contribution of postmortem muscle biochemistry to … 52...steaks, consumers chose guaranteed tender...
TRANSCRIPT
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USDA-ARS U.S. Meat Animal Research Center - ICoMST 2006 Ireland
Contribution of Postmortem Muscle Biochemistry to the Delivery of Consistent Meat Quality with
Particular Focus on the Calpain System
Mohammad Koohmaraie & Geert Geesink
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USDA-ARS U.S. Meat Animal Research Center - ICoMST 2006 Ireland
Presentation Outline
• Tenderness is the eating satisfaction determinant• How to meet consumer expectations?• Tenderness determinants• Where do we go from here?
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USDA-ARS U.S. Meat Animal Research Center - ICoMST 2006 Ireland
Eating Quality Traits
• Tenderness• Juiciness• Flavor• Interaction of tenderness, juiciness and flavor• Tenderness is the most important trait
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USDA-ARS U.S. Meat Animal Research Center - ICoMST 2006 Ireland
Evidence to Support Tenderness Claim
• Consumer surveys– Numerous studies have documented that when
asked, consumers indicate that tenderness is the most important trait affecting satisfaction.
• When provided with a choice between commodity steaks and guaranteed tender steaks, consumers chose guaranteed tender steaks (even at higher price).
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USDA-ARS U.S. Meat Animal Research Center - ICoMST 2006 Ireland
Evidence to Support Tenderness Claim
• The growth of branded beef products in the US (43 and growing – one beef processing company has 6 brands)
• Slice shear force screened steaks • Installation of tenderness prediction
instruments in the US beef processing plants
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USDA-ARS U.S. Meat Animal Research Center - ICoMST 2006 Ireland
Evidence to Support Tenderness Claim
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Tenderness vs PriceTenderness vs PriceTenderness vs Price
2345678
1 2 3 4 5 6 7 8Overall tenderness
Tenderloin steak
Top sirloin steak
Top loin steak
Rump roast
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Value of Tenderness:Cut versus Grade?Value of Tenderness:Cut versus Grade?
$0.00
$2.00
$4.00
$6.00
$8.00
$/lb
.
Prem Choice Choice Select
Tenderloin Strip Top Butt Bottom Round Clod
02/15/02
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USDA-ARS U.S. Meat Animal Research Center - ICoMST 2006 Ireland
Tenderness is Also the Most Variable
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Variation in Sensory Traits of Variation in Sensory Traits of Longissimus SteaksLongissimus Steaks
1 2 3 4 5 6 7 8Sensory panel score
TendernessJuicinessFlavor intensity
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Standard Deviations of Trained Sensory Standard Deviations of Trained Sensory Panel Ratings of Longissimus SteaksPanel Ratings of Longissimus Steaks
.0 .2 .4 .6 .8 1.0
292 Select from 2 plants
308 Select, Low Choice andTop Choice from 2 plants
120 Select and Top Choicefrom 1 plant
SD of sensory rating
FlavorJuicinessTenderness
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Effect of Muscle on Standard Deviations of Effect of Muscle on Standard Deviations of Trained Sensory Panel RatingsTrained Sensory Panel Ratings
.0 .1 .2 .3 .4 .5 .6 .7 .8 .9
Ribeye (LD)Top Round (SM)Top sirloin (GM)
Flat Iron (IS)Mock Tender (SS)Eye of Round (ST)
Top Round (AD)Bottom Round (BF)Shoulder Clod (TB)
Sirloin Tip (RF)Tenderloin (PM)
SD of sensory rating
FlavorJuicinessTenderness
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USDA-ARS U.S. Meat Animal Research Center - ICoMST 2006 Ireland
How to Solve Tenderness Inconsistency?
• Must understand the basis for tenderness
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USDA-ARS U.S. Meat Animal Research Center - ICoMST 2006 Ireland
Tenderness Determinants
1. Connective Tissue (Collagen)2. Sarcomere Length3. Proteolysis
• The relative contribution of each of the above factors is muscle dependent
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Effect of Muscle on Collagen ConcentrationEffect of Muscle on Collagen Concentration
0 1 2 3 4 5 6 7 8 9 10
Psoas major
Gluteus medius
Longissimus
Adductor
Rectus femoris
Triceps brachii
Infraspinatus
Semimembranosus
Biceps femoris
Semimembranosus
Supraspinatus
Collagen concentration of cooked muscle (mg/g)
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Effect of Muscle on Sarcomere LengthEffect of Muscle on Sarcomere Length
1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0
Gluteus medius
Longissimus
Semimembranosus
Biceps femoris
Adductor
Supraspinatus
Semitendinosus
Rectus femoris
Infraspinatus
Triceps brachii
Psoas major
Sarcomere length of cooked muscle, µm
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Effect of Muscle on Proteolysis (Aging)Effect of Muscle on Proteolysis (Aging)
0 10 20 30 40 50 60 70
Psoas major
Supraspinatus
Infraspinatus
Adductor
Rectus femoris
Triceps brachii
Semitendinosus
Gluteus medius
Semimembranosus
Longissimus
Biceps femoris
Percentage of desmin degraded during aging
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5.75.9
Prot.
Collagen
Sarc.
Tend.
Relative merit
Flat Iron (IS)Ribeye (LD)
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5.7
1.80 µm2.25 µm
5.9
Prot.
Collagen
Sarc.
Tend.
Relative merit
Flat Iron (IS)Ribeye (LD)
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4.5 mg/g
5.7
1.80 µm
7.6 mg/g
2.25 µm
5.9
Prot.
Collagen
Sarc.
Tend.
Relative merit
Flat Iron (IS)Ribeye (LD)
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4.5 mg/g
60% degraded
5.7
1.80 µm
7.6 mg/g
22% degraded
2.25 µm
5.9
Prot.
Collagen
Sarc.
Tend.
Relative merit
Flat Iron (IS)Ribeye (LD)
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USDA-ARS U.S. Meat Animal Research Center - ICoMST 2006 Ireland
What is the Source of Variation In These Tenderness Determinants?
• Connective tissue – locomotion/carcass location• Sarcomere length – Carcass hanging and rigor mortis
• Proteolysis - ?• Proteolysis – μ-calpain
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Tough; WBS = 9.0 kgTender; WBS = 2.8 kg
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Sources of Variation in Meat TendernessSources of Variation in Meat Tenderness
1. Exists at slaughter
2. Created during processing/harvest
3. Both
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Biological Basis of Meat TendernessBiological Basis of Meat Tenderness(only Longissimus)(only Longissimus)
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336722402
3
4
5
6
7
8
9
Time Postmortem, h
Shea
r Fo
rce,
kg
12
h kg SD h µm SD0 5.07 .78 0 2.24 .183 5.10 1.05 3 2.00 .236 6.53 1.51 6 1.80 .139 8.26 1.23 9 1.72 .05
12 8.24 1.53 12 1.75 .0624 8.66 2.01 24 1.69 .0972 4.36 .75 72 1.76 .07
336 3.10 .63 336 1.90 .13
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336722402
3
4
5
6
7
8
9
Time Postmortem, h
Shea
r Fo
rce,
kg
12
h kg SD h µm SD0 5.07 .78 0 2.24 .183 5.10 1.05 3 2.00 .236 6.53 1.51 6 1.80 .139 8.26 1.23 9 1.72 .05
12 8.24 1.53 12 1.75 .0624 8.66 2.01 24 1.69 .0972 4.36 .75 72 1.76 .07
336 3.10 .63 336 1.90 .13
Toughness phaseToughness phase
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Rigor Mortis
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2
3
4
5
6
7
8
9
0 24 72 336
Time Postmortem, h
Shea
r Fo
rce,
kg
ShorteningNo Shortening
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336722402
3
4
5
6
7
8
9
Time Postmortem, h
Shea
r Fo
rce,
kg
12
h kg SD h µm SD0 5.07 .78 0 2.24 .183 5.10 1.05 3 2.00 .236 6.53 1.51 6 1.80 .139 8.26 1.23 9 1.72 .05
12 8.24 1.53 12 1.75 .0624 8.66 2.01 24 1.69 .0972 4.36 .75 72 1.76 .07
336 3.10 .63 336 1.90 .13
Tenderization PhaseTenderization Phase
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33672482402
3
4
5
6
7
8
9
Time Postmortem, h
Shea
r Fo
rce,
kg
12
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33672482402
3
4
5
6
7
8
9
Time Postmortem, h
Shea
r Fo
rce,
kg
12
Tough
Tender
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Mechanism of Postmortem Meat Tenderization
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Before agingBefore aging
After agingAfter aging
MFI
Day 1 = 43
Day 7 = 79
Day 21 =83
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Robson et al., 1981Robson et al., 1981
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ToughTough TenderTender
Shear force = 9.0 kg Shear force = 9.0 kg Shear force = 2.8 kg Shear force = 2.8 kg
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NebulinNebulin
DesminDesmin
TroponinTroponin--tt
AlphaAlpha--actininactinin
TitinTitin
VinculinVinculin
Shear Force
Day 1 = 7.5
Day 7 = 4.7
Day 21 = 3.3
Shear Force
Day 1 = 10.9
Day 7 = 10.1
Day 21 = 8.2
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Degradation of key muscle proteins is responsible for postmortem meat tenderization
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The function of these proteins is to maintain structural integrity of myofibrils
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Differences in the rate and extent of degradation of these proteins are responsible for differences in the rate and extent of postmortem meat tenderization.
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Causative Proteolytic SystemCausative Proteolytic System
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Criteria for InvolvementCriteria for Involvement
Protease must:
1. Be endogenous to muscle cell.
2. Be able to reproduce postmortem changes in myofibrils.
3. Have access to myofibrils in situ.
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Potential CandidatesPotential Candidates**
1) The lysosomal proteolytic system
2) The multicatalytic proteolytic system
3) The ubiquitous calpain proteolytic system
*Skeletal muscle calpain or Calpain 3
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Potential CandidatesPotential Candidates**
1. The lysosomal proteolytic system
2. The multicatalytic proteolytic system
3. The ubiquitous calpain proteolytic system
*Skeletal muscle calpain or Calpain 3
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No Direct Role for MCP Because:No Direct Role for MCP Because:
1. It is endogenous
2. It can not degrade myofibrillar proteins
3. It has access to myofibrils in situ
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Because MCP has no detectable effect on myofibrils (EM, SDS - PAGE), it is not involved in postmortem proteolysis and meat tenderization.
Koohmaraie, 1992
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Titin
Myosin
a-Actinin
DesminTroponin-T
28-32 kDaTroponin-T
Effect of µ-calpain and MCP on myofibrillar protein degradation
Koohmaraie, 1992
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Myofibrillar proteins are degraded rapidly in an ATP -dependent manner when isolated, but not when associated
in myofibrils
Solomon and Goldberg, 1996
ProteolysisSubstrates + ATP / - ATP
- ATP + ATPpmol of tyrosine
Myosin 150 415 2.7Actin 110 630 5.7Tropomyosin 220 680 3.1Troponin 415 845 2.0Actomyosin 75 90 1.2Myofibrils 24 40 1.6
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Specific interactions between the myofibrillar proteins appear to protect them from ubiquitin-dependent degradation.
Solomon and Goldberg, 1996
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Current evidence suggests that activity of calpain
(µ-calpain) is the underlying mechanism of meat tenderization that occurs during storage of meat/carcasses at refrigerated temperatures.
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Criteria For InvolvementCriteria For Involvement
Protease must:
1. Be endogenous to muscle cell
2. Be able to reproduce postmortem changes in myofibrils
3. Have access to myofibrils in situ
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Evidence Supporting the Calpain RoleEvidence Supporting the Calpain Role1. Only calpain can exactly duplicate postmortem changes (structural,
protein degradation, etc.).
2. Incubation of muscle strips with or injection of whole carcasses with compounds (CaCl2 and ZnCl2) that activate or inhibit calpains, accelerates or inhibits postmortem proteolysis and meat tenderization.
3. Beta-agonist-induced muscle growth
4. Genetic mutations (callipyge)
5. Calpastatin over-expression
6. Calpain 3 KO
7. μ-calpain KO
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Postmortem Proteolysis is Reduced in Transgenic Mice
Over-expressing Calpastatin
M. P. Kent, M. J. Spencer*, and M. KoohmaraieUSMARC and *UCLA Duchenne Muscular Dystrophy
Research Center
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Tg and NTg mice
Transgenic
Non-transgenic
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Calpastatin Activity
Human calpastatin activity is quantified with 14C-casein as substrate
Day 0 Day 7
NTg (n=5) 0.74 0.51
Tg (n=8) 279 218
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Postmortem Protein DegradationWestern blots of Troponin-T and Vinculin
Troponin -T Vinculin
Non-Transgenic Transgenic
0 1 3 7 0 1 3 7Days PMNon-Transgenic Transgenic
0 1 3 7 0 1 3 7
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Postmortem Protein DegradationDesmin is used as an indicator of postmortemproteolysis.
Non-Transgenic TransgenicDays PM 0 1 3 7 0 1 3 7
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Postmortem Desmin Degradation
Immunodetectable desmin degradation
0
25
50
75
100
0 1 2 3 4 5 6 7
Days Postmortem
% R
emai
ning
ControlTransgenic
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Conclusion
• Calpastatin over-expression in mice clearly supported the previous correlative observations.
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Postmortem Proteolysis in P94/Calpain 3 Knockout Mice
G. Geesink, R. Taylor, and M. Koohmaraie
USMARC and INRA
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Observations that Preclude a Role for P94 in Postmortem Proteolysis
1. p94 is calcium-independent or requires extremely low concentrations of calcium (Sorimachi, et al, 1997).
2. p94 is not inhibited by calpastatin (Sorimachiet al., 1993).
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P94/calpain 3Con KO Con KO Con KO
P94
m-Calpain
Control Knockout
D0 D1 D3 D0 D1 D3
P94
m-Calpain
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Postmortem Changes in Other Muscle ProteinsControl Knockout
D0 D1 D3 D0 D1 D3
Nebulin
Dystrophin
Metavinculin + Vinculin
µ-Calpain
Desmin
Troponin-T
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Quantification of Desmin Degradation
TimePostmortem
Control Knockout
Day 168.9 73.5
Day 3 38.4 33.7
Amount of intact desmin relative to at-death levels (%)
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Conclusion
• Results obtained using p94 KO mice support the hypothesis that p94 does not play any role in postmortem proteolysis.
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µ-Calpain is Essential for Postmortem Proteolysis of
Muscle Proteins
G. Geesink, S. Kuchay, A.H. Chishti and M. KoohmaraieCCL Research, University of Illinois College of
Medicine and USMARC
Postmortem proteolysis of muscle proteins in µ-calpain knockout mice
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Protocol• Male and female control and KO mice were produced
by the U. of Illinois College of Medicine.• Experiment 1 (Western blots): 6 CON and 6 KO
mice. Hind limb muscles were sampled at 0, 1, and 3 days postmortem.
• Experiment 2 (Zymography): 4 CON and 4 KO mice. Hind limb muscles were sampled at 0, 1, and 3 days postmortem.
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Control µ-Calpain knockoutD0 D3 D7 D0 D3 D7
µ-Calpain
Zymography of µ- and m-Calpain
m-Calpain
Autolyzed m-Calpain
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Postmortem Changes in Muscle ProteinsControl Knockout
D0 D1 D3 D0 D1 D3
Nebulin
Dystrophin
Metavinculin + Vinculin
Desmin
Troponin-T
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Quantification of Postmortem Proteolysis
Amount of intact protein at day 3 relative to at-death levels (%)
Protein Control Knockout
Dystrophin 15.1 52.6
Metavinculin 0.8 109.2
Vinculin 64.4 79.2
Desmin 40.1 87.4
Troponin-T 83.8 99.9
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Conclusion
• µ-Calpain is the major proteolytic enzyme that causes postmortem proteolysis of myofibrillar and associated proteins.
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USDA-ARS U.S. Meat Animal Research Center - ICoMST 2006 Ireland
Where Do We Go From Here?
• I hope I have shown you enough evidence to convince you that indeed μ-calpain is responsible for postmortem proteolysis. These are just some examples from a long list of evidence . . . .
• In spite of this unequivocal evidence, we see continued presentation of new hypotheses without any supporting evidence.
• We (reviewers and journal editors) must require supporting evidence that meets the state of the art.
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USDA-ARS U.S. Meat Animal Research Center - ICoMST 2006 Ireland
Where Do We Go From Here?• As a community, for over 100 years we have known
that proteolysis is the cause of postmortem meat tenderization (Hoagland et al.).
• Using state-of-the-art methodology, we have shown that μ−calpain is the cause of postmortem proteolysis.
• We now need to learn the mechanism(s) of μ−calpain regulation in postmortem muscle to enable the industry that we serve to produce a product that is consistently tender.
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USDA-ARS U.S. Meat Animal Research Center - ICoMST 2006 Ireland
ContributorsPast and present members of the laboratory:
Abdallah BabikerAubrey SchroederGeorgianna WhippleDavid KretchmarSteven Shackelford Teresa KendallBrad MorganDean PringleJohn KilleferJuan ArbonaHenriette Van den Hemel-Grooten
Matthew DoumitSteven LonerganCathy ErnstCarol LorenzenEduardo DelgadoMargrethe TherkildsenEva VeisethDan NonnemanGeert GeesinkMatthew KentTommy Wheeler
Sue Hauver – Technician of 16 years
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USDA-ARS U.S. Meat Animal Research Center - ICoMST 2006 Ireland
Contributors/Collaborators
Darrel GollJudith SchollmeyerSteven SeidemanThayne DutsonRobert MerkelHarry MersmannJohn CrouseMarta Fiorotto
Geert GeesinkTommy WheelerSteven ShackelfordRichard TaylorMelissa SpencerS. KuchayA.H. Chishti