5. Post-mortem changes in muscle and its conversion into meat

Muscle metabolism in the living animal

Major function = contraction

ATP is required for:

• Contraction

• Maintaining muscle’s functional integrity

• Produced: free fatty acids; glucose (blood); glycogen (muscle fibres)

Sarcoplams and mitochondria enzyme systems ensure ATP supply to contractile elements ATP required to fuel Ca pump of sarcoplasmic reticulum

Energy metabolism in muscles

• Fed animals have low free fatty acids and therefore glucose is mostly used

• Fasting animals use free fatty acids

• If FFAs and glucose are not enough than glycogen is used

Glycogen and glucose broken down easily by:

• Glycolysis

• Oxidative decarboxylation

• Oxidative phosphorylation

Process of energy metabolism

• Takes place in the sarcoplasm

• Enzymes that catalyse other processes (not glycolysis) are located in the mitochondria

• Operation requires aerobic conditions (=6 oxygen per 1 glucose)

• If there is no oxygen than you can only process energy via glycolysis.

Post-mortem acidification

• Death → O2 supply (including glucose and FFAs) to muscle ceases when blood circulation stops

• Therefore ATP only regenerated through breakdown of glycogen by glycolysis (anaerobic)

• Glycogen broken down → LA accumulates as no longer removed by blood = muscle gradually acidifies

If glycogen is not limiting: LA production ceases when enzyme system will no longer function at low pH

pH change post-mortem

Unstressed animal: pH fall from 7.2 to 5.5

importance of acidification to meat

• Affects: water-holding capacity (WHC) and color

• Influence of lower pH on structure of muscle constituents

• WHC decreases so that drip/exudate is lost - especially if muscle is cut

• Meat relatively dark and translucent in living animal/recently dead to being paler and opaque

How does the water holding capacity change when an animal dies?

• When animal dies → pH decreases & Lactic Acid accumulates

  • This causes pH to fall to isolectric point of myofibrillar proteins causing the electrical charge on these proteins to reach zero

  • Hence proteins lose the electrostatic repulsion that allows them to hold water

• Additionally due to rigor mortis, actin and myosin link irreversibly to form actomyosin cross-bridges.

  • Because they are packed so tightly together, it causes the myofilament lattice to shrink laterally.

Where is the water bound in muscle?

1. Extracellular space outside muscle fibres - contain 5-10% of total water in muscle

2. Intracellular space outside myofibrils - filled with sarcoplasm - contains sarcoplasmic proteins - creatine kinase and myoglobin (red color of drip)

Acidification effect on meat color

• Acidification = protein change

• Change in structure = increase light scattering properties of contractile elements of muscle fibre

  • When animal was still living there was little extracelular space which caused light to be absorbed rather than reflected.

• As myofilament lattice shrinks and extracellular space volume increases structure becomes optically more heterogeneous.

• As a result meat appears paler/lighter

What happens to water when WHC decreases?

• Water is lost from proteins → squeezed out from myofibrils into sarcoplasm increasing “extra-myofibrillar” volume

• Eventually flows out of muscle and when meat is cut will flow out.

What is this figure?

Rigor mortis development

Figure: relationship between ATP depletion and the onset of rigor mortis

Rigor mortis development

Rigor mortis is the post-mortem stiffening and loss of extensibility in muscle tissue as it transforms into meat.

• In livin muscle, ATP binds to the myosin head to cause its dissociation from actin, keeping the muscle relaxed.

• When an animal is slaughtered, oxygen supply ceases and energy metabolism becomes anaerobic.

• As ATP levels drop below 5 mmol * kg-1 actin and myosin bind irreversibly to form permanent actomyosin cross-bridges.

Rigor onset time

Relate to factors affecting level of glycogen and creatine phosphate at death and rate of post-mortem muscle metabolsim

• Affected by stress and exercise

• Rate of development reduced if carcass cooled quicker

Discuss the different types of rigor mortis that can be found?

1. Normal rigor mortis as mentioned :Anaerobic glycolysis converts muscle glycogen into lactic acid, dropping muscle pH from approximately 7.2 down to an ultimate pH of 5.2–5.5. As creatine phosphate (CP) and glycogen are exhausted, ATP resynthesis stops, and permanent actomyosin cross-bridges fix the sarcomere length

2. Alkaline rigor:

   • Occurs when animals suffer severe pre-slaughter exhaustion.

   • This depletes glycogen and CP

   • Therefore after slaughter no LA can be formed

   • Gives DFD appearance

Rigor mortis development and meat quality

• After rigor muscle can no longer shorten (be stretched) and sarcomere and muscle length fixed

• Sarcomere length (reflects myosin and actin overlap) important for tenderness and toughness

• After onset of rigor → no danger of cold/heat shortening or compromising meat texture

Temperature at which rigor mortis is entered

• Above at least 15C, but mostly 35C = less tender meat due to heat shortening causing the denaturation of proteolytic calpains

• At cold temperature = cold shortening

Resolution of rigor and tenderization of meat

• You can not completely resolve it

• Muscle is not etensible again as pre-rigor

• Thick and thin filaments remain locked by mosyin cross-bridges (actomyosin)

• Tenderisation not caused by filaments regaining ability to slide

• Myofibrils structure begins to break down.

Tenderization rate

• Different rates → different recommended ‘ageing’ times

• Keeping meat at refrigerated temperature is expensive

  • Storage, refrigeration, weight loss

• Compromized reached commercially to produce meat with acceptble tenderness in reasonable time.

Recommended conditioning time for pork, lamb and beef

Conditioning/aging

the holding of carcasses or meat cuts at various temperatures above freezing to cause improvements in meat quality

Conditioning process

Tenderization occurs in two phaess:

1. Rapid phase → changes in myofibrillar component

   • Most important

   • Attachments of thin (actin) filaments to z-discs show some breakdown

   • Increase in amount water-soluble nitrogen compounds

2. Slower phase → structural weakening of intramuscular connective tissue

   • Only small changes seen in major connective tissue components such as collagen

Two main sorts of enzymes involved in tenderization

Tenderization comes from proteolytic enzymes in muscles

1. Calpains

   • Though to be more imporant (at leas in red meat species and poultry)

2. Cathepsins

   • May be more important in post-mortem degradation of fish muscle

   • Possibly tenderization meat kept at high temp

Calpains

• Activated by Ca2+ ions

• Maximum activity in neutral to alkaline conditions (pH 7.5)

• Show some activity (±25%) at pHu (pH 5.5, normal meat)

• Degrade: myofibrillar component

  • Actin and myosin

  • Troponin-T

  • Titin (connectin)

  • Desmin

What inhibits calpains

Inhibited by endogenous protein calpastatin - high calpastatin activity reduces extent of proteolysis in muscles

Cathepsins

• Occur in lysosomes and sarcoplasm

• Released from lysosomes post-mortem

• Maximum activity in mildly acidic conditions

Degrade:

• Troponin-T

• Some collagen cross-links

• Mucopolysaccharides of connective tissue ground substance

• Only actin and myosin < pH 5 - unlikely under normal conditions

Calpastatin

• Inhibits calpains

• Calpastatin activity in muscle thought to be more vital than amount of calpains present in controlling development of meat tenderness

• Encouraged efforts to identify alleles of calpastatin gene

• When animals are stressed during slaughter, elevated adrenaline levels will increase calpastatin activity

Why is meat from Bos taurus cattle more tender than the meat from Bos indicus?

-

Evidence of the importance of calpains

• Meat from animals treated with B-adrenergic agonists undergo little/no proteolysis is relatively tough

  • Reduce normal activity of proteolytic enzymes involved in continual process of accretion and breakdown of muscle proteins

  • Reduction in activity persists post-mortem

Different forms of calpains

Two main forms:

1. m-calpain activated at high Ca ion concentration: calpain I and II

2. mu-calpains activated at low Ca ion concnetration: mu- calpain

Which calpain is more crucial regarding post-mortem tenderization?

• mu-calpains

• Activated earier than m-calpains

• correlates with when most breakdown of myofibrilar protein occurs

What is an indicator for the conditioning process?

• One most obvious change that occurs in myofibrillar proteins during ageing of meat from all
  species is breakdown of troponin-T

• Small amount of actin and myosin degraded too

• Most breakdown occurs in proteins associated with z-disc and cytoskeleton - particularly titin (connectin) and dsemin

Key process of tenderisation:

Gradual breakdown of cytoskeleton framework which holds contractile elements (thick and thin filaments) attached and arranged within structure of muscle fibre

Possible action of calpains post-mortem

After exhaustion of ATP and development of rigor mortis: membrane systems of SR and mitochondria no longer sequester Ca ions

• Released into sarcoplasm and bath myofibrils

Increased Ca concentration activates mew-calpains allowing proteolysis

Normally calpains inhibited by being bound to calpastatin

Calpain activity promoted by higher Ca levels → Ca ions remove inhibition

Calpain activity is also promoted by higher pH and temp

Enhance enzyme activity post-mortem by infusing carcass with 0.3 M CaCl

Vitamin D to improve tenderness

Vit D is fat soluble and is essential for calcium and phosphorous metabolism

• Therefore elevates calcium concentration in blood and muscle tissue

• More Ca also stimulates activity of calpains post-mortem

Disadvantages of giving animals vit D to improve tenderness

• May reduce feed intake and growth rate is commercially uneconomic

• Very high dietary intake is toxic