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CHARACTERIZATION OF POSTMORTEM FACTORS REGULATING
PORK COLOR, WATER HOLDING CAPACITY AND TENDERNESS

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Chuck P. Allison

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CHARACTERIZATION OF POSTMORTEM FACTORS REGULATING PORK
COLOR, WATER HOLDING CAPACITY AND TENDERNESS

By

Chuck P. Allison

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Animal Science

2001

ABSTRACT

CHARACTERIZATION OF POSTMORTEM FACTORS REGULATING PORK
COLOR, WATER HOLDING CAPACITY AND TENDERNESS

By

Chuck P. Allison

The rate of biochemical reactions which occur in porcine skeletal muscle
prior to, and immediately following harvest have a dramatic impact on both
quality and quantity of pork produced. The objective of these studies was to
quantify biochemical characteristics of porcine longissimus dorsi (LM) muscle
and determine their relationship to pork color, water holding capacity and
tenderness. Longissimus dorsi muscle samples were excised from carcasses at
20 min postmortem for quantification of phosphofructokinase and pyruvate
kinase capacity. Phosphofructokinase and, pyruvate kinase activities were not
correlated with LM pH, purge, drip loss, or color (P > .31). Tenderness,
measured by Wamer-Bratzler shear (WBS), was optimized by. aging loin chops
for 7 (I. Western blot analysis of desmin from 4 tender (<4 kg) and 4 less tender
(>4.7 kg) samples revealed that desmin degradation paralleled decreases in
WBS through postmortem storage. These data indicate that variation in pork
color and water holding capacity is not associated with variation in glycolytic
enzyme capacity. Additionally, tenderization of most pork loin chops is complete

by d 7 and tenderization coincides with desmin degradation.

This thesis is dedicated to my parents who have always believed in me and given
me the courage to pursue my dreams. This is also for Melissa, who has
sacrificed closeness with her family so that I can further my education, and has
been there with love and encouragement every step of the way.

ACKNOWLEDGEMENTS

This work would have not been possible without the orchestrated help of
so many that I am extremely indebted to. I would first like to thank Dr. Roger
Johnson for allowing me to work on an independent study project under his
direction and for ultimately creating a position within his meat quality program for
me, prior to attending Michigan State University. Through interaction with Dr.
Johnson, I was able to gain an understanding of the meat quality problems that
face the swine industry and an intense desire to learn more.

I would like to thank my major professor, Dr. Matthew Doumit for his
guidance, friendship and for challenging me to think outside the box both in the
laboratory and in the classroom. Additionally, I would like to thank him for taking
a chance and accepting me into his program. Dr. Doumit has gone above and
beyond the call of duty to ensure that I fully understand the principles of meat
science, and I will always be grateful for the opportunity to work under his
direction. I would also like to thank the members of my guidance committee, Drs.
Al Booren, Ron Bates and Gale Strasburg for their valuable input and advice in
my research, graduate program and for this thesis. I would also like to
acknowledge the efforts of Dr. Rob Templeman for his assistance with the design
and analyses of the tenderness study.

I owe a special thanks to Ms. Sharon Debar for her assistance with lab
work. She has given unselfishly of her time to assist in the early morning harvest

of animals and has always been there to make buffers when there was a last

minute change of plans in assays. I certainly appreciate all the time and effort
that she has put into these projects.

I would like to thank Tom Forton, Jennifer Dominguez and the Meat Lab
crew for harvesting and fabricating the pigs used in these studies, as well as
being patient while we took the samples that we needed. I would also like to
thank Dave Edwards and Dr. Bates for raising the animals that were used to
quantify enzyme capacity.

I would like to thank my lab mates, Nick Mesires, John Heller, Matt Ritter
and Jason Scheffler for a lot of fun times, their constructive criticism and their
willingness to help with my research program. I would also like to thank
Courtney Dilley and Jeannine Grobbel who have worked in the lab and whose
willingness to help has not gone unnoticed. I certainly appreciate all of the work
you guys have done.

Lastly, I would like to thank Melissa who has allowed me to follow my
dreams and has been there in the trenches to encourage me along. I will always

be indebted to her for the sacrifices that she has made for me.

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................... viii
LIST OF FIGURES .............................................................................................. ix
LIST OF ABBREVIATIONS ................................................................................. x
INTRODUCTION .................................................................................................. 1
LITERATURE REVIEW ........................................................................................ 3
A. Skeletal Muscle ........................................................................................... 3

1. Skeletal Muscle Contraction .................................................................... 3

2. Postmortem Changes in Skeletal Muscle ........................................ 5

3. Red, Firm and Non-exudative Pork ......................................................... 6

4. Pale, Soft and Exudative Pork ................................................................. 7

5. Dark, Firm and Dry Pork ........................................................................ 10

B. Control of Glycolytic Metabolism ............................................................... 11
1. Phosphofructokinase ............................................................................ 12

2. Pyruvate Kinase ................................................................................... 13

3. Genetic Predisposition .......................................................................... 14

a. Halothane Gene .............................................................................. 14

b. Napole Gene ................................................................................... 16

4. Harvest Procedures .............................................................................. 17

C. Pork Tenderness ....................................................................................... 18
1. Determinants of Meat Tenderness ....................................................... 18

2. Causes of Postmortem Tenderization .................................................. 20
CHAPTER 1 ....................................................................................................... 22
A. Abstract ..................................................................................................... 22
B. Introduction ................................................................................................ 23
C. Materials and Methods .............................................................................. 24

1. Animals and Meat Quality Data Collection ........................................... 24

2. Sample Preparation for Enzyme Assay ................................................ 27

3. Enzyme Assay ...................................................................................... 28

4. In vitro Phosphorylation of Pyruvate Kinase ......................................... 30

5. Data Analysis ........................................................................................ 31

D. Results and Discussion ............................................................................. 32
E. Implications ............................................................................................... 45
CHAPTER 2 ....................................................................................................... 46
A. Abstract ..................................................................................................... 46
B. Introduction ................................................................................................ 47
C. Materials and Methods .............................................................................. 48

vi

1. Animals and Meat Quality Data Collection ........................................... 48

2. Sample Preparation for Analysis of Protein Degradation ....................... 49

3. SDS-PAGE and lmmunoblotting ........................................................... 50

4. Data Analysis ......................................................................................... 50

D. Results and Discussion ............................................................................. 51
a. Wamer-Bratzler Shear .................................................................... 51

b. Analysis of Desmin Degradation ..................................................... 52

E. Implications ............................................................................................... 58
LITERATURE CITED ......................................................................................... 59

vii

LIST OF TABLES
Table 1.1 Timeline for meat quality measurements ........................................... 25

Table 1.2 V"max values, standard deviations and ranges for enzyme capacity by
fraction ............................................................................................... 29

Table 1.3 Least square means and ranges for carcass measurements ............. 33

Table 1.4 Correlation coefficients between temperature, pH, color and water
holding capacity ................................................................................. 37

Table 1.5 Correlation coefficients between glycolytic enzyme capacity and pork
quality traits ....................................................................................... 44

viii

Figure 1.1
Figure 1.2

Figure 1.3

Figure 1.4

Figure 2.1

Figure 2.2

Figure 2.2

LIST OF FIGURES

Longissimus muscle pH decline ........................................................ 34
Postmortem longissimus muscle temperature decline ...................... 36
Relationship between L1 and measure of water loss in longissimus

muscle .............................................................................................. 40

Relationship between 45 min pH and measure of water loss in
longissimus muscle .......................................................................... 42

Means and standard deviations for WBS values measured at 1, 3, 7
and 14 d-postmortem ....................................................................... 53

Western blot detection of desmin degradation using a monoclonal
“D76” antibody (Panel A and B) ....................................................... 55

Western blot detection of desmin degradation using a monoclonal
“D76” antibody (Panel C and D) ....................................................... 57

LIST OF ABBREVIATIONS
ADP - Adenosine Diphosphate
AMP — Adensoine Monophosphate
AMPK - AMP-Activated Protein Kinase
ATP - Adenosine Triphosphate
BSA - Bovine Serum Albumin
CWHC — Centrifuge Water Holding Capacity
DFD - Dark, Firm and Dry
DRIP1 — Suspension Drip Measured from 24 to 48 h postmortem

DRIP6 - Drip loss measured under simulated retail display conditions day 6 to
day 7 postmortem

F-6-P - Fructose-1,6-phosphate

L6 - Minolta (CIE) L” values taken at d 6 postmortem
L7 - Minolta (CIE) L* values taken at d 7 postmortem
LD — Longissimus Dorsi

LDH - Lactate Dehydrogenase

LM - Longissismus Muscle

MCE - Mercaptoethanol

MSU - Michigan State University

MW — Molecular Weight Standards

NADH — Nicotinamide Adenine Dinucleotide, reduced form
NFDM - Non-fat Dry Milk

PEP — Phosphoenol Pyruvate

PK - Pyruvate Kinase

PSE - Pale, Soft and Exudative

RN“ - Rendement Napole
SDS-PAGE - Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

TBS — Tris Buffered Saline

WBS - Warner Bratzler Shear value

xi

INTRODUCTION

With the production of faster growing pigs that yield leaner, heavier
muscled carcasses, pork quality problems have arisen. Selection procedures
have been implemented by seedstock swine producers to eliminate sources of
inferior pork quality in the last ten years. The removal of the halothane gene
from many genetic nucleus herds led scientists to believe that the source of
inferior quality pork had been eliminated. However, it has been estimated that
10-20% of pigs harvested today still produce pale, soft and exudative (PSE) pork
that has unacceptable meat color, water-holding capacity and/or tenderness.

Much research has focused on the causes of PSE meat. It has been well
documented that environmental stressors and high ambient temperatures or
fluctuating daily temperatures increase the occurrence of PSE pork. It is
generally accepted that a rapid postmortem pH decline, while muscle
temperature remains high, is the cause of PSE in pork. However, research to
date has been unable to conclusively establish the factors that are responsible
for the conversion of porcine muscle to PSE meat.

The most immediate approach to reducing the occurrences of PSE pork
is through selection towards halothane resistant breeding stock, careful
management throughout the life of the animal and rapid chilling of the carcass.
However, the ultimate goal is to estainSh a better understanding of the
biochemical processes that are responsible for the variation observed in ultimate

pork quality.

These studies were conducted to: 1) quantify the enzymatic capacity of
regulated steps in glycolysis and determine their relationship to porcine
longissimus dorsi muscle traits (pH decline, color and water-holding capacity);
and 2) quantify the optimal tenderization time of porcine longissimus dorsi
muscle and determine if degradation of desmin is associated with pork muscle
tenderization.

Collectively, these data will provide a better understanding of biochemical
mechanisms responsible for differences in pork quality. This knowledge could
lead to manipulation of biochemical systems that lead to PSE pork muscle.
Thus, allowing suppliers to produce and market animals possessing superior

color, water holding capacity and tenderness.

LITERATURE REVIEW

Skeletal Muscle

Orcutt et al. (1990) demonstrated that skeletal muscle constitutes 37% of
the live weight of pigs. However, skeletal muscle did not evolve for human
consumption, but rather to provide a means of locomotion through carefully
orchestrated contraction and relaxation cycles. The same processes that make
locomotion possible are also responsible for attempting to maintain homeostasis
in the muscle after exsanguination. Therefore, a basic understanding of how
living tissue functions provides insight into the problems that may arise in the
postmortem conversion of muscle to meat.
Skeletal Muscle Contraction

During skeletal muscle contraction, chemical energy, derived from the
hydrolysis of ATP, is converted into mechanical force (Regnier et al., 1995). In
adult, fast-twitch skeletal muscle, the signal for excitation-contraction coupling is
transferred across the triad junction from transverse tubules to-the terminal .
cisternae. This wave of depolarization causes ryanodine receptors to open and
results in release of calcium from the sarcoplasmic reticulum (Coronado et al.,
1994). Calcium diffuses from the terminal cistemae and binds to troponin
molecules. When calcium is bound to troponin, it relieves the inhibition that
tropomyosin exerts on crossbridge formation between actin and myosin. The
activated troponin interacts with tropomyosin causing it to shift its position along

the actin filament. The shift by tropomyosin allows the myosin head to attach to

the actin molecule and initiates a crossbridge cycle (Potter et al., 1995). Pate
and Cooke (1989) suggested the following basic cycle occurred during skeletal
muscle contraction after the actin-myosin crossbridge had formed. Inorganic
phosphate is released from the myosin head and a power stroke results, in which
the myosin head “pulls” the actin filament toward the m-line. The z-lines move
closer together (sarcomere shortening) resulting in the force needed for
locomotion. Simultaneously, ADP is released and ATP attaches to the myosin
head causing dissociation of the actin-myosin complex. Subsequently, the
hydrolysis of ATP to ADP and inorganic phosphate by myosin ATPase initiates a
new cycle. When the stimulus for muscle contraction ceases, the sarcolemma
and the transverse tubules become repolarized. The calcium ATPase pumps the
calcium back into the sarcoplasmic reticulum. Additionally, tropomyosin regains
its original position over the myosin-binding site. In the absence of an
attachment between actin and myosin, tension is not generated and the
stretching imposed by elastic components in the muscles cause the filaments to
slide passively over one another. The muscle returns to the resting state.

White et al. (1964) demonstrated that ATP and its immediate source of
replenishment, creatine phosphate, could be maintained by oxidative
phosphorylation in slow working muscle. However, in rapidly contracting muscle,
the oxygen supply becomes insufficient and ATP must be synthesized

anaerobically via glycolysis.

Postmortem Changes in Skeletal Muscle

Once an animal has been exsanguinated muscle tissues are no longer capable
of generating energy via aerobic methods. The muscle is therefore forced to
switch to anaerobic processes to generate ATP. The biochemical reactions that
occur after exsanguination are largely responsible for the ultimate quality of
meat. The muscles of living pigs are moderately dark in color, firm in texture and
dry in appearance (Briskey, 1964b). The magnitude of change in muscle
characteristics from those of living muscle is a direct result of the rate and extent
of postmortem anaerobic glycolysis, pH decline and temperature decline.

It is generally accepted that the net product of anaerobic glycolysis from
glucose is 2 ATP, 2 lactate and 2 H‘“. Thus, the postmortem demands for ATP
and increased ATP production will result in more hydrogen ion accumulation and
a reduced ultimate pH. The rate of glycolysis is determined by the utilization of
energy in the cell. The major sites of ATP utilization are the myofibrillar (myosin)
ATPase, calcium ATPase and sodium/potassium ATPase. Myosin ATPase
catalyzes the hydrolysis of ATP to ADP and an inorganic phosphate generating
the energy to drive the crossbridge cycle (Bechtel and Best, 1985). The amount
of ATP hydrolyzed by myosin ATPase during a single muscle twitch is
approximately .3 mmoles/l (lnfante and Davies, 1962). The calcium ATPase
functions to resequester calcium into the SR so that relaxation of skeletal muscle
can occur. Bechtel and Best (1985) calculated the amount of ATP to resequester
the calcium released during a single muscle twitch to be about .1 mmoles/I. The

sodium/potassium pump functions to maintain the ion gradient of the cell or a net

negative charge on the inside of the cell. This is accomplished by pumping 3
sodium ions out of and 2 potassium ions into the cell for every ATP molecule
hydrolyzed (Bechtel and Best, 1985).

The extent of glycolysis is related to the amount of glucose or glucose
equivalents that are available to be processed through glycolysis and the stability
of glycolytic enzymes. Monin et al. (1981) demonstrated that glycolytic potential,
3 measure of available substrates and the end product in anaerobic glucose
metabolism, could be used as an indicator of the potential postmortem lactate
formation and consequently H+ accumulation. When muscle ATP stores become
depleted, permanent actin-myosin crossbridges form and the muscle is said to be
in rigor.

Forrest et al. (1963) noted that the ultimate gross morphology of porcine
muscle ranged from pale, soft and exudative to dark, firm and dry in appearance.
The patterns of pH decline (Briskey, 1964a), and/or the pH and temperature
relationships in the muscle prior to the onset of rigor mortis are associated with
the ultimate muscle classification (Sayre and Briskey, 1963). A brief description
of each condition will follow.

Red, Firm and Non-exudative Pork

Many different criteria have been used to predict the conversion of muscle
to red, firm and non-exudative pork (normal pork). Wismer-Pedersen and
Briskey (1961) demonstrated that when glycolysis proceeds at an intermediate
rate, which requires 6-12 hours until lactate production ceases, the muscle

exhibits a grayish pink to red color, moderately firm structure and dry

appearance. In 1963, Sayre and Briskey reported that if the pH of the muscle is
above 6.0 at 2 hours postmortem and temperature is below 35°C at the onset of
n'gor, the muscle would exhibit normal properties regardless of the ultimate pH.
Additionally, they observed highly significant correlations between muscle protein
solubility and water-holding capacity. Scopes and Lawrie (1963) demonstrated
that as the normal ultimate pH of 5.5 is attained, certain sarcoplasmic proteins
are already denatured. These data demonstrated that even under normal
glycolytic conditions, there is a gradual drop in the solubility of sarcoplasmic
proteins and a slight decrease in water-holding capacity of the meat.

The National Pork Producers Council’s Pork Quality Solutions Team
(1998) has developed targets for normal, firm and non-exudative fresh pork
quality. These targets are to be used as ranges for pork longissimus traits
measured at 24 hr postmortem. The solutions team suggests that ultimate pH
range from 5.6 to 5.9 and color range from 3 to 5 visually or when measured
objectively in the range of 49 to 37 (Minolta CIE L). Most importantly, they
suggest that the drip loss not exceed 2.5%.

Pale, Soft and Exudative Pork

Pale, soft and exudative (PSE) pork is often associated with a rapid pH
decline that results in low muscle pH in combination with high muscle
temperature. Fujii et al. (1991) demonstrated that PSE pork is associated with a
mutation in the ryanodine receptor. The mutation appears to result in a
hypersensitive gating of the calcium release channel where channel opening is

facilitated and closing is inhibited. Although it is currently possible to identify pigs

that are homozygous or heterozygous for this mutation, breeds and lines of pigs
that do not possess this genetic defect still produce pork carcasses that exhibit
PSE characteristics (Pommier and Houde, 1993). A survey of 14 packing plants
by Kauffman et al. (1992), revealed that approximately 16% of pork produced
exhibited PSE characteristics. Moreover, the Pork Chain Quality Audit (Cannon
et al., 1996) and the National Pork Producers Council (1991), found that >10% of
all pork carcasses generated in the United States contained PSE meat.

Pale, watery meat develops as a result of excitement of stress-sensitive
pigs before the harvest process, which cause an increase in body temperature
and an accelerated rate of postmortem anaerobic metabolism. This increase in
glycolytic rate causes an increase in hydrogen ion accumulation in the muscle.
The fact that PSE characteristics are hard to induce in pigs that are less
susceptible to stress suggests that there is an innate difference among genetic
lines that causes some pigs to have a greater predisposition to develop an
abnormal pH decline (Gerrard, 1997).

Briskey et al. (1959) demonstrated that, although normal at death,
muscles that were ultimately PSE had significantly lower pH values and higher
muscle temperature at 40 min postmortem. Moreover, the pH values decreased
rapidly to 5.5 or lower, while the temperature remained at 36-41°C. A rapid drop
in pH accompanied by a high temperature results in the denaturation of
approximately 20% of the sarcoplasmic and myofibrillar proteins (Honikel and
Kim, 1986). This helps to explain the undesirable pale color and exudative

characteristics associated with PSE muscle after harvest.

As postmortem muscle pH approaches the isoelectric point of 5.1, the net
protein charge is reduced, as are repulsive forces between myofilaments
(Wrsmer-Pedersen, 1971). This results in a decrease in the spacing observed
between myofilaments. Thus, fresh meat with a lower ultimate pH will have less
net protein charge, decreased myofilament spacing (i.e. less space for water to
be trapped) and less interaction with water, which is a polar substance.

Honikel and Kim (1986) suggested that the wateriness observed in PSE
muscle is determined by breaks in the cell membrane through which fluid can
quickly exude from the cell. Additionally, they observed the myofibrillar protein
myosin in the drip from PSE muscles and suggest that myosin would not be able
to escape the cell if the membranes were intact. According to Offer (1991),
denaturation of sarcoplasmic proteins has a major influence on the increase in
paleness, while denaturation of the myofibrillar proteins is responsible for the
decreases in water-holding capacity. Warriss and Brown (1987) reported a
biphasic relationship between 45-minute postmortem pH, reflectance and drip
loss. These authors demonstrated that muscles with a lower 45-minute pH had
more drip loss and were paler.

Potentially, the most important aspect of PSE pork is the reduced water-
holding capacity of the meat. The inability of meat to bind water affects packer
profitability, functionality and versatility of the product and most importantly
consumer acceptability (T opel et al., 1976). Kauffman et al. (1978) demonstrated
that during transit PSE hams lost three times more moisture than normal hams

and seven times more moisture than dark, firm and dry hams (DFD).

Additionally, moisture losses during curing, smoking and chilling are higher in
PSE hams than those observed in normal or DFD hams. Compared to normal
hams and shoulders, hams and shoulders with severe PSE exhibit 33% and 11%
lower water-holding capacities, respectively (Cannon et al., 1995).

Dark, Firm and Dry Pork

Briskey et al. ( 1959) demonstrated that if there was a very limited pH
decline and rigor mortis took place at a relatively high pH, the muscle remained
dark red in color, firm in texture and dry in appearance. He also noted that if
glycolysis proceeded extremely slowly and rigor mortis occurred over a long
period of time, the same ultimate muscle quality would occur.

Dark, firm and dry pork is often associated with long-term stress that
results in depletion of muscle glycogen prior to harvest. As a result, there is less
glycogen to fuel anaerobic glycolysis, less lactate and hydrogen ion accumulation
and a higher ultimate pH (>6.0). Because of this higher ultimate pH, proteins in
DF D meat have a relatively high net protein charge and undergo minimal protein
denaturation. Both of these factors contribute to an increase in myofilament
spacing (Wismer—Pedersen, 1971), higher water holding capacity and minimal
extracellular fluid accumulation (Kauffman et al., 1994).

Kauffman et al. (1999) subjected eight pork loins representing DF D and
PSE conditions to a series of objective and subjective measures to demonstrate
extremes in meat quality. When compared with PSE loins, DFD loins averaged
1.5 units higher ultimate pH, 4.7% less drip loss and 136% more bound water.

Additionally, DFD loins were darker, firmer and more tender.

10

With all the positive factors associated with DFD pork (is. increased water
holding capacity, decreased light reflectance and decreased protein
denaturation) it would seem that it would be the target of pork producers.
However, there are negatives associated with DFD meat that make it
undesirable. Many of the bacterial species that grow on meat survive within a pH
range of 5.0-8.0 with optimum growth occurring around pH 7.0 (Cannon et al.,
1995). This makes DF D meat a prime target for bacterial growth. Additionally,
the dark appearance of DFD meat does not appeal to the average American
consumer (Kauffman, 1993).

Control of Glycolytic Metabolism

The rate of postmortem glycolysis plays a major role in the ultimate quality
of postmortem tissue. White et al. (1964) and Conn and Stumpf (1966),
indicated that the rate of each enzyme-catalyzed reaction is related to the
concentration of active enzyme, the availability of substrates, coenzymes and
cofactors, the presence of activators or inhibitors, and the temperature and pH
conditions. Scrutton and Utter (1968), stated that availability of substrate or
regulation of catalytic activities of rate limiting enzymes, or both, are probably the
major factors contributing to regulation of glycolytic flux. Two controlled steps in
glycolysis, catalyzed by phosphofructokinase and pyruvate kinase, as well as
antemortem and postmortem factors will be discussed in terms of their regulation

or influence on the ultimate quality of meat.

11

Phosphofructokinase

Phosphofructokinase (PFK) catalyzes the conversion of fructose-6-
phosphate (F-6-P) to fructose-1, 6-phosphate and utilizes one ATP. The
implication that phosphorylation of F-6-P was a rate controlling step in glycolysis
was first made by Passonneau and Lowry (1962). Mansour and Mansour (1962),
demonstrated that PF K limited the rate of glycolysis in liver flukes. These
findings led Sayre et al. (1963) to investigate PFK activity of porcine muscle
extracts in relation to PSE development of the intact muscle. Activity of PF K was
not associated with the rate of postmortem glycolysis or with the physical
properties of the muscle. This would seem to exclude PFK as a contributing
factor to the development of PSE muscle. However, Crabtree and Newsholme
(1972) demonstrated that differences in PFK activity among muscles of different
species reflect demonstrable differences in capacities for glycolytic flux.
Glycolytic flux refers to the rate of flow of intermediates through a metabolic
pathway.

F iber-type associated differences in glycolytic flux capacity, in addition to
differences in ATPase activities among fiber types, have long been associated
with species differences in rate of postmortem pH decline. Connett and Sahlin
(1996) concluded that type II fibers would be expected to have increased
fructose-6-phosphate, which would lead to a slightly greater fraction of PFK being
used at any given state of the phosphate energy system. The absolute rate of
glycolytic flux will depend on the amount of enzyme present in that fiber.

Additionally, PFK activity in vivo has been shown to be influenced by a variety of

12

cellular regulators (Mansour, 1963). Among these are substrate availability,
allosteric control, phosphorylation and dephosphorylation and product
accumulation.
Pyruvate Kinase

Pyruvate kinase (PK) catalyzes the conversion of phosphoenolpyruvate
and ADP to pyruvate and ATP. In a comprehensive study of PK by Schwagele et
al. (1996), skeletal muscle PK from halothane sensitive and normal pigs was
purified. These researchers found that the enzyme from both sources had an
apparent molecular weight of 52 kD and existed as a tetramer in solution.
Although no modifications were observed in the subunit size or the structure of
the enzyme, differences were found in the activity of the isolated enzyme.
Schwagele et al. (1996) found that skeletal muscle PK isolated from halothane
sensitive pigs had four-fold more specific activity as compared to the enzyme
isolated from normal muscle. More importantly, they found a striking difference
in the pH dependency of PK from the two sources of animals. Pyruvate kinase
isolated from halothane sensitive animals retained 70% of its activity when
assayed at pH 5.5, whereas normal muscle retained less than 10% of its activity.
Using isoelectric focusing on polyacrylamide gels, they demonstrated that normal
muscle has two isoforms of PK, whereas a third isoform is present in PSE
muscle. They further demonstrated that the third isoform is highly
phosphorylated and is most likely responsible for the increase observed in
specific activity, reduced Km for phosphoenolpyruvate and a shift in the pH

dependency of PK. The later results in PK being active under the acidic

13

conditions that rapidly occur in PSE muscle. This may allow the continued rapid
accumulation of lactate and hydrogen ions in PSE muscle under conditions that
would result in slow glycolysis in muscles of normal animals. Existence of this
isoform was not determined in living muscle, and it is not known whether this
phosphorylated PK isoform is a causative factor or an artifact of the rapid
glycolysis in PSE muscle. It is currently unknown if similar regulation of PK
occurs in halothane negative animals that subsequently produce PSE pork.
Genetic Predisposition

The genetic makeup of a pig can have a significant influence on the
ultimate quality of the meat. Two genetic mutations that have received
considerable attention are the halothane gene and the Rendement Napole gene
(RN'). Both of these mutations have resulted in deleterious effects on meat
quality.
Halothane Gene

It became clear in the 1960’s that predisposition of pigs to produce PSE
pork had a marked genetic component. Danish and American scientists
discovered that certain breeds contained a large portion of animals that were
prone to produce PSE pork (Briskey, 1964b). Moreover, they found a close
association between animals that produced PSE pork and animals that had
porcine stress syndrome (Briskey, 1964b; Topel et al.,1969; Judge, 1972).
Eikelenboom and Minkema (1974) demonstrated that porcine stress could be

triggered by the anesthetic gas halothane.

14

The halothane mutation has been reported to involve an arginine to
cysteine transition in the SR Ca++ release channel (ryanodine receptor) protein at
residue 615 (Fujii et al., 1991). It has been determined that the mutation results
in a hypersensitive calcium release channel, which results in elevated
sarcoplasmic CaM and increased energy consumption due to crossbridge cycling
(myosin ATPase) and calcium ATPase. The calcium ATPase pump works more
chronically to resequester the excess calcium back to the sarcoplasmic reticulum
lumen. Thus, increased myosin ATPase and Caii-ATPase activity leads to
elevated muscle temperature and accelerated glycolytic metabolism, which often
results in the development of PSE meat.

Pigs that express the halothane gene have been used for their ability to
enhance carcass muscling. However, reduced yields associated with increased
water loss negate improvements in leanness and mass of carcasses. Fresh and
processed meat from halothane-positive animals has been shown to have
undesirable cooking and eating characteristics compared to that of halothane
negative animals (Boles et al., 1991).

In a study by Pommier and Houde (1993), 913 boneless pork loins were
selected in a commercial cutting operation. The loins were selected by a trained
grader to obtain a representative sample of PSE meat (CIE L _>_ 53.5) and a
normally colored control group. The genotype with respect to malignant
hyperthennia was assessed using a restriction endonuclease assay. These
authors conceded that the results from this study clearly illustrate the harmful

effects of the halothane gene in a commercial operation, but added that

15

preharvest management practices may be more important than elimination of
animals that posses the halothane gene. Moreover, they suggested that there
are other genetic predispositions that ultimately influence meat quality that have
not yet been discovered.
Napole Gene

The Hampshire breed has received considerable attention with respect to
the pale color of lean tissue and decreased product yield. Some Hampshire pigs
possess high antemortem levels of muscle glycogen and their muscle undergoes
extended postmortem glycolysis, resulting in a low ultimate pH and a pale color
(Sayre et al., 1963). The corresponding muscle was later termed acid meat and
the gene was referred to as the Rendement Napole gene (Sellier and Monin,
1994). Le Roy et al. (1990) showed that napole yield was 8% higher in normal
pigs than napole gene carriers. Napole yield is measured as the curing and
cooking yield of a 1009 sample of semimembranosus muscle and is used as an
indicator of technological yield. The mediocre meat quality observed in the
Hampshire breed cannot be attributed to halothane sensitivity. It is well
established that the incidence of the halothane gene is essentially zero for the
breed (Webb et al., 1982). Sayre et al. (1963) reported two to three times higher
glycogen levels at harvest in Hampshire-sired pigs versus Chester White and
Poland China breeds. Although the rate of muscle pH decline is similar to that of
pigs with normal meat quality, the ultimate pH is lower than normal.

Until recently the identity of the RN' gene was unknown. Milan et al.

(2000) determined that a mutation in AMP-activated protein kinase (AMPK)

16

resulted in abnormal metabolism of glycogen. When active, AMPK is expected
to inhibit glycogen synthesis and stimulate glycogen degradation. Therefore, the
AMPK mutation in pigs results in elevated muscle glycogen stores and leads to
increased postmortem lactate and hydrogen ion accumulation, resulting in a low
ultimate pH. High levels of glycogen alone do not fully explain the phenomenon
of an extended glycolysis resulting in meat with a low ultimate pH and PSE
conditions (Monin and Sellier, 1985). The Hampshire pigs used by Monin and
Sellier (1985) exhibited a lower ultimate pH that was not associated with a higher
lactate content nor with differences in buffering capacity. Additionally, other
factors, such as enzyme stability at lower pH, must be a key player in the
continuation of glycolytic metabolism in these animals. Thus, it is imperative to
investigate factors regulating the cessation of glycolysis in systems that glycogen
is not limiting.
Harvest Procedures

The stress that is placed on an animal prior to harvest and the events that
occur following exsanguination directly influence meat quality. Various
production methods have been implemented to either reduce stress or alter
metabolism of the animal prior to harvest. Grandin (1994) recommended 24 h
rest period after arrival at the abattoir to allow the animal’s metabolism to return
to resting levels. Holding pens, loading chutes, restrainers and alleyways leading
to the final drive should enhance the natural movement of pigs to minimize pre-

harvest stress.

17

It is important to remember that the main goal in harvesting pigs is to be
as consistent and time efficient as possible. The quicker the heat can be
dissipated from the carcass, while preventing cold shortening, the more likely it
will result in product of acceptable quality. Heat can be removed faster by
minimizing the stun to stick and stun to chill times, and by increasing the intensity
and duration of chilling (NPPC, 2000).

Pork Tenderness

Historically, pork longissimus muscle has been considered to be relatively
tender (DeVol et al., 1988). However, with single trait selection for increased
lean, unfavorable changes have occurred in pork color and water holding
capacity and there is growing concern adverse effects on tenderness may have
also occured. Cameron ( 1990) found that single trait selection for increased
carcass lean resulted in less tasty, less juicy and less tender pork.
Determinants of Meat Tenderness

The relationship between sarcomere shortening and rigor-associated
toughening has been reported for lamb muscle by Wheeler and Koohmaraie
(1994). As muscle enters rigor, permanent actin-myosin crossbridges form. By
24 h postmortem, sarcomere length will be inversely related shear force values.
However, with postmortem storage substantial improvement in tenderness can
be observed with minimal increase in sarcomere length. The increase in
sarcomere length has been shown to be somewhat of an artifact—due to the

methods used to measure this length (Gann and Merkel, 1977).

18

Using electron micrographs, Gann and Merkel demonstrated that the region
where there is myosin-actin interaction stays the same through postmortem
storage, while there is separation adjacent to z-discs.

Conflicting reports exist with regard to the main determinants of pork
tenderness. Wheeler et al. (2000) concluded that the main determinants in
longissimus dorsi tenderness are connective tissue, sarcomere length and
desmin degradation at d 1 postmortem. However, van Laack and coworkers
(2001) concluded that connective tissue, intramuscular fat content and
myofibrillar structure were the main controllers in tenderness.

Pigs are slaughtered at approximately 6 months of age and the immature
connective tissue does not significantly influence pork tenderness (Avery et al.,
1996). It is generally accepted that intramuscular fat content is responsible for
approximately 10-12% of the variation observed in ultimate tenderness. In
contrast, van Laack and coworkers (2001) demonstrated that in Duroc pigs
intramuscular fat accounted for 47% of the variation in Wamer-Bratzler shear
values. Additionally, they found no relationship between intramuscular fat
content and Warner-Bratzler shear values for Berkshire and Hampshire pigs.

Extensive research has been conducted to quantify the relationship
between sarcomere length and pork tenderness. In a random sample of 120
pork carcasses from a commercial harvest facility, DeVol et al. (1988) reported

that in longissimus dorsi, sarcomere length averaged 1.83 pm and ranged from
1.66 to 2.00 pm. In this study, sarcomere length accounted for 7% of the

variation observed in WBS values. Wheeler et al. (2000), suggested that if

19

sarcomere length could be extended to at least 2.0 pm, the longissimus muscle
would be tender regardless of collagen content or proteolysis. The inconsistent
effects of increasing sarcomere length on shear force values indicates the high

degree of interaction between sarcomere length, proteolysis of specific muscle

proteins and connective tissue on tenderness differences (Hostetler et al., 1972).
Wheeler and Koohmaraie (1999) demonstrated that sarcomere length does not

affect the extent of proteolysis in lamb muscle.

Causes of Postmortem Tenderization

Several studies have focused on the causative factors that influence
postmortem tenderization. The improvement in meat tenderness during
postmortem storage at refrigerated temperatures has been known since the turn
of the 20th century. In 1907 Lehmann reported that there was a 30% increase in
tenderness of meat stored for 8 d postmortem (reviewed by Penny, 1980).
Although this improvement can be measured both subjectively and objectively,
the mechanisms responsible for postmortem tenderization are not completely
understood.

There is substantial evidence that suggests proteolysis of key myofibrillar
proteins is responsible for meat tenderization and improvements in tenderness
are mediated by the calpain system (Koohmaraie, 1988, 1992a, 1994). Guroff
(1964) was the first to document the existence of a calcium-and sulfhydryl-
dependent proteinase from rat brain. Meyer et al. (1964) reported the existence
of a similar proteinase in skeletal muscle and it was later purified from porcine

skeletal muscle by Dayton et al. (1976 a,b).

20

The calpain system consists of p-calpain (the form of the proteinase active

at micromolar calcium concentrations), m-calpain (the form of the proteinase
active at millimolar calcium concentrations) and calpastatin (a protein that
specifically inhibits both forms of calpain at their respective calcium
concentrations required for activation; Koohmaraie, 1992b). Based on the results
of numerous experiments, it can be concluded that proteolysis of key myofibrillar
proteins by u-calpain is the underlying mechanism of meat tenderization that
occurs during postmortem storage at refrigerated temperatures (Koohmaraie,
1996).

The main proteins that have been implicated in postmortem tenderization
include the inter- (e.g., desmin and vinculin) and intra-myofibril (e.g., titin, nebulin
and troponin-T) linkages. The function of these proteins is to maintain the
structural integrity of myofibrils (Price, 1991). Thus, the degradation of these
proteins would cause weakening of myofibrils and result in tenderization.
However, phosphorylation of myofibrillar proteins has been shown to reduce
degradation of these proteins by calpains (Di Lisa et al., 1995;2hang et al.,
1998). Phosphorylation of calpastatin (endogenous calpain inhibitor) renders it a
more effective inhibitor of m-calpain than unphosphorylated calpastatin
(Salamino et al., 1994). The relationship between proteolysis of key muscle
specific proteins and pork tenderness is unknown. Moreover, the mechanism(s)

responsible for controlling the rate of postmortem tenderization is currently

unresolved.

21

CHAPTER 1

PORK QUALITY VARIATION IS NOT EXPLAINED BY GLYCOLYTIC ENZYME
CAPACITY

ABSTRACT

My objective was to determine if increased glycolytic enzyme capacity is
associated with rapid postmortem pH decline that leads to inferior pork color and
water-holding capacity. Duroc (n=16) or HAL-1843 free Pietrain (n=16) sired gilts
were harvested within a two-week period. Temperature of the longissimus
muscle (LM) was logged continuously from 45 min to 22 h postmortem at 5 min
intervals and LM pH was measured at 20, 45, 180 min and 22 h postmortem.
Temperature of LM at 45 min postmortem was negatively correlated with 45 min
pH (P < .05). Longissimus muscle L* values for chops at 24 h postmortem
ranged from 49.6 to 60.2. Fluid loss (Purge) ranged from .79 to 9.91% in
vacuum packaged loin sections stored at 4°C from d 1 to d 6 postmortem. After
purge determination, two 2.5-cm-thick loin chops were cut and allowed to drip in
a simulated retail case at 4°C overnight. Drip loss ranged from .3-1.8%. All
measures of fluid loss were correlated to all L* values (d 1, 2, 6 and 7
postmortem; P < .01). Phosphofructokinase and pyruvate kinase activities in LM
sarcoplasmic fractions were quantified using coupled enzyme assays.
Phosphofructokinase and pyruvate kinase activities were not correlated with LM
pH, purge, drip loss, or color (P > .31). These data indicate that variation in pork
color and water holding capacity is not associated with variation in the capacity of

enzymes to catalyze regulated steps of glycolysis.

22

INTRODUCTION

Pigs that are homozygous HAL-1843 positive and heterozygous carriers
have been used for their ability to increase the amount of lean tissue in a
carcass. However, expression of this mutated gene (ryanodine receptor, Ca++
release channel) also increases the risk of developing pale, soft and exudative
(PSE) meat (Monin et al., 1981). Although it is currently possible to identify pigs
that are HAL-1843 homozygous and carriers, breeds and lines of pigs that do not
possess this genetic defect still produce pork carcasses that exhibit PSE
characteristics (Pommier and Houde, 1993). The biochemical mechanisms that
contribute to PSE pork are not completely understood. However, it is known that
the rate and extent of pH decline influence meat color and water-holding
capacity. Rate and extent of pH decline in muscle may be determined by the
rate of glycolysis, buffering capacity, and/or glycogen storage (Milan et al., 2000).

Bendall (1973) and Warriss et al. (1989) demonstrated that characteristics
of postmortem pH decline are determined by the physiological state of muscle at
the time of stunning. Antemortem stress and anaerobic postmortem conditions
lead to accumulation of muscle H+ via glycolysis. Rapid postmortem glycolysis
causes a rapid pH decline while muscle temperature is relatively high. This leads
to denaturation of muscle proteins and a subsequent loss of water-binding
capacity (Briskey, 1964b). Meat that has undergone a rapid postmortem pH
decline is generally paler in color and more exudative than meat that has

undergone a gradual pH decline.

23

Two major regulatory steps in glycolysis are those catalyzed by the
enzymes phosphofructokinase (PFK) and pyruvate kinase (PK). Sayre et al.
(1963) reported that PFK and phosphorylase activity in vitro were not associated
with rate of pH decline in Iongissismus dorsi muscles of Hampshire, Poland
China and Chester White pigs. However, Schwagele et al. (1996) demonstrated
that muscle from halothane sensitive pigs had four times more total PK activity
than control pigs. Additionally, Schwagele et al. (1996) showed that PK purified
from muscle of halothane sensitive pigs lost only 30% of its PK activity,
compared to a loss of >90% activity of PK purified from control pig muscle, when
PK was assayed at pH 5.5 rather than pH 7.0. These authors demonstrated that
PK in LM of halothane sensitive pigs was more active and less pH labile due to
phosphorylation of the enzyme. It is unknown if similar regulation of glycolysis
contributes to PSE meat in HAL-1843 free pigs. Thus, my hypothesis is that
increased capacity of rate-limiting glycolytic enzymes contributes to rapid
glycolysis and leads to inferior pork color and water-holding capacity.

MATERIALS AND METHODS
Animals and meat quality data collection
Sires from Duroc and HAL-1843 free Pietrain lines were used to
inseminate Yorkshire and F1 Yorkshire-Landrace gilts. Progeny (n=32) were
raised in uniform conditions at the Michigan State University (MSU) Swine
Teaching and Research Farm. Four Duroc and four HAL-1843 free Pietrain sired
gilts were harvested on each of four days within a two-week period at the MSU

Meat Laboratory. All procedures were performed in accordance with guidelines

24

Table 1.1. Timeline for meat quality measurements.

 

 

 

 

 

 

 

 

 

| | TIME POSTMORTEM .
IMEASUREMENTSI 20MIN | 45mm | 180MIN IDAY1j DAY 2 [DAY 6 | DAY7 |
pH IpH20min pH45min pH180min pH22h
TEMP 20 min 45min .1! 22h
COLOR L1 L2 L6 L7
DRIP Loss DRIP1 DRIP6
cwr-rc xx
PURGE x .1! x

 

 

 

 

25

set forth by the MSU Animal Use and Care Committee.

A time line for data collection is shown in Table 1.1. Loin muscle area,
10"1 and last rib backfat thickness, and subjective color, firmness and marbling
were determined according to current NPPC guidelines (NPPC, 2000) after
chilling the carcasses for 22 h at ~2°C. Temperature of the longissimus muscle
(LM) adjacent to the last rib was measured at 20, 45, 180 min and 22 h
postmortem using a hand held temperature probe. From 45 min to 22 h
postmortem, temperature was logged every five min using DeltaTRAK FlashLink
Dataloggers (Pleasanton, CA). Longissimus muscle pH was measured at 20, 45,
180 min and 22 h postmortem with a portable pH meter equipped with a
puncture-type combination pH electrode (Model 1140, Mettler-Toledo, Woburn,
MA). At these same times postmortem, samples were also obtained for
subsequent measurement of pH using the iodoacetate method (Bendall, 1973).
Initial samples (20 min postmortem) were taken midway between the last rib and
the cranial edge of the ilium on the left side of the carcass. Subsequent LM
samples (45 min, 180 min and 22 h) were taken approximately one inch cranial
to the previous sampling site. Longissimus muscle samples were cut into 0.5
cm3 pieces, frozen in liquid nitrogen, and stored at -80°C. At 24 h postmortem, a
section of loin was removed from the right side of the carcass between the 11th
rib and the last lumbar vertebra. From the cranial edge of the loin section,
duplicate 10 9 samples were obtained and used to determine water-holding
capacity by high-speed centrifugation (CWHC; 40,000 x g for 30 min) at 24 h

postmortem (Honikel and Hamm, 1994). Two 2.5 cm thick loin chops were

26

removed from between the 12th and last rib of the section. These chops were
used to determine color (15 min bloom time; ClELab L*, a*, b", 065, 2° standard
observer and 50 mm orifice) at 24 (L1) and 48 h (L2) postmortem with a Minolta
chromameter (CR-310; Ramsey, NJ). Drip loss was determined by suspending
chops from 24 to 48 h postmortem at 4°C (DRIP1; Honikel and Hamm, 1994).
The remaining sections were vacuum packaged at 24 h postmortem in Cryovac
shrink bags using a Multivac machine (Koch, Type AG 800) and stored at 4°C
until d 6 postmortem. The difference between d 6 and 24 h loin weight was
divided by 24 h weight and expressed as purge. After determination of purge,
two 2.5-cm-thick loin chops were cut from the cranial end of the loin section and
allowed to bloom for 15 min before color determination (L6). Chops were
allowed to drip for 24 h at 4°C on styrofoam trays with a moisture-impermeable
ovenIvrap to simulate retail conditions. Drip loss (DRIP6) and color (L7) were
recorded on d 7 postmortem.
Sample Preparation for Enzyme Assam

Longissimus muscle samples used to determine glycolytic enzyme
capacity were obtained at 20 min postmortem as described above. Chemicals
used in muscle extraction and enzyme assays were purchased from Sigma
Chemical Company (St. Louis, MO). Frozen LM samples (1 g) were
homogenized in 10 volumes of ice-cold extraction buffer (75 mM KCI, 10 mM
KHzPO4, 2 mM MgClz, 2 mM EGTA, pH 7.0, containing 50 mM sodium fluoride, 2
mM phenylmethylsulfonyl fluoride, and 6 mg/l of leupeptin). Homogenization was

performed on ice using a Polytron (Brinkman, Westbury, NY) for 2 x 20 sec

27

bursts (setting 4). The homogenate was fractionated into sarcoplasmic and
myofibrillar components by centrifugation at 10,000 x g for 15 minutes. The
supernatant fluid was saved and the pellet was resuspended in 10 ml of
extraction buffer and centrifuged again at 10,000 x g for 15 minutes. The two
supernatant fluids were combined. Protein concentration of supernatant fluid
was determined using the bicinchoninic acid protein assay kit (Pierce, Rockford,
IL). In preliminary experiments, I found that the sarcoplasmic fraction
(supernatant) contained > 95 % of the PK and PFK activity (Table 1.2). Thus, I
used the sarcoplasmic fraction for quantification of PK and PF K activity.
Enzyme Assay

Pyruvate kinase activity was measured using a coupled enzyme assay
described by Schwagele et al. (1996). The assay was adapted so that it could be
performed in a 96-well microtiter plate. The total reaction volume was 210 pl,
which consisted of 3 mM magnesium acetate, 50 mM KCI, 0.2 mM EDTA, 0.2
mg/ml bovine serum albumin (BSA), 30 mM imidazole (pH 7.0), 1mM adenosine
diphosphate (ADP), 0.7 mM phosphoenolpyruvate (PEP), 0.1 mM nicotinamide
adenine dinucleotide, reduced form (NADH), 2 Ulml lactate dehydrogenase
(LDH) and 10 pl of a 1:10 dilution of sarcoplasmic fraction. Activity was
measured by following the oxidation of NADH to NAD" at 340 nm on a
VERSAmax plate reader (Molecular Devices, Sunnyvale, CA) at 22° C. The
assay was linear from 0 to 50 s. Assays performed in the absence of substrate
(PEP) contained less than 1% of sample activity, demonstrating that conversion

of NADH to NAD“ was coupled to PK activity. Pyruvate kinase activity was

28

Table 1.2. Vmam values (pmol/min/ml), standard deviations and ranges for enzyme

capacity by fraction (n=6). All fractions are diluted to be comparable to
the crude homogenate.

 

 

 

 

 

 

 

 

 

| TRAIT I MEAN | SD | RANGE
Pyruvate Kinase C T
Crude homogenate 252.97 13.1 1 233.80-274.4
Supernatant 239.93 15.45 27120-31095
Washed Pellet 11.62 4.36 588-1656
Phosphofructokinase
Crude homogenate 147.25 22.46 115.45-17980
Supernatant 142.92 9.00 13460-15905
Washed Pellet 8.92 5.03 3.16-17.46

 

 

 

 

 

 

29

calculated from the Vmax that was determined from the SOFT max PRO software
(Molecular Devices, Sunnyvale, CA) and expressed as pmolelmin-mg'1 protein.
Samples were simultaneously assayed in triplicate and the intra-assay coefficient
of variation was <4%. To approximate activity at ultimate postmortem pH this
assay was also repeated with the pH of the buffer lowered to 5.5 with HCI.

Phosphofructokinase was quantified using a coupled enzyme assay
described by Scopes (1977). This reaction was performed in a 96-well microtiter
plate that contained a total reaction volume of 210 pl of 3 mM magnesium
acetate, 50 mM KCI, 0.2 mM EDTA, 0.2 mg/ml BSA, 30 mM Tris base (pH 8.0), 1
mM fructose-6-phosphate (F-6-P), 0.2 mM NADH, 1 mM adenosine triphosphate
(ATP), 0.2 mM PEP, 2 units each of PK and LDH/ml and 10 pl of a 1:4 dilution of
sarcoplasmic fraction. The assay was performed at 22°C and was linear from 0
to 180 s. Assays performed in the absence of substrate (F-6-P) contained
approximately 27% of the total activity. This could be attributable to F-6-P, ADP
or ATPases present in the sample. Therefore, negative control assays with no F-
6-P were performed for each sample. The V"...x from control assays were
subtracted from the V"...x for assays performed with F-6-P. The total activity is
expressed as pmole/min-mg'1 protein. Samples were simultaneously assayed in
triplicate and the intra-assay coefficient of variation was <2%.

In vitro Phosphorylation of Pyruvate Kinase

An attempt was made to phosphorylate PK in crude supernatant fluid

using the procedure outlined by Schwagele et al. (1996) for purified PK. Briefly,

the phosphorylation reaction was performed in 200 pl of 5 mM Bis-Tris, pH 6.5,

30

50 pM ATP, 5mM magnesium acetate, 20 mM KCl containing 200 pg equivalent
of protein and 4 units of protein kinase. Prior to use, 1 mg of cAMP-dependent
protein kinase A was reconstituted in 200 pl of dithiothreitol (50 mglml) and
allowed to incubate at room temperature for 10 minutes. The samples were
incubated overnight at 30° C in a water bath. The enzymatic activity was
quantified by assaying the original sample and the phosphorylated sample
simultaneously as described above. I was unable to show an increase in the
enzyme capacity of PK after in vitro phosphorylation. Vmax values measured at
pH 5.5, following phosphorylation showed a >88% reduction in specific activity
when compared to its non-phosphorylated counterpart (data not shown). No
attempt was made to verify if PK was phosphorylated with this method. It is
possible that other sarcoplasmic proteins were preferentially phosphorylated,
instead of PK, under the conditions described.
Data Analysis

Data were analyzed using a mixed model that contained the random main
effect of harvest date and the fixed main effect of genetic line. Least squares
means by sire line of meat quality traits were compared using a protected least
significant difference test (Freud and Wilson, 1997). Pooled within-class
correlation coefficients were calculated for meat quality data with enzyme

capacity by ordinary least squares analysis of variance for a randomized

complete block design.

31

RESULTS AND DISCUSSION

The lines for this study were selected based on previous research
demonstrating a predisposition to produce progeny with differences in
postmortem metabolism, pork color and water holding capacity. It has long been
known that HAL-1843 positive pigs are prone to produce PSE meat. Schwagele
et al. (1996) demonstrated that PK activity is higher in halothane sensitive pigs
and that increased PK activity is due to enzyme phosphorylation. These authors
suggested that elevated PK activity and greater enzyme stability under acidic
conditions contributed to the incidence of PSE in these animals. The current
study was designed to determine if capacity of PK and PFK explains variation in
meat quality of pigs that are HAL-1843 free.

Pietrain-sired hogs produced lighter weight carcasses with larger loin
muscle areas than Duroc-sired hogs at a similar age (Table 1.3). No differences
in pH, color, water-holding capacity and glycolytic enzyme activity were found
between sire groups (P > 0.06). Therefore, meat quality data and their
relationship to glycolytic enzyme activity are presented and discussed collectively
for all pigs.

The portable pH meter and the iodoacetate method of measuring pH
produced similar results (r = 0.83). Additionally, similar correlations existed
between meat quality traits and pH values obtained by either method (data not
shown). Therefore, only pH values obtained using the portable pH meter are
reported. The iodoacetate method has been shown to produce unavoidable

alkalinization that leads to pH values approximately 0.1 pH unit higher than the

32

Table 1.3. Least square means and ranges for carcass

 

 

 

 

 

 

 

 

 

 

 

 

 

 

measurements
BACKFAT

SIRE n LIVEWT ch 10TH RIBILAsr RIB LEA LENGTH
LINE Ike)“ Ike)” (mm) (mm) (cm’)*** (cm)**
Duroc 16 134 103 19.8 30.7 51.5 66.2

RANGE 122-142 91-106 12.7-25.4 17.8-40.6 41.5-60.6 83.2-91.2
Pietrain 16 126 98 16.5 27.4 61.5 64.0

RANGE 115-141 86-101 10.2-25.415.2-38.1 52.9-73.2 79.2-86.9

 

** Least square means differ, P<.01
*** Least square means differ, P<.001

33

 

7.00

 

6.80 .--.___fi-_F_E —-_ ”ZA- - d f--. - -- Z __.m*-_.w2, .-..---____.

6.60

 

6.40

 

6.20

 

pH

 

6.00 ~1>~—~—--‘F - ~~

5.80

 

 

5.60 .

 

5.40 ,,

 

5.20

 

 

 

 

Figure 1.1:

Time (Hours postmortem)

Longissimus muscle pH decline. Measurements were taken with a
portable pH meter and puncture type probe adjacent to the last rib.
Vertical lines at data points represent means 1 SD.

34

actual pH (Bendall, 1973; Dutson, 1983). I also found that pH values obtained
using the iodoacetate method were approximately 0.1pH unit higher than those
obtained with a portable pH meter.

Figure 1.1 shows the rate and extent of postmortem pH decline.
Relatively large standard deviations associated with mean pH values within the
first 3 h postmortem reflect differences in the rate of pH decline among
longissimus muscles. The range in LM pH was 5.8 to 6.7, 5.6 to 6.6 and 5.3 to
6.4 at 20 min, 45 min and 3 h postmortem, respectively. In this study, differences
in ultimate pH (22 h postmortem) were relatively small (Figure 1.1; range 5.4 to
5.6).

Figure 1.2 depicts the temperature decline in LM measured with a portable
temperature probe at 20 min, 45 min, 3 h and 22 h postmortem, and measured
continuously from 45 min postmortem to 22 h postmortem. Although the
temperature decline curves among carcasses are generally parallel, differences
in initial muscle temperature exist. Differences in muscle temperature at 45 min
postmortem may result from variation in initial body temperature, heat produced
during the conversion of glycogen to lactic acid and/or the hydrolysis of creatine
phosphate and ATP (Bendall, 1973). I observed an inverse relationship between
temperature at 45 min postmortem and pH at 45 min postmortem (Table 1.4).
This relationship has also been reported by McCaw et al. (1997).

Several measures of color and water-holding capacity were made. Day 1
and d 2 measures were performed on different chops than d 6 and d 7.

However, the four chops were within approximately a 13 cm region of the same

35

45

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time (Hours postmortem)

i port-1515166591356“ ‘ ‘T:.—'H'I§Eé§1md"uai—”_._‘ “_ mammal“ — :— TDELTKTRKKW?”

 

Figure 1.2. Postmortem Iogissimus muscle temperature decline. Means 1
standard deviations of temperature measured using a hand-held
temperature probe are represented by (I). Average temperature
decline measured using the dataloggers is indicated by (O). The
individual carcass that had the highest temperature (0) and the
carcass with the lowest temperature (+) are illustrated to show the
range in temperature observed.

36

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37

loin section. Different measures of water-holding capacity (DRIP1, CWHC,
PURGE and DRIP6) and color (L1, L2, L3 and L6) were highly correlated to each
other (Table 1.4). Longissimus muscle L* values were positively correlated with
water loss (Figure 1.3). Both water loss and L* value were inversely related to
LM pH from 20 to 180 min postmortem (Table 1.4). Of the pH measures, 45 min
pH was most highly correlated with water loss and color (Table 1.4). This
supports conclusions made by Sayre and Briskey (1963) and Sellier and Monin
(1994), that the rate of pH decline, as measured by 45 min pH, influences the
degree of protein denaturation during rigor onset. The relationships between LM
water loss and 45 min pH are graphically depicted in Figure 1.4. Although
different measures of water loss did not give the same absolute number, samples
with superior or inferior water-holding properties were identified by all methods of
measuring water-holding capacity or water loss used in this study (Figure 1.3 and
1.4). Although no severe PSE pork was observed among the samples in the
current study, the variation in pork color and water-holding capacity was of
practical importance to packer profitability and consumer acceptance.

Twenty-two-hour pH was not related to early postmortem measures of
color or water-holding capacity (Table 1.4). Warriss and Brown (1987),
demonstrated that ultimate pH was related to reflectance and exudate, but only
accounts for about 15% of the variation in these traits. In this study, the lack of a
relationship between 22 h pH and water-holding capacity or L* values at d 1 and
2 may be attributed to the lack of variation in ultimate pH.

A modest correlation (r~-.4) between 22 h pH and L* at d 6 and 7 was

38

Figure 1.3. Relationship between L1 and measures of water loss in longissimus
muscle. Panel A: Water-holding capacity (I) was measured by high-
speed centrifugation (CWHC). Panel B: Purge (A) was measured on
boneless loin section from d 1 to d 6 postmortem stored at 4°C. Drip
1 (*) was assessed using the suspension method from d 1 to d 2.

Drip 6 (9) was measured on duplicate chops under simulated retail
display case conditions from d 6 to d 7.

39

% WATER LOSS

% WATER LOSS

25.00

 

20.00

 

 

15.00

 

 

 

 

 

 

10.00 .—i—_r . L_-_ - . - __._____ _ _. ——*—_—---——1

5.00. -. H 2-2--- - 2722 _-___-____u____u_-2 -. .____

 

 

 

0.00
49.00 51.00 53.00 55.00 57.00 59.00 61.00

L1

 

12.00

 

 

 

 

10.001~___"_._-______-_ _- I _ A‘. -22.- ,2. .-._._

8.00

6.00 ._

 

4.00 .

2.00 I

 

 

 

 

 

49.00 51.00 53.00 55.00 57.00 59.00 61.00

40

Figure 1.4. Relationship between 45 min pH and measures of water loss in
longissimus muscle. Panel A: Water-holding capacity (I) was
measured by high-speed centrifugation (CWHC). Panel B: Purge
(A) was measured on boneless loin section stored at 4°C from d 1 to
d 6 postmortem. Drip 1 (*) was assessed using the suspension
method from d 1 to d 2. Drip 6 (9) was measured on duplicate chops
under simulated retail display case conditions from d 6 to d 7.

41

% WATER LOSS

% WATER LOSS

25.00

20.00

15.00

10.00

5.00

0.00

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5.40

12.00

10.00

8.00 .

6.00 .

4.00

2.00 .

0.00

5.60 5.60 6.00 6.20 5.40 6.60
45 MINUTE pH

6.80

 

 

 

 

 

 

 

 

 

 

 

 

 

5.40

5.60 5.60 6.00 6.20 6.40 6.60
45 MINUTE pH

42

6.80

observed (Table 1.4). It is possible that slight differences in ultimate pH may
impact long term color stability. Indeed, myoglobin stability is reduced at lower
pH and higher temperatures. During normal rigor development, reflectance
begins to increase rapidly below pH 6.2 (Warris and Brown, 1987). The negative
correlation observed between 22 h pH and L” at day 6 and 7 indicates that pork
with a lower ultimate pH becomes lighter during postmortem storage.

Coupled enzyme assays were used to quantify total activity of PF K and
PK in the sarcoplasmic fraction of longissimus muscles. Since these assays
were conducted under controlled conditions in vitro, it should be recognized that
activity data reflect the in vitro capacity of these enzymes to catalyze their
respective reactions, rather than in vivo activity. It is also worth noting that the
practice of measuring Vmax values under optimal conditions in vitro and relating
them to in vivo flux rates is based on sound theoretical framework (Newsholme
and Crabtree, 1986). The in vitro activity of PFK and PK ranged from .32 to .67

and 3.8 to 6.4 pmole/min-mg'1 protein, respectively. These activities are similar

to those previously reported for porcine LM (Schwagele et al., 1996; Rosochacki
et al., 2000). I observed no correlation between glycolytic enzyme capacity and
LM pH, color or water-loss (Table 1.5). In contrast, Schwagele et al. (1996)
demonstrated that PK in LM of halothane sensitive pigs, which are prone to
producing PSE meat, was more active and less pH labile due to phosphorylation
of the enzyme. My findings indicate that a similar regulation of glycolysis does
not contribute to the variation observed in pork quality of HAL-1843 free pigs. I

also measured activity of PK at pH 5.5 and observed a loss of >88% PK activity

43

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in all LM samples at this pH compared to activity measured at pH 7.0. These
observations are similar to the reduction in activity seen by Schwagele et al.
(1996) for control pigs, but much greater than the 30% loss in PK activity
observed for halothane sensitive pigs in that study. This provides additional
evidence that phosphorylation-dependent stabilization of enzyme activity, as
observed by Schwagele et al. (1996) for halothane sensitive pigs, is not a key
factor regulating variation in pork quality of HAL-1843 free pigs. My findings
support data previously reported by Sayre et al. (1963) who demonstrated that
differences in specific activity of glycolytic enzymes was not related to ultimate
meat quality. My findings are also consistent with those of Scopes et al. (1974),
who showed that different concentrations of PFK and PK did not affect the rate at
which glycolysis proceeded in vitro. I conclude that in HAL-1843 free pigs, LM
color and water-holding capacity are not associated with the capacity of rate-
limiting glycolytic enzymes.
IMPLICATIONS

In this study, L* value was positively correlated to all measures of water
loss. Forty-five min postmortem pH was most highly correlated to water loss.
Differences in glycolytic enzyme capacity do not appear to be responsible for the
variation observed in pork quality of HAL-1843 free pigs. Additional research is
necessary to identify other biochemical factors, such as allosteric enzyme
regulators, physiological buffering compounds, or mechanisms regulating ATP
utilization in muscle, that influence the rate of pH decline and consequently

govern pork quality.

45

CHAPTER 2
ANALYSIS OF POSTMORTEM TENDERIZATION IN PORCINE LONGISSIMUS
DORSI MUSCLE

ABSTRACT
My objective was to quantify the rate and extent of postmortem tenderization in
porcine longissimus dorsi (LD) muscle and determine if proteolysis of desmin
corresponds to mechanical measures of tenderness. Berkshire (n=32) and
Yorkshire (n=16) sired pigs were harvested on two days at a commercial abattoir.
Four 5.72-cm sections of the LD were removed at d 1 from the 11th rib to the 3rd
lumbar vertebrae. Loin sections were randomly assigned to aging treatments of
1, 3, 7 and 14 d, vacuum packaged and stored at 4°C. After storage, two 2.5-cm
thick chops were cooked to an internal temperature of 71°C on Farbenivare Open
HearthTM broilers. Chops were cooled overnight at 4°C and three 1.27-cm
diameter cores per chop were sheared with a Wamer-Bratzler Shear (WBS)
machine. No differences in WBS values were observed between breed or loin
location (P>.05). Shear values decreased (P<.0001) from 4.1 kg at d 1 to 3.6 kg
and 3.2 kg at d 3 and d 7, respectively. Chops aged for 7 and 14 d had similar
WBS values (P>.05). Western blot analysis of desmin from 4 tender (54 kg) and
4 less tender (34.7 kg) samples at d 1 revealed that desmin degradation
paralleled decreases in WBS. Intact desmin was typically undetectable in tender
samples by d 7 and in less tender samples by d 14. Tenderization of most pork
loin chops was completed by d 7, however some chops exhibited additional

tenderization and desmin degradation between 7 and 14 d postmortem.

46

INTRODUCTION

The overall eating quality of a product will influence repeat purchase
decisions of today’s consumer. Of these quality traits, tenderness is usually
accepted as the most crucial (Koohmaraie, 1996). Several studies have shown
that various factors in production and processing up to the point of retail display
affect tenderness (Wood et al., 1994). Single trait selection for increased
carcass lean has resulted in less tasty, less juicy and less tender pork (Cameron,
1990). Additionally, it has been known for almost a century that postmortem
storage of product at refrigerated temperatures will improve tenderness
(reviewed by Penny, 1980). Although these improvements in tenderness can be
measured both subjectively and objectively, the mechanisms that control these
improvements are not clearly defined (Koohmaraie, 1991).

During postmortem storage of meat, numerous changes occur in skeletal
muscle, which result in loss of structural integrity of the tissue. This loss of
structural integrity is thought to be responsible for meat tenderization and the
principal mechanism being limited to the proteolysis of myofibrillar proteins
(Koohmaraie, 1992b). It has also been documented that meat tenderization
results from the postmortem degradation of myofibrillar proteins by calcium-
dependent proteases, or calpains (Goll et al., 1992). Increased levels of
calpastatin have been shown to inhibit degradation of myofibrillar proteins by
calpains (Koohmaraie et al., 1995).

Desmin is an intermediate filament protein that is part of the cytoskeleton

of nearly all animal cells (Robson, 1989;1995). It has been demonstrated to

47

have a subunit molecular weight of 52 kD (lp et al., 1985). Immunoelectron
microscope localization studies indicated that desmin encircles the Z-line and
radiates out perpendicularly to the myofibril axis to ensnare and connect adjacent
myofibrils (Richardson et al., 1981). Desmin has also been shown to link
myofibrillar Z-Iines to the cell membrane skeleton (Robson, 1995). Additionally,
desmin “knockout” mice have been shown to exhibit severe disruption of muscle
cellular organization (Milner et al., 1996).

In preliminary experiments, degradation of desmin, but not degradation of
calpastatin or filamin was associated with improved tenderness at d 7
postmortem. The objective of this study was to quantify the rate and extent of
postmortem tenderization in porcine longissimus dorsi (LD) muscle and
determine if proteolysis of desmin corresponds to mechanical measures of
tenderness at 1, 3, 7 and 14 d postmortem

MATERIAL AND METHODS
Animals and Meat Quality Data Collection

Sires from Berkshire and Yorkshire lines were used to inseminate F1
Yorkshire-Landrace gilts. Progeny were raised in uniform conditions at the MSU
Swine Teaching and Research Farm. Berkshire (n=32) and Yorkshire (n=16)
sired pigs were harvested on two days at a commercial abattoir. Bone-in loin
sections were removed from the carcass at d 1 and transported to the MSU Meat
Laboratory. Internal temperature of the meat was monitored during transport, as
was the temperature of the ice chest. Internal temperature of loin sections never

exceeded 4°C and the ice chests never exceeded 5°C. Sections were trimmed

48

of external fat and removed from the bone. Four 5.72-cm sections of the LD
were cut from the 11th rib to the 3'“ lumbar vertebrae. The sections were
randomly assigned to aging treatments of 1, 3, 7 and 14 d, vacuum packaged
and stored at 4°C. After respective storage times, two 2.5-cm-thick chops were
cooked to an internal temperature of 71°C on Farberware Open HearthTM broilers.
Temperature was monitored continuously using copper-constantan
thermocouples (10.2 cm in length, .081 cm in diameter and accuracy i 0.1°C;
Omega, Stamford, CT), and chops were turned once when the internal
temperature reached 40°C. Chops were cooled overnight at 4°C and three 1.27-
cm diameter cores per chop were sheared with a Wamer-Bratzler shear (WBS)
machine.
Sample Preparation for Analysis of Protein Degradation

Frozen longissimus muscle samples (19) were homogenized using a
Polytron (Brinkman, Westbury, NY) for 1 x 20 sec burst (setting 4) in 10 volumes
of ice cold 50 mM tris, 10 mM EDTA, pH 8.3, containing 2 mM
phenylmethylsulfonyl fluoride, and 6 mg/l of leupeptin. Five hundred pl of the
crude homogenate were mixed with 500 pl of 2x treatment buffer without MCE
(125mM Tris pH 6.8, 4% SDS, 20% glycerol) by repeated pipeting. Samples
were heated at 50°C for 20 min, then mixed again by repeated pipeting, heated
for 5 min and centrifuged at 16,000 x g for 20 min. The supernatant fluid was
removed and protein concentration was determined using the bicinchoninic acid
protein assay kit (Pierce, Rockford, IL). Samples were then mixed with an equal

volume of 2x treatment buffer with 10% MCE and heated at 95°C for 3 min.

49

SDS-PAGE and lmmunoblotmig

Desmin was resolved by SDS-PAGE (Laemmli, 1970) on .75 mm-thick
10% (37.5:1 acrylamidezbisacrylamide) separating gels with 4% (37.5:1) stacking
gels. Electrophoresis of proteins was performed at 200 V for approximately 45
min at room temperature in BIO-RAD Mini Protean ll gel assemblies. Proteins
were electrophoretically transferred to lmmobilon-P membranes (Millipore,
Bedford, MA) in buffer containing 25 mM Tris, 193 mM glycine, and 15%
methanol (T owbin et al., 1974). Lanes containing molecular weight markers
were stained with amido black. To prevent non-specific antibody binding,
membranes were incubated in blocking buffer (10% non-fat dry milk (NFDM) in
Tris-buffered saline [TBS] containing 20 mM Tris pH 7.4, 137mM sodium
chloride, 5 mM potassium chloride and .05% Tween-20) for 1 h. Membranes
were incubated in primary D76 hybridoma supernatant followed by an alkaline
phosphatase conjugated anti-mouse lgG diluted 1:1000 (Sigma) for 45 min. The
D76 hybridoma, developed by DA. F ischman, was obtained from the
Developmental Studies Hybridoma Bank and developed under the auspices of
the NICHD and maintained by The University of Iowa, Department of Biological
Sciences, Iowa City, IA 52242. All antibodies were diluted in blocking buffer.
Membranes were washed three times with blocking buffer after each incubation.
Antibody binding was visualized by exposure to BClP/NBT (Bio-Rad).
Data Analysis

Data were analyzed using a mixed model that contained the random main

effect of the individual pig and the fixed main effects of breed, day (1, 3, 7 and 14

50

postmortem), location, sex and harvest date along with the two way interactions
of breed‘day, breed’location, breed‘sex, day‘location, day'sex and location*sex
Least square means by sire line were compared using a protected least
significant difference test (Freud and Wilson, 1997). None of the interaction
terms were found to be important (P>.5). Therefore, only the main effect of day
was evaluated.

RESULTS AND DISCUSSION

Warner-Bratzler Shear

 

No differences were observed between the two genetic lines with respect
to tenderness for any of the aging treatments (P>.2) and data were pooled by
day. Shear values decreased (P<.0001) from 4.1 kg at d 1 to 3.6 kg and 3.2 kg
at d 3 and d 7, respectively (Figure 2.1). Chops aged for 7 and 14 cl had similar
WBS values (P>.05; Figure 2.1). In contrast, van Laack et al. (2001)
demonstrated that there was a significant improvement in tenderness from d 7 to
14 postmortem. My'data are consistent with those reported by Koohmaraie et al.
(1991 ), who demonstrated that maximum tenderness in pork is achieved by d 5
postmortem. Although I did not measure tenderness at d 5, maximum
tenderness is achieved between 3 and 7 d postmortem in this study.
Collectively, these data show that storage beyond 7 d postmortem does not
improve tenderness of pork. A slight numerical decrease in WBS was observed
from d 7 to 14 postmortem. This appears to result from continued tenderization

of some samples (change in WBS from d 7 to 14 ranged from 0 — 1.65 kg). It is

51

unclear why tenderization occurs more slowly in some samples or why
proteolysis continues to result in additional tenderization in these samples.
Analysis of Desmin Degradation

To quantify the relationship between proteolysis and WBS values, a
subset of samples were chosen. Western blot analysis of desmin was performed
on samples from 4 tender (5 4.0 kg) and 4 less tender (> 4.7 kg) chops at d1
postmortem. Figure 2.2 depicts the Western blot analysis of desmin and the
corresponding WBS values. The disappearance of intact desmin corresponds to
decreases in WBS values through d 7 postmortem. At d 1 postmortem intact
desmin was observed in all samples regardless of WBS value (Figure 2.2 panel
A). This helps to explain the low correlation reported by Wheeler et al. (2000)
between tenderness and percent desmin degraded by d 1 postmortem. At d 3
postmortem the divergence of desmin degradation between the tender and less
tender samples can begin to be observed (Figure 2.2 panel B). By (I 7
postmortem, further degradation of desmin can be observed in more tender
samples and more intact desmin is present in the samples that measure less
tender (Figure 2.2 panel C). However, by d 14 there is very little intact desmin
observed in any of the samples and a range in WBS values of 2.3 - 3.5 kg
(Figure 2.2 panel D). It is unclear why there is continued degradation of desmin
after d 7 with only minor improvements in WBS values. These data provide
evidence that there are other factors that need to be elucidated to more fully

understand postmortem tenderization.

52

 

60

 

50 777.7 77-- -77.- .7 7 7 _.. _7 _—

 

4_O -

3.0 ‘r—

was (kg)

2.0 -

 

 

 

00‘

Day 1 Day 3 Day 7 Day 14

Figure 2.1. Means and standard deviations for WBS values measured at d 1, 3,
7 and 14 days postmortem. Means with different letters differ (P <
.0001)

53

Figure 2.2. Western blot detection of desmin degradation using a monoclonal
“D76” antibody. The corresponding WBS values are shown at the
bottom of each western blot. Molecular weight standards (MW) are,
from top to bottom, as follows: myosin, 200 kD; B-galactosidase, 116
kD; phosphorylase b, 97 kD; bovine serum albumin, 66 kD; hen egg-
white ovalbumin, 45 kD; bovine carbonic anhydrase, 31 kD; soybean
trypsin inhibitor, 21.5 kD. Panel A: desmin measured at d 1
postmortem. Panel B: desmin measured at d 3 postmortem. The
bottom arrow indicates a putative degradation product of desmin.

54

 

   

- - -<—Desmin

WBS(kg) 2.7 5.0 3.0 3.7 4.0 4.4 4.7 3.1

55

Figure 2.2. Western blot detection of desmin degradation using a monoclonal
“D76” antibody. The corresponding WBS values are shown at the
bottom of each western blot. Molecular weight standards (MW) are,
from top to bottom, as follows: myosin, 200 kD; B—galactosidase, 116
kD; phosphorylase b, 97 kD; bovine serum albumin, 66 kD; hen egg-
white ovalbumin, 45 kD; bovine carbonic anhydrase, 31 kD; soybean
trypsin inhibitor, 21.5 kD. Panel C: desmin measured at d 7
postmortem. The bottom arrow indicates an putative degradation
product of desmin. Panel D: desmin measured at d 14 postmortem.
The bottom arrow indicates a putative degradation product of desmin.

56

 

 

 

Desmin

 

57

IMPLICATIONS

Tenderness of pork products plays a key role in repeat purchase decisions
of consumers. At this point, retailers are unable to segregate pork products and
guarantee the consumer a tender product. Based on these data, it does not
appear that measurement cf myofibrillar protein degradation at d 1 postmortem
reflects ultimate tenderness of the pork product. These data do however,
suggest that proteolysis can be measured after d 3 postmortem. Detection of
tenderness early postmortem would allow for strategies to be implemented to
“guarantee” a tender product. Until the strategies can be implemented, pork loin
chops should be aged at least 7 d postmortem to optimize tenderness for the

consumer.

58

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