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.
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• 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Unpublished results!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! !!!!