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Effects of muscle-specific oxidative stress on cytochrome c release and oxidation-reduction potential properties Yiling Ke, Rachel M Mitacek, Anupam Abraham, Gretchen G. Mafi, Deborah L. VanOverbeke, Udaya DeSilva, and Ranjith Ramanathan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01735 • Publication Date (Web): 10 Aug 2017 Downloaded from http://pubs.acs.org on August 10, 2017

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Journal of Agricultural and Food Chemistry

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Effects of muscle-specific oxidative stress on cytochrome c release and oxidation-reduction

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potential properties

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Yiling Ke, Rachel M. Mitacek, Anupam Abraham, Gretchen. G. Mafi, Deborah L. VanOverbeke, Udaya DeSilva, Ranjith Ramanathan*

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Department of Animal Science, Oklahoma State University, Stillwater, OK 74078, USA

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* Corresponding author. TEL: +1 405-744-9260; Fax: +1 405-744-4716.

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EMAIL: [email protected] (R. Ramanathan).

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ABSTRACT

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Mitochondria play a significant role in beef color. However, the role of oxidative stress in

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cytochrome c release and mitochondrial degradation is not clear. The objective was to determine

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the effects of display time on cytochrome c content and oxidation-reduction potential (ORP) of

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beef longissimus lumborum (LL) and psoas major (PM) muscles. PM discolored by day 3

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compared with LL. On day 0, mitochondrial content and mitochondrial oxygen consumption

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were greater in PM than LL. However, mitochondrial content and oxygen consumption were

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lower (P < 0.05) in PM than LL by day 7. Conversely, cytochrome c content in sarcoplasm was

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greater on days 3 and 7 for PM than LL. There were no significant differences in ORP for LL

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during display, but ORP increased for PM on day 3 when compared with day 0. The results

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suggest that muscle-specific oxidative stress can affect cytochrome c release and ORP changes.

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Keywords: Beef color, myoglobin, metmyoglobin reducing activity, mitochondria, oxidation-

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reduction potential

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INTRODUCTION

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Consumers associate the bright-red color of beef with freshness and wholesomeness.

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Myoglobin is the primary sarcoplasmic protein responsible for beef color. Depending on the

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redox state of the heme and the ligand bound, myoglobin exists in three forms.1 Ferrous heme

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with no ligand attached results in purple color due to predominant deoxymyoglobin, while

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oxymyoglobin gives the characteristic bright-red color. The oxidation of both oxymyoglobin and

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deoxymyoglobin leads to metmyoglobin formation or discoloration. Approximately 15% of meat

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is discounted in price due to discoloration.2 Hence, characterizing the factors associated with

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myoglobin oxidation is important to minimize the losses due to discoloration.

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Metmyoglobin accumulation is muscle-specific and depends on the inherent muscle

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biochemistry. Longissimus lumborum (LL) is classified as a color-stable muscle while psoas

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major (PM) is a color-labile muscle.3-5 Among the various factors that affect color stability, the

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role of mitochondria has been investigated by numerous researchers.6-9 Mitochondrial activity

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can influence myoglobin redox state via oxygen consumption and metmyoglobin reduction.6,7,9

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However, oxidative processes that affect mitochondrial function can impact beef color.10

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Immediately after animal harvest, blood flow ceases, and the muscle metabolism changes

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from aerobic to anaerobic. This can significantly affect muscle homeostasis, increase oxidative

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stress,11 and degeneration of mitochondria.12 In healthy cells, cytochrome c is the major electron

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carrier between complex III and IV of the electron transport chain. The level of cytochrome c

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content in sarcoplasm is an indicator of mitochondrial damage or outer membrane breakdown.13

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The concentration of mitochondria is greater in type I (predominant red fibers) muscles

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than in type II (white fibers). The proportion of red and white fibers varies between different

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muscles in a beef carcass. PM muscle has greater red fiber content than LL muscle. Thus, it is



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possible that the oxidative changes will be greater in PM than LL muscles. However, limited

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studies have determined the effects of muscle type and display time on cytochrome c level in

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relation to beef color. The oxidation-reduction potential (ORP) measurements can predict

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oxidative stress in tissue.14 Hence, the quantification of ORP changes with display time will

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provide a better understanding of the muscle-specific differences in oxidative stress. Therefore,

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the objective of this study was to determine the effects of display time and muscle type (beef LL

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and PM) on changes in mitochondria, cytochrome c content, and ORP.

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MATERIALS AND METHODS

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Raw materials and processing

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Eight USDA paired Select beef LL (IMPS #180)15 and PM from market age animals were

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collected within 48 h of harvest from a commercial packing plant. The muscles were transported

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on ice to the Food and Agricultural Products Center at Oklahoma State University. Following

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overnight storage, five 2.5-cm-thick steaks were cut from the anterior end of each muscle and

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randomly assigned to days 0, 1, 3, 5, and 7. Steaks were placed (N = 16 steaks on each day; n = 8

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from LL and n = 8 from PM) on foam trays with absorbent pads, over-wrapped with a PVC film

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(oxygen-permeable polyvinyl chloride fresh meat film; 15,500–16,275 cm3 O2/m2/24 h at 23 °C,

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E-Z Wrap Crystal Clear Polyvinyl Chloride Wrapping Film, Koch Supplies, Kansas City, MO).

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After packaging, steaks were placed in a coffin-style open display case maintained at 2 °C ± 1

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under continuous lighting (1612 to 2152 lx, Philips Deluxe Warm White Fluorescent lamps;

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Andover, MA; color rendering index = 86; color temperature = 3000 K). All packages were

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rotated daily to minimize the variances in light intensity or temperature caused by the location.

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Following surface color measurements, steaks designated to days 0, 3, and 7 were cut in half. The first half was used to measure metmyoglobin reducing activity (MRA) and muscle 4 ACS Paragon Plus Environment

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oxygen consumption, and the second half was used to quantify ORP, cytochrome c content, pH,

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lipid oxidation, and for mitochondrial studies. The steak half assigned to MRA and muscle

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oxygen consumption was then bisected parallel to the oxygenated surface to expose the interior

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of the steak (resulting in two interior pieces). The first piece was used to measure MRA and the

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second piece was used to measure muscle oxygen consumption. Before taking samples from the

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second half, a pH probe was used to measure muscle pH. Following pH measurements from the

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second half, approximately 50 g of muscle samples visually devoid of fat and connective tissue

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were vacuum packaged and assigned for mitochondrial studies. Another 10 g of the sample that

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contained displayed surface and interior was used for lipid oxidation measurements. Two 5 g

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samples were collected for cytochrome c and ORP measurements, vacuum packaged and kept at

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-80 °C until use. Mitochondrial studies, cytochrome c, pH, MRA, muscle oxygen consumption,

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and ORP measurements were conducted on days 0, 3, and 7. Fresh tissue samples were used for

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oxygen consumption, mitochondrial quantification using protein content, and lipid oxidation

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studies; however frozen tissue was used for mitochondrial quantification using DNA-method.

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Proximate and pH analysis

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Proximate analysis was conducted using an AOAC-approved (Official Method 2007.04)

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near-infrared spectrophotometer (FOSS Food Scan™ 78800; Dedicated Analytical Solutions,

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DK-3400 Hillerod, Denmark). Compositional values were reported on a percent (%) basis. pH

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was determined by inserting the pH probes at four different locations within a steak using a

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Mettler Toledo SevenGo pH meter.

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Surface color

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Surface color was measured on days 0, 1, 3, 5, and 7 at three locations using a

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HunterLab MiniScan XE Plus spectrophotometer (HunterLab Associates, Reston, VA, USA) 5 ACS Paragon Plus Environment


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with a 2.5-cm diameter aperture, Illuminant A, and 10° standard observer. Reflectance at

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isobestic wavelengths from 400 – 700 nm was used to quantify myoglobin redox forms on the

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surface of steaks. Reflectance at 474, 525, 572, and 610 nm was converted to K/S values using:

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K/S = (1-R)2 / 2R. These values were then substituted into following equations1 to calculate the

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percentage of oxy- and metmyoglobin.

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% oxymyoglobin = [K/S610÷K/S525 for 100% metmyoglobin - K/S610÷K/S525 for sample]

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[K/S610÷K/S525 for 100% metmyoglobin - K/S610÷K/S525 for 100% oxymyoglobin]

% metmyoglobin = [K/S572÷K/S525 for 100% oxymyoglobin - K/S572÷K/S525 for sample] [K/S572÷K/S525 for 100% oxymyoglobin - K/S572÷K/S525 for 100% metmyoglobin]

K and S indicate absorption and scattering coefficient, respectively.

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Myoglobin reference standards were prepared using the extra steaks not used in the study.

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Percentage myoglobin values were also used to calculate muscle oxygen consumption and MRA.

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Muscle oxygen consumption

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Muscle oxygen consumption was determined indirectly as the changes in oxymyoglobin

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level. In the current study, a modified procedure of Madhavi et al.16 was used to determine

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muscle-specific oxygen consumption on days 0, 3, and 7. Following surface color measurements,

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the steak was bisected parallel to the oxygenated surface to expose the interior of steak, resulting

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in two interior pieces. The first interior (the section that was exposed to air) was used for oxygen

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consumption studies and the second interior was used for MRA studies. Freshly cut LL or PM

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muscle samples were allowed to oxygenate for 60 min at 4 ˚C, vacuum packaged, and scanned

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twice on the bloomed surface to measure oxymyoglobin. Oxygen consumption (measured by

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conversion of oxymyoglobin to deoxymyoglobin) was induced by incubating samples at 30 ˚C


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for 30 min. The surface color was scanned using a Hunter Lab Miniscan XE Plus

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spectrophotometer for reflectance from 400 to 700 nm to determine the initial % oxymyoglobin.

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Samples were rescanned immediately to determine remaining surface oxymyoglobin.1 To

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calculate % muscle oxygen consumption, the following equation was used: (% pre-incubation

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surface oxymyoglobin - % post-incubation surface oxymyoglobin).

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Metmyoglobin reducing activity

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Metmyoglobin reducing activity was determined on days 0, 3, and 7 of display.17

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Samples from the interior of steak halves were submerged in a 0.3% solution of sodium nitrite

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(Sigma, St. Louis, MO) for 20 min to facilitate metmyoglobin formation, and then removed,

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blotted dry, vacuum packaged (Prime Source Vacuum Pouches, 4 mil, Koch Supplies Inc.,

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Kansas City, MO), and scanned with a HunterLab MiniScan XE Plus spectrophotometer to

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determine pre-incubation metmyoglobin values.1 Each sample was incubated at 30 °C for 2 h to

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induce metmyoglobin reduction. Upon removal from the incubator, samples were rescanned to

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determine the percentage of remaining surface metmyoglobin. The following equation was used

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to calculate %MRA: [(% surface metmyoglobin pre-incubation - % surface metmyoglobin post-

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incubation).

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Mitochondrial content quantification

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Mitochondrial content in LL and PM muscles during storage were quantified as total

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yield and as mitochondrial DNA.

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Total mitochondrial content in longissimus and psoas muscles

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A differential centrifugation method was used to isolate mitochondria from LL and PM muscles.8 Mitochondrial protein content was determined using a bicinchoninic acid protein



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assay, and reported as mg/g of tissue.18 Briefly, 10 g of minced tissue devoid of fat and

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connective tissue was washed twice with 250 mM sucrose and suspended in 20 mL of

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mitochondrial isolation buffer [250 mM sucrose, 10 mM HEPES, 1 mM EDTA, and 0.1% BSA,

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pH 7.2]. The suspension was stirred slowly and hydrolyzed with protease (protease/tissue, 0.5

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mg/g of tissue) for 20 min; the pH was maintained between 7.0 and 7.2. After proteolytic

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digestion, the suspension was diluted to 300 mL with mitochondrial isolation buffer and

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homogenized using a Kontes Duall grinder (Vineland, NJ) followed by a Wheaton Potter-

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Elvehjem grinder (Millville, NJ). The homogenate was centrifuged at 900 x g for 20 min with a

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Sorvall refrigerated RC-5B centrifuge (Thermo Fisher Scientific, Waltham, MA), and the

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resulting supernatant was again centrifuged at 14,000 x g for 15 min. Mitochondrial pellets were

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washed twice and suspended in mitochondrial suspension buffer (250 mM sucrose, 10 mM

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HEPES, pH 7.2). All steps were performed between 0 - 4 °C.

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Quantification of mitochondrial DNA

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A 274 bp amplicon of bovine mitochondrial cytochrome b gene (gb|JX472274.1|;

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forward primer – 5’GACCTCCCAGCTCCATCAAACATCTCATCTTGATGAAA3’, reverse

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primer – 5’CTAGAAAAAGTGTAAGACCCGTAATATAAG3’) was used to evaluate

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mitochondrial copy number19 and a 119 bp amplicon based on the 18S rRNA gene (forward

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primer - 5’TTCGAACGTCTGCCCTATTAA3’, reverse primer –

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5’GATGTGGTAGCCGTTTCTCAGG3’) was used as the normalizer. Total DNA was isolated

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using a QIAamp DNA Mini Kit (Qiagen, Valencia, CA) and manufacturer’s instructions with

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following modifications. Approximately, 0.3 g of the ground muscle in liquid nitrogen was used

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for further processing. The PCR assay was validated first by assessing the primer efficiencies via

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performing a standard curve analysis using 7-log dilutions of template DNA. The specificity of


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the assay was determined by ensuring a single amplicon and by sequence analyzing several

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amplicons to confirm that they represent cytochrome b gene. Paired LL and PM muscles were

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assayed for mitochondrial cytochrome b gene and 18S rRNA using an SYBR green1 reporter

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assay (Roche Diagnostics, Indianapolis, IN). The reaction mixture contained forward and reverse

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primer, master mix (Roche Diagnostics, Indianapolis, IN), and template DNA. Cycling

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conditions were; 95 °C for 10 min followed by 50 cycles of 94 °C for 15 s, 58 °C for 15 s, and

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72 °C for 20 s. The relative mitochondrial copy number was calculated using the comparative Ct

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method as previously described.20 For the two muscle types, the mitochondrial cytochrome b Ct

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was normalized to the 18S gene. The validity of the 18S rRNA gene as a normalizer was

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ascertained by ensuring that the Ct values for each sample for the normalizer fell within ±1

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amplification cycle.

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Mitochondrial oxygen consumption

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Mitochondrial oxygen uptake was measured using a Clark oxygen electrode6,7 (polarizing

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voltage of 0.6 V and an 8 mL incubation chamber). The reaction components were added to the

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incubation chamber, which was maintained at 25 °C by a water jacket and Lauda RE120

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circulating water bath (Westbury, NY). The chamber was stirred with a 10 mm Teflon-covered

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bar at 600 rpm. The electrode was attached to a Rank Brothers digital model 20 oxygen

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controller (Cambridge, England) and connected to a computer and data logger. Oxygen

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consumption was recorded over time at pH 5.6 by suspending mitochondria in incubation buffer

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(250 mM sucrose, 5 mM KH2PO4, 5 mM MgCl2, 0.1 mM EDTA, 0.1% BSA, and 20 mM maleic

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acid) with the addition of succinate (10 mM) as a substrate. The oxygen consumption rate was

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calculated based on the method of Estabrook.21

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Quantification of cytochrome c The cytochrome c content in sarcoplasm was quantified using a sandwich-ELISA assay

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per manufacturer’s instructions with following modifications (Abcam, Cambridge, UK). To

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measure cytochrome c content in sarcoplasm, 100 mg of either LL or PM muscle was

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homogenized in 900 µL of buffer (10 mM HEPES, pH 7.2) using a Wheaton Potter-Elvehjem

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tissue grinder. The homogenates were centrifuged at 900 x g for 10 min. The supernatant was

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then centrifuged again at 14,000 x g for 10 min. Mitochondria sediment at 14,000 x g, and the

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supernatant was used to determine cytochrome c content in the sarcoplasm. The protein

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concentration of the supernatant was determined using a BCA assay. The samples were

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transferred to a 96-well microplate and were incubated for 3 h. Following incubation, the plate

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was washed using TRIS washing buffer. Cytochrome c detector bovine specific antibody

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solution was added to each well before another one-hour incubation time. The liquid was

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discarded, and the plate was washed again. The horseradish peroxide solution was added, and the

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microplate was incubated for one hour. The plate was washed five times before 200 µL

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development solution was added to each well. The absorbance of each well in the microplate was

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measured at 600 nm every 90 s for 30 min at room temperature using a kinetic program. A

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standard curve was plotted with known cytochrome c and expressed as µg cytochrome c in per

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gram tissue.

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Oxidation-reduction potential

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A RedoxSys analyzer was used to measure the ORP status in muscles. This machine

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measures the balance of oxidants and reductants, providing a direct measurement of oxidative

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stress using electrochemical principles.22 Briefly, 100 mg meat sample from each muscle type on

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days 0, 3, and 7 after postmortem was homogenized in 50 mM phosphate buffer at pH 7.4 using


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a handheld homogenizer. The homogenate was centrifuged at 12,000 x g for 5 min. 20 µL of the

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supernatant was applied on the sensor attached to RedoxSys analyzer reader (Aytu BioScience,

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Inc, Englewood, CO) and ORP status was recorded as millivolt (mV). A lower number

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represents a higher antioxidant status. This technique has been used to measure oxidative stress

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status in serum and plasma.22,23

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Lipid oxidation Thiobarbituric acid reactive substances values were utilized to indicate lipid oxidation.

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blended with 25 mL trichloro acetic acid (TCA) solution (20%) and 20 mL distilled water.

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Samples were homogenized using a Sorvell Omni tabletop mixer (Newton, CT) for 1 min and

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filtered through a Whatman (#1) filter paper. One mL of filtrate was mixed with 1 mL of

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thiobarbituric acid (TBA) solution (20 mM), incubated in a boiling water bath for 10 min. The

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samples were cooled and the absorbance at 532 nm was measured using a Shimadzu UV-2600

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PC spectrophotometer (Shimadzu Inc., Columbia, MD, USA). Blank samples for the

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spectrophotometer consisted of 2 mL TCA/distilled water (1:1 v/v) and 2 mL TBA solution.

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Statistical analysis

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From each steak, 5 g of the sample that contained both interior and displayed surface was

The experimental design was a split-plot, where muscles (LL and PM) from each animal

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served as a block in the whole plot (n = 8). In the subplot, a steak was assigned a day of the

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display (0, 1, 3, 5, or 7 for surface color measurements and 0, 3, and 7 for biochemical studies).

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For surface color, mitochondrial-, and biochemical studies, the fixed term included muscle type,

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display time, and their interaction. The random terms included animal (Error A) and unspecified

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residual error (Error B). Type-3 tests of fixed effects were performed using the Mixed Procedure

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of SAS (version 9.3, SAS Institute Inc; Cary, NC). Least square means for protected F-tests (P < 11 ACS Paragon Plus Environment


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0.05) were separated by using the pdiff option (least significant differences) and were considered

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significant at P < 0.05 level.

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RESULTS AND DISCUSSION

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Effects of muscle type on surface color and biochemical properties

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The relationship between oxidative stress, mitochondria, and cell death have been

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investigated extensively in medical research.25,26 However, limited information is currently

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available on muscle-specific oxidative stress on mitochondrial degeneration. There were no

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significant differences in proximate compositions between two muscles (Table 1). In the current

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study, PM had greater % metmyoglobin and lower redness (P < 0.05) compared with LL on day

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3 (Table 2). LL had a stable red color than PM during display (changes in % metmyoglobin level

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in LL and PM between day 0 and 7 were 28.4 and 78.3, respectively). A lower color stability in

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PM was characterized by lesser (P < 0.05) MRA than LL on days 0, 3, and 7 (Table 3). However,

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PM had greater (P < 0.05) muscle oxygen consumption on day 0, while LL had greater oxygen

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consumption on days 3 and 7 than PM (Table 3). There were no differences (P > 0.05) between

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muscle types for lipid oxidation on day 0 of the display, however, PM had greater lipid oxidation

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(P < 0.05) on days 3 and 7. Various studies have reported color labile nature of PM during

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display than LL.3-5 The authors speculated that differences in fiber type and greater oxygen

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consumption attributed to rapid discoloration. In the present research, we focused on how

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oxidative changes can affect mitochondrial degradation and cytochrome c level in sarcoplasm to

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understand the lesser color stability of PM.

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Mitochondrial oxygen consumption Mitochondrial oxygen consumption was greater for PM on day 0 than LL (Table 4). This

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suggests that there are inherent differences in oxygen consumption capabilities per mg

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mitochondria between muscle types. Previous research also reported a greater oxygen

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consumption in isolated bovine PM mitochondria27 and in steaks28 compared with LL. Greater

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oxygen consumption ability of PM mitochondria and greater mitochondrial content may be

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responsible for increased oxygen consumption in PM steaks. However, the overall change in

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mitochondrial oxygen consumption from day 0 to day 7 for LL was less (P < 0.05) than PM

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(changes in mitochondrial oxygen consumption in LL and PM between day 0 and 7 were 7.8 and

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27.3, respectively).

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Immediately after animal harvest, cells try to maintain homeostasis for energy production;

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however, the lack of blood flow leads to failure of adaptive mechanisms. These changes can

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affect the pro- and antioxidant balance, leading to accumulation of reactive oxygen species

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(ROS). Tricarboxylic substrates such as citrate and malic acid are utilized faster in PM than that

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in LL.29 Both citrate and malate are intermediates in the tricarboxylic acid cycle and can be used

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for mitochondrial oxygen consumption. Postmortem bovine muscle can use succinate, pyruvate,

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and malate for oxygen consumption.6,7 With a less efficient antioxidant system, mitochondrial

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utilization of substrates for oxygen consumption can lead to greater ROS and increased oxidative

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changes which can trigger lipid oxidation. Both complexes I and III are the potential sites of

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ROS.30,31 Pre-incubation of HNE (a secondary lipid oxidation product) with bovine heart

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mitochondria decreased mitochondrial oxygen consumption.10 The reaction between

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mitochondrial protein and lipid oxidaiton products can be partially accounted for lower oxygen

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consumption in PM than LL mitochondria on day 7. Although mitochondrial oxygen



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consumption was greater on day 0, an increased oxidative stress combined with mitochondrial

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degeneration may be responsible for greater changes in oxygen consumption between day 0 and

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7 for PM than LL.

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Oxidation-reduction potential

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There was a significant muscle type and display time interaction for ORP (P < 0.001).

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PM had a greater ORP on day 0 (indicative of electron donating proteins or compounds) than LL

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(Figure 1). The ORP measurements using the RedoxSys implies the net balance of oxidants and

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reductants, providing a direct measure of oxidative stress.22 Mitochondria contain several

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antioxidant proteins and electron accepting compounds. Hence initial ORP values were higher in

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PM than LL (a lower number indicates a greater antioxidant potential). Interestingly, ORP values

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for LL did not change significantly during display. Although the mechanism for stable ORP in

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LL is not clear, we speculate that a greater MRA and a lower muscle oxygen consumption may

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have limited oxidative changes. ORP changes between days 0 and 3 were greater for PM than LL.

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This suggests that the antioxidant defense mechanisms lose its efficacy more rapidly in PM than

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in LL. In support, Joseph et al.4 reported that PM has a lower amount of antioxidant proteins than

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LL in sarcoplasm, which makes it more susceptible to oxidative stress and buildup of free

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radicals and secondary lipid oxidation products, leading to faster discoloration. Lipid oxidation

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products can bind with lactic acid dehydrogenase and myoglobin to make muscles more prone to

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discoloration.25,26 In support, redness and % metmyoglobin accumulation were greater in PM

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than LL between days 1 and 3.

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Mitochondrial and cytochrome c content changes

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On day 0, PM muscle had a 2.55-fold greater mitochondrial DNA content than LL (Table 4). On day 0 of display, the average mitochondrial protein content in LL and PM were 0.21 and 14 ACS Paragon Plus Environment

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0.38 mg/g of muscle, respectively (P = 0.02). This represents approximately a 1.80-fold

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difference in mitochondrial content between two muscles. PM has predominant red fibers32 than

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LL. Mohan et al.3 reported approximately a 1.21-fold higher mitochondrial concentration in beef

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PM compared with LL muscle in 10-day aged samples using cytochrome c oxidase activity.

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Previous research also demonstrated that mitochondrial enzyme levels were lower in rabbit and

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guinea pigs white muscles than in red muscles.33,34 However, in the present study mitochondrial

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content in PM was lower than LL on days 3 and 7. This demonstrates that the mitochondrial

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degradation was greater in PM than LL.

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Mitochondria are the first organelle subjected to oxidative damage from free radicals,

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which can trigger mitochondrial degradation.11 Although the mechanism of muscle-specific

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differences in mitochondrial degradation in postmortem muscle is not clear; we speculate the

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effects of oxidative stress on mitochondria from various disease conditions such as Alzheimer's

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or Parkinson's diseases from the medical research. Both inner and outer mitochondrial

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membranes are composed of phospholipids. Further, oxidative phosphorylation occurs within

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the mitochondrial inner membrane which can be a source of ROS formation. Hence,

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mitochondrial membranes are very prone to lipid peroxidation. Previous research demonstrated

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that incubation of secondary lipid oxidation product such as HNE with bovine heart

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mitochondria increased vacuolation and fragmentation.10 In normal mitochondria, the outer

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membrane is more permeable due to porin proteins than the inner membrane; however, the

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oxidative condition can increase permeability and fluidity of the inner membrane. 35,36 This can

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lead to release of cytochrome c from the inner membrane to the outside. There were no

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significant differences in cytochrome c content in sarcoplasm of LL muscle with increased

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storage time, but the cytochrome c content increased for PM (Figure 2). In healthy cells,



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cytochrome c is located within the mitochondrial inner membrane and also interact with

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cardiolipin (a diphosphatidylglycerol lipid present in the mitochondrial membrane).37

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Physiologically, greater levels of free cytochrome c in intermembrane of mitochondria indicate

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mitochondrial damage by ROS.12 Several proapoptotic stimuli can trigger permeabilization of the

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outer mitochondrial membrane and promote mobilization of cytochrome c from the inner

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membrane and cardiolipin38,39 to sarcoplasm. Currently, limited knowledge is available on the

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factors that trigger the release of cytochrome c to sarcoplasm in postmortem muscles. Since there

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were significant differences between muscle types, a systemic analysis of changes in

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mitochondrial- and cytochrome c content can be used as a potential tool to predict color stability.

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As the results suggest, mitochondrial degradation caused by oxidative stress can

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influence meat color stability. Various studies have demonstrated the role of mitochondria in

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color stability6-7,9 and in bloom development.40-43 A greater oxygen consumption may be

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detrimental to color stability and also for the formation of a bright-red color. Hence, controlling

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mitochondrial oxygen consumption via postharvest techniques such as packaging or storage

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temperature can minimize discoloration. A moderate mitochondrial activity might be beneficial

349

for a brighter-red steak that lasts for a longer time.

350

In summary, PM had a shorter color stability than LL. In addition to the role of oxygen

351

consumption and MRA, oxidative stress has muscle-specific effects on mitochondrial

352

degradation and cytochrome c release. PM had greater mitochondrial degradation and less

353

antioxidant capacity than LL with increased display time. Greater oxidative stress in PM may be

354

responsible for cytochrome c release and lower mitochondrial content in PM than LL. Therefore,

355

characterizing the factors responsible for mitochondrial degradation postmortem may help to

356

understand the muscle-specific differences in color stability.


Page 17 of 28


357

ABBREVIATIONS USED

358

LL, longissimus lumborum; PM, psoas major; ORP, oxidation-reduction potential; MRA,

359

metmyoglobin reducing activity; AOAC, Association of Official Analytical Chemists; IMPS,

360

Institutional Meat Purchase Specifications; USDA, United States Department of Agriculture;

361

PVC, polyvinyl chloride; HNE, 4-hydroxy-2-nonenal

362

ACKNOWLEDGMENT

363

Appreciation is expressed to the RedoxSys/Aytu Biosciences for providing the oxidation-

364

reduction potential meter.

365

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366 367

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472


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473


Figure captions

474 475

Figure 1

476

Effects of muscle type and display time on oxidation-reduction potential properties

477 478 479 480

A lower value represents greater antioxidant capacity. a–c

Least squares means with a different letter differ (P < 0.05); standard error bars are indicated at each time point. LL = longissimus lumborum; PM = psoas major

481 482 483

Figure 2

484

Effects of muscle type and display time on cytochrome c content in sarcoplasm

485 486 487

a–d

Least squares means with a different letter differ (P < 0.05); standard error bars are indicated at each time point. LL = longissimus lumborum; PM = psoas major

488 489 490 491



492

Page 22 of 28

Figure 1

Oxidation reduction potential (mV)

295 290

LL

PM

a a

a

285 280

c

c

275 270

b

265 260 255 250 0

3

7

Display time (d) 493 494


Page 23 of 28

495


Figure 2 0.16 LL

PM

Cytochrome c content (µg/g tissue)

0.14

d c

0.12 0.10

b a a

0.08

a

0.06 0.04 0.02 0.00 0

3 Display time (d)

7

496 497 498



499

Table 1

500

Proximate composition and pH of LL and PM muscles Trait

LL

PM

SE

P-value

Moisture (%)

70.5

70.4

0.97

0.94

Protein (%)

21.7

20.9

0.4

0.28

Fat (%)

5.9

7.3

1.3

0.51

pH

5.57

5.61

0.02

0.13

Page 24 of 28

501 502

LL = longissimus lumborum; PM = psoas major

503

SE = standard error

504


Page 25 of 28


505

Table 2

506

Least square means for the effects of muscle type and display time on surface color Trait a) a* values (redness)

Muscle LL PM

0 30.6a,x 29.5a,y

Display time (day) 1 3 5 b,x a,x 33.4 30.1 28.4c,x 32.4b,y 20.3c,y 15.4d,y

b) % metmyoglobin

LL PM

7.2a,x 5.6a,x

7.2a,x 10.5b,x

12.5b,x 65.2c,y

21.4c,x 77.5d,y

7 26.4d,x 13.1e,y 35.6d,x 83.9e,y

507 508

LL = longissimus lumborum; PM = psoas major

509 510

For both a* value and % metmyoglobin: display time, P < 0.0001; muscle effect, P < 0.0001; display time x muscle, P < 0.0001

511 512

x-y

Means in a column with a different letter differ (P < 0.05)

513

a-e

Means in a row with a different letter differ (P < 0.05)



514

Table 3

515 516

Least square means for the effects of muscle type and display time on MRA, oxygen consumption, and lipid oxidation

Page 26 of 28

517

519 520

SE518 Display time (day) 0 3 7 x,a x,b 3.2 a) MRA (%) LL 65.4 60.4 54.4 x,c y,a y,b y,b PM 55.4 30.4 26.4 x,a x,b 3.1 b) Oxygen consumption (%) LL 57.4 50.4 42.4 x,c y,a y,b y,c PM 68.5 45.6 34.5 x,a x,b 0.009 c) Lipid oxidation LL 0.031 0.052 0.077 x,c PM 0.038 x,a 0.072 y,b 0.124 y,c LL = longissimus lumborum; PM = psoas major; MRA = metmyoglobin reducing activity; SE = standard error

521 522

For both MRA and oxygen consumption: display time, P < 0.0001; muscle effect, P < 0.001; display time x muscle, P < 0.001

523 524

Lipid oxidation is indicated as thiobarbituric acid reactive substances and measured as absorbance at 532 nm.

525

x-y


526

a-c



Page 27 of 28


527

Table 4

528

Comparison of mitochondrial content and oxygen consumption in beef longissimus and psoas muscles during display Parameters Mitochondrial content

Mitochondrial oxygen consumption (nanomoles of oxygen consumed/min per mg of mitochondria)

LL

PM

SE

P-value

Mitochondrial protein yield, d 0 Mitochondrial protein yield, d 3 Mitochondrial protein yield, d 7 Relative mitochondrial DNA content, d 0

0.21 x,a 0.19 x,ab 0.15 x,b 1.00

0.38 y,a 0.24 x,b 0.12 x,c 2.55

0.03

0.002

0.28

0.01

d0 d3

30.2 x,a 26.5 x,ab

42.5 y,a 32.4 x,b

3.2

0.001

d7

22.4 x,b

15.2 y,c

529 530 531 532

Mitochondrial protein yield represents total mitochondria mg of protein per gram of tissue isolated from paired LL and PM muscles by differential centrifugation; mitochondrial DNA content was reported as a relative fold change in comparison to paired PM and LL muscles.

533

For both mitochondrial content and mitochondrial oxygen consumption: display time, P < 0.0001; muscle effect, P < 0.001

534

SE = standard error; LL = longissimus lumborum; PM = psoas major

535

x-y


536

a-c


537 538



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