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Muscle is an elegant biological system with mechanisms in place to control calcium release and storage. At death, calcium ions slowly diffuse from storage in the sarcoplasmic reticulum (SR) to the sarcoplasm where they activate the calcium-dependent proteases (the calpains) and enhance tenderness. It is a well-known fact that feeding cattle diets containing high levels of polyunsaturated fatty acids (PUFA) [like wet distillers grains plus solubles (WDGS)] increase PUFA levels in beef. Previous research has shown that beef from cattle fed WDGS tended to be more tender than beef from cattle fed corn or WDGS with dietary antioxidants. It is possible that PUFA content of the SR membrane will increase as a consequence of feeding WDGS, which will predispose the SR membrane to more rapid post-rigor oxidization. This would result in calcium being released earlier post-rigor than normal, which would activate calpains earlier and enhance tenderness. Feeding antioxidants might add stability to the SR membrane, slowing release of calcium post-rigor and delaying and/or reducing proteolysis and tenderization.
Feeding WDGS provides an excellent model to generate beef samples with varying degrees of oxidation capacity, as does feeding antioxidants. Different antioxidants provide different mechanisms of scavenging and reducing free radicals. Vitamin E (VitE) works through cell membrane stabilization as well as inhibition of phospholipid degradation. Synthetic antioxidants such as Agrado (Ag; a mixture of tert-Butylhydroquinone and ethoxyquin) work through protection of the feed against oxidative degradation during storage. When fed in combination with VitE, synthetic antioxidants added to the feed clearly have a sparing effect on VitE level. Chickens fed synthetic antioxidants have a significantly higher VitE content in the plasma compared to chickens fed a feed that was not stabilized with synthetic antioxidants. It is possible that synthetic antioxidants present in feed might still be active in the gastrointestinal tract, protecting VitE from oxidative deterioration. On the other hand, research from South Dakota State University concluded that there was no synergistic effect between VitE and ethoxyquin in a study of beef cattle supplemented with VitE as well as a combination of the VitE and ethoxyquin. These confounding results exploit the need for additional further study at the cellular level. Studies that use diet (corn vs WDGS) and antioxidants (VitE, Ag, and a combination of both) should provide a wide range of samples for evaluation of post-harvest changes in beef that affect eating quality.
The objectives of this study were to:
This trial consisted of eight different combinations of diets and antioxidant treatments. Pens (2 per treatment) contained ten steers each, for a total of 160 British × Continental steers. There were two diets (corn or 30% WDGS) and four antioxidant treatments: (1) 50 IU of VitE throughout the entire study (control), (2) 1500 IU of VitE/head/day for the first 51 days and 500 IU of VitE/head/day for the rest of the 106 day feeding period, (3) 300 ppm of Ag/head/day for the first 51 days and 150 ppm of Ag/head/day for the rest of the 106 day feeding period and (4) a combination of 1500 IU of VitE/head/day and 300 ppm of Ag/head/day for the first 51 days and 500 IU of VitE/head/day and 150 ppm of Ag/head/day for the rest of the 106 day feeding period. This treatment design was analyzed as a 2x4 factorial arrangement.
Steers were harvested at a commercial packing plant. After 48 hours of postmortem chilling, 80 out of 160 designated carcasses with equal numbers of carcasses from each pen were selected (10 carcasses from each treatment). The carcass selection was based on treatment and quality grade (USDA Choice). Strip Loins were collected from the designated carcasses, vacuumed-packaged and transported to the Loeffel Meat Laboratory at the University of Nebraska-Lincoln.
Subprimals from the left side of each carcass were aged for 2 and 7 days, and subprimals from the right side of each carcass were aged for 14 days. After aging, each Strip Loin was fabricated into two steaks for tenderness measurements and three steaks to be used for lab samples for each aging period. Steaks were cut from the anterior to the posterior end of the Strip Loin. Upon fabrication, all steaks were packaged in oxygen-permeable packaging and subjected to retail display conditions. During each day of retail display, a five-person trained panel consisting of graduate students in the Department of Animal Science at the University of Nebraska-Lincoln subjectively evaluated discoloration of each steak as a percentage (0 – 100%) of total surface area. Samples for tenderness assessments and free-calcium concentrations were obtained on day 0 and 7 of retail display of each aging period, and samples for oxidation assessments were obtained on day 0, 4 and 7 of retail display of each aging period. For SR membrane fatty acids, phospholipids and lipid profile analysis, samples were obtained at day 0 of retail display after 14 days of aging.
Tenderness was evaluated using a mechanical measure of shear force (Warner-Bratzler shear force). Free-calcium concentrations were evaluated via inductively coupled plasma spectroscopy, and lipid oxidation was evaluated via the thiobarbituric acid reactive substances assay. SR was isolated through differential centrifugation, and the SR fatty acids were quantified via gas chromatography.
Fatty acid analyses revealed distinct differences among fatty acid profiles of SR from cattle fed different diets. In general, SR from cattle fed WDGS had higher concentrations of 15:0, 17:0, 18:1 trans, 18:2, total PUFA and total unsaturated fatty acids (UFA) compared to SR from cattle fed corn. Feeding WDGS also lowered concentrations of 14:0, 16:0, 16:1 and 18:1 vaccenic acid and total saturated fatty acids (SFA) and tended to lower concentrations of 18:0, 18:1 oleic acid and total monounsaturated fatty acids (MUFA). There was no clear pattern of alteration of fatty acid profiles from any antioxidant treatment. Long chain PUFAs: 20:3, 20:4, 20:5, 22:4, 22:5 were unaffected by the diets.
No differences existed in discoloration among steaks of different treatments from day 0 to 5. At day 6 and 7 of the retail display period (Figure 1), steaks from steers supplemented with VitE or the combination of VitE and Ag were significantly less discolored than steaks from steers not supplemented with VitE. However, it is important to note that there was no difference in discoloration among steaks due to diet (Corn vs. WDGS) or synthetic antioxidant only (Ag vs. no Ag) during days 6 and 7 of retail display.
Lipid oxidation values followed a similar pattern with the discoloration score numerically, but there was no statistical differences in lipid oxidation among steers fed a 30% WDGS diets supplemented with antioxidants or not. Reductions in oxidation rates due to VitE and/or Ag supplementation were observed at all three retail display periods only when steers were fed corn diets. VitE + Ag supplementation was most effective in suppressing oxidation, followed by VitE only and then Ag only supplementation. When looking at simple diet treatment effects without any antioxidant supplementation, steaks from corn-fed steers had higher lipid oxidation values compared to steaks from steers fed 30% WDGS.
No differences in objective tenderness and free-calcium concentration among steaks from steers of any treatment groups or any of the aging and display periods. A difference in objective tenderness and free-calcium concentration was expected as previous work has shown steaks from steers fed WDGS were more tender and had more free calcium than steaks from cattle fed corn or WDGS with dietary antioxidants.
Regarding the proposed mechanism of tenderization, this study revealed that feeding WDGS increased PUFA content in SR. Furthermore, this experiment showed that feeding 30% WDGS and two types of antioxidants had no effect on tenderness and free-calcium concentration. Discoloration and lipid oxidation were significantly suppressed by VitE supplementation and minimally suppressed by Ag supplementation, but these two measurements were likely not indicators of membrane stability.
Although the results did not fully support the hypothesis, it is too early to drop consideration of this proposed mechanism completely. Previous research, which showed differences in tenderness and free-calcium concentration, was based on feeding 50% WDGS. The 30% WDGS fed in this study might be insufficient to drive the proposed oxidation process and distinguish WDGS from corn. One contradictory finding in this study is that beef from steers fed only corn was more oxidized than beef from steers fed WDGS in all display periods. It is possible that feeding distillers grains caused a vitamin E-sparing effect by synthesizing antioxidant peptides and thus alleviating the oxidative stress induced by oxidized lipids in WDGS. Finally, measuring lipid oxidation on muscle tissue is likely not the best way to measure SR membrane oxidation. A sensitive, simple and reliable method to detect lipid oxidation in an extremely small sample volume needs to be applied for direct measurement of SR membrane oxidative status. Although the true mechanism and time course of membrane oxidation and calcium release are still unclear, these results provide a conceptual foundation for new research perspectives on meat tenderization.
Figure 1. Discoloration of Strip Loin Steaks (M. Longissimus lumborum) from steers fed a corn diet or a diet of corn and 30% wet distillers grains plus solubles (WDGS), supplemented with or without vitamin E (E) or Agrado (Ag) or a combination of E and Ag displayed for 7 days under retail conditions. *Steaks from steers supplemented with E or the combination of E and Ag were significantly less discolored than steaks from steers not supplemented with E.
Figure 2. Mr. Michael D. Chao preparing purge for free calcium extraction.