Degree Type

Dissertation

Date of Award

2011

Degree Name

Doctor of Philosophy

Department

Animal Science

First Advisor

Elisabeth Huff-lonergan

Abstract

During the postmortem storage there are numerous changes in the protein profile of beef. It is well documented that some of these changes directly affect the structure of the myofibril during postmortem aging. Our hypothesis was that there are alterations to proteins during postmortem storage that affect tenderness development. By identifying these alterations, these proteins can be used as indicators of tenderness and can aid in the identification of consistently tender beef.

The first experiment was designed to define the biochemical differences that govern tenderness and palatability of economically important muscles from the beef round using muscles with known tenderness differences. At 24 h postmortem, the longissimus dorsi (LD), gracillus (GR), adductor (AD), semimembranosus (SM), sartorius (SAR), vastus lateralis (VL), and vastus intermedius (VI) muscles were removed from ten market-weight beef cattle. Sensory and biochemical characteristics were determined in each muscle and compared with the LD. This study showed that the AD and VL had poor sensory traits compared to the LD. However, the GR, SAR, and VI had sensory traits similar to the LD. The AD was also an important example of how understanding the biochemical characteristics of a muscle can aid in the utilization of that muscle. The most notable attribute of the AD was its lack of change in protein degradation during aging. The AD had no detectable change in the abundance of the 30 kDa degradation of troponin T from one to 14 days postmortem, and no significant change in tenderness. Postmortem aging of this muscle provided no added benefit as it did in many of the other muscles evaluated. In most beef round muscles, postmortem proteolysis provided a good indication of the postmortem tenderization occurring during aging.

Results from our first study suggested that proteolysis is the key process in indicating postmortem tenderness and that the AD had little proteolysis from one to 14 days postmortem. The lack of change in protein degradation in the AD provided a model to determine the changes in protein abundance over time in a muscle with little proteolysis from one to 14 days postmortem. Once these changes were determined, they were then compared to the changes in protein abundance over time in a muscle with greater proteolysis (LD) from one to 14 days postmortem to identify the proteins that may play a role in postmortem tenderization. So, this second experiment was designed to determine the identity of proteins that are altered during the postmortem aging process by using muscles that differ in tenderness and postmortem protein degradation. The AD (n=5) was chosen because it shows very little change in sensory and instrumental tenderness during 14 days of aging and the LD (n=5) was chosen because it showed a large increase in sensory and instrumental tenderness. A highly soluble sarcoplasmic fraction and a less soluble, crude myofibrillar fraction were extracted from each muscle. Two-Dimensional Difference In Gel Electrophoresis (2-D DIGE) was used to compare samples from one and 14 days postmortem within the same muscle and protein fraction. This study illustrated that both glyceraldehyde-3-phosphate dehydrogenase and beta-enolase are not expected to be robust indicators of tenderness across muscles because alterations to the relative abundance of these proteins were not directly related to the amount of tenderization that occurred in both the AD and LD during postmortem storage. In addition, the usefulness of creatine kinase M-type as an indicator of tenderness was unclear because it undergoes both degradation and solubility changes during postmortem storage. Postmortem modifications, through proteolysis or solubility changes, of the structural proteins found to change in relative abundance during aging in the LD (actin, myosin heavy chain, myomesin-2, tropomyosin alpha 1 chain and α-actinin-3) can play a key role in postmortem tenderization by disrupting the structure of the myofibril. As a result, actin, myosin heavy chain, myomesin-2, tropomyosin alpha 1 chain and α-actinin-3 are anticipated to be good indicators of tenderness across muscles.

While actin, myosin heavy chain, myomesin-2, tropomyosin alpha 1 chain and α-actinin-3 appeared to be important to the process of postmortem tenderization, it was also important to verify that differences in these proteins could be detected between high and low star probe samples. Therefore, the next study was designed to identify proteins in bovine longissimus dorsi that differed in steaks from high or low star probe samples. The LD was removed from ten cattle at 24 hours postmortem, and the two with the highest (average kg of force = 6.57) and lowest star probe values (average kg of force = 3.75) at 14 days postmortem were designated the high and low star probe samples, respectively. Using 2-D DIGE to compare the protein profile of the sarcoplasmic fraction of high and low star probe samples of the LD, proteins were identified that may have potential as indicators of tenderness. Of the proteins identified in the previous study as changing in relative abundance over time, myomesin-2, tropomyosin alpha 1 chain, and actin were identified as being more abundance in the sarcoplasmic fraction of low star probe samples compared to high star probe samples. Therefore, myomesin-2, tropomyosin alpha 1 chain, and actin are important to the process of postmortem tenderization and can be detected between high and low star probe samples. Because of this, these proteins have the greatest potential to become indicators of postmortem tenderness.

In addition to myomesin-2, tropomyosin alpha 1 chain, and actin, both phosphoglucomutase 1 (PGM1), and myosin light chain 1 (MLC1) differed in relative abundance in the sarcoplasmic fraction between high and low star probe samples. Two spots identified as MLC1 and four of the five spots identified as PGM1 were more abundance in the low shear samples compared to the high star probe samples. One spot of PGM1 was identified as more abundant in the high star probe samples compared to the low star probe samples. Of all the proteins identified as differing in relative abundance between high and low star probe samples, MLC1 was determined to be a candidate protein for postmortem tenderization because it had the most significant difference between the high and low star probe groups. The second portion of this study was designed to determine the extent to which μ-calpain activity could be responsible for the appearance of MLC1 in the highly soluble sarcoplasmic fraction during aging and examine the rate of the appearance of MLC1 in the highly soluble fraction during incubation with μ-calpain. Myofibril incubation with μ-calpain revealed that μ-calpain catalyzes the rapid release of MLC1 from the myofibril. One hypothesis suggested by Hayashibara and Miyanishi (1994) was that MLC1 may provide stability to the actomyosin complex by keeping actin and myosin in close proximity and that the rapid release of MLC1 into the highly soluble sarcoplasmic fraction may influence tenderness by disrupting the stability of the actomyosin complex.

Of all the proteins identified as differing in relative abundance between high and low star probe samples, PGM1 offered the best model to investigate the effects on posttranslational modifications on postmortem tenderization. Because four of the five spots identified as PGM1 were more abundant in the low star probe samples and one of the five spots identified as PGM1 was more abundant in the high star probe samples, it indicates that the individual isoforms of PGM1 were as important to tenderness as the overall amount of PGM1. Because posttranslational modifications (phosphorylation) play a key role in the regulation of the PGM1 activity, the final study was designed to identify differences in phosphorylation of PGM1 in samples that differed in star probe value. 2-Dimensional PAGE (13 cm, pH 4-7; 8% acrylamide gel) was used to compare the sarcoplasmic fraction of the LD at one day postmortem in the high and low star probe groups. This study showed that there was a greater abundance of phosphorylated protein in the most alkaline of the phosphorylated PGM1 spots in high star probe samples compared to low star probe samples. This increase in phosphorylation may indicate increased activation and/or activity of PGM1. More importantly this study illustrated the importance of investigating posttranslational modification in the search for biomarkers because the least phosphorylated isoform of PGM1 had a greater abundance of phosphorylated protein in steaks from the high star probe samples while all other isoforms of PGM1 did not differ in the abundance of phosphorylated protein between steaks from the high and low star probe samples. This suggests that the posttranslational modifications of proteins may be as important as total protein in determining postmortem tenderization.

In conclusion, myomesin-2, tropomyosin alpha 1 chain, actin, and myosin light chain 1 show promise as potential indicators of tenderness. Because variation in posttranslational modifications have effects on protein activity, cellular locations and signaling pathways, the investigation into the posttranslational modifications of any candidate proteins may help in understanding usefulness of these proteins as biomarkers and the mechanism of tenderization during postmortem storage of meat.

DOI

https://doi.org/10.31274/etd-180810-3107

Copyright Owner

Mark Joseph Anderson

Language

en

Date Available

2012-04-30

File Format

application/pdf

File Size

194 pages

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