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

In a living animal, what is the major function of muscle, and how is the ATP needed to support it produced and used?

Major function = contraction

ATP is required for:

• Contraction

• Maintaining the muscle's functional integrity

• Fuelling the Ca²⁺ pump of the sarcoplasmic reticulum (relaxation)

ATP is produced from:

• Free fatty acids

• Glucose (from blood)

• Glycogen (stored in muscle fibres)

How supply is ensured: enzyme systems in the sarcoplasm and mitochondria deliver ATP to the contractile elements.

Key idea: fuel (fatty acids, glucose, glycogen) → ATP via sarcoplasm and mitochondria → powers contraction, Ca²⁺ pump and muscle integrity

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

Aerobic energy metabolism in muscle (post-mortem context: why it stops after death)

• Occurs in the mitochondria (the Krebs cycle and electron transport chain), after glycolysis in the sarcoplasm

• Requires oxygen: 6 O₂ per 1 glucose

• Yields far more ATP than glycolysis alone (about 30-32 vs. 2 ATP per glucose)

• No oxygen → only glycolysis → pyruvate becomes lactate → pH falls

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 on meat quality

• 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 that is relatively dark and translucent in living animal will become paler and opaque when dead

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)

3. Space within myofibrils - about 80% of water

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: if there was high stress before death than glycogen and creatine phosphate were used up meaning that ATP cannot be regenerated after death = rigor moris.

  • However if the animal was relaxed during death: then there would still be a lot of creatine phosphate and glycogen allowing the regeneration for ATP for a little while = later rigor mortis.

• Rate of development reduced if carcass cooled quicker: at a temperature between 10C and 15C enzyme activity is minimized and ATP is used up very slowly. Any colder SR stops working = no calcium ion. Any higher ATPase works really fast and rigor mortis occurs very quickly

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 extensible 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.

Why do different tenderization rates matter commercially, and how is the problem handled?

• The issue: meat tenderizes at different rates, so each type needs a different recommended ageing time.

  Why long ageing is a problem: keeping meat at refrigerated temperature is expensive because of:

  • Storage costs

  • Refrigeration costs

  • Weight loss (moisture loss during ageing)

  The solution: a compromise is reached commercially, producing meat with acceptable tenderness in a reasonable time rather than waiting for maximum tenderness.

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

What are the steps in the 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 least in red meat species and poultry)

   • Are released upon increase in Ca2+ concentration

   • optimal pH: neutral to alkaline (7.5)

   • Released early post-mortem

2. Cathepsins

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

   • Tenderizes meat more than calpains at higher temperatures.

   • Are released from lysozymes, when lysozyme membrane are distrupted post-mortem

   • Optimal pH: acidic

   • Released later post-mortem

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?

• Bos Taurus cattle have more post-mortem proteolysis due to less calpastatin

What evidence shows that calpains are important for meat tenderization?

• β-agonists reduce the activity of proteolytic enzymes (including calpains) that drive normal muscle protein turnover (accretion and breakdown)

• This reduced activity persists post-mortem

• Result: little or no proteolysis during aging, so the meat stays relatively tough

Different forms of calpains

Two main forms:

1. m-calpain (calpain II) activated at high Ca ion concentration

2. μ-calpains (calpain I) activated at low Ca ion concnetration: μ- calpain

Which calpain is more crucial regarding post-mortem tenderization?

• μ-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 μ-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