ABSTRACT
SURIAATMAJA, DAHLIA. Mechanism of Meat Tenderization by Long-Time LowTemperature Heating. (Under the direction of Dr. Tyre C. Lanier).
The tougher texture of the lesser desirable cuts of beef is primarily attributable to higher
content of collagen and/or cross-linked collagen. Many chefs, and some recent scientific
studies, have suggested that long-time, low-temperature (LTLT) isothermal (sous vide)
heating can considerably tenderize such meat and still retain a steak-like (rather than pot
roast) texture. We investigated the effects of extended LTLT heating at 50 – 59 °C as a
pretenderizing treatment for a tougher beef cut (semitendinosus, eye of round), and compared
this to a bromelain-injection meat tenderizing treatment (‘meat tenderizer’ addition with no
preheating). Because our intended application envisioned finish cooking by grilling of these
pretenderized steaks at restaurants, we subsequently grilled all steaks to 65 °C internal
(medium done). To counteract water losses associated with extended LTLT heating, all
steaks were pre-injected with 15% of a salt/phosphate solution. Tenderization, as monitored
by a slice shear force (SSF) measurement, did not occur at 50 °C but did initiate above 51.5
°C (isothermal). At 56 °C, a LTLT temperature chosen for safe treatment of steaks, a tender
yet steak-like texture was obtained after 24 hr heating. Sodium dodecyl polyacrylamide
electrophoresis (SDS-PAGE) did not indicate proteolysis of myosin heavy chain, as would
have been expected if cathepsin (protease) had been active during heating. Instead, a
pronase-susceptability assay indicated a high association of tenderization with the conversion
of collagen to a partially denatured amorphous (‘enzyme-labile’) form, suggesting that this
partial denaturation of collagen is mainly responsible for the tenderizing effect. This
occurred at much lower temperature than has previously been reported for the initiation of
collagen denaturation (>60 °C) by differential scanning calorimetry, which likely is
accounted for by the longer time of LTLT heating. Bromelain injection also produced
tenderization as indicated by SSF measurements, but these steaks evidenced mushy texture
and uneven appearance. Thus LTLT at 56 °C can be an effective means of tenderizing lesstender cuts of beef in a central location, for distribution and finish grilling at restaurants, and
simultaneously it pasteurizes meat which has been injected with salt/phosphate to enhance
yield and succulence. This is accomplished without detriment to meat color such that these
steaks can be finish grilled at restaurants to almost any desired degree of doneness with no
compromise in food safety.
© Copyright 2013 Dahlia Suriaatmaja
All Rights Reserve
Mechanism of Meat Tenderization by Long-Time Low-Temperature Heating
by
Dahlia Suriaatmaja
A thesis submitted to the Graduate Faculty of
North Carolina State University
in partial fulfillment of the
requirements for the degree of
Master of Science
Food Science
Raleigh, North Carolina
2013
APPROVED BY:
_______________________________
Tyre C. Lanier
Committee Chair
______________________________
Dana J. Hanson
________________________________
Fletcher Arritt
________________________________
Clint Stevenson
DEDICATION
I dedicate this thesis to my dear and loving husband, Daniel, for all of his supports and
patience throughout the course of my graduate program. To my beloved daughters: Natalie,
who assisted me with biochemistry, and Melissa, for her understanding that sometimes I had
to miss her tennis tournaments.
ii
BIOGRAPHY
Dahlia Suriaatmaja was born and grew up in Jakarta, Indonesia. She went to Singapore for
her secondary school and completed the GCE “O” level. She attended Grade 13 High School
in Toronto, Canada. Dahlia went to University of Southern California to study Chemical
Engineering for her undergraduate degree and participated in undergraduate research with
Dr. Theodore Tsostsis.
After completing a B.Sc in chemical engineering, Dahlia went back to Indonesia and worked
for a coatings company. She began her work in quality control and then moved to the product
development and purchasing divisions. After 10 years of employment, Dahlia took time off
to concentrate on homeschooling her two daughters. During this time she helped start a dry
cleaning business with her husband. Later, she developed a proprietary chemical cleaner for
dry cleaning and marketed the chemical cleaner “Stain Free” for dry cleaning purposes.
Dahlia later moved to the United States with her husband and two children. Her love for food
brought her to culinary studies at Alamance Community College in Burlington NC, which
then led her to study food science at NCSU. Dahlia enrolled as graduate student and
developed an interest in the LTLT (sous vide) cooking method introduced to her by Dr. Tyre
Lanier who has been her mentor in this MS research project.
iii
ACKNOWLEDGMENTS
I would like to express my gratitude to many individuals who have given me guidance and
support in my work. I am thankful for the opportunity to do this thesis and for the grace of
God to complete it.
I owe my deepest gratitude to my advisor, Dr. Tyre Lanier, for his excellent guidance,
support and patience. As my mentor, he has taught me to think positively, think deeper, and
to question thoughts and ideas. I would like to thank my thesis committee for their support.
Dr. Dana Hanson has been kind enough to share his knowledge of the beef industry. Dr.
Fletcher Arritt has taught me to be always concerned with food safety in my work and Dr.
Clint Stevenson has given me encouragement. I also would like to thank Dr. Jason Osborne,
for his advice on statistics.
I am grateful to Dr. Jonathan Allen for his kindness, advice and support. I would like to thank
Dr. Christopher Daubert and Dr. K.P. Sandeep for all of the advice related to my research. I
want to appreciate Penny Amato for her assistance in the lab and Karl Hedrick for his help
with lab equipment. Many thanks to Evelyn Durmaz, Ruth Watkins, Paige Luck, Chris
Pernell, Luis Pena, Yvette Pascua, and Travis Tennant for all the supports.
I wish also to thank friends who have supported me, Maria Granados, Nihat Yavuz, Ron
Sequira, Theresa Wiggins, and Yunhee and John Gray.
iv
TABLE OF CONTENTS
LIST OF TABLES ............................................................................................................... viii LIST OF FIGURES ............................................................................................................... ix Chapter 1 . Literature Review ............................................................................................... 1 Introduction: meat texture and succulence .......................................................................... 1 Instrumental Measurement of Tenderness/Toughness in Meat ......................................... 3 Structure and composition of muscle as affecting meat texture ......................................... 4 Muscle Structure and Composition....................................................................................... 4 Meat Proteins .......................................................................................................................... 5 Sarcoplasmic Proteins ............................................................................................................ 6 Myofibrillar Proteins .............................................................................................................. 7 Connective tissue proteins .................................................................................................... 10 Altering meat structure to modify tenderness/toughness of cooked meat ....................... 11 Rigor mortis, sarcomere contraction and ageing effects on toughness ............................ 12 Role of added (exogenous) proteases/activators in meat tenderization ........................... 14 Blade tenderization ............................................................................................................... 16 Effects of Cooking on Meat Texture ................................................................................... 17 Isothermal Heating Effects................................................................................................... 17 Tenderization of Meat during Isothermal Heating, Long Time/Low Temperature
(LTLT) ................................................................................................................................... 20 Ramp Heating Effects ........................................................................................................... 23 Sous Vide (LTLT) Cooking .................................................................................................. 25 v
Food Safety Considerations in LTLT Processing of Meat ................................................ 26 Summary and implications for future development .......................................................... 28 Tables ..................................................................................................................................... 31 Figures.................................................................................................................................... 32 References .............................................................................................................................. 44 Chapter 2 . Beef Steak Tenderization by Long Time, Low Temperature Heating ....... 52 Introduction ........................................................................................................................... 52 Material and Methods .......................................................................................................... 54 Materials ................................................................................................................................ 54 Methods.................................................................................................................................. 54 Experiment 1. Initiation Temperature for Tenderization................................................. 54 Experiment 2. Effect of salt/phosphate solution enhancement on cook loss during LTLT
heating .................................................................................................................................... 56 Experiment 3. Comparison of tenderization by LTLT heating vs. by injection of
bromelain ............................................................................................................................... 57 Sample preparation for chemical analysis.......................................................................... 58 pH determination of raw meat prior to treatments ................................................... 59 Cook loss determination ........................................................................................... 59 Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)
measurement ............................................................................................................. 59 TCA precipitation absorbance value ........................................................................ 60 Modified slice shear force (SSF) measurement of meat tenderness......................... 60 vi
Separation of collagen fractions based on solubility and pronase susceptibility ..... 61 Statistical analysis ................................................................................................................. 62 Results and Discussion.......................................................................................................... 62 Experiment 1. Initiation temperature for tenderization. ........................................... 62 Experiment 2: Effect of salt/phosphate solution enhancement on cook loss during
LTLT heating............................................................................................................ 66 Experiment 3. Comparison of tenderization by LTLT heating (56 °C only) vs. by
injection of bromelain............................................................................................... 67 Changes in SSF......................................................................................................... 68 Changes in Collagen ................................................................................................. 69 Evidence of proteolysis ............................................................................................ 69 Cook Yields .............................................................................................................. 71 Effects on Meat Color .............................................................................................. 71 Conclusions ............................................................................................................................ 71 Acknowledgement ................................................................................................................. 72 Figures.................................................................................................................................... 73 References .............................................................................................................................. 86 vii
LIST OF TABLES
Table 1-1. Relationship between number of Hp cross-links and tenderness (Lepetit, 2007). 31 viii
LIST OF FIGURES
Figure 1-1. Schematic diagram illustrating the effect of swelling with aid of a salt/phosphate
solution, due to charge repulsion of myofibrils. Adapted from Offer & Knight (1988). 32 Figure 1-2. Organization of striated muscle. Adapted from Tortora, Funke, & Case (2001). 33 Figure 1-3. Sarcolemma and basal lamina. Adapted from Huijing (1999). ............................ 34 Figure 1-4. Schematic diagram of sarcomere skeletal protein. Adapted from Au (2004). ...... 35 Figure 1-5. Schematic diagram of cold shortened meat (1) penetration of Z line and (2)
rejection by Z line. Adapted from Marsh & Carse (1974). ............................................ 36 Figure 1-6. Relative activity of enzymes on three fractions of meat. Adapted from Kang &
Rice (1970). ..................................................................................................................... 37 Figure 1-7. Relative percentage change of (a) fiber diameter and (b) fiber length with time at
various temperatures. Adopted from Hostetler & Landmann (1968). ............................ 38 Figure 1-8. The effect of isothermal heating at different temperatures for one hour:
toughening occurs in two separate phases. Adapted from Davey (1974)........................ 39 Figure 1-9. Tensile strength of perimysial connective tissue isolated from semitendinosus
muscle after 1 hour heating at different temperatures. Adapted from Christensen,
Purslow, & Larsen (2000). ............................................................................................. 40 Figure 1-10. (a) Changes in shear force value during ramp heating and, (b) percentage change
in diameter, with a fast heating rate (>3 °C/min). Data is reconstructed from Table 2
(Li, Zhou, & Xu, 2010). .................................................................................................. 41 Figure 1-11. Percentage change of fiber diameter or length with slow heating rate ( < °1.3
C/min). Adapted from Hostetler & Landmann (1968).................................................... 42 Figure 1-12. The effect of non-isothermal heating on shear values (A) and percentage of
soluble hydroxyproline with heating 0.3 °C/min. Adapted from Penfield (1973). ......... 43 Figure 2-1. SSF apparatus ....................................................................................................... 73 Figure 2-2. Schematic flow chart of pronase susceptibility assay (Powell, Hunt & Dikeman,
2000) ............................................................................................................................... 74 Figure 2-3. Effect of LTLT on SSf when semitendinosus muscle was held isothermally at 24
hr and 48 hr ..................................................................................................................... 75 Figure 2-4. Effect of LTLT on cook loss when semitendinosus muscle was held isothermally
at 24 hr and 48 hr ............................................................................................................ 76 ix
Figure 2-5. Effect of LTLT heating at 50 °C, 51.5 °C, and 53 °C on SSF of semitendinosus
muscle ............................................................................................................................. 77 Figure 2-6. Effect of LTLT heating at 50 °C, 51.5 °C, and 53 °C on cook loss on
semitendinosus muscle.................................................................................................... 78 Figure 2-7. Effect of 15% salt/phosphate (S/P) injection after LTLT heating at 53 °C / 24 hr
on semitendinosus muscle............................................................................................... 79 Figure 2-8. Effect of SSF of semitendinosus muscle on LTLT at 56 °C / 24 hr , bromelain
and control after grilling to 65 °C ................................................................................... 80 Figure 2-9. Effect of cook yield of semitendinosus muscle on LTLT at 56 °C/24 hr,
bromelain and control grilled to 65 °C internal temp. .................................................... 81 Figure 2-10. Photograph of ST muscle sliced steaks obtained from LTLT at 56 °C and
bromelain treatment grilled to 65 °C intenal temp.......................................................... 82 Figure 2-11. Relative content of three measured collagen fractions of LTLT 56 °C/24 hr,
bromelain, and control grilled to 65 °C internal temp. ................................................... 83 Figure 2-12. Effect of LTLT at 56 °C/24 hr, bromelain and control on MHC proteolysis
measured by SDS PAGE ................................................................................................ 84 Figure 2-13. Effect of LTLT at 56 °C/24 hr, bromelain, and control measured by TCA
absorbance at 280 nm...................................................................................................... 85 x
Chapter 1. Literature Review
Introduction: meat texture and succulence
The major factors affecting consumer acceptability of cooked meat are tenderness,
succulence, and flavor (Horsfield & Taylor, 1976). Sensory analysis has shown that these
three factors are assessed independently of each other by consumers (Harries, Rhodes, &
Chrystall, 1972) and among these, tenderness is the primary factor that influences consumer
purchase decisions (Boleman et al., 1997). This review will focus primarily on factors
affecting steak tenderness, with some attention also given to the attribute of succulence.
The term ‘succulence’ refers mainly to the moisture/juiciness/lubrosity aspects of cooked
meat, and would relate to the ease of swallowing chewed meat (Horsfield & Taylor, 1976).
Moisture loss during cooking will thus adversely affect succulence. The juiciness/succulence
that comes from melting of the intramuscular fat content of meat is also an important factor
(José, Maria, & Carlos, 2009). For example, the extreme succulence of Wagyu beef from
Japan is associated with its very high degree of marbling/fat content. The most pleasant
sensations seem to derive from oleic acid in the triglycerides, whereas linoleic acid is deemed
to be less pleasant (Cyranoski, 2008). Succulence in very lean cuts of beef will however be
mainly determined by its water holding properties, since the fat component will be low or
missing.
1
Tenderness, or its counterpart, toughness, is a multi-dimensional quality comprised of the
related sensory attributes of resilience, resistance and chewiness (Horsfield & Taylor, 1976).
As will be subsequently discussed in more detail, tenderness/toughness is mostly determined
by the concentration and composition of connective tissue proteins, because most meats
offered commercially are properly handled and aged so as to insure desirable attributes of the
myofibrillar proteins or sarcomeres (Kemp, Sensky, Bardsley, Buttery, & Parr, 2010).
Interestingly, the water holding properties of meat affect not only meat succulence, but
indirectly can have some effect on tenderness as well, since the density of both the
myofibrillar and the connective tissue matrices increase as water is lost from meat,
effectively increasing resistance to biting through the meat. An extreme example of this
effect is the tougher texture of beef jerky. The major fraction of meat is water, 85% of which
is held in the inter-filament spacing of meat myofibrils (Offer & Knight, 1988). The filament
lattice separation is controlled by pH and ionic strength that determines the electrostatic
repulsive forces between the filaments. The addition of salt (typically sodium chloride)
and/or phosphates will thus increase the inter-filament spacing as shown in Figure 1-1,
allowing more room for water to be held by capillary forces within the meat. With salt
addition the thick filament in raw meat may also be partially depolymerized into myosin
molecules which, upon heating, may aggregate to form a gel. Water is also held tightly
within this gel fraction (Offer & Knight, 1988). Thus injection/tumbling of meat pieces with
salt/phosphate solutions can enhance water binding, and increase cook yields, adding to meat
succulence and possibly impart a slight tenderizing effect as well (Smith, Fletcher, & Papa,
1991).
2
Instrumental Measurement of Tenderness/Toughness in Meat
This approach to measuring tenderness/toughness is generally preferred in research studies
because sensory analysis is time consuming and expensive (King, Wheeler, Shackelford, &
Koohmaraie, 2008) and thus not suitable for testing large numbers of samples (Shackelford
& Wheeler, 2009). Various instrumental methods and geometries have been used. Probe
type penetrometers (Swatland, 2006; Tressler, Birdseye, & Murray, 1932) and the AlloKramer shear/ compression test (Cavitt, Youm, Meullenet, Owens, & Xiong, 2004) have
been used for this purpose, but the Warner Bratzler Shear Force test (WBSF) (Sullivan &
Calkins, 2011; Wheeler, Shackelford, & Koohmaraie, 1997) has been more widely used. For
WBSF testing, cooked meat samples are chilled overnight prior to coring. Cylindrical cores
of 1.27 cm diameter are removed parallel to the orientation of the meat fibers with either a
manual or automated coring device. Cores are sheared perpendicular to the muscle fiber
direction with a V-notch blade attached to universal texture testing machine at room
temperature (Warner, 1952; Wheeler, Koohmaraie, & Shackelford, 1995). A similar test,
termed the Slice Shear Force (SSF) measurement, substitutes a single horizontal descending
blade for the V-shaped blade of the WBSF cutting head to enable slicing of a greater surface
of steak area (slices of steak rather than cored samples). SSF data can be compared to WBSF
data via the following equation (Shackelford & Wheeler, 2009):
WBSF = (0.1063 * SSF) + 2.2718
Somewhat similar to this is a slicing blade device termed the Meullenet Owens Razor Shear
(MORS), which instead employs a sharp craft razor blade. It has primarily been used to
3
measure the tenderness of poultry meat (Cavitt, Youm, Meullenet, Owens, & Xiong, 2004).
Since most poultry meat derives from extremely young animals, its tenderness is largely a
factor of myofibrillar protein configuration, not connective tissue proteins.
For beef muscles cooked by a standardized protocol, a WBSF value between 3.9 and 4.6 kgf
are categorized as “slightly tender” (Shackelford, Morgan, Cross & Savell, 1991). However,
according to Smith et al. (1982) consumer satisfaction is obtained at WBSF < 4.1 kgf
(Sullivan & Calkins, 2011). There is considerable variation in toughness among different
muscles within an animal, and even within individual muscles depending on the sampling
location (Rhee, Wheeler, Shackelford, & Koohmaraie, 2004).
Structure and composition of muscle as affecting meat texture Muscle Structure and Composition
In general, mammalian muscle contains about 75% water, 19% protein, 2.5% lipid, 1.2%
carbohydrate, and 2.3% non-protein substances (Lawrie, 2006). The proteinaceous structures
are most important to imparting the textural properties of meat, and these also determine the
water holding properties of meat.
A thin layer of connective tissue called the endomysium surrounds bundles of myofibrils,
each of which is composed of bundles of myofilaments, to form a single muscle cell, termed
the muscle fiber. Bundles of muscle fibers are in turn surrounded by a connective tissue layer
termed the perimysium to form muscle fiber bundles which are also grouped together
4
forming larger secondary bundles, surrounded by an outer connective tissue layer termed the
epimysium (Figure 1-2). Directly beneath the endomysium layer is the basal lamina, which
consists of two layers; the lamina densa and lamina lucida (Figure 1-3). The lamina densa
forms a layer overlaying the endomysium, but the collagen fibrils of the endomysium cannot
penetrate it. The lamina lucida borders on the sarcolemma or cell membrane. The
sarcolemma and basal lamina are thus attached to one another and can only be separated by
collagenase and/or acidic conditions.
Meat Proteins
The primary protein component of the connective tissues depicted in the two previous figures
(Figure 1-2 and Figure 1-3) is collagen; wheras the myofilaments of the myofibrils are
primariy myosin and the actin (the ‘myofibrillar proteins’). Meat proteins are commonly
grouped into three categories on the basis of their solubility characteristics: (1) those soluble
only in salt/high ionic strength solutions (the myofibrillar proteins; chiefly myosin and actin),
(2) those easily soluble in water or low ionic strength (the ‘sarcoplasmic proteins’, which
includes most enzymes and heme proteins) and (3) those largely insoluble in high or low
ionic strength solutions (connective tissue proteins; chiefly collagen). Myofibrillar proteins
comprise 50 to 55% of the total protein content, sarcoplasmic proteins constitute
approximately 30 – 34% and the remaining 10 – 15% are connective tissue proteins.
Myofibrillar and connective tissue proteins are fibrous in nature, being rod-shaped molecules
of twisted polypeptide chains, whereas sarcoplasmic proteins are globular in conformation
(Starsburg, Xiong, & Chiang, 2008).
5
Proteins under normal (physiological) conditions of pH and temperature remain in the native
(folded) conformation. This conformation is related to the hydrophobicity, polarity and the
steric hindrance of the amino acid side chains. Changes in the thermodynamic stability, such
as may be caused by heating or a change in solvent conditions, can cause denaturation
(unfolding) of proteins (Kauzmann, 1959). Heating, which leads to rupture of hydrogen
bonds and strengthening of hydrophobic interactions, results in both denaturation and
subsequent aggregation of proteins, since aggregation is also thermodynamically favorable.
Whereas globular proteins tend to expand upon denaturation, fibrous proteins generally
contract on cooking since dissolution of their folded helices leads to their compacting in a
more globular form (Tornberg, 2005). Heat denaturation of proteins may thus effect changes
in meat texture, and each type of protein in meat can affect texture differently as it denatures
(Martens, Stabursvik, & Martens, 1982; Penfield, 1973).
Sarcoplasmic Proteins
The sarcolemma encompasses the cytoplasm of the muscle cell called the sarcoplasm. The
major proteins dispersed in the cytoplasm are myoglobin and many enzymes in lysosomal
compartments, dispersed throughout the sarcoplasm. Lysosomal enzymes include acidic
proteases known as cathepsins with optimum activity within the pH range 2.5 to 6.0. Several
different cathepsins have been identified, including cathepsins A, B, C, D, E, and L, but only
cathepsin B, D, and L are known to degrade muscle proteins (Mikami, Whiting, Taylor,
Maciewicz, & Etherington, 1987). Cystatin, an endogenous inhibitor of cathepsin, binds to
cathepsins L and B and thus may inactivate their catheptic activity in meat (Wang & Xiong,
6
1999) while cathepsin D, an aspartate proteinase, apparently has no inhibitor in the lysosome
(Etherington & Bardsley, 1995). Cathepsins B and D degrade actin and myosin in vitro
(Okitani, Matsukura, Kato, & Fujimaki, 1980) while cathepsin L degrades actin, myosin, αactinin, troponin and tropomyosin (Mikami, Whiting, Taylor, Maciewicz, & Etherington,
1987). Cathepsin D has also been hypothesized to attack collagen postmortem (Judge &
Aberle, 1982) but these authors provided no evidence of this.
Another lysosomal protease that is optimally active at more neutral pH is calcium-dependent
protease (CDP), better known as calpain. There are two types of calpain, which require
different optimal calcium concentrations; m-calpain requires calcium at millimolar levels
while μ-calpain requires calcium at micromolar concentrations (Camou, Marchello, Thompson, Mares, & Goll, 2007). Endogenous inhibitors, calpastatin, are also present in the
sarcoplasm. μ- calpain is most likely to activate during ageing because its optimal calcium
concentration is most likely to be attained post-mortem (Goll, Thompson, Li, Wei, & Cong, 2003). Meat tenderization induced by calpain is caused by disruption of the Z-lines of the
myofibrils during early postmortem ageing; there is no known effect on connective tissues
however. The amount of proteolytic activity occurring during the ageing period is regulated
by pH, temperature and calcium concentration (Ceña, Jaime, Beltrán, & Roncalés, 1992).
Myofibrillar Proteins
Myofibrillar proteins are the main component of the myofibrillar contractile structure
(Figure 1-4). The main such proteins are actin and myosin, the main myofilament proteins.
7
Regulatory myofibrillar proteins include tropomyosin, troponin complex, actinin, C-protein,
α- and β- actinin, M-protein and other minor proteins. Cytoskeletal myofibrillar proteins
are titin, nebulin, desmin and other minor protein that support the myofibrillar structure
(Huff-Lonergan, Mitsuhashi, Beekman, Parrish, Olson, & Robson, 1996). Myofibrils contain
repeating units called sarcomeres, separated by boundary zones termed the Z-line. The
striated appearance of the myofibrils is due to the contractile components of sarcomeres, the
myofilaments. Myofilaments consist of thick filaments, which constitute the dark (A) band
and thin filaments, which constitute the light (I) band. The thick filament is also referred as
the myosin filament as it contains myosin as the main protein. The middle of the thick
filament, or the H-zone, is composed of myosin rods with the globular heads external. The
center of the H-zone is the M line. The thin filaments are also commonly referred to as actin
filaments as actin is its main protein constituent. One end of the thin filament is attached to
the Z-line while the other is oriented towards the A band. The overlapping space between
thick and thin filaments is important as it affects hydration, tenderness, myosin extraction,
juiciness and water holding capacity of the meat (Lawrie, 2006). In the relaxed state, the H
band and the I band are at maximum length. For contracted muscle the actin filaments moves
toward the center of the H band; minimizing the I band and the H band. The length of the A
band is not affected by contraction, but the I band is shortened during contraction. In tightly
contracted or cold shortened meat the overlap can be as much as 50%. This tightly contracted
condition can contribute markedly to meat toughness, independent of the connective tissue
content or degree of collagen cross-linking (Kinsman, Kotula, & Breidenstein, 1994).
8
The third myofilament present is known as the gap filament or intermediate filament, which
is a fine line that runs parallel to the thick and thin filaments, connecting the Z-disk to the Mline of a sarcomere. This filament thus binds the thick filament to the Z-line. The main
protein is titin, which has a molecular weight of about 1 million Daltons. Titin can be
degraded during ageing by calpain as demonstrated by SDS-PAGE. However, this
degradation alone seems not to markedly influence meat tenderness (Fritz, Mitchell, Marsh,
& Greaser, 1993).
Myosin, which constitutes 50% of all myofibrillar protein, has a long rod-like asymmetrical
structure that is 65 Å wide, 1600 Å long and 40 Å deep with a molecular weight of 500 kD.
In 8 M urea, myosin disassociates into 6 polypeptides containing 2 heavy chains each of 200
kD and 4 light chains of 20 kD (x2), 15 kD and 25 kD. The two heavy chains form a double
helix rod. One end of the rod is attached to 2 globular heads, S1 and S2. The S1 head
protrudes to the thin filament to bind with actin. The long tail interacts with others to form
the backbone of the thick filament.
Actin constitutes 22% of the total myofibrillar protein. In skeletal muscle, actin exists in the
form of double helical filaments made of polymerized globular monomers, each being 43
kDa in size. Thin filaments each contain about 400 actin molecules and provide a binding site
which allows myosin to temporarily bind during muscle contraction or permanently bind
following rigor mortis.
9
Connective tissue proteins
The main extracellular connective tissue proteins are collagen, reticulin and elastin, which
are insoluble proteins. Those which most affect meat texture are collagen and elastin. Elastin
is present in only small amounts though its quantity varies among different muscles.
There are 12 types of collagen identified found in intramuscular, skin, tendon, bone, dentine,
cartilage, disc, basement membrane, cartilage, vascular, and skin. The collagen types are
categorized into three major groups; those related to meat tenderness are (Bailey, 1989):
•
Fibre-forming collagen (types I, II, III and V)
•
Non fibre-forming collagen (type IV)
•
Filamentous collagen (type VI and VII)
The collagen molecule is composed of three polypeptide chains, or α-chains. Individual
chains are unstable but when three α- chains entwine, they form a stable triple helix. The α
-chain is a left-handed helix with three amino residues per turn, but the trimer or
tropochollogen, is a stable right-handed helix. Each α-chain is comprised of more than
1000 amino acids and has molecular weight of 100 kD. The repeating unit of collagen is
made up of Gly-X-Y, in which X and Y are most often proline and hydroxyproline but can
also be other amino acids, with the exception of tryptophan. The collagen molecule contains
approximately 33% glycine, 12% proline, and 11% hydroxyproline. Hydroxyproline is found
in both elastin and collagen and in the enzyme acetylcholinesterase but since collagen is by
10
far a more abundant constituent of meat, hydroxyproline content is often used to quantify
collagen content (Bailey, 1989).
Connective tissue proteins are often said to contribute a ‘background’ toughness in the sense
that the toughness they contribute to meat is not related to handling or storage of the meat
post-mortem. This toughness can be related to both the quantity of collagen in a particular
muscle and/or to the prevalence of mature covalent cross-links in that collagen (Lepetit,
2007) (Table 1-1). Cross-links decrease the tendency of collagen to convert to soluble gelatin
during cooking in the presence of water (Hill, 1966).
Altering meat structure to modify tenderness/toughness of cooked meat
As was mentioned in the introduction, both connective tissue and myofibrillar proteins can
affect tenderness/toughness in beef, though in those cuts of meat typically considered to be
tough, or in meat of older animals (Tornberg, 2005), connective tissue seems primarily
responsible for meat toughness (Shimokomaki, Elsden, & Bailey, 1972). Thus a naturally
tender cut of beef is largely so because (a) its myofibrils are not tightly contracted
(sarcomeres shortened) and/or the Z lines of sarcomeres have been weakened due to calpain
activity during aging, and (b) the content and/or extent of cross-linking of collagen in its
connective tissues is sufficiently low. Each of these factors will subsequently be explained in
some detail, and effects of exogenous treatments, such as proteolytic enzyme applications,
11
heat-induced chemical/structural changes, and mechanical disruption of natural muscle
structure, will be discussed as agents to tenderize otherwise tough cuts of beef.
Rigor mortis, sarcomere contraction and ageing effects on toughness
Post-mortem processing and storage conditions can play an important role in meat tenderness
as affected by the myofibrillar structure. Kemp, Sensky, Bardsley, Buttery, & Parr (2010)
and Dransfield (1977) stated that the sarcomere length varies according to carcass posture
during rigor mortis. As previously discussed, shortened sarcomeres contribute to less tender
meat. Another possible means of inducing shorter sarcomeres is cold shortening. After
death, muscle undergoes anaerobic glycolysis, causing lactate build up which leads to the
decline of meat pH from 7.4 to around 5.5. During this period the supply of ATP decreases
which leads to the formation of actomyosin cross bridges. When the excised muscle is too
rapidly chilled to below10 °C while the meat pH is higher than 6.2, contraction of the
sarcomeres can occur (Marsh & Leet, 1966). This occurs because at lower temperature the
sarcoplasmic recticulum cannot supply enough calcium for muscle relaxation (Whiting,
1980) yet the muscle still has an ample supply of ATP. This results in muscle shortening by
35 - 40% (Locker, 1963) which causes cooked meat to be tougher in texture (King, Wheeler,
Shackelford, & Koohmaraie, 2008; Marsh & Carse, 1974). When muscle has shortened by
35%, actin myosin filaments overlap causing the myosin filaments to either penetrate the Zline or the myosin filaments to crumple as shown in (Figure 1-5). Thus cold-shortened meat
would also have an increased cross section density of connective tissue, causing a higher
12
tension and shrinkage during heating (Dransfield & Rhodes, 1976). The phenomenon of cold
shortening is independent of an animal’s age (Bouton, Harris, & Ratcliff, 1981).
Aging of meat post-rigor during chilled storage is necessary because meat toughens at rigor
due to formation of the permanent bond between myosin and actin. Ageing usually is
maximized at about 12 days of storage at 4 °C holding. The tenderization that occurs during
ageing is due to proteolytic activity by calpains that disrupts the myofibrillar structure at the
Z line of sarcomeres (Kinsman, Kotula, & Breidenstein, 1994). Calpain activation starts
when the amount of calcium released is high enough to initiate proteolytic attack. At death,
the muscle is at pH 7.5 to 8.0, gradually dropping to an ultimate level of 5.4 - 5.5 due to the
formation of lactic acid during glycolysis in the absence of oxygen. While the optimum
activity of calpain is said to be at pH 7.2 – 8.2, some workers have suggested that 15-25% of
maximum activity remains even at pH 5.5- 5.9. Calpain degrades desmin, which weakens the
binding of α- actinin to the Z disk, tropomyosin, titin, and M line proteins (Huff-Lonergan,
Mitsuhashi, Beekman, Parrish, Olson, & Robson, 1996). The calpain system has however no
effect on the integrity of actin and myosin myofilaments (Penny, 1980).
Cathepsins may possibly also take part in the ageing or conditioning process, especially when
the pH has decreased and the temperature is elevated. Yates, Dutson, Caldwell, & Carpenter
(1983) showed that degradation of myosin and troponin T increased at 37 °C. However,
Koohmaraie, Whipple, Kretchmar, Crouse, & Mersmann (1991) showed that cathepsin,
which though it may be active up to 70 °C during cooking, does not degrade myofibrillar
13
protein at 2 °C (chilled storage). Thus under normal conditions of ageing, cathepsin likely
plays no role in tenderization of meat and the bulk of evidence suggests that calpain alone is
responsible for tenderization during refrigerated ageing (Dayton, Goll, Zeece, Robson, &
Reville, 1976; Dayton, Schollmeyer, Lepley, & Cortés, 1981; Nowak, 2011).
Role of added (exogenous) proteases/activators in meat tenderization
One approach to meat tenderization is the addition of proteolytic enzymes. Those which have
been investigated to date include plant extracts, fungal proteases and bacterial proteases
(Foegeding & Larick, 1986). Plant extract proteases are most commonly used commercially,
primarily papain (papaya extract), bromelain (pineapple extract), and ficin (fig extract).
Others studied for use include actinidin (kiwi extract) and zingiber (ginger extract)
(Christensen et al., 2009; Ha, Bekhit, Carne, & Hopkins, 2012). Cucumis (cucumber extract)
and ginger have been trialed to tenderize tough buffalo meat (Naveena & Mendiratta, 2004).
Papain has a broad substrate activity against both myofibrillar proteins and connective tissues
(Ashie, Sorensen, & Nielsen, 2002; Kang & Rice, 1970). Using azocoll as a substrate,
Foegeding & Larick (1986) showed that papain has optimal activity at 60 - 70 °C; thus this
enzyme is most active during meat cooking. Etherington (1984) also found some activity of
papain on meat at cold storage temperatures. A meat temperature of 80 °C is required for its
inactivation; so often residual activity can occur post-cooking in steaks. Thus use of papain
14
can easily cause over- tenderization and result in a mushy meat texture (Ashie, Sorensen, &
Nielsen, 2002).
Bromelain belongs to the cysteine plant proteinases that are known to attack myofibrillar
proteins. It also is most active during cooking of meat, having an optimum temperature range
of 60 - 70 °C (Foegeding & Larick, 1986). It shows homology to mammalian cysteine
proteinases such as cathepsin B, L, H and calpains. The tenderization mechanism of
bromelain is not clearly defined though there is evidence that bromelain has a greater activity
towards collagen as compared to papain, and as compared with its activity towards
myofibrillar proteins (Figure 1-6) (Etherington & Bardsley, 1995; Kang & Rice, 1970).
The application of exogenous enzymes to the meat surface or by injection into the meat, even
with subsequent tumbling or massaging, does not really produce an even distribution of the
added protease at the cellular level throughout the meat piece being treated. Enzymes are
proteins, which on the cellular level are quite large and do not easily travel through meat
membranes (Carvajal-Rondanelli, 2003). Therefore, the proteolytic effect is necessarily
concentrated and localized at the point of injection or application, which is another possible
contributor to the development of mushy texture in cooked meats, which have been treated
by plant enzymes.
Calcium chloride injection of meat, even into the live animal’s blood stream or into meat
prerigor, can be carried out to better activate m-calpains during ageing and thereby enhance
15
meat tenderization (Wheeler, Koohmaraie, & Shackelford, 1997). However, calcium
chloride has a metallic taste that is often objectionable unless the meat is properly seasoned
to mask the off-flavor.
Blade tenderization
Blade tenderization (BT) is commonly practiced by the meat industry to partially overcome
myofibrillar- or connective tissue-associated toughness by severing the muscle in multiple
locations, while attempting to minimize visual indication of this damage/tenderization. Sharp
needles/blades are used to pierce the meat, which physically cut or tear the tissues at preset
intervals. The USDA is presently promulgating rules to require retail labeling of meat treated
by BT for retail and food service because such steaks could be contaminated internally with
pathogenic bacteria as the needles/blades pierce through the meat tissue (U.S. Department of
Agriculture, Food Safety and Inspection Service, 2013). USDA suggests that BT steaks
should be cooked to at least 65 °C internal (coldest spot) for safe consumption, meaning that
such steaks could not be presented to consumers in the ‘rare’ or ‘medium rare’ level of
doneness (Alfaro, 2013; Tewari & Juneja, 2007).
The physical damage caused to the muscle structure by BT treatment also seems to facilitate
greater cook loss during heating (Smith, Fletcher, & Papa, 1991). Consumers are able to
differentiate the tenderness of mechanical tenderized steak from that of naturally tender
steaks (Maddock, 2008).
16
Effects of Cooking on Meat Texture
The effects of cooking on meat texture are quite complex and do not always result in
tenderization. Time, temperature, and heating rate are the three variables which ultimately
determine how cooking affects meat texture.
Kinetic studies on the effects of temperature on changes in meat chemistry and
tenderness/waterholding are most typically carried out by isothermal heating of meat over a
range of temperatures. Under these conditions one would expect that changes might occur at
much lower temperatures than may be observed for ramp cooking, especially when compared
to heating at the relatively high rates (5 - 10 °C / min) that are typically used in differential
scanning calorimetry (DSC) (Leikina, Mertts, Kuznetsova, & Leikin, 2002). Most
commercial cooking of meats is conducted by relatively slow ramp heating (0.5 to 3 °C/min),
necessitated by the relatively large thickness of most commercial meat products and physical
limitations of heat transfer in commercial cookers. Many reported laboratory studies have
however employed relatively fast heating rates, >3 °/min (Li, Zhou, & Xu, 2010), which are
difficult to achieve commercially except when very thin meat pieces are being heated. The
following discussion of cooking (heating) effects on proteins and meat structure, and the
resulting effects on meat texture and water holding properties, will address heating under
these three different conditions.
Isothermal Heating Effects
Shrinkage of muscle fibers in diameter is first observed at about 37 °C during isothermal
heating. A greater degree of diameter shrinkage occurs at 45 °C, whereupon shrinkage in
17
length seems to only initiate (Figure 1-7) (Hostetler & Landmann, 1968). Both length and
diameter shrinkage increase with higher temperatures of isothermal heating. This study also
noted that cook losses increased in accordance with muscle fiber shrinkage, as would be
expected.
Interestingly, extending the isothermal heating time beyond about 30 min does not seem to
induce much additional shrinkage in these fibers in either dimension (Hostetler & Landmann,
1968). This is counter-intuitive, since one would suppose that the activation energy for
changes in the meat proteins responsible for shrinkage would have been reached at the lowest
temperature where shrinkage is first detectable. Subsequently then, since proteins would
continue to denature, shrinkage would be expected to proceed at some rate proportional to
the temperature, the rate constantly increasing to a maximum at some higher temperature.
Maximum shrinkage of the fibers might be reached only when proteins were fully denatured
and the maximal amount of water had been expelled from the fibers.
Somewhat corresponding to this data, Davey (1974) measured toughening of meat (measured
by shear testing) during isothermal heating (Figure 1-8). Compared to raw meat, isothermal
heating at temperatures of 40 - 50 °C produced a significant toughening of fibers which they
attributed to myofibrillar protein denaturation. In the range of 50 – 65 °C the fibers became
more tender during isothermal heating, but when heated at 65 - 75°C fibers were again
observed to become more tough. The authors attributed the toughening at 65 - 75 °C to
collagen shrinkage. Presumably the logic of assigning the toughening that occurred at 40 - 50
18
°C to myofibrillar protein denaturation, and the toughening that occurred at 65-75 °C to
collagen shrinkage, was that when measured by DSC (at high heating rates) myofibrillar
proteins denature at temperatures somewhat lower than does collagen (Martens, Stabursvik,
& Martens, 1982).
However, Christensen, Purslow, & Larsen (2000) reported hardening of isolated perimysium
at 40 - 50 °C during isothermal heating (one hour) (Figure 1-9). According to Lewis &
Purslow (1989) de-crimping of collagen (presumably due to its denaturation) most likely
begins at 40 °C. Collagen is arranged in a ply formation and the de-crimping increases the
collagen density which in turn increases the collagen strength (Lewis & Purslow, 1989).
Similarly, Field, Pearson, & Schweigert (1970) demonstrated that the epimysium contracts
during hardening and muscle with a more contracted epimysium exhibits a higher shear force
value.
Since Li & Zhou (2010) measured denaturation onset temperatures of endomysium and
perimysium to be 50 and 62 °C, respectively, by DSC (at a heating rate of 10 °C/min), it
would be expected that under isothermal conditions, endomysium would denature at perhaps
an even lower temperature than does perimysium, but certainly by the point at which
perimysium apparently denatures and hardens (Christensen, Purslow, & Larsen, 2000).
Based on this evidence it becomes difficult to assign changes in meat texture and
morphology below 50 °C to any particular proteins or meat structures. Presumably either
myofibrillar proteins, or collagen, or both could undergo denaturation and contribute to meat
19
shrinkage, toughening and water loss during isothermal holding at these temperatures.
Clearly something different occurs in the 50 - 65 °C range that contributes to meat
tenderization and this will be discussed further in the next section.
Field, Pearson, & Schweigert (1970) demonstrated that hardening of muscle due to
contraction occurs even up to 90 °C and this likely is not caused by myofibril denaturation
because those proteins would have completely denatured at much lower temperatures. Fiber
length continuously decreases from 50 °C to above 80 °C when heated non-isothermally; this
may be another indicator that collagen is predominantly contributing to texture changes at
higher temperature. Toughening in the 65 -75 °C range (Beilken, Bouton, & Harris, 1986;
Christensen, Purslow, & Larsen, 2000; Davey, 1974) has however only been measured
during an isothermal heating time of 1-2 hr; longer times in this temperature range still lead
to tenderization when sufficient time ensues (Machlik & Draudt, 1963).
Tenderization of Meat during Isothermal Heating, Long Time/Low Temperature
(LTLT)
In the first 2 hr or so of heating at 50 - 59 °C there is no significant shear force decrease in
tougher muscles; indeed meat toughens during such heating (Machlik & Draudt, 1963). This
also has been attributed to initial shrinkage of the collagen induced by the heating treatment
(Finch & Ledward, 1972). The degree of shrinkage and toughening during this initial phase
of heating was found to depend on the concentration of mature cross-links in the muscle
(Lepetit, 2007).
20
M. Christensen, L. Christensen, Ertbjerg, & Aaslyng (2011) reported there is no decrease of
shear force with pork ST after heating 5 hours at 53 °C isothermal. The shear force decreased
only after cooking was extended for 17 hours and decreased even further when heating was
at 58 °C isothermal. This suggests that tenderization is ongoing with extended cooking time.
Beilken, Bouton, & Harris (1986) reported that in meat of very old animals the shear force
decreased at 50 °C and 55 °C after an extended heating time of 24 hr with further decreases
noted upon holding for 48 hr at these temperatures. This decreased toughness is very
significant when cooking temperature is elevated to 60 °C with holding for 24 - 48 hr.
Beilken, Bouton & Harris (1986) suggested that this tenderization noted between 50 - 60 °C
at longer times of heating is due to weakening of connective tissue (Bouton & Harris, 1981;
Bouton, Harris, & Ratcliff, 1981). Beilken, Bouton, & Harris (1986) found the meat of
young animals begins to tenderize at 50 °C while that of very old animals tenderizes more
readily at 60 °C.
Snowden & Weidemann (1977) found that isolated collagen fibrils heated at 60 °C exhibited
a partially amorphous melted fraction under electron microscopy. This amorphous melted
fraction was also shown to be susceptible to becoming soluble when subjected to pronase
digestion. Extended cooking at 60 °C for one hour increased the fraction susceptible to
pronase digestion. This suggests that isothermal cooking at 60 °C caused partial denaturation
of cross-linked collagen, which did not result in its solubilization unless subjected to
subsequent pronase digestion.
21
Powell, Hunt, & Dikeman (2000) used this approach to measure the pronase susceptibility of
collagen after isothermal heating at 50, 60 and 70 °C. Tenderization increased as the pronase,
or ‘enzyme’, labile fraction (ELF) increased. These treatments produced only a small amount
of soluble collagen, suggesting that tenderization is caused mostly by weakening of partially
denatured (cross-linked) connective tissue (Bouton & Harris, 1972; M. Christensen, L.
Christensen, Ertbjerg, & Aaslyng, 2011; Lewis & Purslow, 1989).
M. Christensen, L. Christensen, Ertbjerg, & Aaslyng (2011) associated the decrease of meat
toughness during LTLT with a measured increase in residual cathepsin activity. However,
Wang & Xiong (1999) showed there is no breakdown of myosin heavy chain measurable by
SDS-PAGE in bovine heart during cooking at 50 °C which argues against a role of cathepsin
activity in tenderization over this range. The authors concluded that while the literature
suggests cathepsin activity does exist in beef which could be active over this temperature
range, natural inhibitors also exist which negate its effects.
Machlik & Draudt (1963) showed that collagen must shrink (denature) before tenderization
can occur and noted a maximum rate of tenderization at 60 - 65 °C. Beilken, Bouton &
Harris (1986) noted that cook loss at 60 °C after 24 hours was almost double that of the cook
loss at 55 °C /24hr.
22
Ramp Heating Effects
Hostetler & Landmann (1968), who observed shrinkage of muscle fibers at 37 °C during
isothermal heating (Figure 1-7) also found that during slow ramp heating (1.3 °C/min) of
isolated muscle fibers the fiber diameter starts to shrink at 37 °C such that the diameter
decreased to about 84% of its original width upon reaching 40 °C (Figure 1-7a).
While they could detect initial shrinkage of muscle fiber length at 45 °C during isothermal
heating (Figure 1-7b), this was not evident during ramp heating until a temperature of 50+ °C
was attained (Figure 1-11). It is thus apparent that ramp heating can delay the onset of many
events, which may onset at lower temperatures when measured under isothermal heating
conditions.
When Laakkonen, Wellington, & Sherbon (1970a) measured water losses during very slow
ramp heating (0.1 °C/min) they detected an increase also beginning at 37 °C, in agreement
with the shrinkage of muscle fibers noted by Hostetler & Landmann (1968). As previously
discussed, these associated changes in muscle morphology and water holding (and texture,
noted previously) likely reflect the combined effects of heat-induced denaturation in
myofibril and connective tissues.
The shrinkage in length of muscle fibers noted at 50+ °C during slow (1.3 °C/min) heating by
Hostetler & Landmann (1968) (Figure 1-11) was accompanied by a loss in birefringence
between 54 - 56 °C, which they attributed to denaturation of the myofibrils. It is possible
however that changes in birefringence could also have indicated denaturation of collagen
23
surrounding the myofibrils at this temperature since the melting of collagen also results in
loss of birefringence (Engel, 1970).
In agreement with the isothermal studies of Beilken, Bouton, & Harris (1986) which showed
tenderization of beef over the 50 - 60 °C range, Penfield (1973) also reported decreases in
shear value of meat when slowly ramp heated (0.3 °C/min) from 50 - 60 °C, which correlated
with a corresponding increase in hydroxyproline solubility (Figure 1-12). This also suggests
that tenderization over this range primarily corresponds to changes in collagen of the
connective tissues, though only a 5% solubilization in muscle having a high percentage of
cross-linked collagen was reported by Powell, Hunt, & Dikeman (2000). As previously
discussed, they noted that only partial denaturation of collagen, resulting in pronase
susceptibility but not solubilization, is mainly associated with the tenderization occurring
over this temperature range. And while residual collegenolytic activity was observed by
some workers (Laakkonen, Wellington, & Sherbon, 1970a; 1970b) after heating meat in this
range, it is not clear if this actually results in hydrolysis of collagen, as was previously
discussed (Laakkonen, Wellington, & Sherbon, 1970a; 1970b).
Interestingly, when a very fast rate of heating is used, there is no tenderization effect noted in
the temperature range 50 - 65 °C. Li, Zhou, & Xu (2010) found that meat is constantly
increasing in shear force value when rapidly heated (> 3 0C/min) to endpoint temperatures
ranging from 40 to 90 °C (Figure 1-10a). Muscle fiber diameter decreased dramatically over
the same temperature range (Figure 1-10b). Thus clearly sufficient time within the
24
temperature range 50 - 65 °C is required for the tenderization reaction(s) to occur, and fast
heating does not allow for tenderization to proceed.
The significant toughening that occurred above 65 °C (Figure 1-10a) might not be expected
to derive from collagen denaturation since this is above the temperatures at which epimysium
and perimysium were found to totally denature as measured by DSC (Brüggemann, Brewer,
Risbo, & Bagatolli, 2010; Li, Zhou, & Xu, 2010; Martens, Stabursvik, & Martens, 1982).
Actin denatures over the range of 66 - 73 °C as measured by DSC (Martens, Stabursvik, &
Martens, 1982), but such a jump in meat toughness seems unlikely from this event alone.
These authors (Li, Zhou, & Xu, 2010) concluded that this toughening at high temperatures is
due to collapse of the epimysial/perimysial structures as manifested by the significant
decrease of fiber diameter. Perhaps this decrease in fiber diameter is actually reflecting
water losses, and drying out of the meat is inducing a stronger texture. Paul, McCrae, &
Hofferber (1973) concluded that toughening at 70 °C is probably due to hardening of
myofibrillar protein and the effect of tenderness cannot be completely explained by increases
in heat labile collagen (Field, Pearson, & Schweigert, 1970).
Sous Vide (LTLT) Cooking
Sous vide as translated from French means simply “under vacuum”. It is one specialized
method of LTLT cooking wherein the food (meat) is vacuum packed in a plastic pouch and
heated in a controlled temperature water bath. The method allows for maximum heat transfer
rate while also preventing contamination of the food by the water or during subsequent
25
chilled/frozen handling. Thus, if the food is pasteurized as well as tenderized by the LTLT
treatment, that state of pasteurization is maintained until the seal of the package is broken.
This method of LTLT cooking is favored by many chefs, some of whom have made specific
claims that meat is tenderized by the treatment when it is conducted within certain
parameters. For example, Myhrvold, Young, & Bilet (2011) in their seminal book Moderist
Cuisine noted that “weakening the [collagen] mesh is the job of cooking. To do that,
collagen fibers must be heated with sufficient moisture; the minimum temperature varies
from one kind of meat to another, but is about 52 °C for most red meats.”
Food Safety Considerations in LTLT Processing of Meat
The greatest concerns with LTLT processing of meats derive from the possible growth and/or
toxin production of Clostridium botulinum, Clostridium perfringens, and Baceillus cereus
because they are able to survive low heat treatments. They are gram positive, spore-forming
bacteria, and all can cause acute food poisoning. They produce toxins, which are harmful to
the human intestinal track and generally cause even more severe illness in elderly or healthcompromised persons. C. botulinum and C. perfringens are anaerobic bacteria, whereas B.
cereus is a facultative bacteria. Beside these spore-forming bacteria, Listeria monocytogenes,
Salmonella spp, Staphylococcus aureus and Escherichia. coli are non-spore forming and
facultative bacteria that also can impose illness (Jay, 2005; Tewari & Juneja, 2007) . C.
botulinum grows most rapidly at 12 to 50 °C (Jay, 2005). The spores of C. botulinum are heat
resistant requiring a temperature in excess of 100 °C for destruction. C. perfringens grows
most rapidly from 20 to 52 °C with an optimum temperature from 37 to 45 °C and optimum
26
pH ranging from 5 to 9 (Jay, 2005; Willardsen, Busta, Allen, & Smith, 1978) . B. cereus has
an optimum growth region from 5 - 50 °C and optimum pH range of 4.9 – 9.3 (Jay, 2005). To
control the growth of these spore forming bacteria an LTLT heated product should be rapidly
cooled afterwards to < 4 °C to prevent spore germination and inhibit growth of vegetative
cells (Ghazala, Ramaswamy, Smith, & Simpson, 1995). The growth of vegetative cells can
be stopped by heating and the minimum treatment as set by the U.S. Department of
Agriculture, Food Safety and Inspection Service (1999) for pasteurization (6.5 D reduction,
Salmonella) is 54.4 °C for 112 min.
This U.S.D.A., F.S.I.S. (1999) recommended process is not adhered to by chefs of many
leading restaurants today. For example, the very popular chef-authored manual on sous vide
cooking Moderist Cuisine (Myhrvold, Young, & Bilet, 2011) suggested that a 6.5 D
reduction of Salmonella can be accomplished at a lower pasteurization temperature, longer
time: 52 °C for 314 min. However, according to to Willardsen, Busta, Allen, & Smith
(1978), C. perfringens is able to grow at up to 52 °C. The authors of Modernist Cuisine did
however inject a disclaimer in their book, saying “ This book cannot and does not substitute
for legal advice about food regulations in the United States as a whole or in any U.S. legal
jurisdiction. Nor can we guarantee that following the information presented here will prevent
foodborne illness. Unfortunately, the many variables associated with food contamination
make eliminating all risk and preventing all infections virtually impossible. We cannot accept
responsibility for either health or legal problems that may result from following the advice
27
presented here. If you operate a commercial establishment and serve food to the public,
consult the rules and health regulations in your area”.
Summary and implications for future development
It seems clear from this review of literature that any tenderization that may occur at
temperatures below 50 °C (such as during refrigerated ageing) is not likely the result of any
changes to connective tissue/collagen. Indeed, isothermal heating at temperatures below 50
°C actually seems to toughen meat and connective tissue and no tenderization occurs
afterwards despite very long holding times (Bouton & Harris, 1981; Christensen,
Christensen, Ertbjerg, & Aaslyng, 2011). From the evidence it would appear that collagen
may denature (with extended time of isothermal heating) at temperatures as low as 40 °C
(Christensen, Purslow, & Larsen, 2000), resulting initially in its contraction and shrinkage.
This likely also contributes to water loss from the meat, which may be accentuated by
denaturation of the myofibrillar proteins at 40 - 50 °C. If calpains or cathepsins are active at
these temperatures there should be some degree of tenderization detectable as they degrade
myofibrillar proteins, but this has not been reported to date.
At >50 °C, however, it would appear that the activation energy for some new change in meat
structure is initiated. From the evidence presented thus far in the literature this seems most
likely to involve a particular type of unfolding/denaturation specific to cross-linked collagen,
which can be measured as ELF (pronase susceptible) and which dramatically softens tough
connective tissue. This reaction appears to occur at all temperatures between just above 50
28
°C up to the mid 60s °C and possibly higher. But this tenderization reaction is never apparent
when meat is heated at a rapid heating rate to an internal endpoint of 65 °C or higher, such as
occurs in normal grilling of beef steaks.
The beef industry does fully cook and simultaneously tenderize many tough cuts of meat
(high in cross-linked collagen content) but typically at temperatures above 65 °C and for
sufficient time to convert most collagen to gelatin. Such meat is uniformly brown in color to
its core because of the higher temperatures and times used. In home food preparation the
crock pot is often used to achieve similar tenderization of tough beef cuts; this is carried out
near 80 °C (Sundberg & Carlin, 1976) and typically results in a ‘pot roast’ texture (meat falls
apart) rather than a ‘steak-like’ texture typical of grilled meat.
LTLT heating of meat in the 50 – 60 °C range might result in a less cooked internal
appearance, offering a means of tenderizing meat without fully cooking it, allowing for
subsequent grilling to almost any desired level of internal ‘doneness’ at restaurants. Clearly
however there would be a concern that using LTLT to merely tenderize meat would result in
excessive water loss, with accompanying loss of meat succulence. However, if LTLT
heating were carried out under the correct parameters, the parcooked meat would be
pasteurized. This would allow for the enhancement of the meat by injected solutions to
improve its yield and succulence. LTLT heating by the sous vide method (vacuum packaged
meat in a water bath) would facilitate both the heating and the subsequent safe
29
storage/handling of the product until the package was opened and steaks were grilled at
restaurants.
30
Tables
Table 1-1. Relationship between number of Hp cross-links and tenderness (Lepetit, 2007).
Muscle
Q
n0(Hp)
Q n0 (Hp)
Tenderness
Biceps Femoris
3
0.5
1.50
1
Semimembranosus 3
0.5
1.5
≥1
Gluteus Medius
2.77
0.35
0.97
2
Longissimus dorsi
1.86
0.36
0.67
3
Psoas Major
1.41
0.45
0.63
4
Q, collagen content of muscles (%), n0 (Hp), Hydroxylxylpyridinoline (mole/mole
collagen)
31
Fig. 4, is therefore that lateral expansion of the filament lattice results in
more water being incorporated into the myofibrils in the spaces between
filaments and, since this water is firmly held, the meat is able to retain
more water. Conversely, lateral shrinkage of the filament lattice expels
water from the myofibrils and, since this expelled water is less-firmly
Figures
held, it can be lost more readiJy from
the meat.
gains (swelling)
..
losses (shrinking)
• • • •
• • • •• •• ••
• •••• •• •• •
• ••
• •••
• •• •• • • • •••
• ••••••••••••
• • • •
••••••••••
• • • • •
• •
•
FIG.
••••
4. Hypothesis explaining the origin of changes in water-holding of meat.
Figure
theaeffect
of swelling
of a salt/phosphate
On the1-1.
left Schematic
hand side isdiagram
shown illustrating
schematically
transverse
sectionwith
of a aid
myofibril,
with thick filaments (large circles) and thin filaments (small circles) seen endon,
fonning
a hexagonal
lattice.ofOnmyofibrils.
the right hand
side,from
the Offer
same &
myofibril
is
solution,
due to
charge repulsion
Adapted
Knight (1988).
shown with an expanded lattice. that during or acid-induced swelling
the thick filaments may depolymense, but the thm filaments would become
further apart.348 Reproduced by pennission of the Royal Society of Chemistry.
32
Figure 1-2. Organization of striated muscle. Adapted from Tortora, Funke, & Case (2001).
33
Figure 1-3. Sarcolemma and basal lamina. Adapted from Huijing (1999).
34
Figure 1-4. Schematic diagram of sarcomere skeletal protein. Adapted from Au (2004).
35
(1)
(2)
FIG. 2. Configuration of actin and myosin filaments and Z-lines at (a) 95% stretch,
(b) 33% Figure
stretch,1-5.
(c)Schematic
24% stretch,
(d)of24%
(e)(1)
35%
shortening.
Onlyand
the
diagram
cold shortening,
shortened meat
penetration
of Z line
(2)
longitudinal dimensions are to scale. Bent thick filaments in (e) are diagrammatic only,
rejection
by Z line.ofAdapted
& Carse
(1974).
to indicate
(i) penetration
Z-lines,from
(ii) Marsh
rejection
by Z-lines.
tips starting to appear on the other side and a number of the filaments do indeed
appear to have reached this stage.
(e) At 35% shortening (S.L. 1.37 pm) actin tips reach the ends of those myosin
filaments which have successfully penetrated the Z-lines from neighbowing sarcomeres
(Fig. 2e, i) . If we assume that polarity considerations apply to cross-linkage formation
in rigor as they appear to in contraction (Huxley, 1963) then the two filament species
(their ends on the point of overlap and their structures correctly polarized for interaction) can now form rigor linkages. The entire structure thus becomes firmly crossbridged throughout its length.
With these critical values defined, we are in a position to attempt an elementary
36
DEGRADATION
OF ME
desirable tendern
RE
PAPAIN
WATER SOLUBLE
RHOZYME
INSOLUBLE
P-II
SALT SOLUBLE
FIGIN
FRAOTION
FRACTION
0
m
FRAGTION
TRYPSIN
GROMELAIN
PERCENT
Fig, l-Relative
activity
DIGESTED
of enzymes
on three fractions
of meat.
Figure 1-6. Relative activity of enzymes on three fractions of meat. Adapted from Kang &
Rice (1970).
due to a longer period at its optimum
temperature range.
Numerous attempts have been made to
correlate chemical and physical changes
taking place in specific structural components of meat during post mortem
aging with tenderization. As pointed out
in the literature, modification
of connective tissue is desirable to reduce toughness
of meat since the quantity and degree of
cross linkage of connective tissue affects
tenderness of meat (Cover and Smith,
1956; Irvin and Cover, 1959; Go11 et al.
1964a and 1964b), and yet little or no
change in connective tissue has been
observed during natural aging, (Steiner,
1939; Wierbicki et al., 1954; de Fremery
and Streeter, 1969) whereas a convincing
body of evidence indicates changes within
the fibrous components of muscle during
aging. Davey and Gilbert (1968a and
1968b) showed that aging processes are
associated with an increased extractability of actin as well as with the disruption
of Z bands in myofibrils. A similar change
in fine structure, i.e., disruption
of Z
bands, was caused by a brief treatment of
myofibrils
with trypsin (Stromer et al.,
1967).
The proteolytic enzymes tested in our
experiment
showed hydrolytic
activity
both on myofibrillar
protein and on
connective tissue, and the degree of
hydrolysis of these fractions varied with
the type of enzyme used. Collagenase,
bromelain
and trypsin
degraded the
stroma fraction more strongly than the
myofibrillar
fraction. In contrast, papain,
Rhozyme P-l 1 and ficin showed more
activity on the myofibrillar
fraction than
on the other. These differences in hydrolytic characteristics
may lead us to a
better understanding of mechanisms of
enzymatic tenderization
and to a more
Cover, S. and Smit
two methods
scores, shear fo
tent of two cut
312.
Davey. C.L. and G
meat tenderness
ability
of myofi
aging. J. Food
Davey, C.L. and G
meat tenderness
lar proteins
e
aging. J. Food
de Fremery,
D. an
ization of chic
alkali-insoluble
post-mortem
a
Goll.
D.E.,
Hoeks
1964a. Age asso
nective tissue.
lagenase. J. Foo
Goll,
D.E..
Hoeks
1964b.
Age a
muscle connect
increasing temp
Habeeb, A.F.S.A.
amino groups
sulfonic acid. A
Hegarty,
G.R., Br
1963. The rela
protein
characte
derness. J. Food
Irvin, L. and Cove
heat on the c
muscles. Food
Miyada,
D.S. an
hydrolysis
of
teolytic enzymes
Steiner. G. 1939.
beef muscle at
Hyg. 121:193.
Stromer,
M.. Goll,
Morphology
of
cle and the effec
rigor myofibrils.
Wang, H., Weir, C.
B. 1957. The
izers on the
beef.
Proc.
N
American Meat
Wierbiki.
E.. Kunk
Deatheraae.
F.E
derness & ‘prote
mortem aging.
Ms. received
12/19
cepted 6/16/70.
37
Bureau
of Commercial
Fisheries,
Technological
Laboratory,
of the myofilaments denature, the immobile water is freed,
but after this,
escapes from the intermyofibrillar
space, and carries with
crease in fiber
it some soluble sarcoplasmic proteins. This material, visible
uring the next
in our observations as amorphous particles, appeared
tle change in
around the entire fiber, not at the cut ends only. The fiber
peratures (53,
simultaneously became narrower, this process being essenber width
(23
PHOTOMICROGRAPHIC
STUDIES OF MUSCLE FIBER FRAGMENTS.
1.A69
tially complete
at 53°C.
f heating, with (a)
Results of our time-temperature studies on isolated fibers
(Fig. 2) also showed that the diameter decreased in a
very little
esed
photoelectric
manner parallel to the loss of water-holding capacity as
37scale,
and while
45’C.
demonstrated by Hamm et al. (1962). Thus the decrease
the intime
alues
Fig. re4
in diameter may be explained as the early stages of heat
after
At
dicate this.
clearly
denaturation where unfolding of peptide chains has ochebirefringence
first 9 min
curred, causing a loss of water-holding capacity but little
o 25 min, with
disturbance in the parallel array of myofilaments.
of the heating
The changes in length of fibers do not conform to this
hortening took
behavior pattern, but instead resemble more closely the
over 5characmin.
nkage
behavior described by the curves Hamm et al. (1960) have
scle
fibers that
apy shows
shown for the decrease orTIME
loss (MIN.)
of the acidic groups. Fiber
, the birefrinlength
as heat
shortening
doesi not
untilfragmelcts
the temperature
Fig. 2. Change
widthbegin
of fiber
zvith time at reaches
differmuscle heat
fiber
creasing
cfkt
tenzperatzrres.
(b) 50” or more, when birefringence (Fig. 4) is noticeably
me
isotropic
by
n width
began
ued to 45°C.
the time they reached 55 to 6O”C, while others retained
ease in width
some birefringence at 66 to 6S”C.
omplete, since
d.
DISCUSSION
d very slowly
THE CHANGESin muscle fibers observed in these experito 55°C. Bements seem to be related most closely to the water-holding
nts shortened
( 61.C
properties of muscle proteins as described
by0 37’C
Hamm et
al.
STAGE
riginal
length.
. WIDTH
9 45-c
0 69-C
(1960) and Hamm et al. (1962). AsTEMP.
proteins coagulate
0 LENGTH
resulted
in an
= 53-c
I# 77-c
after
s 0 being denatured by heat, their water-holding capacity
ber length.
40 as drip
50 during 60
70
80
is lost. HammIO states 20that the30juice lost
ired temperaTIME (MIN.)
this process is that water held
immobile by the myofilasmall (2.5%)
Fig.
3.butChange
[clkgth
fiber
fragrrzc?kts When
zvitlk the
tivrc proteins
trt diffcrments,
is notin water
ofof hydration.
fragments
with
7°C.
Another
elkt temperatures.
of the myofilaments denature, the immobile water is freed,
but after this,
escapes from the intermyofibrillar
space, and carries with
crease in fiber Figure 1-7. Relative percentage change of (a) fiber diameter and (b) fiber length with time at
it some soluble sarcoplasmic proteins. This material, visible
uring the next
in our observations as amorphous particles, appeared
tle change in various temperatures. Adopted from Hostetler & Landmann (1968).
around the entire fiber, not at the cut ends only. The fiber
eratures (53,
simultaneously became narrower, this process being essenber width (23
tially complete at 53°C.
heating, with
Results of our time-temperature studies on isolated fibers
(Fig. 2) also showed that the diameter decreased in a
sed very little
manner parallel to the loss of water-holding capacity as
37 and 45’C.
demonstrated by Hamm et al. (1962). Thus the decrease
the time re38
in diameter may be explained as the early stages of heat
after this. At
denaturation where unfolding of peptide chains has oce first 9 min
curred, causing a loss of water-holding capacity but little
25 min, with
disturbance in the parallel array of myofilaments.
of the heating
The changes in length of fibers do not conform to this
hortening took
Cooking toughness in beef
60
935
-
50a,
-
9
a,
40-
r
L
L
m
30-
5
20
-
10 -
30
40
50
60
70
80
Cooking temperature ("C)
90
100
Figure 2. The two-phase effect of cooking temperature on shear-force values. Standard deviations
are given by vertical lines. Each point is the mean of 8 to 16 determinations from the muscles of four
bulls.
Figure 1-8. The effect of isothermal heating at different temperatures for one hour:
toughening occurs in two separate phases. Adapted from Davey (1974).
30
50
70
90
Codting temperature ("C )
Figure 3. The relationship of the second toughening phase to shrinkage and weight loss. Curve 1,
hear-force values (mean of 6 determinations); curve 2, shrinkage along the fibres; curve 3, weight loss.
other two changes of cooking-shrinkage and weight loss were identified with the
second phase (Figure 3). The onset of shrinkage along the fibres of unshortened muscle
occurred in parallel with toughening. Further shrinkage was slight once maximum
oughness was achieved. The temperature for half change in toughness was 67 to 68 "C
and for shrinkage, 70 "C.
39
*+ ,-.$!%"&!"& "% /0+ 1 *"/% 23$"&3" 44 567778 97:;97<
V:M
C16> V> ;./36$% '& 0$3%17$ "#$/+136 %0#$360. '& 4$#1(8%1/7 *'33$*01K$ 01%%5$ &#'( "$$& !"#$%"&'$&(!)! <10. 13*#$/%136 *''+136 0$(4$#/05#$> `/75$%
/#$ 61K$3 /% ($/3% a ^>b> B&c-DE>
Figure 1-9. Tensile strength of perimysial connective tissue isolated from semitendinosus
!"#$% &#'( "$$& !"#$%"&'$&(!)!) *''+$, &'# - . /0 ,1&2
muscle after
1 hour%0#$360.
heating'&at0.$different
&$#$30 0$(4$#/05#$%)
0.$ "#$/+136
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=:! ; 0.$ "#$/+136 %0#$360. '&
13*#$/%$ 0'
=:! ;> ?8
Larsen
(2000).
0.$ !"#$% </% /44#'@1(/0$78 9:A 6#$/0$# 0./3 0.$
"#$/+136 %0#$360. '& !"#$% &#'( #/< ($/0 BC16> DE>
F.$%$ #$%570% /#$ 13 /6#$$($30 <10. G505361 $0 /7>
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5371+$78 0./0 0.$ (/#+$, 13*#$/%$ 13 %1367$ (5%*7$ !"#$
0'56.3$%% '"%$#K$, /0 0$(4$#/05#$% /"'K$ 9:! ; 1%
,1#$*078 ,5$ 0' *'33$*01K$ 01%%5$> F.1% 130$#4#$0/01'3 1%
&5#0.$# %544'#0$, "8 0.$ !3,136 0./0 !"#$% 7/*+136
$3,'(8%15( ,' 3'0 %.'< ,1I$#$30 "#$/+136 %0#$360.%
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/7>) -HH9E> O'<$K$#) 0.$ 13*#$/%$, "#$/+136 %0#$360. '&
!"#$% %$$3 .$#$ <10. 13*#$/%136 0$(4$#/05#$ 1% ('#$
71+$78 ,5$ 0' .$/0 ,$3/05#/01'3 '& /*013 /3, '0.$# %/#2
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P:Q=:! ; /3, /"'50 9M! ;) #$%4$*01K$78 B;.$36 R S/##1%.)
-HPHT U//++'3$3) -HPVE> F.$%$ %05,1$% 13,1*/0$ 0./0 0.$
(8'!"#177/# *'(4'3$30 '& <.'7$ ($/0 *'30#1"50$% ('%0
'& 0.$ %$*'3, #1%$ 13 *''+$, ($/0 0'56.3$%%> O'<$K$#)
0.$ 4'%%1"17108 '& 13,1#$*0 $I$*0% '& *'77/6$3'5% *'(2
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(/8 ,#1K$ '50 W51, &#'( 0.$ (5%*7$ !"#$%) */33'0 "$
temperatures.
Adapted from Christensen, Purslow, &
$@*75,$,>
X3 *'30#/%0) 0.$ 0$3%17$ "#$/+136 %0#$360. '& 4$#1(82
%15( </% &'53, 0' 13*#$/%$ &#'( #/< 0' M:! ; *''+$,
($/0 /3, ,$*#$/%$, /0 .16.$# *''+136 0$(4$#/05#$%
BC16> VE> F.$%$ !3,136% /#$ 13 /6#$$($30 <10. 4#$K1'5%
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*$30#/01'3 '& *'77/6$3 !"#$% 4$# 5310 <1,0. /3, .$3*$
0.$ 35("$# '& 7'/, "$/#136 !"#$%Z$7$($30%> J0 0$(2
4$#/05#$% /"'K$ M:! ; 4#'6#$%%1K$ 7'%% '& 0.$ *#1(4
%0#5*05#$) %.#13+/6$ '& 0.$ *'77/6$3 !"#$% /3, 13*#$/%$,
%'75"171%/01'3 '& *'77/6$3 '**5#%) <.1*. 0.$3 ,$*#$/%$%
0.$ 7'/, "$/#136 /"17108 /3, 0.$#$"8 0.$ 0$3%17$ "#$/+136
%0#$360. BU$<1% R S5#%7'<) -H=HE> F.$ #$%570% %.'< 0./0
0.$ *./36$% '& <.'7$ ($/0 0'56.3$%% 54'3 *''+136 /0
0$(4$#/05#$% "$7'< 9:! ; /44$/# 0' #$W$*0 0.$ 0.$#(/7
*./36$% '& 0.$ 4$#1(8%1/7 *'33$*01K$ 01%%5$>
C#$$[136 /3, %5"%$\5$30 0./<136 <177 */5%$ %'($
($*./31*/7 /3, *.$(1*/7 *./36$% 13 0.$ (5%*7$ 01%%5$
,5$ 0' &'#(/01'3 '& 1*$ *#8%0/7% 13 130#/2 /3, $@0#/2
*$7757/# 7'*/01'3% B]$K13$) ?$77) U'K/00 R ;.#8%0/77)
-HH9T ^/+/0/) Y%.1,/) G'#10/ R _/6/0/) -HHME> F.1%
($*./31*/7 ,/(/6$ */3 */5%$ %'($ <$/+$3136 '& <.'7$
40
(a)
80 WBSF (N) 70 60 50 40 30 20 10 30 40 50 60 70 80 90 100 Temperature ( C ) % of 5iber diameter change (b)
100 80 60 40 20 0 0 20 40 60 80 100 Temperature ( C ) (b)
Figure 1-10. (a) Changes in shear force value during ramp heating and, (b) percentage change
in diameter, with a fast heating rate (>3 °C/min). Data is reconstructed from Table 2 (Li,
Zhou, & Xu, 2010).
41
place and was essentially complete in a little over 5 min.
When viewed under polarized light, muscle fibers appeared brilliantly birefringent. Upon heating, the birefringence gradually decreased (Fig. 4) for all muscle fiber
fragments observed. Some fragments became isotropic by
01
’
30
Fig. 1. Change
increas;lzg
I
40
Zrt
width
I
I
50
60
TEMPERATURE “C
trnd
Imyth
of
fiber
temperature.
.
WIDTH
0
LENGTH
70
fragments
80
with
behavior pattern, but in
behavior described by the
shown for the decrease o
shortening does not beg
50” or more, when bire
s 0
IO
Fig. 3. Change
elkt temperatures.
Figure 1-11. Percentage change of fiber diameter or length with slow heating rate ( < °1.3
C/min). Adapted from Hostetler & Landmann (1968).
42
20
in
[clkgth
olume
40 11975)
ess
H
wo
95
ess
at
alnd
eemail,
to
he
nt
deres
deal.
ifi40
50
60
70
ociEND POINT TEMPERATURE(“C)
udFig. Z-The
effecr of end point temperature
on shear values IA) and
hat
percentage of hydroxyproline
solubilized
IS).
ST
as
Figure 1-12. The effect of non-isothermal heating on shear values (A) and percentage of
he
ed
soluble hydroxyproline with heating 0.3 °C/min. Adapted from Penfield (1973).
erAs end point increased. solubilization
Solubilization
of hydroxyproline
ars
of hydroxyproline increased (P < 0.01) as
s a in heated ST cores
ed
Solubilization of hydroxyproline val- shown in Figure 2. An increased solubiliues are given in Table 1. Heating rate had zation of collagen in ST with increasing
de- an influence on solubilization in cores. At internal temperature was reported by
m- the slower rate without respect to end Paul et al. (1973).
ans point, 5.92 * 0.96% of the hydroxyproin- line was solublized. This value was greater Relationship between hydroxyproline
°C (P < 0.01) than the 4.67 + 0.70% solu- solubilization and shear values
blized with the fast rate of heating.
There was a significant relationship be-
analysis
IC yield.
43
of variance
of shear values,
solubiliration
F Values
of hydroxyproline,
proteolytic
enzyme
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Chapter 2. Beef Steak Tenderization by Long Time, Low Temperature Heating
Introduction
Tenderness is a primary determinant of beef consumer acceptance (Boleman et al., 1997).
Except when the meat has been cold-shortened or improperly aged, a tough beef steak is
likely to have a relatively high collagen content and/or a high percentage of cross-linked
collagen in its connective tissues (Lepetit, 2007).
Typically tougher meat cuts are somewhat tenderized to improve palatability by either blade
tenderization (Maddock, 2008; Pietrasik & Shand, 2011) or the use of exogenous proteolytic
enzymes (‘meat tenderizers’) (Etherington & Bardsley, 1995; Sullivan & Calkins, 2010).
Neither approach is capable of producing steaks from tougher beef cuts that compare in
palatability to naturally tender steaks however (Maddock, 2008). Enzyme tenderization is
particularly prone to producing an objectionably mushy texture in steaks arising from
difficulty in evenly distributing the enzymes within muscle tissue (Carvajal-Rondanelli,
2003) and/or residual activity following cooking (Ashie, Sorensen, & Nielsen, 2002).
Beilken, Bouton, & Harris (1986) showed that tenderness of a typically tough beef muscle,
semimembranosus, increases during holding at 55 °C or above, which they attributed to
weakening of the connective tissues (Beilken, Bouton, & Harris, 1986). Christensen,
Purslow, & Larsen (2000) showed that while isolated perimysium from semitendinosus (ST)
muscle increased in fracture stress after one-hour isothermal treatment at 40 or 50 °C, a
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decrease in fracture stress occurred upon incubation at higher temperatures. Snowden &
Weidemann (1978) found that, upon sufficient heating, a relatively large fraction of crosslinked collagen, while remaining insoluble, nonetheless becomes readily susceptible to
digestion by pronase. Powell, Hunt, & Dikeman (2000) found that this pronase susceptibility
correlated with increasing tenderness of ST when heated from 50 to 70 °C.
Moisture losses from beef are initiated at relatively low temperatures, caused by shrinkage of
collagen and the sarcomere in combination with lowered water holding capacity of denatured
myofibrils (Hostetler & Landmann, 1968; Laakkonen, Wellington, & Sherbon, 1970a). Both
heating time and temperature affect water loss from meat. For example, Bouton & Harris
(1986) also noted that cook loss after holding at 60 °C for 24 hours was almost double that at
55 °C for the same holding time.
The objectives of the present study were to first pinpoint the minimum temperature at which
tough beef steaks can be tenderized during isothermal heating, and then to explore the
mechanism whereby this tenderization proceeds, and how water loss is affected as well. The
desired heating process would be sufficient to insure safe processing yet gentle enough to
minimize color change (relatively long time, low temperature LTLT). Thus the research
could lead to development of a method for tenderizing beef steaks that does not rely on blade
tenderization or the use of exogenous proteolytic enzymes (‘meat tenderizer’ enzymes) and
therefore allow for finish cooking of steaks to any desired degree of doneness by grilling in
restaurants.
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Material and Methods
Materials
USDA choice grade beef psoas major (PM) and semitendinosus (ST) muscles, aged by
chilling 14 - 21 days post-mortem, were obtained from a local supplier. The pH ranged 5.4 –
5.6 in the muscles selected. PM was used as a ‘naturally tender’ control for comparison while
ST was chosen for tenderization experiments because it is a representative ‘tough’ beef cut
and because it is largely free of heavy bands of fat or connective tissue and therefore more
homogeneous in composition, thus reducing variability in texture testing (Huffman, Miller,
Hoover, Wu, Brittin, & Ramsey, 1996; Shackelford, Morgan, Cross, & Savell, 1991; Sullivan
& Calkins, 2011). Bromelain (Enzeco ® bromelain 240) was supplied by Enzyme
Development Corp. (New York, NY) and stored at 4 °C until used. Sodium tripolyphosphate
(STPP, Brifisol ® STPNEW) was obtained from B.K. Giulini Corp. (Simi Valley, CA) and
non-iodized salt (NaCl) was purchased from the supermarket.
Methods
Experiment 1. Initiation Temperature for Tenderization
This experiment explored the changes in meat texture during LTLT cooking as an effect of
heating temperature (isothermal) at one or two long heating times. Both PM and ST muscles
were trimmed of visible surface fat and cut to steaks of approx. 1.25 cm thickness. ST steaks
for this experiment were obtained from a single intact muscle. Prior to finish grilling, ST
steaks (3 per treatment) were initially (experiment 1a) isothermally heated at 50, 53, 56, or 59
54
°C (+/- 0.5 °C) for 24 or 48 hr (times are determined subsequent to the internal temperature
attaining within 1.5 °C of the water bath temperature, denoted as “time zero”). Additionally 3
PM and 3 ST steaks remained refrigerated prior to grilling to serve as ‘tender’ and ‘tough’
controls. For LTLT heating, steaks were weighed and vacuum packaged in Cryovac bags
(product 97390 type B 620) using a JVR Busch RB 0016 DIZ0 vacuum sealer (Maulburg
Germany) prior to heating in a water bath. To monitor meat temperature, a thermocouple (Ktype, Omega Engineering, Stamford CN) was inserted into the center of one additional steak,
which was placed in an open bag, weighted with a bottom clip to hold it immersed in the
water bath while the bag top was kept above the water bath, to effectively evacuate the
package. All steaks were completely submerged in water baths, which were isothermally
controlled at each of the predetermined temperatures. Steaks were placed within a divided
rack to ensure bags did not touch during cooking. Following the LTLT treatments the steaks
were immediately chilled in ice water and held overnight before grilling. All steaks were
grilled on a unit with two hot plates (top and bottom, Breville) preheated to 177 °C to an
endpoint internal temperature of 65 °C, as measured within one steak (the grill held 4 steaks
per grilling pass). A temperature of 65 °C was chosen as the end point of grilling to represent
a ‘medium’ done steak since, following removal from the grill at this point, the internal
temperature actually reached 70 – 71 °C before the center of the steak cooled (American
Meat Science Association, 1995).
Experiment 1a was regarded as only preliminary to experiment 1b and thus was not
replicated. Experiment 1b was carried out identically to experiment 1a except a more narrow
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range of water bath temperatures was used (50, 51.5 and 53 °C) (+/- 0.5 °C) and heating time
was 24 hr only. A randomized block design was used wherein 3 separate ST muscles were
used, each being portioned into 18 steaks. The steaks within each of the three muscles were
randomly assigned to one of the three temperature treatments.
All steaks in experiment 1 (a and b) were subsequently evaluated for cook loss and slice
shear force (texture) as explained later.
The hypothesis for this experiment is that meat tenderizes at the temperature range 50 – 59
°C.
Experiment 2. Effect of salt/phosphate solution enhancement on cook loss during LTLT
heating
A single intact ST muscle was cut into two portions after trimming as described previously.
One portion (approx. 3/5 of the muscle) was injected with 16% (w/w, above the initial
weight) of a 6% NaCl+ 2% STPP (sodium tripolyphosphate) solution so as to achieve a
target meat concentration of 0.83 % NaCl and 0.28 % STPP. The remaining portion was not
injected (control). Both injected and non-injected portions were tumbled (packaged
separately in sealed bags but tumbled separately) for 30 min at 3 °C in a Polymaid tumbler
(Key Laboratories, Inc., Largo, Fl) of 20 cm dia., 20 cm depth at 24 rpm. Following brief (23 hr) exposure to -20 °C to firm the portions, the injected portion was then cut into 9 steaks
of 1.25 cm thickness. These were packaged as in experiment 1 and randomly (3 per
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temperature) subjected to 0 (as previously defined), 24 and 48 hr heating at 53 °C (+/- 0.5
°C), then immediately cooled in an ice water bath. The non-injected portion was sliced into
only 6 steaks of the same thickness and cooked identically. Cook loss was measured as
described later. This experiment was not replicated as it was only done in confirmation of
accepted industry practice for improvement of cooked yield.
Our hypothesis is that salt phosphate can be used to increase cook yield during treatment.
Experiment 3. Comparison of tenderization by LTLT heating vs. by injection of
bromelain
This experiment was conducted similarly to that of experiment 1 except that all ST steaks
were tenderized by LTLT treatment only at 56 °C for 24 hr Also all muscles used in this
experiment were enhanced by injection (prior to heat treatment) with a 15% salt/phosphate
solution as in experiment 2 so as to minimize cook loss during the LTLT treatment.
Additionally, some steaks were derived from muscle portions to which bromelain, a widely
used meat tenderizing enzyme, had been added to the salt/phosphate solution prior to
injection and tumbling. In order to better understand the mechanism of tenderization
occurring, steaks in this experiment also received more extensive evaluation subsequent to
LTLT treatment and grilling, including not only cook loss and SSF (texture) measurements,
but also analysis for evidence of myosin heavy chain proteolysis and changes to the collagen
component of the meat which may have occurred during the treatments. Informal sensory
evaluations of the grilled steaks from this experiment were also conducted.
57
Thus three main treatments, prior to grilling of all steaks, were compared: (a) LTLT
treatment at 56 °C for 24 hr, (b) bromelain injection to a final concentration 14.5 ppm, and
(c) an untreated control. For each replicate of the experiment (3 total replications) one whole
ST muscle was used. The muscle was cut into approximately three equal portions, which
pertained to each of the three treatments mentioned above. After injection (treatments a and b
only), all portions (a, b and c) were separately tumbled (packaged within separate bags) for
30 minutes at 3 °C as in experiment 2. Following brief (2-3 hr) exposure to -20 °C to firm the
portions, five steaks were then cut from each of the three portions for subsequent evaluations,
3 of these being grilled and evaluated for cook loss and SSF while the remaining 2 were
vacuum packaged and finish cooked in a 100 °C water bath to the same internal temperature
as by grilling (65 °C) so as to avoid losses of liquid that would interfere with the chemical
analyses conducted on these (heating time for both grilling and water bath cooking was
essentially identical).
The hypothesis for this study is that at >50 C something initiates as the activation energy is
met, most likely some particular type of unfolding specific to crosslinked collagen, which
can be measured as ELF and which dramatically softens tough connective tissue.
Sample preparation for chemical analysis.
Following finish cooking to 65 °C in boiling water to simulate grilling, sample pouches were
immediately plunged in ice water for 30 min. Pouches were then stored in at -20 °C for 5
days prior to freeze drying (Model 75035, Labconco Corp., Kansas City, MO) to a moisture
58
content to below 7%. Freeze dried samples were powdered with a food processor (MiniPro,
Black & Decker) for 5 min followed by a mortar and pestle. Moisture content of wet samples
prior to drying and of freeze dried samples were measured according to AOAC (1995).
pH determination of raw meat prior to treatments
A digital pH meter (Fisher model 420 digital with Accunet probe) was used to measure pH of
raw meat at room temperature. Only muscles in the pH range 5.4 to 5.6 are used. 10 mg of
meat samples are chopped coarsely mixed with 90 mg of deionized water until homogenized.
The homogenized solution was measured for pH.
Cook loss determination
Cook loss from the untreated, raw (‘green’ weight) was determined as the difference in
weight before and after treatment for grilled samples. Following grilling each sample was
patted dry with a paper towel and weighed.
cook loss %= weight after treatment-­‐weight before injection
x 100%
weight before injection
Measurements were made for at least 3 steaks per treatment.
Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) measurement
The protein concentration of each treatment for SDS-PAGE was determined by measuring
the difference in absorbance 280 nm and 260 nm, diluting the soluble sample with DI water
in a 1:20 ratio. Details of the sample preparation are described in Ruilova-Duval (2009) and
59
Segel (1976). SDS-PAGE was used to assess myofibrillar proteolysis by measuring
disappearance of myosin heavy chain (MHC). The procedure for SDS-PAGE was conducted
according to manufacture’s instruction with Bis-Tris Mini Gel (Invitrogen Inc.). Gels were
scanned using densitometry (Alpha Innotech Corp., San Leandro, CA) to determine protein
band degradation. The density of the MHC band was expressed as a percentage of the density
of all bands in each lane.
TCA precipitation absorbance value
The TCA precipitation and salt soluble extraction procedure is determined according to Joo,
Kauffman, Kim and Park (1999) and Zipprerer’s (1972) procedure. Briefly, 20% w/v of FD
sample is extracted with 0.3 M potassium chloride phosphate buffer (pH 7.0). Optical density
readings were recorded at 280 nm using spectrophotometer (Spectronic 1001 Bausch &
Lomb Rochester, NY).
Modified slice shear force (SSF) measurement of meat tenderness
A slight modification of the SSF procedure of Shackelford, Wheeler, & Koohmaraie (1999)
was used to measure the relative tenderness of samples. Figure 2-1 illustrates the
methodology used for SSF. The only modification made was the use of samples less than one
inch in thickness at the time of grilling. This modification was made in order to minimize the
come up time to reach the designated isothermal heating temperature (time to reach time zero
of heating) and also to maximize the number of steaks that could be obtained from any one
intact muscle.
60
Separation of collagen fractions based on solubility and pronase susceptibility
Total collagen was measured by a modification of the method of Hill (1966) by first
dissolving 0.125 g of freeze-dried sample in 10 ml 6N HCl. This was then digested with 6 N
HCl for 14 hours at 130 °C. Two ml of this was then neutralized with KOH solution and the
volume adjusted to 50 ml volume with addition of water. The procedure of Blumenkrantz &
Asboe-Hansen (1975) was then used to assay hydroxyproline using a single toluene
extraction. A conversion factor of 7.25 was used to convert readings to collagen content.
The acid hydrolysis was conducted in duplicate and the hydroxyproline assay was read in
triplicate.
The soluble collagen or Ringer soluble fraction (RSF) was determined using a modification
of published methods (Hill, 1966; Powell, Hunt, & Dikeman, 2000). Briefly, 0.125 g of
freeze dried sample was mixed with 2.5 ml of quarter-strength Ringer’s solution which had
been warmed to 50 °C. The solution was held for 2 hr at room temperature to allow the
freeze-dried sample to fully rehydrate. The sample was then vortexed and centrifuged at
6,000 x g for 10 min. The supernatant was collected and the residue was re-extracted as
before. The supernatants were then combined and digested with 10 ml of 6 N HCl for 14
hours at 130 °C for hydroxyproline assay as previously described.
Powell, Hunt, & Dikeman’s (2000) procedure was used to quantitate the relative
percentaages of total unalterable fraction (TUF) and enzyme labile fraction (ELF) except that
pronase digestion was carried out by shaking for 16 hours with shaking, stopped by
61
centrifugation, then centrifuged for 2 hr at 20,000 x g. The supernatant was then decanted
and the residue transferred carefully to a glass test tube with the aid of added distilled water
for acid digestion and hydroxyproline determination. This determined the TUF; ELF was
calculated as total collagen minus TUF+RSF.
Figure 2-2 is a schematic of the procedure used to assess the relative percentages of TUF,
ELF and RSF fractions.
Statistical analysis
Experiments 1b and 3 were conducted using a randomized complete block design with three
replicates. The least square means procedure of the Proc general linear models method
(SAS, Cary NC, version 9.2) was used to separate the means for each treatment.
Results and Discussion
Experiment 1. Initiation temperature for tenderization.
SSF results (Figure 2-3) showed that ST muscle held isothermally at 50 °C for up to 48 hr
toughened with no subsequent tenderization. At 53 °C some tenderization was noted, and
when the heating temperature was increased to 56 °C a greater tenderization was noted.
Increasing the temperature to 59 °C did not seem to induce additional tenderization however.
Except for 53 °C, much greater change was noted in SSF during the first 24 hr of heating
than in the second 24 hr.
62
It is clear that LTLT heating at 53 °C or above could produce tenderization of ST comparable
that of a naturally tender muscle (PM) without a tenderization treatment. 48 hr heating at 53
°C produced a SSF almost equal to the PM control. A similar low SSF was achieved after
only 24 hr isothermal heating at 56 or 59 °C.
Cook loss in these same samples appeared to increase with increasing temperature (Figure
2-4) over the initial 24 hr of isothermal heating, with little further increase in the subsequent
24 hr of heating. Cook losses were quite large, ranging from about 25 to 36% after 24 hr of
isothermal heating.
To more precisely determine the temperature of onset of tenderization (isothermal heating,
24 hr minimum), the experiment was repeated using heating temperatures over a more
narrow range (Figure 2-5). The SSF data clearly revealed that tenderization initiated within
the narrow range of 50 - 51.5 °C.
Only small changes in SSF were measured at the zero time of heating (point at which the
core of steaks was within 1.5 °C of the bath temperature) as compared to raw steaks (control)
(Figure 2-3 and Figure 2-5). The heating time required to reach this ‘zero time’ temperature
was about 15 minutes. At this point presumably myofibrillar proteins would be highly
denatured since Penny (1967) saw denaturation of myosin within 10 min at temperatures as
low as 35 °C. Several workers have attributed toughening of meat during heating at
temperatures below 60 °C primarily to myofibrillar denaturation (Davey & Niederer, 1977;
63
Martens, Stabursvik, & Martens, 1982), so it seems odd that little or no toughening was
measurable at time zero in these steaks.
In the first 2 hr or so of heating over the entire range 50 - 59 °C meat is known to toughen in
texture (Machlik & Draudt, 1963). This has been attributed to initial shrinkage of collagen
induced by the heating treatment (Finch & Ledward, 1972). The degree of shrinkage and
toughening during this initial phase of isothermal heating was found to depend on the
concentration of mature cross-links in the muscle (Lepetit, 2007). According to Lewis &
Purslow (1989) de-crimping of collagen (presumably due to its denaturation) most likely
occurs well below 50 °C. Collagen is arranged in a ply formation and the de-crimping
increases the collagen density, which in turn increases the collagen strength (Lewis &
Purslow, 1989; McCormick, 1999). Similarly, Field, Pearson, & Schweigert (1970)
demonstrated that the epimysium contracts during hardening and muscle with a more
contracted epimysium exhibits a higher shear force value.
Christensen, Purslow & Larsen (2000) later confirmed that perimysium toughens markedly
when heated isothermally for one hour at 40 °C, becoming even stronger when incubated at
50 °C. Since Li and Zhou (2010) measured denaturation onset temperatures of endomysium
and perimysium to be 50 and 62 °C, respectively, using DSC which employs a fast heating
rate (10 °C/min), it would be expected that under isothermal conditions endomysium would
denature at an even lower temperature than does perimysium, but certainly by the point at
64
which perimysium apparently denatures and hardens at 40 °C (Christensen, Purslow, &
Larsen, 2000).
Thus it seems likely that the toughening of ST meat that occurred during extended heating at
50 °C (Figure 2-3 and Figure 2-5) is primarily attributable to collagen shrinkage. This
shrinkage also likely gives rise to the expelling of water from muscle during LTLT heating,
measurable as cook loss (Figure 2-4 and Figure 2-6). Water is held within muscle primarily
by capillarity, within the myofibrils (Offer & Knight, 1988). The myofibrils are attached to
the basal lamina, which contains type IV collagen (Bailey, 1989). When mysosin denatures it
aggregates, freeing water to migrate from the myofibril (Offer & Knight, 1988). As explained
previously, the perimysium and endomysium apparently begin to denature at temperatures as
low as 40 °C and this eventually leads to their contraction and the expelling of water, now
freed from the capillarity of an intact myofibrillar structure, from the meat.
The initiation of tenderization upon holding at temperatures above 50 °C (Figure 2-3 and
Figure 2-5) likely indicates some dramatic change over extended heating time in the collagen
of a tougher meat cut such as ST. Many workers have considered this to be a type of
‘accelerated aging’ (Dutson, 1983); that is, a result of proteolytic attack (in this case
primarily from endogenous cathepsins, since calpains are largely inactivated at temperatures
above 37 °C (Goll, Thompson, Li, Wei, & Cong 2003). However the dramatic onset of
tenderization within such a narrow temperature range (50 – 51.5 °C), and above 50 °C, does
not seem to correlate with known temperature activity ranges of cathepsins present in beef
65
muscle. For example, beef cathepsins are reported to have an activity range extending from
35 °C up to only 55 – 60 °C (Draper & Zeece, 1989; Okitani, Matsukura, Kato, & Fujimaki,
1980). Therefore, a tenderization reaction which only initiates at 50 °C would not seem to
implicate cathepsins. Wang & Xiong (1999) presented evidence that argued against a role of
cathepsin activity in tenderization over this temperature range. The authors concluded that
while the literature suggests cathepsin activity does exist in beef which could be active over
this temperature range, natural inhibitors also exist which negate its effects.
Snowden & Weidemann (1978) showed that upon sufficient heating a relatively large
fraction of cross-linked collagen, while remaining insoluble, nonetheless becomes readily
susceptible to digestion by pronase. Powell, Hunt, & Dikeman (2000) found that this
pronase susceptibility correlated with increasing tenderness of ST when heated from 50 to 70
°C. This would indicate that there might be partial denaturation of cross-linked collagen that
leads to a molten globule structure, which is less tough than the native cross-linked state, but
nonetheless still insoluble.
Experiment 2: Effect of salt/phosphate solution enhancement on cook loss during LTLT
heating.
The large cook losses observed in ST meat after LTLT heating at 50 - 59 °C (Figure 2-4)
would likely be unacceptable when using the LTLT for meat tenderization in commercial
practice. Enhancement of meat by injection/tumbling with solution of NaCl and phosphates
is a well accepted practice for reducing cook losses in meat (Smith & Young, 2007) and
66
derives from the ability of added salt and phosphate to increase the charge on neighboring
myofibrils, promoting their separation and thus allowing more room for water uptake (Offer
& Trinick, 1983; Offer & Knight, 1988). When ST muscle chunks were injected to 15%
above green weight and tumbled prior to LTLT heating at 53 °C isothermal for 24 hr, this
enhancement treatment resulted in reduction of cook losses in the steaks sliced from about
32% to only 6% (Figure 2-7). For subsequent work on this LTLT approach to tenderization
(experiment 3) all meat was injected with this same salt/phosphate solution prior to any
further tenderizing treatment.
Experiment 3. Comparison of tenderization by LTLT heating (56 °C only) vs. by
injection of bromelain.
LTLT treatment was conducted at 56 °C since, per the results of experiment 1, this effected
significant tenderization of meat and represents a safe temperature of treatment that could be
considered for commercial application. The U.S. Department of Agriculture, Food Safety
and Inspection Service (1999) requires beef to be cooked at minimum temperature of 54.4 °C
for at least 112 minutes to achieve a minimum lethality of 6.5 log reduction of Salmonella.
This temperature also insures virtual elimination of vegetative cells of C. perfringens (Juneja,
Marks, Huang, & Thippareddi, 2011; Willardsen, Busta, Allen, & Smith, 1978).
67
Changes in SSF
As expected from the results of Experiment 1, significant decreases (P < 0.05) were noted in
SSF of grilled steaks, which had received the LTLT treatment as compared to the control
(Figure 2-8). Likewise SSF of the bromelain-treated steaks decreased upon grilling, to a level
not statistically different from the LTLT treatment in SSF (P < 0.05). Informal tasting of
these steaks revealed a quite uneven and more mushy texture in steaks treated by bromelain
injection as compared to by LTLT (Figure 2-10), the latter having a more consistent steaklike texture.
This result was obtained despite that the bromelain treatment used (14.5 ppm) being that
suggested by Sullivan (personal communication, 2013) as the maximum which would effect
significant tenderization without any negative textural characteristics (typically, mushiness).
However, proper distribution of an injected plant-derived protease like bromelain within
muscle is quite difficult to achieve, even when the solution is injected via multiple locations
and tumbling is employed, as in the present experiment. This is because proteases are
proteins, which are too large in molecular weight to easily move through muscle membrane
(Carvajal-Rondanelli, 2003). The result then is a localized tenderizing effect at the points of
injection where the enzyme solution pools, resulting in a localized mushy texture.
68
Changes in Collagen
The soluble collagen fraction in steaks cooked to 65 °C internal (about 6-10% of total
collagen) did not differ significantly due to treatment (Figure 2-11, P < 0.05). The TUF
fraction decreased significantly (p < 0.005) accompanied by a significant increase in the
ELF fraction (P < 0.01) in LTLT steaks as compared to both the bromelain treatment and the
control (the latter two treatments did not differ significantly in ELF or TUF (P < 0.05)). This
suggests that cross-linked collagen only partially denatures during LTLT, to a form that
becomes pronase-susceptible, and that it is this conversion of collagen from the native to an
intermediate molten globule form (not to fully denatured gelatin), which may be largely
responsible for tenderization of ST meat (Powell, Hunt, & Dikeman 2000).
Evidence of proteolysis
No evidence of marked proteolytic degradation of myosin heavy chain (MHC) was noted for
any of the steak treatments by SDS-PAGE (Figure 2-12). Some decrease in the MHC band
could be noted for the LTLT and bromelain treatments but the change was small (lsd = 0.01)
as compared to the control. A slight increase of TCA absorbance (possible evidence of
peptide production by proteolysis) was observed during LTLT (Figure 2-13, lsd = 0.1291)
but not in the bromelain-treated steaks, as compared to the control.
Laakkonen, Wellington, & Sherbon (1970b) postulated that tenderization during LTLT
heating could be due to catheptic activity. M. Christensen, L. Christensen, Ertbjerg, &
Aaslyng, (2011) associated the decrease of meat toughness during LTLT with a measured
69
increase in residual cathepsin activity after heating; however, they did not directly measure
proteolysis of meat proteins in this case. Wang & Xiong (1999) however suggested that
cystatin, a cathepsin inhibitor inhibitor, binds to cathepsin L and B in beef muscle,
preventing catheptic activity in intact meat during heating.
We were particularly surprised that the bromelain treated steaks, which clearly decreased in
SSF during grilling and which exhibited a mushy texture, did not show evidence of MHC
breakdown by SDS-PAGE. However, Foegeding & Larick (1986) and Kang & Rice (1970)
both presented evidence that bromelain preferentially degrades collagen as opposed to
myofibrillar protein. Although collagen degradation is not easily measured by SDS-PAGE it
seems likely that tenderization of ST by bromelain in this experiment was largely the result
of collagen degradation by the enzyme since marked tenderization occurred and yet no MHC
degradation was noted.
Clearly a role of autolytic activity by cathepsins in the tenderization by LTLT heating cannot
be absolutely ruled out by the present evidence. However, cathepsins are known to be quite
active against myosin and other myofibrillar proteins (Draper & Zeece, 1989; Okitani,
Matsukura, Kato, & Fujimaki, 1980) such that, if this was the chief mechanism of this
tenderization by LTLT heating, more marked degradation of MHC would have been
expected in the LTLT treated steaks. Lack of evidence for MHC degradation in bromelain
70
treated steaks is more easily explained by the greater affinity of bromelain for collagen than
for myofibrillar proteins (Foegeding & Larick 1986; Kang & Rice 1970).
Cook Yields
The pH of ST meat after salt/phosphate injection was about 5.8 (data not shown); this
increase in meat pH is partially reflective of the increase in water holding capacity effected
by salt/phosphate injection. As expected, the cook yields for all treatments were reasonably
high and no significant differences were noted between treatments (Figure 2-9, p < 0.05).
Effects on Meat Color
The visually assessed color of LTLT steaks after grilling was quite pink, indicating that the
LTLT treatment, though rigorous enough to produce a pasteurized product with marked
increase in tenderness, was yet sufficiently mild to still allow grilling to a ‘medium’ degree
of doneness, which is desired by many restaurant consumers.
Conclusions
Tenderization of ST meat initiates at temperatures just above 50 °C for isothermal heating.
The present evidence would suggest that tenderization results primarily from the partial
unfolding of cross-linked collagen as revealed by a significant increase in ELF, while
71
conversion to gelatin (RSF) was not related to grilled steak tenderness. Proteolysis of MHC
in these samples was slight at most, and thus it would seem unlikely that catheptic-induced
autolysis could explain the marked increase in meat tenderness induced by LTLT at 56 °C/24
hr It remains unclear why this heat-induced tenderization reaction is so dramatically switched
on at just above 50 °C (isothermal) and further study is needed to better understand the
changes that occur in cross-linked collagen in the 50 – 59 °C range of isothermal (LTLT)
heating.
The practical implications of this work are that LTLT heating can possibly provide a good
way to more uniformly and consistently tenderize tough, lower valued cuts of meat to be
suitable for grilling than by blade tenderization or the application of plant-derived meat
tenderizing enzymes. Enhancement with salt and phosphate helps retain the yield and
succulence of meat during the LTLT treatment, which besides tenderizing the meat provides
a pasteurization treatment to the injected meat. Because the meat color appears not to be
strongly affected by LTLT at 56 °C for up to 24 hr, tenderization by this method assures the
safety of a pasteurized product regardless of the final degree of doneness to which the steak
is grilled.
Acknowledgement
The authors gratefully acknowledge the contribution of Enzyme Development Corporation
for providing Bromelain® 240 for this present work.
72
Figures
Figure 2-1. SSF apparatus
73
Figure 2-2. Schematic flow chart of pronase susceptibility assay (Powell, Hunt & Dikeman,
2000)
74
40!
50!
SSF, kgf!
53!
30!
56!
ST ctrl grilled!
59!
20!
PM ctrl grilled!
10!
0!
0!
24!
48!
Hold time, hour!
Figure 2-3. Effect of LTLT on SSF when semitendinosus muscle was held isothermally at 24
hr and 48 hr.
75
Cook Loss!
40%!
35%!
50 C!
30%!
53 C!
25%!
56 C!
20%!
59 C!
15%!
10%!
5%!
0%!
0!
24!
Hold Time, hr!
48!
Figure 2-4. Effect of LTLT on cook loss when semitendinosus muscle was held isothermally
at 24 hr and 48 hr.
76
50 C 50!
51.5 C 40!
SSF, kgf!
53 C 30!
Raw!
20!
10!
0!
0!
24!
Holding time, hours!
Figure 2-5. Effect of LTLT heating at 50 °C, 51.5 °C, and 53 °C on SSF of semitendinosus
muscle
77
Cook loss after 24 hrs (%)!
50°C
35!
51.5°C
30!
53°C
25!
20!
15!
10!
5!
0!
Cooking temperature (°C)!
Figure 2-6. Effect of LTLT heating at 50 °C, 51.5 °C, and 53 °C on cook loss on
semitendinosus muscle
78
Injected with S/P!
Cook loss after 24 hrs (%)!
Non injected!
40%!
20%!
0%!
Treatments!
Figure 2-7. Effect of 15% salt/phosphate (S/P) injection after LTLT heating at 53 °C / 24 hr
on semitendinosus muscle
79
Sousvide!
Bromelain!
Control!
Slice shear force, kgf!
30!
b 25!
20!
15!
a 10!
a 5!
0!
Treatment!
Figure 2-8. Effect of SSF of semitendinosus muscle on LTLT at 56 °C / 24 hr , bromelain
and control after grilling to 65 °C
80
Sousvide!
Bromelain!
Cook yield after 24 hr
treamten, %!
Control!
100!
80!
60!
40!
20!
0!
Treatment!
Figure 2-9. Effect of cook yield of semitendinosus muscle on LTLT at 56 °C/24 hr,
bromelain and control grilled to 65 °C internal temp.
81
Figure 2-10. Photograph of ST muscle sliced steaks obtained from LTLT at 56 °C and
bromelain treatment grilled to 65 °C internal temp.
82
Percentage of total collagen RSF 100%!
ELF 80%!
60%!
b b Bromelain!
Control!
TUF a 40%!
20%!
0%!
LTLT!
Figure 2-11. Relative content of three measured collagen fractions of LTLT 56 °C/24 hr,
bromelain, and control grilled to 65 °C internal temp.
83
MHC percentage of total
protein, %!
15
LTLT!
Bromelain!
Control!
10
b!
ab!
a!
5
0
Treatment!
Figure 2-12. Effect of LTLT at 56 °C/24 hr, bromelain and control on MHC proteolysis
measured by SDS PAGE
84
TCA absorbance at 280 nm!
LTLT!
1.5!
Bromelain!
b!
a!
Control!
a!
1!
0.5!
0!
Treatment!
Figure 2-13. Effect of LTLT at 56 °C/24 hr, bromelain, and control measured by TCA
absorbance at 280 nm
85
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