Date of Award
Master of Science
Steven M Lonergan
The average consumer is unaware of the characteristics of pork products that will constitute high quality meat. Labels on pork products have the opportunity to define those characteristics for retailers, chefs, purveyors, or consumers, yet those labels are not implemented consistently and accurately throughout the pork industry. Juiciness, texture, and color can be quantified through water holding capacity, mechanical tenderness, and color measurements. The developments of those characteristics rely on rate and extent of pH decline. The first hypothesis of this study is that subtle differences in ultimate pH can result in significant differences in pork quality. Identification of commonalities in the conversion of muscle to meat through skeletal muscle metabolism, therefore, may allow more accurate predictions for the rate and extent of pH decline that regulates pork quality.
The rate and extent of pH decline relies on glycolytic flux as well as glycolytic capacity in the conversion of muscle to meat. There is a great deal of evidence that glycogen content in muscle does impact the extent of pH decline. However, more recent investigations have determined that glycolytic flux is controlled by two main factors. First, the amount of fructose 1-phosphate that passes phosphofructokinase (PFK), a major rate-limiting enzyme. Second, mitochondrial function in the early postmortem period. Further research is required to identify the enzymes or substrates that determine glycolytic capacity. Therefore, the second hypothesis of this work is that glycolytic metabolites and proteins have differing abundances at 45 minutes postmortem that can be used to identify day 14 pH and meat quality.
Longissimus dorsi samples in the current study were a subset of a larger study involving two genetic lines of pigs divergently selected for residual feed intake. The objective of the current experiment was to identify metabolomic and proteomic markers for different ultimate pH (14 d postmortem) from pork longissimus dorsi samples, to understand the role of energy metabolism in ultimate pork quality. Chops for quality and proteomic analysis were removed and aged 14 d postmortem for pH and quality measurements. Two groups of chops were classified into normal d 14 pH (pH, μ=5.59, 5.53-5.67; NpH, n=10) and low d 14 pH (pH, μ=5.42, 5.38-5.45; LpH, n=10). Marbling, water holding capacity (purge loss % & cook loss %), tenderness (intact desmin & star probe), and color (Hunter L, a & b and visual color score) were used to evaluate quality on chops.
Samples were excised at 45 min postmortem and snap-frozen in liquid nitrogen then homogenized into a powder for further metabolomic and proteomic quantification. Nontargeted metabolomics through gas chromatography- mass spectroscopy was used to quantify metabolites involved in glycolysis or the citric acid cycle at 45 min postmortem. Furthermore, two-dimensional difference in gel electrophoresis and mass spectrometry measured differing abundances of proteins that were soluble in low ionic strength buffer form 45 min postmortem samples. Western blotting was used to quantify protein abundances of soluble PFK, peroxiredoxin-2, and AMP deaminase-2 (reduced & non-reduced).
Classification on ultimate pH resulted in significantly different pH at 14 d postmortem (P < 0.01), a trending difference at 24 hr postmortem (P = 0.10) and no difference at 45 min postmortem (P = 0.64). Quality measurements resulted in improved water holding capacity (purge loss %, NpH: 2.40, LpH: 3.84, P < 0.01; cook loss %, NpH: 22.81, LpH: 20.21; P = 0.03), color (Hunter L: NpH: 47.63, LpH: 50.58, P < 0.01; Hunter b: NpH: 6.74, LpH: 7.58; P = 0.02), tenderness (Star Probe: NpH: 5.36, LpH: 6.31; P < 0.01) and protein degradation (day 7 intact desmin: NpH: 1.78, LpH: 3.39; P = 0.02; day 14 intact desmin: NpH: 1.43, LpH: 2.36; P = 0.06). visual marbling and hunter a were not significantly different (P > 0.10). Significant metabolites that were more abundant in the NpH classification included pyruvate (P = 0.01) and malate (P = 0.01). Those more abundant in the LpH classification included fructose 6-phosphate (P = 0.08) and lactate (P = 0.09). Selected proteins were all significantly higher in the NpH samples and were involved in contraction (α-actin), glycolysis (glyceraldehyde 3- phosphodehydrogenase (GAPDH), fructose bisphosphate aldolase (ALDO), pyruvate kinase (PK), ß enolase and lactate dehydrogenase (LDH)) and heat stress regulation (α 2-heat shock glycoprotein, and heat shock protein 70 (HSP70)).
Western blot analysis of peroxiredoxin-2 showed a greater abundance at 45 min postmortem in the NpH group (P = 0.02). Total PFK decreased from 45 min to 14 d postmortem (P < 0.01), while the total reduced AMP deaminase increased from 45 min to 14 d postmortem (P = 0.03). No significant differences were identified between the NpH and LpH groups for 45 min PFK or reduced and non-reduced AMP deaminase-2, nor 14 d peroxiredoxin and reduced AMP deaminase-2.
Classification based on day 14 pH resulted in improved quality in the NpH group that could be used to identify key metabolic processes in the conversion of muscle to meat. First, more abundance of lactate in the LpH samples supports the hypothesis that lactate accumulation will contribute to ultimate pH, however 45-minute postmortem pH was not different in the current study. Furthermore, the accumulation of pyruvate in the NpH samples coincides with a greater abundance of PK in the NpH samples. Greater abundance of chaperone proteins and peroxiredoxin-2 at 45 min postmortem may indicate that mitochondria and proteolysis enzymes in the normal pH group, maintained improved function. Identification of α-actin in the sarcoplasmic, soluble fraction of the NpH group in conjunction with a greater accumulation of fructose 6-phosphate in LpH may signify protein-protein interaction during the onset of rigor mortis that contribute to extended postmortem activity of PFK, despite limited differences in PFK abundance.
It can be concluded that metabolic enzymes play an important role in postmortem pH decline. Early postmortem quantification of proteins and metabolites will allow for more targeted future investigations into the mechanisms behind pH and quality developments. The current work brings into question the variability of glycolytic protein abundance vs. activity and mitochondrial stability in postmortem muscle.
Elizabeth Ailene Zuber
Zuber, Elizabeth Ailene, "Metabolomic and proteomic features of fresh pork loin that vary in ultimate pH and pork quality" (2020). Graduate Theses and Dissertations. 18438.