Metabolic and proteomic pathways in muscle involved in beef tenderness development

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2022-05
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Schulte, Matthew David
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Huff-Lonergan, Elisabeth
Lonergan, Steven M
Hansen, Stephanie L
Stalder, Kenneth J
Prusa, Kenneth J
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Animal Science
Abstract
Consumers desire high-quality and tender beef products and are willing to pay a premium for greater quality products. However, consistent and accurate predictability of beef tenderness is not currently available in the beef industry. Prediction of and understanding of the biological mechanisms behind tenderness development have been assessed through proteomic analysis, but consistent predictive biomarkers have not been identified. Along with a desire for high-quality and tender beef products, consumers are becoming more knowledgeable about the sustainability of production practices used in the beef industry, such as rapid growth strategies like nutritional supplementation. The influence of rapid growth on beef quality and tenderness development is not fully understood. Therefore, the objective of this experiment was to identify the impact of rapid growth on postmortem tenderness development and identify pathways impacted by and products produced that can be indicators of beef tenderness. The first experiment was conducted to identify the impacts of supplementation of ractopamine hydrochloride (RH) and supranutritional zinc (Zn) on tenderness development of extended aged beef longissimus thoracis muscle and postmortem protein degradation. It was hypothesized that RH and Zn supplementation would influence the tenderness development of extended aged steaks. Supranutritional Zn supplementation resulted in greater (P=0.04) calpain-1 76-kDa band autolysis at 2 d postmortem but only numerically lesser WBSF values at 7, 14, and 28 d postmortem. Supranutritional Zn supplementation also resulted in greater (P=0.05) hot carcass weights and greater (P<0.01) ribeye areas than cattle not supplemented with supranutritional Zn. Feeding RH resulted in greater WBSF values at 7 (P=0.02), 14 (P=0.01), and 28 (P=0.01) d postmortem, which was attributed to lesser desmin degradation at 7 (P=0.02), 14 (P<0.01), and 28 (P<0.01) d postmortem and lesser (P<0.01) calpain-1 76-kDa band autolysis at 2 d postmortem. These results link nutritional supplementation strategies known to impact animal growth to the rate of postmortem tenderness development, and the biochemical mechanisms to control the rate of tenderness development need to be more fully defined. The second experiment was designed to test the hypothesis that supplementation of RH and supranutritional Zn would influence tenderness development. Supplementation of supranutritional Zn trended for lesser (P=0.0638) pH values at 6 h postmortem and a trend for lesser (P=0.0585) Warner-Bratzler shear force (WBSF) values at 1 d postmortem compared to muscle from cattle not supplemented Zn. Supplementation of RH resulted in greater (P=0.04) pH values at 6 h postmortem and greater (P<0.01) WBSF values at 1 d postmortem compared to muscle from cattle not supplemented RH. Samples from steers supplemented supranutritional Zn-only had the lowest pH at 6 h postmortem and lowest WBSF values at 1 d postmortem compared to samples not supplemented supranutritional Zn-only. The opposite was seen in muscle from cattle supplemented RH-only, which had the greatest pH at 6 h postmortem and greatest WBSF values at 1 d postmortem than muscle from cattle not supplemented RH-only. These differences in WBSF values were explained by postmortem protein degradation, where muscle from cattle supplemented with RH had lesser (P=0.05) desmin and lesser (P=0.04) troponin-T degradation at 1 d postmortem and greater WBSF values compared to muscle from cattle not supplemented RH. Although supranutritional Zn supplementation did not significantly influence protein degradation at 1 d postmortem, muscles from cattle supplemented Zn-only did have numerically greater troponin-T degradation at 1 d postmortem than samples not supplemented Zn-only. This study linked nutritional supplementation to postmortem metabolism that impacted pH decline and tenderness development through postmortem protein degradation. Supplementation of Zn and RH to beef cattle demonstrates a clear impact on metabolic properties of muscle that influence pH decline and the rate of postmortem tenderization. The second objective was to identify early postmortem sarcoplasmic protein (abundance and post-translational modifications) and metabolite variations that could explain pH decline and tenderness development differences from cattle supplemented RH and supranutritional Zn. In muscle samples from steers supplemented supranutritional Zn-only, sorbitol was more abundant at 1 d postmortem than all other treatments. This increased abundance of sorbitol may be related to the use of the polyol pathway and excess glucose in the muscle due to the influence of zinc to stimulate glucose uptake by the muscle and glucose oxidation, like insulin. Use of the polyol pathway may have limited ATP production and thus hastened pH decline. The muscle from supranutritional Zn-only supplemented animals had a lesser adenosine monophosphate deaminase 1 at 1 d postmortem than all other treatments, which could be related to accelerated lactate accumulation and limited postmortem glycolysis. Muscle samples from steers supplemented supranutritional Zn-only had a lesser abundance of soluble myosin regulatory light chain 2 at 1 h postmortem compared to all other treatments. A lesser abundance of soluble myosin regulatory light chain 2 at 1 h postmortem could influence actomyosin cross-bridge formation and rigor development in postmortem muscle. A greater abundance of structural proteins (troponin-T and myosin-7) at 1 h postmortem in the soluble protein fraction was identified in muscle samples from steers supplemented RH (CON-RAC and SUPZN-RAC) compared to muscle from CON-NO. A greater abundance of structural proteins could be evidence of greater protein turnover in muscle from cattle supplemented RH due to the known function of RH to increase protein accretion. Additionally, these soluble proteins could be impacting the observed slower rate of pH decline in muscle from RH supplemented animals by impacting rigor formation. A greater abundance of phosphorylated isoforms of phosphoglucomutase-1, ATP synthase subunit beta, and malate dehydrogenase at 1 h postmortem in the phosphoproteome analysis in the CON-RAC treatment compared to all other treatments could be influencing postmortem glycolysis, and pH decline as the muscle from RH-only fed animals had the greatest pH values at 6 h postmortem. Differences in energy metabolism enzymes, metabolites, and structural proteins in the sarcoplasmic protein fraction may explain the differences in postmortem pH decline and tenderness development at 1 d postmortem. The final investigation into the relationship between pH decline and tenderness development was conducted because pH decline was related to tenderness development in the previous study. The objective of this experiment was to test the hypothesis that early postmortem variations in the muscle metabolome, proteome, and phosphoproteome could explain differences in pH at 6 h postmortem and lower WBSF values at 1 d postmortem of samples classified by pH values at 6 h postmortem. Longissimus thoracis muscle samples were classified by 6 h pH into low (LpH) and high (HpH) pH classifications. The LpH classification had a lesser pH value at 1, 3, 6, and 24 h postmortem and lesser WBSF values at 1 d postmortem compared to the HpH classification. This lesser WBSF value in the LpH classification was explained by greater postmortem proteolysis (greater calpain-1 autolysis, desmin degradation, and troponin-T degradation) at 1 d postmortem. The LpH classification had a greater abundance of energy production enzymes and metabolites, which are related to glycogenolysis, glycolysis, and the tricarboxylic acid cycle, at 1 h postmortem. A lesser abundance of heat shock protein beta 1 at 1 h postmortem and a greater abundance of cytochrome c at 1 d postmortem was identified in the sarcoplasmic protein fraction of the LpH classification. This difference in apoptotic related proteins could demonstrate earlier apoptosis occurring in the LpH classification. The LpH classification also had a greater abundance of several proteasome-related proteins at 1 h postmortem in the sarcoplasmic protein fraction that could be influencing postmortem protein degradation through the 20S proteasome subunit. A more rapid pH decline is linked to an earlier onset of postmortem proteolysis through multiple proteolytic systems (calpain, apoptotic, and proteasome), resulting in more tender products at 1 d postmortem. In conclusion, a hastened pH decline was linked to early tenderization due to greater postmortem proteolysis through multiple proteolytic systems (calpain, apoptotic, and proteasome). This hastened rate of pH decline may be influenced by greater use of the polyol pathway and greater rates of glycolytic metabolism due to greater metabolic enzyme abundances. Rapid metabolism early postmortem in beef muscle shows the potential to dictate earlier tenderization of products for earlier marketing. Supranutritional Zn supplementation may enhance the pH decline of the longissimus thoracis muscle and enhance earlier tenderization.
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