Proteomic features associated with tenderness of aged pork loins

Abstract

<p>Tenderness plays a large role in the definition of meat quality and consumer purchasing decisions. The objectives for this study were to determine differences in proteolysis and sarcoplasmic proteomes that are attributed to variation in tenderness of aged pork loins with similar pH, lipid, and color measurements. Loins (n = 159) were collected one day postmortem from carcasses of Duroc-sired crossbred commercial pigs and aged for approximately 9 – 11 days. Chops (2.54 cm) were collected evaluated for purge, cook loss, pH post-aging, visual color and marbling, Hunter L, a, and b, sensory, star probe measurements (kg), and total lipid. Two libraries of samples with different star probe values were assembled. Final selection limited samples to be within specified ranges for ultimate pH (5.54 – 5.86), marbling score (1.0 – 3.0), and percent total lipid (1.61 – 3.37%). A low star probe group (n = 12, 4.95 kg) and high star probe group (n = 12, 7.75 kg) were utilized for further testing. Data was collected using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blots, two dimensional difference in gel electrophoresis (2D-DIGE), and mass spectrometry. The use of SDS-PAGE and Western blots quantified the amount of degradation of the proteins troponin-T, desmin, filamin, and titin in classified sample groups, as well as the rate of autolysis of calpain-1. SDS-PAGE was also used to evaluate predominate myosin heavy chain isoforms to determine fiber type differences in the classification groups. Sarcoplasmic protein abundance and modification of proteins was evaluated by using 2D-DIGE. Two experiments utilizing 2D-DIGE were performed 1) using an 11 cm immobilized pH gradient (IPG) pH 4 – 7 strip and 2) using a 24 cm IPG pH 6 – 9 strip. Protein spots that differed in abundance were identified from the 2D-DIGE experiments with the use of mass spectrometry.</p> <p>Calpain-1 was completely autolyzed in both high and low star probe samples, demonstrating calpain-1 was potentially active to some extent in all samples. Extreme differences in proteolysis were exhibited in whole muscle samples where low star probe samples had more troponin-T (P < 0.01), desmin (P < 0.01), and filamin degradation (P < 0.01). Both low and high star probe myofibrillar samples showed degradation of the large protein titin, but select high star probe samples also exhibited intact bands of titin. Results from the 2D-DIGE experiments showed high star probe samples had significantly more metabolic, stress response, and regulatory protein abundance compared to low star probe samples. Specifically, the stress response protein peroxiredoxin-2 was more abundant in high star probe samples in 2D-DIGE experiment one (P ≤ 0.01, 2 spots) and one-dimensional Western blot confirmations (P = 0.02). Low star probe samples showed significantly more degradation of the structural protein desmin as determined in 2D-DIGE experiment one (P < 0.01) and one-dimensional Western blot confirmations (P < 0.01). These results demonstrate there were extreme proteolytic differences that contributed to measured tenderness of low and high star probe samples. The proteins peroxiredoxin-2 and desmin were found in the soluble protein fraction of muscle as being differentially expressed in classification groups and show potential to be utilized as biomarkers to classify tough and tender aged pork products.</p>