Contribution of postmortem muscle biochemistry to … 52...steaks, consumers chose guaranteed tender steaks (even at higher price). USDA-ARS U.S. Meat Animal Research Center - ICoMST

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


Presentation Outline

• Tenderness is the eating satisfaction determinant• How to meet consumer expectations?• Tenderness determinants• Where do we go from here?


Eating Quality Traits

• Tenderness• Juiciness• Flavor• Interaction of tenderness, juiciness and flavor• Tenderness is the most important trait


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).



• 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



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

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


Tenderness is Also the Most Variable

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

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

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


How to Solve Tenderness Inconsistency?

• Must understand the basis for tenderness


Tenderness Determinants

1. Connective Tissue (Collagen)2. Sarcomere Length3. Proteolysis

• The relative contribution of each of the above factors is muscle dependent

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)

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

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

5.75.9

Prot.

Collagen

Sarc.

Tend.

Relative merit

Flat Iron (IS)Ribeye (LD)

5.7

1.80 µm2.25 µm

5.9

Prot.

Collagen

Sarc.

Tend.

Relative merit


4.5 mg/g

5.7

1.80 µm

7.6 mg/g

2.25 µm

5.9

Prot.

Collagen

Sarc.

Tend.

Relative merit


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



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

Tough; WBS = 9.0 kgTender; WBS = 2.8 kg

Sources of Variation in Meat TendernessSources of Variation in Meat Tenderness

1. Exists at slaughter

2. Created during processing/harvest

3. Both

Biological Basis of Meat TendernessBiological Basis of Meat Tenderness(only Longissimus)(only Longissimus)

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

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

Rigor Mortis

2

3

4

5

6

7

8

9

0 24 72 336

Time Postmortem, h

Shea

r Fo

rce,

kg

ShorteningNo Shortening

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

33672482402

3

4

5

6

7

8

9

Time Postmortem, h

Shea

r Fo

rce,

kg

12

33672482402

3

4

5

6

7

8

9

Time Postmortem, h

Shea

r Fo

rce,

kg

12

Tough

Tender

Mechanism of Postmortem Meat Tenderization

Before agingBefore aging

After agingAfter aging

MFI

Day 1 = 43

Day 7 = 79

Day 21 =83

Robson et al., 1981Robson et al., 1981

ToughTough TenderTender

Shear force = 9.0 kg Shear force = 9.0 kg Shear force = 2.8 kg Shear force = 2.8 kg

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

Degradation of key muscle proteins is responsible for postmortem meat tenderization

The function of these proteins is to maintain structural integrity of myofibrils

Differences in the rate and extent of degradation of these proteins are responsible for differences in the rate and extent of postmortem meat tenderization.

Causative Proteolytic SystemCausative Proteolytic System

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.

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

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

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

Because MCP has no detectable effect on myofibrils (EM, SDS - PAGE), it is not involved in postmortem proteolysis and meat tenderization.

Koohmaraie, 1992

Titin

Myosin

a-Actinin

DesminTroponin-T

28-32 kDaTroponin-T

Effect of µ-calpain and MCP on myofibrillar protein degradation

Koohmaraie, 1992

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

Specific interactions between the myofibrillar proteins appear to protect them from ubiquitin-dependent degradation.

Solomon and Goldberg, 1996

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.

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

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

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

Tg and NTg mice

Transgenic

Non-transgenic

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

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

Postmortem Protein DegradationDesmin is used as an indicator of postmortemproteolysis.

Non-Transgenic TransgenicDays PM 0 1 3 7 0 1 3 7

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

Conclusion

• Calpastatin over-expression in mice clearly supported the previous correlative observations.

Postmortem Proteolysis in P94/Calpain 3 Knockout Mice

G. Geesink, R. Taylor, and M. Koohmaraie

USMARC and INRA

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).

P94/calpain 3Con KO Con KO Con KO

P94

m-Calpain

Control Knockout

D0 D1 D3 D0 D1 D3

P94

m-Calpain

Postmortem Changes in Other Muscle ProteinsControl Knockout

D0 D1 D3 D0 D1 D3

Nebulin

Dystrophin

Metavinculin + Vinculin

µ-Calpain

Desmin

Troponin-T

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 (%)

Conclusion

• Results obtained using p94 KO mice support the hypothesis that p94 does not play any role in postmortem proteolysis.

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

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.

Control µ-Calpain knockoutD0 D3 D7 D0 D3 D7

µ-Calpain

Zymography of µ- and m-Calpain

m-Calpain

Autolyzed m-Calpain

Postmortem Changes in Muscle ProteinsControl Knockout

D0 D1 D3 D0 D1 D3

Nebulin

Dystrophin

Metavinculin + Vinculin

Desmin

Troponin-T

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

Conclusion

• µ-Calpain is the major proteolytic enzyme that causes postmortem proteolysis of myofibrillar and associated proteins.


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.


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.


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


Contributors/Collaborators

Darrel GollJudith SchollmeyerSteven SeidemanThayne DutsonRobert MerkelHarry MersmannJohn CrouseMarta Fiorotto

Geert GeesinkTommy WheelerSteven ShackelfordRichard TaylorMelissa SpencerS. KuchayA.H. Chishti

Documents

Contribution of postmortem muscle biochemistry to … 52...steaks, consumers chose guaranteed tender steaks (even at higher price). USDA-ARS U.S. Meat Animal Research Center - ICoMST