjected to high partial pressures of oxygen by pulling a vacuum on each tube and then flushing the tube with oxygen for 24 h before further evaluation. Sulfmyoglobin and hydroperoxymetmyoglobin were prepared according to the protocol outlined by Michel (1938) and Morey et al. (1973), respectively.
Spectrophotometric Analysis.
Following storage in the high-oxygen environment, 1 mL of each solution replicate was analyzed using the Hewlett Packard spectrophotometer (HP89532R UV-Visible spectrophotometer; Wilmington, DE) to obtain absorbance values from 430 to 670 nm. Protein concentration was determined according to Bradford (1976). Protein concentrations were used to calculate absorbance/µg of protein ratios for statistical analysis.
Statistical Analysis.
Ratios of absorbance/µg of protein and changes in absorbance/µg of protein ratios from two sequential wavelengths were analyzed using the GLM procedure of SAS. The model included the presence of Mb/MSC, VAC, CuSO4, H2O2, and AlOH as fixed main effects. A pairwise t-test was used to separate means when the model demonstrated a significant main effect ( = 0.05).
Phase II. In Vitro Reactions.
In vitro solutions of two or more of Mb, H2O2, 1 ppm of copper (Cu), 0.3 ppm of sodium sulfide (Na2S), 0.3 ppm of sodium sulfite (Na2SO3), and/or 0.3 ppm of sodium sulfate (Na2SO4) were made and are outlined in Table 2. Each solution was replicated three times in a 2 x 2 factorial arrangement. Solutions were mixed at one of two pH levels (pH 5.5 or 7.2) and were subjected to one of two partial pressures of oxygen (high partial pressure or environmental pressure) for 24 h before further evaluation.
Spectrophotometric Analysis.
Following storage in the appropriate environment, 1 mL of each solution replicate was analyzed using the Hewlett Packard spectrophotometer (HP89532R UV-Visible spectrophotometer; Wilmington, DE) to obtain absorbance values from 640 to 670 nm. Protein concentration was determined as previously described. Protein concentrations were used to calculate absorbance/µg of protein ratios for statistical analysis.
Statistical Analysis.
Ratios of absorbance/µg of protein and changes in absorbance/µg of protein ratios from two sequential wavelengths were analyzed using the GLM procedure of SAS. The model included the presence of Mb, Cu, Na2S, Na2SO3, Na2SO4, and H2O2 as fixed main effects. A pairwise t-test was used to separate means when the model demonstrated a significant main effect ( = 0.05).
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Results and Discussion
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Experiment 1. Determination of the Pharmaceutical Product
Injection-site lesions were visible in 87% of the chucks. Both steaks from each of the two chucks obtained from the steers vaccinated with Product A and one pair of steaks from a chuck obtained from a steer vaccinated with Product B exhibited the green discoloration after 48 h of boxed storage. After an additional 3 d of boxed storage and 3 d of simulated retail display, no additional steaks revealed the green discoloration in the muscles of the chuck, indicating that discoloration occurs in a relatively short period of time after the muscle surface is exposed to the high-oxygen environment. Both products generating green discoloration of injection-site lesions included Xtend III as a mineral oil-based adjuvant in the product. Results indicated that certain pharmaceutical products could cause the discoloration, whereas others may not. These differences in products may be a result of product formulation and/or a result of the immune response elicited in the steers when treated with various products.
Experiment 2. Effect of Vitamin E Supplementation
The results of Exp. 1 indicated that the green discoloration could be recreated with Product A or B. Given these data, Product A was used to vaccinate 50 cattle in order to determine whether the supplementation of vitamin E could be used to eliminate or reduce the oxidation reaction that was occurring in the high-oxygen, modified-atmosphere packages.
Carcass Characteristics.
There were no differences in carcass characteristics between the control and vitamin E-supplemented groups of fed steers. Average carcass traits for control and Vitamin E-treated steers, respectively, were: hot carcass weights, 366.4 ± 25.0 kg and 372.0 ± 26.6 kg; longissimus muscle areas, 87.0 ± 6.5 cm2 and 84.8 ± 7.7 cm2; fat thickness, 1.3 ± 0.3 cm and 1.4 ± 0.3 cm; USDA yield grade, 2.83 ± 0.46 and 3.09 ± 0.49; and marbling score, 354.8 ± 41.6 (Slight 54) and 380.4 ± 48.7 (Slight 80).
Boxed Storage and Retail Display.
The percentages of chuck steaks with a green injection-site lesion at each retail boxed storage and retail display time, by treatment, are presented in Figure 1. No steaks exhibited the greening reaction until 48 h of boxed storage time. At the end of boxed storage, there was no difference (P = 0.95) in the percentage of steaks in the treatment vs. the control group exhibiting greening characteristics from control (48%) and treated (44%) steers (Figure 1). Following 96 h of retail display, 80% of steaks from control and 72% of steaks from treated steers exhibited the greening reaction at the site of the injection (Figure 1). The percentage of steaks that initially showed the greening characteristics in boxed storage vs. first showing the greening characteristics during retail display are presented in Table 3. The difference in average time (Table 3) when lesions turned green between chuck steaks from control and vitamin E-supplemented steers did not differ (P = 0.10), indicating that green discoloration was not delayed in the vitamin E supplementation group. Of those steaks that eventually exhibited greening, 39.5% (n = 8 steaks from the control group, n = 7 steaks from the vitamin E group) of the steaks did not have a visible lesion at the time of packaging (data not presented in tabular form). Sixty-five percent (n = 15) of the steaks that did not have a visible lesion (n = 23) at the time of high-oxygen, modified-atmosphere packaging turned green.
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Figure 1. The percentage of chuck steaks exhibiting the greening reaction at each color evaluation stratified by control (CONTROL) vs. treatment groups (VIT E). BS represents the evaluations during boxed storage; RD represents the evaluations during simulated retail display. No differences were detected between control vs. treatment groups at any time period.
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Table 3. Overall percentages of chuck steaks exhibiting the greening reaction, percentage of steaks exhibiting greening that turned green during boxed storage, percentage of steaks exhibiting greening that turned green during retail display, and the least squares means for hours until greening stratified by control vs. vitamin E-supplemented-groupsa
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Color attributes (L*, a*, and b*) of injection-site lesions did not differ (P > 0.05) before packaging or after retail display between steaks for control and treated steers (Table 4). However, the difference in a* between muscle tissue and injection-site lesion tissue after retail display was greater (P < 0.05) in steaks from control vs. treated steers (Table 4). The difference between a* values at the location of the injection-site lesion and normal tissue after display was 6.55 ± 1.29 for the control steers vs. 5.71 ± 1.29 for the treatment steers (Table 4). From an objective perspective, these values indicated that discoloration of lesions when compared to normal tissue was more visible in steaks from the control steers than for steaks from the vitamin E-supplemented group.
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Table 4. Least squares means for CIE L*, a*, and b* color attributes and mean differences between muscle color and lesion color after retail display stratified by control and vitamin E-supplemented steersa
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Thiobarbituric acid values were higher (P < 0.05) in steaks from control steers at the site of the green injection-site lesion and at a point 6 cm away from the injection-site lesion, than in steaks from treated steers. Steaks from control steers had TBA values of 1.576 ± 0.100 and 0.947 ± 0.050 at the injection-site lesion and at a point 6 cm away from the lesion, respectively. Values for TBA in steaks from treated steers were 0.400 ± 0.090 and 0.239 ± 0.050 at the site of the lesion and 6 cm away, respectively. Steaks from vitamin E-supplemented steers are indicative of diminished lipid oxidation compared to steaks from control steers. Since there was less lipid oxidation in green injection-site lesions of treatment steaks than in control steaks, the green discoloration may not be related to lipid oxidation.
George et al. (1995b) documented that hydroxyproline concentrations were elevated at the center of injection-site lesions compared with undamaged muscle tissue. Hydroxyproline levels were elevated (P < 0.05) at the location of greening vs. a point 6 cm away from the greening site in steaks from the control and treated groups, indicating that the greening reaction occurred at the location of the injection-site lesion.
Histopathologic Evaluation.
Figure 2 illustrates a green injection-site lesion after retail display and microscopic views of a cross section of normal muscle tissue and a green injection-site lesion. There were no histopathologic differences in injection-site lesions due to vitamin E supplementation. Lesions were characterized by expansion of fascial planes by fibrosis, macrophages, small lymphocytes, and lesser numbers of multinucleate giant cells, plasma cells, and eosinophils. Macrophages and multinucleate giant cells often surrounded or contained variably sized, clear, round vacuoles, interpreted as a lipid component of adjuvant. Fibrosis and inflammatory cellular infiltrate often dissected into the adjacent endomysium entrapping atrophied and regrenerating muscle fibers. George et al. (1995a,b) reported similar histopathologic findings in injection-site lesions administered at branding and weaning, as well as at sites 2.54 and 5.08 cm away from the center of injection-site lesions, indicating that tissue damage is not limited to the visible injection-site lesion, but is common around the lesion site.
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Figure 2. Characteristic color of the greening reaction observed in injection-site lesions after being packaged in a high-oxygen environment (a). Photomicrograph of normal muscle in cross section, stained with hematoxylin and eosin, illustrates normal fibers and little connective tissue (arrows) (b). Bar = 100 µm. Photomicrograph of injection-site lesion stained with hematoxylin and eosin (c). Pre-existent tissue planes are expanded by sheets of inflammatory cells enclosing lipid droplets (heavy arrow). Fibrosis dissects into adjacent muscle tissue entrapping atrophied myofibers (thin arrow). Bar = 50 µm.
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Experiment 3. Determination of the Greening Reaction
The objective of Exp. 3 was to determine the pigment or chemical reaction responsible for the greening of injection-site lesions observed in Exp. 1 and 2. In order to determine the potential reaction occurring, the pigment extracted from green injection-site lesions was compared to characteristics of in vitro solutions.
Characteristics of Green Injection-Site Lesions.
Figure 3 displays absorbance characteristics of green injection-site lesions of steaks obtained from control and vitamin E-supplemented steers in Exp. 2. The absorbance/µg of protein ratio of the green pigments from injection-site lesions from control and treated steers displayed, on average, a 164.5 and 621.3% increase (P < 0.05) as the wavelength changed from 654 nm to 656 nm, respectively. The absorbance/µg of protein for the lesions from control and treated steers declined (P < 0.05) 75 and 109% as the wavelength changed from 656 nm to 658 nm, respectively. In addition to previously documented absorbance/µg of protein ratio peaks at 617 nm and 589 nm (Nicol et al., 1970; George and Irvine, 1952, respectively), the absorbance/µg of protein ratios of sulfmyoglobin and hydroperoxymetmyoglobin showed an additional peak at 656 nm. The changes observed in the absorbance/µg of protein in the pigments extracted from green injection-site lesions matched the changes in absorbance/µg of protein observed in the sulfmyoglobin and hydroperoxymetmyoglobin pigments. The absorbance/µg of protein ratio of sulfmyoglobin increased 52.8% as wavelength changed from 654 nm to 656 nm, and then declined 125.3% as the wavelength changed from 656 nm to 658 nm. A similar trend was observed for hydroperoxymetmyoglobin where the absorbance/µg of protein increased 15.4% as wavelength changed from 654 nm to 656 nm, and then decreased 38.8% as wavelength changed from 656 nm to 658 nm. The greening reaction observed in lesions under a high-oxygen, modified-atmosphere environment also caused an increase in the absorbance/µg of protein ratio from 654 nm to 656 nm, followed by a decline in the ratio from 656 nm to 658 nm.
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Figure 3. Ratios of absorbance/µg of protein for green injection-site lesions from chucks from control steers and from vitamin E-supplemented steers. C.Les = lesions from chucks from control steers; E.Les = lesions from chucks from steers supplemented with vitamin E.
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Phase I.
Given the characteristics of the pigments from green injection-site lesions, solutions were prepared and absorbance values determined in order for comparisons between green injection-site lesions and solutions to be made. The absorbance/µg of protein ratio for Phase-I solutions demonstrated that, in comparison to the characteristics of the green injection-site lesions, the green pigment was not a result the presence of AlOH. The green characteristics of extracted pigments also were not typical of in vitro solutions that included muscle tissue (MSC, MSC+CuSO4, MSC+VAC, MSC+H2O2, MSC+CuSO4+VAC, and MSC+AlOH), but were typical of some in vitro solutions generated with myoglobin, CuSO4, H2O2, and Mb+CuSO4 (Figure 4). The percentage of change as wavelength changed from 654 nm to 656 nm, as well as the percentage of change as wavelength changed from 656 nm to 658 nm, for each of the in vitro solutions exhibiting characteristics similar to those of extracted pigments from green injection-site lesions are presented in Table 5. Based on the results from Phase I, the green pigment denoted by an increase in the absorbance/µg of protein ratio as the wavelength changed from 654 to 656 nm resemble those most closely obtained by solutions of Mb mixed with CuSO4 or H2O2.
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Figure 4. Ratios of absorbance/µg of protein for green injection-site lesions and in vitro color reactions produced under high partial pressures of oxygen. H2O2 = hydrogen peroxide, HPMb = hydroperoxymetmyoglobin, Mb = myoglobin, MbCu = myoglobin plus copper sulfate, SMb = Sulfmyoglobin, Lesion = green lesions supplied by a cooperating packer.
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Table 5. Comparison of the percent change in spectrophotometric absorbance/µg of protein ratio as wavelength changed from 654 nm to 656 nm and from 656 nm to 658 nm in Phase I in vitro solutions and green lesions
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Phase-I in vitro color reactions revealed that green discoloration could not be created with solutions with muscle, but could occur in a solution with Mb. The Mb+CuSO4 solution generated a green product that most resembled sulfmyoglobin and/or hydroperoxymetmyoglobin.
Phase II.
Based on the results of Phase I, Phase II was conducted to evaluate the effects of Mb, H2O2, pure, elemental copper, and three different sources of sulfur (sulfide, sulfite, or sulfate) on the change in absorbance from 654 to 658 nm to determine if greening could be detected. The absorbance/µg of protein ratio for Phase-II products demonstrated that, in comparison to the characteristics of the lesions, myoglobin had to be present in the solution for the green reaction to occur. Further analysis indicated that the solutions that exhibited a positive percentage of change in the absorbance/µg of protein ratio as wavelength changed from 654 to 656 nm and a negative percentage of change as the wavelength changed from 656 to 658 nm were: Mb+Na2SO4, Mb+Cu+H2O2, Mb+SO3, Mb+Cu+Na2S, and Mb+Cu+Na2SO3, all at pH 7.2 under atmospheric partial pressures of oxygen; Mb+Na2SO4, Mb+Na2S, and Mb+Na2SO3, all at pH 7.2 under high partial pressures of oxygen; and Mb+Cu+Na2SO4 at pH 5.5 under high partial pressures of oxygen (Table 6). The percentage of change in the absorbance/µg of protein ratio as wavelength changed from 654 to 656 nm, as well as from 656 nm to 658 nm for each of the solutions, is presented in Table 6.
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Table 6. Comparison of the percentage of change in spectrophotometric absorbance/µg of protein ratio as wavelength changed from 654 to 656 nm and from 656 to 658 nm in Phase II in vitro solutions and green lesions
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Based on the data from Phase II, the greening reaction observed in lesions in modified-atmosphere packages is more likely related to the presence of sulfate and copper than to the presence of hydrogen peroxide when mixed with Mb. The addition of Na2S, Na2SO3, or Na2SO4, individually, to Mb under neutral pH conditions resulted in absorbance characteristics typical of the green pigments produced in Phase I, and of the green pigments extracted from the lesions in this study. The presence of Cu, Na2SO4, and Mb at a low pH under high ppO resulted in absorbance/µg of protein ratios similar to those of the green pigments produced in Phase I of Exp. 3. Given the conditions under which the discoloration occurs, the combination of Cu and Na2SO4 with myoglobin could potentially be the cause of the green pigment. Because Cu may be present at an injection-site lesion as a result of the immune response, it seems likely that sulfate could be present in the pharmaceutical product.
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Implications
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Results indicated that use of two commercially available products could result in green injection-site lesions when given as an intramuscular injection to steers from 37 to 60 d before slaughter. Although shown to help maintain beef color stability, this study indicated that vitamin E supplementation to steers will not prevent the green discoloration. Results also indicated that incomplete visibility of lesions upon cutting, and before packaging poses difficulties at the packer/processor level. The greening reaction observed in injection-site lesions of chucks could be a result of an interaction between myoglobin, copper, and sulfate. Before recommendations relative to early detection of lesions before green discoloration and/or reformulation of pharmaceutical products can be made, further investigation of the causative agents in the chemical reaction of green discoloration is needed with an emphasis on sulfur and copper compounds.
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Footnotes
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1 Use of products in this publication does not imply endorsement/criticism by Colorado State University or criticism/endorsement of similar products not mentioned. For a detailed list of products used in the study, please contact the authors. 
2 Present Address: Department of Animal Science, University of Minnesota, St. Paul 55108 (phone: 612-624-2405; fax: 612-625-1283; E-mail: droeber{at}umn.edu).
3 Correspondence—phone: 970-491-5826; fax: 970-491-0278; E-mail: Keith.Belk{at}ColoState.edu.
Received for publication January 23, 2003.
Accepted for publication April 15, 2003.
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Abstract
Introduction
