Lactic Acid in Muscle and its Effects on Meat Quality

Eero PuolanneDepartment of Food Technology/

Meat TechnologyUniversity of Helsinki

Helsinki, Finland

Thank you!

• It is an honor and extraordinary pleasure to me to be the recipient of AMSA international Award, and also to have this presentation for you. I do appreciate this very much indeed!

• I also thank the organizers for an excellently organized meeting, fine meeting venue, good food, and finally, for the cozy casual atmosphere!

• I and my wife as well, we are so happy to visit this great country again and meet old friends and make new.

Warning!

• English is not my native language• This PowerPoint presentation and

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The research groupDepartment of Food Technology

Department of Basic Veterinary Sciences /biochemistry

Prof. Eero Puolanne PhD Marita Ruusunen MSc Maria Kylä-Puhju

Prof. Reeta Pösö MSc Katri Sepponen

Motto• Life is to detain the entropy of solar energy• All species try to proliferate and fill their

ecological niche• The strength of the molecules of an organism are

tested billions of times per second• The functions of molecules and the whole

biological system is also tested constantly• In nature, the organisms are optimally balanced• The safety margins decrease the ability to

compete• Farm animals, do they have a harmonic growth

and balanced physiology?

* White fiber * Intermediate fiber

* Red fiber

Porcine M. masseter

Largest, 715 mm2Smallest, 447 mm2

Thinnest, 3.9 mm Thickest, 5.7 mm

Femur crossection areas of 165 d old pigs

Unpublished results!

pH is the Key Factor -Animal Welfare/Meat Quality• A vast flow of information constantly flowing in

the the bodies of fauna• Consciousness is the capability of neural network

to pick up relevant information, slow it down and “put light on it”

• If something is “causing a danger to the life” of the individual, pain and/or distress will occur.

• Our duty is to provide to the animals circumstances, where there is no unnecessary pain/distress

• In a living muscle the pH is 7.2-7.4 …. 6.2– high stress/low pH causes pain and distress

pH is the Key Factor -Animal Welfare/Meat Quality• In a postmortal muscle the pH is 7.2-7.4 … 5.5 … 5.0• Perimortal effects (time, temperature, pH) on

tenderness development, color, and drip and water-binding

• the effects of pHu on rigor/post rigor meat tenderness, color, water-binding (salt-phosphate effects)

• !!!! pH differences of about 0.2-0.3 units cause large differences originating

• !!!! from 10-20 mmol/kg lactic acid or !!!! 5-10 mmol/kg glucose, only

3.0 4.0 5.0 6.0 7.0 8.0

WBC

pH

NaCl

w/o NaCl

The Glycogen• Glycogen, I.8% or 100 mmol/kg (20 … 150

mmol/kg) expressed as glucose equivalents• Serves as energy reservoir that is independent of

blood supply• Would provide energy for 20 s to 15 min ???

depending very many factors, e.g. oxygen status, type of exercise and/or stress

The Glycogen Molecule• Glycogenin, MW 37,300• Branched chains of 13 glucose units• 1,4-bonds, or 1,6-bond at fourth and eighth units• minimizes volume, maximizes non-reducing ends

and available glucose

(PhD Kaisa Immonen, 2000)

1,6-branch

Glycogenin

Prolycogen and macroglycogen

• !!!! Proglycogen, 6-7 tiers, MW 300,000 to 575,000, 64-128 non-reducing ends, ca 10% protein

• !!!! Macroglycogen, 12-13 tiers, MW 5-10 mo• 1000 to 2000 non-reducing ends, ca. 0.4% protein

• !!!! 20-25 phosphorylase tetramers (or 40-50 dimers)/molecule

(PhD Kaisa Immonen, 2000)

Lactic acid

Glycolytic potential

(PhD Kaisa Immonen, 2000)

(PhD Kaisa Immonen, 2000)

II III

I

P.M. glycogenolysis• Where is ATP needed? • Calcium from sarcoplasmic reticulum -->

shortening reactions --> ATP is consumed• The key enzyme of glycolysis

(phosphofructokinase) is inhibited by low pH• Other reasons?• Pre mortem … post mortem?• PSE?• pH 5.7-5.8 is a pivot point

– onset of rigor mortis, if not earlier– the rigor is eventually different (stronger) when

setting on at low pH?

Phosphorylase• 2% of the total protein in muscles• 40-50 dimers or 20-25 tetramers/glycogen molecule• Each one is bound at two points to a (unbranched)

A-chain• One is active and the other is a regulatory binding

site• Cleaves glucose-1-P from the non-reducing end

VERY quickly, until the fourth unit from the branching point

• !!!! When the uppermost tier (whatever it is) is full,theoretically, 34.6% glucose of the glycogen molecule will be available

(PhD Kaisa Immonen, 2000)

Phosphorylase

Phosphorylase

• !!!! When glycogen content is 100 mmol/kg, ca 35mmol/kg glucose-1-P -->70 mmol/kg lactic acid, which means more than 1 unit drop in pH

• in living muscle this enough for fast energy needs• !!!! Macroglycogen is used more in aerobic stress,

proglycogen more in anaerobic stress and post mortem

• The mechanisms may involve the allostericactivation of phosphorylase b kinase by Ca++

(breakdown of PG) and phosphorylase b by AMP (MG)

The Glycogen DebranchingEnzyme

• Bifunctional: transferase and glucosidase• !!!! Transferase moves three glucose units to a B

chain• !!!! 1,6-glucosidase cleaves 1,6-glucosyl unit as

free glucose (8% of the residues)• activity about 10% of that of phosphorylase• !!!! may limit the breakdown rate?• Effects of pH and temperature? • Living muscle/ post mortem muscle?• Residual glycogen 10-20 mmol/kg?

(PhD Kaisa Immonen, 2000)

Phosphorylase

Transferase1,6-glucosidase

The Formation of Lactic Acid• Glucose --> 2 lactic acid + 2-3 ATP• The anaerobic production of energy• Stimulated by mental (adrenalin) or physical

(Ca++, AMP) stress or both• 0 (?) to 80% of energy production

– aerobically 2 to 40 mmol ATP/kg*min– anaerobically 0 to 250 mmol ATP/kg*min

• !!!! E.g. in pigs produce constantly?

Lactate Dehydrogenases

• Tetramers of muscle type (M) and/or heart-type (H)

• H4 (LDH-1) H3M (LDH-2) … M4 (LDH-5)• !!!! At a high rate of glycolysis LDH-5 favors the

reduction of pyruvate to lactic acid, especially in light muscles

• In heart and !!!! in dark muscles LDH-1 converts lactic acid to pyruvate

• LDH-activity, M/H percentages

Monocarboxylate Transporters !!!! !!!! !!!!

• Prof. Reeta Pösö’s group• pKa of lactic acid is 3.86, i.e. at physiological pH

<0.1% undissociated acid• !!!! Only undissociated form is able to cross the

membranes• The key enzyme of glycolysis

(phosphofructokinase) is inhibited by high concentrations of protons

*

Monocarboxylate Transporters• Monocarboxylate transporters (MCT) transport

lactate to the direction of lactate gradient• Nine types found as far, and the most studied are

– MCT1 (in aerobic muscles)– MCT2 ( low Km and Vmax)– MCT4 (high Km and Vmax, in anaerobic muscles)

• !!!! Prof. Reeta Pösö’s group has been able to show a high content of MCT2 in porcine muscles.

• !!!! May be a house-keeping protein for continuous production of lactate?

High capillarization qnd oxygen supply

Low capillarization and oxygen supply *

Lactate is utilized aerobically!!!!!!! !!!! !!!!

• Lactic acid is converted to pyruvate in muscles, heart and non-contracting tissues (especially in liver and in red blood cells)

• LA contains over 90%of the energy of the glucose• LA is consumed (--> CO2) or stored as glucose

(glycogen)• !!!! This takes place aerobically, only• !!!! The formation of glycogen in liver uses ATP

that has been produced aerobically

Glycogen

(AEROBIC!)

*

The Buffering Capacity• ββββ = dA/dpH (A = the amount of acid/base)• The buffering capacity is usually ca 50 (40-60)

mEquivalents/kg*pH• In light muscles higher than in dark muscles • Myofibrillar proteins ca 20-25 mE/kg*pH (pH 5.5 -->)• Soluble components 10 (pH 5.5) to 30 (pH 6.8)

mE/kg*pH• Lactic acid relevant only in low pHs (below 5)• !!!! In muscles, 70-80 mmol LA/kg needed to lower

the pH from 7.2 to 5.5 (3 µmol/kg in water!)

(PhD Riitta Kivikari, 1996)

(PhD Riitta Kivikari, 1996)

The Rate of ATP Consumption and Production• ATP content in the living muscle is ca 8-10

mmol/kg• The consumption varies from 2-3 (at rest) to 200-

300 (maximal) mmol/kg*min– aerobically 2 to 40 mmol ATP/kg*min– anaerobically 0 to 250 mmol ATP/kg*min

• !!!! Theoretically, the drop in pH from 7.2 to 6.2 would require ca 60 mmol/kg LA, i.e. to produce 60 to 90 mmol/kg ATP, which is consumed in 15 to 30 seconds

Sources of ATP• Glycolysis 70-80 mmol/kg___________________________________• Pork myoglobin 0.06 ---- 0.3 mmol/kg• Beef 0.1 ---- 1.0 mmol/kg• 1 mmol O2 ----> 6 mmol ATP• ---> 0.4 ….. 6 mmol/kg ATP• Hemoglobin?• Creatine phosphate …. 20-25 mmol/kg• I.e. up to 1/3 of that of glycolysis• Delay the onset of glycolysis• Slow down the rate of glycolysis• Difference pigs and broilers / cattle and sheep• Cooling rate: drip and cold shortening

What have we done?• The liver cannot possibly receive and convert 150

to 900 g lactic acid in a short period of time• Neither does the blood• Therefore, lactic acid is used in muscles

aerobically, preferably in red and intermediate fibers, sooner or later

• Beef animals and sheep may not have problems• !!!! In (many) porcine and poultry muscles there

now are very little (10 … 0%) red and intermediate fibers and very poor capillarization to get oxygen

• !!!! Cardio-respiratory capacity is also low• !!!! What else we should expect than PSE-like meat?• !!!! What about animal welfare?

Research needs"More quantitative physiological data on

carbohydrate metabolism needed" The fate of lactic acid: where does it go in

stress, how it is utilized/stored?"MCTs?" Proglycogen, macroglycogen vs.

phosphorylase?" Buffering capacity?"Muscle status in stress and at death?"Muscle and species differences!!!! Dead or alive: lactic acid is there! !!!!