Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
Animal Science
Non Technical Summary
The US food industry loses approximately $1 billion due to discoloration of meat. Our long-term goal is to better understand the role of postmortem meat biochemistry to increase the body of knowledge in meat color to minimize the losses due to discoloration. The overall goal of the current proposal is the acquisition of Seahorse XFp oxygen analyzer to enhance mitochondrial research capabilities in meat quality studies. Mitochondrial function and glycolysis play critical roles in a variety of vital cellular processes, including cellular activation, proliferation, differentiation, cell death, and disease progression. Various meat science studies have reported that metabolites present in postmortem muscle can influence mitochondrial function and beef color. Currently, Clark Oxygen electrode is used to measure mitochondrial function. Acquiring Seahorse XFp Oxygen analyzer will substantially increase research capabilities in mitochondrial research by studying cellular respiration and glycolytic activity. Further, advantages include less amount of tissue, automation, analyzing multiple samples at a time, and less time for analysis. Characterizing the role of metabolites in postmortem muscle can significantly enhance the role of mitochondria in beef color. In the present day U.S. beef retailing, marketability of individual whole-muscle cuts are increasing. Therefore, accomplishing the objectives will help to advance the fundamental knowledge related to muscle-specific biochemical mechanisms as it affects meat color stability and also to formulate strategies to enhance shelf life.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
The specific objectives are:1) To acquire Seahorse XFp oxygen analyzer to better understand mitochondrial function.2) To characterize the muscle-specific and quality defect associated mitochondrial functional differences in beef muscles during storage.
Project Methods
Intended research activities with Seahorse Oxygen Analyzer.Objectives: To characterize the muscle-specific (experiment 1) and quality defect (experiment 2) associated mitochondrial functional differences in beef muscles during storage.MethodsProposed activity and plan - experiment 1: Longissimus lumborum and psoas major muscles from 10 carcasses (48 h postmortem) will be used to compare two muscles of different color stability (n = 10 for each muscle; N = 20 total subprimals). Each longissimus and psoas muscle will be fabricated into eight 1.91-cm thick steaks. Steaks will be used for mitochondrial isolation, biochemical studies, and color measurement during seven-day storage at 2°C under retail display conditions.Proposed activity and plan - experiment 2: Mitochondria will be extracted from normal-pH and dark-cutting bovine longissimus lumborum muscles. Longissimus is an economically important muscle marketed as top loin steak or New York strip, and it is often used as a representative muscle of a carcass in meat color research (Kim et al. 2006; Suman et al., 2009). The longissimus at the 12th/13th rib interface is the location used on beef carcasses to assess USDA Quality Grades; thus the location where dark-cutting characteristics are determined.For both experiments, following methodologies will be used.Materials and methodsc.1. Raw material and sample allocation: For both experiments, precautions will be taken during loin collection, transportation, and sample allocation. Normal-pH (approximate mean pH = 5.6) and dark-cutting beef (approximate mean pH > 6.4) longissimus lumborum muscles from 10 carcasses will be collected 48 h postmortem or immediately after grading at a USDA-approved packing plant. All precautions will be taken during retail display and color measurements as described in American Meat Science Association guidelines (2012).c.2. Color and pH measurements: Steak color will be measured using a HunterLab MiniScan XE Plus spectrophotometer (English et al., 2016). Both reflectance spectra from 400 to 700 nm (10 nm increments) and CIE L* and a* will be measured in triplicate for each steak and subsamples will be averaged for statistical analyses. A greater L * and a* value indicate brighter red color. Also, pH will be recorded using a meat pH meter.c.3. Oxygen consumption of steaks: Postmortem oxygen consumption will be measured using a method similar to English et al. (2016). Deoxygenated beef steaks will be allowed to oxygenate at 1°C for 30 minutes. Samples will then be vacuum packaged, incubated at 4°C for 30 minutes, and the decrease in oxymyoglobin content will be used to assess oxygen consumption.c.4. Characterizing mitochondrial functional differences: Mitochondrial isolation: Mitochondria will be isolated using steaks on days 0, 2, 4, and 6 of storage (Lanari et al., 1991). Thirty-five grams of minced tissue will be washed twice with 250 mM sucrose, and then mixed with 70 mL of isolation buffer (pH 7.4). To liberate mitochondria, samples will be homogenized with a tissue grinder. Samples will be centrifuged twice and the resulting pellet will be collected, gently washed with buffer, and suspended in mitochondrial suspension buffer.c.5. Quantification of mitochondrial oxygen consumption: A Seahorse oxygen analyzer will be used to assess the activity of mitochondrial electron transport chain complexes at pH 5.6 (typical postmortem muscle pH) and 6.4 (dark-cutting pH). Three mitochondrial electron transport chain complex substrates will be compared, including glutamate-malate (5 mM/0.5 mM; complex I), succinate (5 mM; complex II), and ascorbate-TMPD (6 mM/0.3 mM complex IV), with the addition of mitochondrial inhibitors such as antimycin A and rotenone.