changes and possible relationship to postnatal growth

(1)

The role of postmortem biochemical and structural The role of postmortem biochemical and structural changes and possible relationship to postnatal

changes and possible relationship to postnatal growth

growth

Giuseppe Bee Giuseppe Bee

Agroscope

Agroscope Liebefeld Liebefeld - - Posieux Posieux , Research Station ALP , Research Station ALP

Water

Water - - holding holding capacity capacity of of meat meat : :

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Why is minimizing drip loss important Why is minimizing drip loss important

- - costumer expects good pork quality costumer expects good pork quality

- small variability in quality

- pork should be consistently tender and juicy

- - what do we observe? what do we observe?

- results from ALP studies

muscles ^N drip loss

(after 24 h) minimum maximum standard deviation

longissimus muscle ⁶⁰⁴ 4.82% 4.82% 1.10% 13.91% 3.08%

semitendinosus muscle 313 2.91% 2.91% 0.92% 11.18% 1.81%

biceps femoris 33 4.32% 4.32% 1.38% 9.61% 1.93%

rectus femoris 223 1.85% 1.85% 0.88% 5.90% 0.80%

semimembranosus muscle 33 5.22% 5.22% 2.22% 13.34% 2.51%

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Location of water in the muscle Location of water in the muscle

- - Lean muscle contains 75% of water Lean muscle contains 75% of water

- - Majority of the water within the structure of the Majority of the water within the structure of the muscle cells

muscle cells

- Within the myofibrils (~ 85% of the water) - Between the myofibrils

- Between the myofibrils and the sarcolemma - Between myofibers

- Between muscle bundles

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- - Bound Bound water water

- 0.5 g water/g protein Æ ¹ / ₈ of total water

- - Entrapped Entrapped (immobilized) (immobilized) water water

- held by steric effects and/or by attraction to bound water

- - Free Free water water

- water whose flow from the tissue is unimpeded

3 possible forms of water in the muscle 3 possible forms of water in the muscle

Majority of this water affects Majority of this water affects

DRIP LOSS DRIP LOSS

http://upload.wikimedia.org/wikipedia/commons/6/69/Watermolecule.png

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Drip loss

Drip loss - - cascade of events (part 1) cascade of events (part 1)

glycogen

lactic acid

meat pH pH drops

conversion of conversion of muscle to meat muscle to meat

approaching approaching protein

protein pI p I

(p ( pI I of myosin = 5.4) of myosin = 5.4)

net charge of net charge of proteins = 0

proteins = 0

⁺ ⁺

+ + - -

- -

+ +

+ + - -

- -

+ +

+ + - -

- -

+ +

+ + - -

- -

+ +

+ + - -

- -

O HH

Reduced

Reduced space space within within the the myofibril myofibril Shrinkage

Shrinkage of of myofibrillar lattice myofibrillar lattice

O HH

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Drip loss

Drip loss - - cascade of events (part 2) cascade of events (part 2)

Shortening of

Shortening of sarcomere sarcomere

Clark et al., 2002

Annu. Rev. Cell Dev. Biol. 18:637-706

higher proportion of water higher proportion of water

in the

in the I I- -band band

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Postmortem proteolysis Postmortem proteolysis

relationship to drip loss

3 h pm 3 h pm

6 h pm 6 h pm

9 h pm 9 h pm

24 h pm 24 h pm 3 h pm

3 h pm

6 h pm 6 h pm

9 h pm 9 h pm

24 h pm 24 h pm

Lawson, 2004 Meat Sci. 68:559-566

no no

proteolysis proteolysis proteolysis

proteolysis

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Cell shrinkage and drip loss Cell shrinkage and drip loss

Schäfer et al., 2002 Meat Sci. 61:355-366

extrafascicular extrafascicular area area extrafibre

extrafibre area area

variation in

variation in extrafascicular extrafascicular area area accounted for 39% accounted for 39% of of variation in drip loss

variation in drip loss variation in

variation in extrafiber extrafiber area area accounted for

accounted for 46% 46% of variation of variation in drip loss

in drip loss

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Calpain system Calpain system

major role in regulating proteolysis of muscle major role in regulating proteolysis of muscle proteins under postmortem conditions

proteins under postmortem conditions

2 major isoforms

(best characterized)

μ- μ -calpain calpain

m m -calpain - calpain

degrade the same set of proteins

calpastatin calpastatin

Goll et al., 2003 Physiol. Rev. 83:731-801

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μ μ - - calpain and postmortem proteolysis calpain and postmortem proteolysis

μ- μ -calpain calpain

m- m -calpain calpain

desmin desmin

Geesink et al., 2006 J. Anim. Sci. 84:2834-2840

μ- μ -calpain calpain

80 kDa

78 kDa

76 kDa

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h postmortem

1 6 12 24

pH

5.4 5.6 5.8 6.0 6.2 6.4 6.6

LM PM SM

h postmortem

1 6 12 24

°C

0 5 10 15 20 25 30 35 40

LM PM

Differences among muscles

SM

Differences among muscles

h postmortem

0.5 0.8 1.0

pH

5.4 5.6 5.8 6.0 6.2 6.4 6.6

Melody et al., 2004 J. Anim. Sci. 82:1195-1205

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LM LM SM SM PM PM

80 kDa

μ μ - - calpain autolysis calpain autolysis

45 min 45 min

80 kDa 78 kDa

80 kDa 78 kDa 76 kDa

LM LM SM SM PM PM Western-blot Casein zymography

24 h

6 h 6 h

(13)

Titin and nebulin Titin and nebulin

Clark et al., 2002

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Localization of

Localization of μ μ - - calpain calpain

LM LM SM SM PM PM

80 kDa

80 kDa 78 kDa

80 kDa 78 kDa 76 kDa

Bright bands in the immunofluorescent images indicate where μ-calpain

monoclonal antibodies were located.

Fluorescent image

Phase-contrast image

24 h 24 h

6 h 6 h

45 min

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Drip loss and desmin degradation Drip loss and desmin degradation

Drip loss

24 h 96 h

0.5%

1.0%

1.5%

2.0%

2.5%

3.0%

3.5% LM

PM SM

relative abundance of the

desmin degradation product

postmortem

0.75 h 6 h 24 h

relative abundance

0.05 0.15 0.25 0.35 0.45 0.55 0.65

LM PM SM

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Intermediate filament protein

Intermediate filament protein - - desmin desmin

Clark et al., 2002

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pH decline and drip loss pH decline and drip loss

pH decline

minutes postmortem

45 180 360 1440

pH

5.4 5.6 5.8 6.0 6.2

6.4 H-pH

L-pH

Drip loss

day after storage

0 1 2 5

drip loss

1%

2%

3%

4%

5%

H-pH L-pH

Bee et al., 2007 Meat Sci. 76:359-365

Loin samples selected at this time point

drip loss (d of storage)

-0.49

-0.49 -0.51 -0.51

pH 2 5

pearson

pearson correlation coefficients correlation coefficients

45 min 45 min

-0.56

-0.56 -0.60 -0.60 6 h 6 h

-0.62

-0.62 -0.75 -0.75

24 h 24 h

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Proteolysis and drip loss Proteolysis and drip loss

day of storage

H-pH

L-pH

desmin

desmin immunoblots immunoblots

0 1 2 5

Intact desmin degradation

day after storage

0 1 2 5

Relative abundance of intact desmin (%)

40 50 60 70 80 90 100 110 120

H-pH L-pH

degradation of desmin degradation of desmin

Bee et al., 2007 Meat Sci. 76:359-365

drip loss (d of storage)

0.41 0.41 0.45 0.45 0.42 0.42

1 2 5

pearson

pearson correlation coefficients correlation coefficients

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Proteolysis and drip loss Proteolysis and drip loss

degradation of talin degradation of talin

intact talin degradation

day after storage

0 1 2 5

Relative abundance of intact talin (%)

0 20 40 60 80 100 120 140

H-pH L-pH

day of storage

H-pH

L-pH

talin

talin immunoblots immunoblots

0 1 2 5

Bee et al., 2007 Meat Sci. 76:359-365

drip loss (d of storage)

0.35 0.35 0.60 0.60 0.51 0.51

1 2 5

pearson

pearson correlation coefficients correlation coefficients

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Costameric protein

Costameric protein - - talin talin

Clark et al., 2002

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Intermediate filament protein

Intermediate filament protein - - vinculin vinculin

Clark et al., 2002

intact vinculin degradation

day after storage

0 1 2 5

Relative abundance of intact vinculin (%)

40 50 60 70 80 90 100 110

H-pH L-pH

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Proteolysis and drip loss Proteolysis and drip loss

autolysis of

autolysis of μ μ- -calpain calpain

μ-calpain subunit

(80 kDa) and

its

large subunit autolysis

products (78 and 76 kDa)

80 kDa 78 kDa 76 kDa

relative abundance

0 20 40 60 80 100

H-pH L-pH

H H -pH - pH L- L -pH pH

80 kDa 78 kDa 76 kDa

0.45 0.48 0.59

desmin talin drip loss

(1 d)

pearson

pearson correlation coefficients correlation coefficients

80 kDa

0.59 0.73 0.58

78 kDa

-0.57

-0.57 -0.66 -0.66 -0.63 -0.63

76 kDa

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Cytoskeletal

Cytoskeletal attachment and drip loss attachment and drip loss

Lawson, 2004 Meat Sci. 68:559-566

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Intermediate filament protein

Intermediate filament protein - - integrin integrin

Clark et al., 2002

(25)

Cytoskeletal

Cytoskeletal attachment and drip loss attachment and drip loss

low drip loss

high drip loss

3 h 6 h 9 h 24 h

Lawson, 2004 Meat Sci. 68:559-566

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Cytoskeletal

Cytoskeletal attachment and drip loss attachment and drip loss

β β- -integrin integrin cytoplasmic

cytoplasmic domains domains

m- m -calpain calpain

coco--localization of localization of β

β--integrin integrin

&

mm--calpaincalpain

Prerigor Rigor

Lawson, 2004 Meat Sci. 68:559-566

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Integrin degradation and drip loss Integrin degradation and drip loss

Zhang, 2006 Meat Sci. 74:578-585

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Relative abundance of intact integrin

(LM)

0.5 h 24 h 72 h

relative abundance

0.0 0.1 0.2 0.6 0.8 1.0 1.2 1.4 1.6

1.8 Small litter Large litter

LS; P = 0.08 BtW; P = 0.65 LS x BtW; P = 0.33

Large

Large litter litter Small litter Small litter

0.5 h 24 h 72 h

Relative abundance of intact integrin

(STD)

0.5 h 24 h 72 h

relative abundance

0.0 0.1 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

1.8 Small litter Large litter

Proteolysis of integrin Proteolysis of integrin

relative abundance relative abundance of intact integrin of intact integrin Drip loss

Drip loss 0.5 h 0.5 h 24 h 24 h 72 h 72 h

LM LM - - 0.12 0.12 - - 0.34 0.34 -0.16 - 0.16

STD STD 0.07 0.07 0.16 0.16 - - 0.31 0.31

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Relationship between drip loss and postnatal Relationship between drip loss and postnatal

growth growth

- - Differing growth rate induced by specific feeding Differing growth rate induced by specific feeding strategies might affect proteolysis?

strategies might affect proteolysis?

greater proteolysis

less proteolysis

higher μ-calpain activity

higher μ-calpain:calpastatin ratio

Thaw loss

%

8 9 10 11 12

13 AA

RA RR

Kristensen et al., 2002 J. Anim. Sci. 80:2862-2871

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μ μ - - calpain activity calpain activity

μ-calpain activity

(casein zymography)

hours postmortem

0.5 24.0

S tandardized cont our quant it y

0.8 1.0 1.2 1.4 1.6

AA RA RR

P-values

Feeding regimen: 0.03 Birth weight: 0.86 Time postmortem: < 0.01

μ-calpain m-calpain

AA AA RA R A RR RR AA AA RA R A RR RR

LM after 48 h

Feeding strategy

AA RA RR

Drip loss, %

1 3 5 7

LM

Feeding strategy

AA RA RR

Shear force values, kg

0.0 3.2 3.6 4.0 4.4

ab

a

b

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Lean growth efficiency and drip loss Lean growth efficiency and drip loss

pH decline

minutes postmortem

15 30 45 1440

pH

5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8

control selected

Lonergan et al, 2004 Meat Sci. 68:559-566

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changes and possible relationship to postnatal growth

The role of postmortem biochemical and structural The role of postmortem biochemical and structural changes and possible relationship to postnatal