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LECTURE NOTES FOR THE UNDERGRADUATE

COURSE (2013-2014 Batch)

LPT 321 MEAT SCIENCE (1+1)

Dr. K. DUSHYANTHAN, M.V.Sc., Ph.D.,


PROFESSOR
DEPARTMENT OF LIVESTOCK PRODUCTS TECHNOLOGY
(MEAT SCIENCE),
MADRAS VETERINARY COLLEGE,
CHENNAI 600 007
2016

STRUCTURE AND COMPOSITION OF MUSCLE


Meat - composed primarily - muscle, associated connective tissues like
adipose tissue (fat), bone and cartilage as well as epithelial and nervous
tissues and connective tissue proper.
Skeletal muscle - principal source of muscle tissues in meat. Small amount
of smooth m
Muscle - also present in meat.
The muscle + connective tissues = to the qualitative and quantitative
characteristics of meat.
Meat is not synonymous with muscle, even though it is made mostly of
muscle.
The skeletal muscles constitute - about 35-65% of the carcass weight of meat
animals.
In addition - consists of some smooth muscle, primarily - component of blood
vessels.
Cardiac muscle - confined solely to the heart.
These muscles constitute about 30-45% of the live weight or 35-60% of the
carcass weight of meat animals.

SKELETAL MUSCLE
The skeletal muscle - as striated muscle due to - transverse
banding pattern observed microscopically -also known as
voluntary muscle.
These are attached directly to bones - some are attached to
ligaments, fascia, cartilage and skin - only indirectly to bones.
More than 600 muscles in the animal body - vary in shape, size
and activity.
The specific characteristics - muscle - related to its function.
Each muscle - covered - a thin connective tissue sheath continuous - connective tissue constituents - course - interior
-muscle.
Nerve fibres & blood vessels enter and exit the muscle -connective
tissue networks providing - muscle within an innervating system vascular bed for nutrient supply and water removal

MUSCLE FIBRE
The structural unit of skeletal muscle tissue - muscle
fibre.
Highly specialized cell
Muscle fibres constitute 75-92 % - total muscle volume.
Connective tissues, blood vessels, nerve fibres &
extracellular fluid - major volume - remaining volumes,
- extracellular fluid.
Muscle fibres - long, unbranched, threadlike cells, which
taper slightly at both ends.
Vary considerably in diameter, ranging from 10 m to
more than 100 m (1 m micrometer = one millionth
of a meter), within the same species and even within the
same muscle.

SARCOLEMMA

Membrane surrounding - muscle fibre - Sarcolemma.


The prefix sarco derived from the Geek words
sarx or sarcos - mean flesh and
lemma - Greek word for husk.
Composed of protein and lipid material.
Sarcolemma - elastic; endures tremendous distortion during contraction, relaxation
and stretching.
Structure, composition and properties - identical to plasmalemma (cell membrane) of
other cells of the body.
Periodically - length - fibre, and around - circumference, invagination of the
sarcolemma forms - network - tubules - called transverse tubules.
Usually called as T system or as T tubules.
Motor nerve fibre endings terminate - sarcolemma - myo-neural junction;
prefix myo - the Greek word mys, meaning muscle.
The myoneural junction - site - endings - motor nerve fibre - implanted in small
invaginations of the sarcolemma.
Structures present - myoneural junction form a small mound - surface of the muscle
fibre.
The entire complex is known as the motor end plate.

SARCOPLASM
The cytoplasm of muscle fibres -sarcoplasm.
Intracellular colloidal substance all organelles suspended.
Water constitutes - 75-80 percent - sarcoplasm.
Sarcoplasm - skeletal muscle contains
lipid droplets,
variable quantities of glycogen granules,
ribosomes,
numerous proteins,
Non-protein nitrogenous compounds and
a number of inorganic constituents.

NUCLEI
Skeletal muscle fibres - multinucleated due to
tremendous variation in their length - number of nuclei
per fibre is not constant.
A fibre several centimeters long - several hundred nuclei
- regular distribution every 5 m along its length,
except near tendinous attachments where they increase
in number and are more irregularly distributed.
The number of nuclei - increases - vicinity - myoneural
junction.
In mammalian muscle nuclei - located - periphery fibre, just beneath - sarcolemma.
Ellipsoidal in shape - longest axis oriented parallel to the
long axis of the fibre.

MYOFIBRILS
An organelle unique to muscle tissue.
Long thin, cylindrical rods 1 to 9 m in diameter.
Bathed by the sarcoplasm - extend the entire length - muscle fibre.
Muscle fibre - 50 m diameter -from 1000 - 2000 (more) myofibrils.
Cross-sections - myofibrils well-ordered array of dots - two
distinct sizes - myofilaments (within the myofibrils).
The myofilaments - thick and thin filaments - myofibril.
longitudinal section - thick filaments - aligned parallel to each
other - arranged in exact alignment across the entire myofibril.
Thin filaments - aligned across - myofibril - parallel - each other thick filaments.
Arrangement & thick filaments overlapping - certain regions along
- longitudinal axes - accounts - characteristic banding - striated
appearance - myofibril.
This banding effect - takes form - alternative light and dark areas
- term striated muscle in reference to skeletal muscle.

Within light and dark bands of the myofibrils - areas - different density
Light band - singly refractive viewed - polarized light - described as isotropic - called the I band;
Broad dark band - doubly refractive - or anisotropic- in polarized light designated the A band.
A band - much denser than - I band.
Both the A and I bands - bisected by - thin lines.
I band - bisected - dark thin band - Z line.
The unit - myofibril between two adjacent Z lines - a SARCOMERE.
Sarcomere includes both an A band and the two half I bands located on
either side - A band.
Sarcomere - repeating structural unit of the myofibril - basic unit
events -muscles contraction-relaxation cycle occur.
Sarcomere length - not constant dimensions & I band - are
dependent on the state of contraction - time the muscle is examined.
In - central region - A band - area - slightly less density than remainder band - lighter region - called - H zone.
Narrow dense band - M line - bisects - center - A band.
A region - low density appears within - H zone on either side - M line pseudo H zone.

MYOFILAMENTS
Thick & thin bands differ in their dimensions - chemical composition - properties &
position within the sarcomere.
Thick filaments - 14-16 nm (nanometers) in diameter & 1.5 m long - constitute the A
band sarcomere consist myosin - referred to - myosin filaments. Held in transverse and
longitudinal register by thin cross bands located periodically along the length and by cross
connections in the centre of the A band.
The alignment of these cross connections in the centre of the A band corresponds to the
transverse density characteristics of the M-line.
Thin filaments - 6-8 nm in diameter - extend approximately 1.0 m on either side - Z line constitute I band sarcomere - consist primarily - actin - referred to - actin filaments.
H zone - less dense than - rest - A band - centre region between the ends of the opposing
actin filaments.
Width - H zone varies - state of contraction - muscle.
Densest area - A band - on the either side - H zone - both the actin and myosin filaments present.
I band contains only - thin actin filaments - least dense band of the entire myofibril.
Myosin filaments - H zone region - sarcomere - oriented - definite hexagonal pattern six
thin filaments surround each thick filament.

Z - LINE ULTRASTRUCTURE
Longitudinal section - actin filament on one side - Z line lies between
two actin filaments - opposite side - Z line.
Indicating - actin filaments per se do not pass through - Z line.
Actin filaments - believed - terminate at - Z line.
Ultra-thin filaments - Z filaments - constitute - material - Z line &
they connect - actin filaments on either side of it.
Near - Z line - each actin filament connects to four Z filaments that
pass obliquely through - Z line.
Each - 4 Z filaments - connects - actin filament - adjacent sarcomere.
Structural arrangement - Z line - each actin filament - one sarcomere
oriented - centre - four actin filaments - next sarcomere.
Longitudinal sections - offset (or oblique) arrangement - Z filaments
results - characteristic zigzag pattern - Z line & explains why an
actin filament on side - Z line appears - lie between two actin
filaments on either side of it.

PROTEINS OF THE MYOFILAMENTS


Proteins actin and myosin constitute 75-80% myofibril & remaining - regulatory proteins.
So named - direct or indirect regulatory functions adenosine triphosphate-actin-myosin complex.
Other regulatory proteins - tropomyosin, troponin, two
M proteins -actinin, C protein and -actinin listed decreasing order of concentration - myofibril.
Proteins - thick and thin filaments - regulatory proteins
are referred to as myofibrillar proteins.
The regulatory proteins - associated - various elements
- myofibril. Tropomyosin, virtue - imino group
contributes - folding among polypeptide chains that
results in the formation of a globular (spherical) shaped
molecule approximately 5.5 nm in diameter.

Globular molecule referred - G-actin (globular actin) and


as such constitutes the monomeric (single molecule) form
of actin.
The fibrous nature of the actin filament occurs because of
the longitudinal polymerization (linking) of G-actin
monomers to form F-actin (fibrous actin).
In F-actin, the G-actin monomers are linked together in
strands, much like the beads on a string of pearls.
Two strands of F-actin are spirally coiled around one
another to form a super helix that is characteristic of
the actin filament.
From this helical arrangement of G-actin monomers, the
actin filaments overall diameter of approximately 6-8 nm
can be readily visualized.
The isoelectric pH (pH of minimum electrical change and
solubility) of actin is approximately 4.7.

Myosin - 50-55% - myofibrillar protein & characterized - high


proportion - basic & acidic amino acids making - highly charged
molecule.
Actin - possesses a relatively low charge. The isoelectric pH of
myosin is approximately 5.4. Myosin, with lower proline content
than actin, has a more fibrous nature.
Structure - myosin molecule - elongated rod shape - thickened
portion at one end.
Thickened end - myosin molecule - usually referred - head region
& long rod-like portion - backbone - thick filament - tail region.
Portion - molecule between - head & tail regions are called the
neck.
Head region - molecule is double headed & projects laterally
from the long axis - filament.
Myosin is subjected to the proteolytic (protein breakdown) action
of the enzyme trypsin, it is split near the neck into two fractions
that differ in molecular weight; light meromyosin and heavy
meromyosin.

In centre - A band - either side - M line - myosin filament contains tail portion - myosin molecules without - globular heads.
This region within H zone - either side - M line - pseudo H zone.
Polarity - myosin filaments - such that heads on either side - bare
central region - A band - oriented - oblique angle away - M line.
Protruding heads - functionally active sites - thick filaments - muscle
contraction - myosin heads form cross bridges - actin filaments.
During muscle contractions each myosin head attaches - G-actin
molecule - actin filament.
Formation - cross bridges between through - interaction - actin &
myosin filaments produces - chemical complex - actomyosin.
Formation - actomyosin results - rigid & relatively inextensible
condition - muscle.
Actomyosin - major form - myofibrillar proteins - found - pm muscle
& rigidity - following death (rigor mortis) largely due - complex.
Transient compound - living animal since cross bridges between
actin & myosin filaments - broken during - relaxation phase contraction cycle. (Cross bridges - nonexistent in muscle when it is at
rest.)

GOLGI COMPLEX
Golgi complex - located - sarcoplasm near nuclei - consist - flattened vesicles apparently function - concentrating & packaging apparatus - products metabolic production line - cell.
Muscle fibre multinucleated - numerous Golgi complexes.
Vesicles - golgi complex resemble - membranes - sarcoplasmic reticulum.
SMOOTH MUSCLE
Present - greatest quantity - walls - arteries, lymph vessels & gastrointestinal &
reproductive tracts - involuntary nature - long, unevenly thickened - centre &
tapering - both - sides.
Myofibrils - homogenous & do not show alternating dark & light bands like skeletal
muscle.
No Z or M-lines - SR - not much developed. Smooth muscle - poorly - blood
CARDIAC MUSCLE
Possesses - unique property - rhythmic contractility - continues - early embryonic
life until death. Resemble characteristic properties - both skeletal and smooth
muscle - also involuntary.
Fibres - rounded to irregular - shape & give off branches - mixed up - nearby fibres.
Nucleus - placed - centre - fibre. Myofibrils depict striations similar to skeletal
muscle.
Sarcoplasm shows numerous & more mitochondria than - skeletal or smooth
muscle.
Intercalated discs are present at the position of Z-lines.

CONVERSION OF MUSCLE TO MEAT


Conversion of 'Muscle' to 'Meat' - result - series - biochemical biophysical
changes initiated - muscle - death - animal due - stoppage - blood
circulation.
Immediate change - bleeding - consumption - oxygen supply - muscle
resulting - inhibition - cytochrome system & oxidative phosphorylation.
Sarcoplasmic ATP depletes the ATP levels - increase the inorganic
phosphate - stimulates - breakdown - glycogen to lactic acid.
Ineffectual resynthesis - ATP by anaerobic glycolysis cannot maintain - ATP
level and drops - actomyosin forms & inextensibility - rigor mortis ensue.
Loss - extensibility reflecting actomyosin formation proceeds slowly at first
(the delay period) then - great rapidity (the fast phase), extensibility
remains constant - low levels.
The time - onset - rigor - given temperature depends most directly - level ATP.
Synthesis of ATP from creatine phosphate & ADP or by glycolysis cannot
maintain - ATP level - too long.
Higher glycogen content - muscle can prolong - time - some extent - not
indefinitely.

Rigor Mortis
The first and most considerable post-mortem change which occurs in
muscle is Rigor mortis - Characterised
by hardening & contraction - voluntary muscles
by a loss in transparency - surface - muscle causing dullness and
by stiffening of the joints.
It is followed - slight increase - temperature - carcass to 1.5C or
more above normal - beef carcasses & temperature gradually drops surrounding atmosphere.
Onset - rigor - accompanied - lowering - water holding capacity
Due to drop in pH & consequent approach - muscle proteins - their
isoelectric point or due - denaturation of sarcoplasmic proteins
Even when rigor mortis occurred at high pH - loss - water holding
due - disappearance - ATP & consequent formation - actomyosin.
Addition - ATP - post-mortem muscle - March Bendal factor - still
operating restores - WHC towards - in vitro level.
Once rigor - complete, the muscle remains placid & inextensible
provided - kept free from - contamination; - no resolution - rigor.

Contd

The lowered ATP levels - difficult - maintain - structural integrity - proteins.


The lowering pH - accumulation - lactic acid also makes liable - denature.
Denaturation - frequently accompanied - loss of power - bind water & fall - pH
causes - myofibrillar proteins approach - isoelectric point. Both - cause
exudation.
Denaturation - sarcoplasmic proteins - makes - liable - attack cathepsin Enzymes - capable - breaking down even collagenous connective tissue - muscle
& cause tenderisation - meat during aging.
The breakdown proteins - peptide & amino acids & accumulation - various
metabolites from glycolytic & other processes afford - rich medium - bacteria.
Absence - microbial spoilage - the holding - unprocessed meat above the
freezing point 1.5C is known as Conditioning, Ageing, Ripening and /or
Tenderization - long been associated - an increase - tenderness & flavour.
Accumulation - lactic acid & lower level - muscle pH - important postmortem
change - conversion - muscle - meat.
Rate & extent - pH decline variable - influenced - species - food animal,
various pre-slaughter factors, environmental temperature, etc.,
Gradual decline continues - approximately pH 7 - living muscle during first
few hours (5-6 hours) & there - little drop - next 15-20 hours - ultimate pH range of 5.5 5.7.

Rate of pH decline - enhanced - high environmental temperature. Low


ultimate pH - desired - check - proliferating microorganisms - storage.
Sharp decline - postmortem pH - before - dissipation - body heat through
carcass chilling - cause denaturation of muscle proteins - Muscles depict
pale, soft and exudate (PSE) condition.
Contrary - muscles - maintain a consistently high pH during postmortem
conversion - meat depict a dark, firm and dry (DFD) condition - Both the
conditions are undesirable.
Pre-rigor meat - quite tender - its toughness increases until rigor mortis
completed - continues - tough - some more time.
Resolution - rigor due denaturation, degradation - aging meat - tender.
During conversion to meat - muscle - susceptible - invading
microorganisms - body defence mechanism stops operating & membrane
properties altered - Rapid decline - muscle pH - causes denaturation collagenous connective tissue.
Only after the biochemical changes - takes place muscle converted to
meat. That meat is tender and fit for consumption.
Essential to allow the process of Conditioning, Ageing, ripening or
tenderisation unprocessed meat kept above the freezing point (-1.5C) in
the absence of microbial spoilage.

POSTMORTEM CHANGES

Slaughter of food animal - Series of physical and chemical changes several hours or even days
Resulting in the conversion of muscle to meat
Immediate loss of oxygen supply - muscle due to exsanguination
(bleeding)
Stored oxygen in myoglobin depleted - inhibition - aerobic pathway citrate cycle - cytochrome system.
Store - creatinine phosphate (CP) used - rephosphorylation - ADP to ATP
(creatine phosphate + ADP = ATP + creatine) - soon exhausted.
Energy metabolism - shifted - anaerobic pathway resulting - breakdown glycogen - lactic acid.
This - continues till all - glycogen stored - muscle - exhausted.
Resynthesis - ATP - anaerobic pathway - not enough - maintain - required
ATP level & - depletes - formation - actomyosin resulting - onset - rigor
mortis.
Important changes - take place during postmortem period are

Loss of Homeostasis
Postmortem glycolysis and pH decline
Rigor Mortis
Loss of Protection from Invading Microorganisms
Degradation due to Proteolytic Enzymes
Loss of Structural Integrity

Loss of Homeostasis
o Homeostasis mechanism system - physiologically balanced
internal environment - helps - body - cope up - stresses - oxygen
deficiency, extreme variation in temperature, energy supply, etc., is
lost
o Homeostasis - controlled - nervous system - ceases - 4-6 minutes
after bleeding.
o In absence - blood supply - loss - body heat and temperature starts
declining.
Postmortem glycolysis and pH decline
Absence - oxygen, anaerobic glycolysis leads - formation - lactic
acid - glycogen reserves:

Accumulation - lactic acid lowers down - muscle pH - important


postmortem change - conversion - muscle - meat.
Rate & extent - pH decline variable - influenced - species - food
animal pre-slaughter factors, environmental temperature, etc.

In most species - gradual decline continues - pH 7 living muscle - first few hours (5-6 hours) & - little
drop - next 15-20 hours giving - ultimate pH range - 5.5 5.7.
Rate - pH decline - enhanced - high environmental
temperature.
Low ultimate pH - desired - check - proliferating
microorganisms during storage.
Sharp decline - postmortem pH - before dissipation - body heat through carcass chilling cause denaturation - muscle proteins.
Muscles depict pale, soft and exudate (PSE) condn.
Contrary - muscles - maintain - consistently high pH
during postmortem conversion to meat depicts dark, firm and dry (DFD) condition.
Both the conditions are undesirable.

Rigor Mortis
Contraction - muscles after death - another important
postmortem change - process - conversion - muscle - meat.
Very well-known - particular level or concentration - ATP
complexed - Mg++ - required - breaking - actomyosin bond &
bringing - muscle - relaxed state & as drops permanent
actomyosin cross bridges begin - form & muscle gradually
becomes less & less extensible under - externally applied
force.
Period immediately following exsanguination - actomyosin
formation proceeds very slowly - first & muscle - relatively
extensible & elastic - period - delay phase - rigor mortis.
Actomyosin formation picks up & muscle begins - loose
extensibility - phase - fast or onset phase - rigor mortis.
All - creatine phosphate (CP) depleted - ADP - no longer phosphorylated ATP - muscle becomes quite inextensible &
stiff.
Stage marks - completion - rigor mortis - rapid.

Normal meat
PSE meat

DFD meat

Postmortem pH decline - very slow or very fast - onset &


completion - rigor mortis - rapid.
Onset - rigor mortis - enhanced - ambient temperature
above 20C.
Phenomenon - rigor mortis resembles - muscle contraction
- living animal muscle except - rigor mortis - irreversible
under normal conditions.
Resolution - rigor mortis takes place due - microbial
degradation - muscle structure - due course - time.
Pre-rigor meat - quite tender - toughness keeps increasing until rigor mortis - completed.
Continues - tough - some more time - resolution - rigor due
- denaturation or degradation or aging - meat again
becomes tender.
Onset - rigor mortis - accompanied - decrease - water
holding capacity.
True even when rigor mortis takes place - high pH due disappearance - ATP & consequent formation - actomyosin

Loss of Protection from Invading Microorganisms


During postmortem period - body defence mechanism stops operating
& membrane properties - altered.
During conversion meat - muscle - quite susceptible - invading
microorganisms
Except - low pH - most - other pm changes favour bacterial growth.
Handling precautions - necessary - prevent contamination - meat.

Degradation due to Proteolytic Enzymes


Several autolytic lysosomal enzymes called cathepsins - remain
inactive - living muscle tissue - activated - muscle pH declines.
Enzymes initiate - degradation - muscle protein structure.
Catheptic enzymes - capable - breaking down - collagenous connective
tissue muscle & cause tenderisation - meat during aging

Loss of Structural Integrity


Postmortem alteration - membrane properties initiates - degradation muscular proteins.
Progressive disruption - myofibrillar structure
Resolution - rigor mortis - known - occur due - disintegration - Z-line
structure.
Rapid decline - muscle pH - denaturation - collagenous connective
tissue

NUTRITIVE VALUE OF MEAT


Meat - nutritious food - fully digestible - appealing - eyes & pleasing - sense
olfaction
Attributed - its abundant high quality proteins, essential fatty acids (EFA),
important minerals & B-complex group of vitamins.
Meat Proteins
Meat conc. source - proteins - far superior - plant proteins - very high Biol. Value.
Lean meat cuts - 16.5-20% proteins - rich - essential amino acids (EAA) not
synthesis - body & deficient diet will lead - protein malnutrition
Myofibrillar & sarcoplasmic proteins - very high quality contain EAA.
Connective tissue proteins - lower levels - tryptophan & sulphur containing amino
acids. Collagen - essentially poor - lysine content.

Meat Fats
Contain ample amount EFA & nutritional demand - body easily met i/m fat
Caloric value - fat - meat - attributed - fatty acids - triglycerides. Number - calories
- lean meat - less than - derived - equal weights - many other foods.
Caloric value - particular meat depends - amount - fat - meat cuts. Important FA meat fat - oleic acid (unsaturated FA) followed - palmitic and stearic acids
(saturated FA).
EFA in human diets - linoleic, linolenic, and arachidonic acids.
Pork & organ meats - good sources of linoleic & linolenic acids. Excess dietary
linoleic acid is converted to arachidonic acid in human body to meet its demand.

Phospholipids - components - cell wall & mitochondria & play vital role - cellular
metabolism Meat fat - contains some quantity of cholesterol and blood cholesterol level
increases after ingestion of cholesterol in food. Well known that our body is capable of
synthesizing more cholesterol than is normally ingested. Organ meats - remarkable high
cholesterol content as compared to skeletal meat.

Minerals
Meat - good source - all minerals except calcium - in close association - lean tissues in meat
Potassium - most abundant followed by phosphorus. Meat - good source of iron - required synthesis haemoglobin, myoglobin & certain enzymes - plays vital role maintaining good
health. Human body - limited capacity to store iron mainly - liver - a part of regular dietary
intake. Meat provides this important mineral in a form that is easily absorbed in the
system.

Vitamins
Lean meat - excellent source - B-complex group - vitamins - only traces fat-soluble vitamins
- restricted - body fat. Vitamin C - absent - lean meat - certain organs contain it - minor
quantities. Among - B-complex group of vitamins thiamine, riboflavin & niacin
concentrations - quite high
Pork surpasses several meats - B-complex vitamins are concerned - lean pork has 5-10 times
mote thiamine than other meats. Monogastric animals - pigs intake of vitamins in feed is
directly reflected in their tissues.
Several organ meats - less protein & fat - skeletal meats - more economical sources of
protein & vitamins than retail cuts of skeletal meats Liver - rich source iron, riboflavin,
niacin & vitamin
Nutritive value skeletal & organ meats - properly utilised to alleviate the malnutrition.

FRAUDULENT SUBSTITUTION OF MEAT AND ITS


RECOGNITION
The differentiation of the muscle and fat of animals is of
importance in connection with the possible substitution of
inferiorandattimesrepugnantmeatforthatofgoodquality.
The substitutions practiced - horseflesh for beef, chevon for
mutton, mutton for venison, beef for mutton and occasionally
thefleshofthecatforhareorrabbit.
Not difficult to differentiate the flesh and fat - animals in the
carcassformorinjointsbymeansofanatomicalconfirmation.
Recognition of horseflesh or other meats in minced or in
sausage form depends on tests of chemical or biological
nature.
Inthehandlingofmeatsandpreparationofmeatfoodproducts
attempts are sometimes made to substitute meat of lesser
quality for that of higher quality with the object of deceiving
public and gain more profit. The level of the mixing may vary
from1to99%.Ifitis100%itissubstitution.

Various types of meat being substituted as mentioned above - differentiate


or recognize by three methods.
They are
o Physical or physiological methods.
o Chemical tests and
o Biological tests
These methods are also classified as follows:
o Physical and chemical techniques
Fatty acid pattern,
Histidine di-peptides and
Direct probe mass Spectrometry.
o Immunological techniques
Agar Gel Immuno Diffusion (AGID)
Counter Immuno Electrophoresis (CIE)
Enzyme Linked Immuno Sorbent Assay (ELISA)
o Electrophoretic Techniques
Polyacrylamide gel electrophoresis (PAGE)
SDS Polyacrylamide gel electrophoresis (SDS PAGE)
Polyacrylamide gel isoelectric focussing (PAGIF)
Agarose isoelectric focussing (AGIF)
o DNA Assay Techniques

Physical Methods
Anatomicaldifferences-eachspecies-carcass&appearance-
muscle&fatcolour,odour,textureandtaste-provided-general
differencebetweenspecies-earlierdays-foodanalysis.
Canbeattempted-providedmeats-formofjointsandincarcass
form
Carcasses of Different Species of Food Animals
1. Horse
Neckandthebonesoflimbsarelongerthantheox.
Sternumofhorseiscanoeshaped.
Nodiarthrodialjointbetweenthefirstandsecondsternalribs.
Thereare18pairsofribsandarenarrowerthanthoseofox.
2. Bull
o Massivemuscles-neck&shoulder&hindquarters-well-bred
animals.
o Neck&Ligamentum nuchae-thicker&strongerthan-ox.
o Anteriorpartischio-pubicsymphysis-welldeveloped&forms
adistincttubercle.Inguinalcanalsarepatent.

3. Ox
Lesser muscular development - bull - neck and shoulder
region.
Evencovering-fat-exterior.
Scrotalfat-prominent,nodular&moreorlesspointed.
Pelvis is narrow and usually contains a relatively large
quantityoffat.Fatisusuallyplentifuloverthekidneysand
alongthesub-lumbarregion.
4. Cow
Thigh - less rounded, very noticeable - hind quarters
(sunkenround)pelvis,anteriortubercularpelvisbroader-
ox.
Udderpresent-removedtriangularareaofattachmentis
noticeable on each side of midline of the abdominal wall.
Heifers - udder - slightly developed and consists chiefly of
fat.
Inoldcowstheudderissoft,spongy,roundandpendulous.

5. Sheep
Sheep carcass (whether or ewe) characterised abundant & even distribution S/C fat.
Ram carcass - distinguished by great muscular
development - region neck & shoulders ligamentum nuchae - large and strong - neck is thick
& inguinal canals are patent.
6. Goat
Goats are long and lean - little S/C fat, kidney fat
abundant even in poor carcasses.
S/C connective tissue - sticky nature & during skinning
loose hairs - skin - adherent - S/C tissue & cannot be
removed completely. Pelvis of goat is long and narrow.
7. Hog

Carcass - pig cannot easily be mistaken - any other animal most countries - skin is left - carcass. But even when the
skin is removed there should be no difficulty in identification

DIFFERENTITATION OF CARECASSES OF FOOD ANIMALS


Differentiation of Carcasses of Horse and Ox
Carcassofthehorseandoxmaybedifferentiatedbythefollowing
details
1. In the horse - unusual length - sides is noticeable, together
withthegreatmusculardevelopmentofthehindquarters.
2. The thoracic cavity is longer in the horse; this animal
possesses18pairsofribs,whereastheoxhas13pairs.
3. Theribsinthehorsearenarrowerbutmoremarkedlycurved.
4. Thesuperiorspinousprocessesofthefirstsixdorsalvertebrae
are more markedly developed in the horse and are less
inclinedposterior.
5. In the forequarter, the ulna of horse extends only half the
lengthoftheradius;intheoxitisextendedandarticulateswith
thecarpus.
6. In the hindquarter, the femur of the ox possesses no third
trochanter; the fibula is only a small pointed projection, butin
thehorseitextendstwothirdthelengthofthetibia.

7. In the horse the last three lumbar transverse


processesarticulate-eachother-sixtharticulating-
similarmannersacrum-donotarticulateintheox.
8. The horse carcass shows considerable development
of soft yellow fat beneath the peritoneum, especially
in the gelding and mare, but in the stallion the fat is
generallyofalightercolourandalmostwhite.Inthe
ox the kidney fat is always firmer, whiter and more
abundantthaninthehorse.
9. Horseflesh is a dark red, initially brown or reddish
brown on exposure to atmosphere the colour turns
bluish.Marblingisabsentinhorsemeat;itisfirmbut
sticky in nature due to high glycogen content.
Horsemeat has a pronounced sweet taste, repulsive
odour and well defined muscle fibre. Beef lack the
bluishtinge.

CHEMICAL METHODS OR TESTS


Consistofthedeterminationof
Thecontentofglycogeninflesh,
Thepercentageoflinoleicacidinfat,
The amount of iodine absorbed by unsaturated fatty acids in fat
and
Therefractiveindexoffat.

1. Test for Glycogen Content of Meat


Horsefleshisricherthanthefleshofotherfoodanimalsinglycogen.
Inhorsemeatglycogenisfoundinlargequantitiesirrespectiveofage.
Horse0.5to1.0%
Beef-0.0to0.5%
Porkandmutton-nil.
Disadvantages
1. Thefleshshouldbetestedforthecontentofglycogensoonafterthe
slaughterasitdisappearsfromthefleshquickly.
2. Liver of all food animals especially pig liver contains more glycogen
whentheyareusedinsausagemakingitgivesahighpercentage.So
caremustbetakenininterpretationofresults.

2. Linoleic Acid Test


Identification of horse fat when admixed with beef or lard or suet -
demonstratingthepresenceof1to2%oflinoleicacid
Otheranimalfats-notpresentinproportionhigherthan0.1%.
3. Iodine Value
Based - amount - iodine absorbed - unsaturated fatty acids present - fat &
varies-differentanimals.Theiodinevaluesofanimalfatsare
Horsefat-71to86
OxFat-38to46
SheepFat-35to46
PigFat-50to70.
The iodine value of ox and sheep are to some extent found to be closely
identical.
Hence,itisnotpossibletoconfirmthetypeofmeatlegally.
4. Refractive Index
Fatisliquefiedbyheatandthusconvertedintooilanditsrefractiveindexis
estimated.
Allliquids&oilspossessspecificrefractiveindex&estimatedbytheaidof
refractometer.
Horsefat-53.5
Oxfat-notabove40
Pigfat-notabove51.9

BIOLOGICAL METHODS
Fool-proofmethodsinjudicialenquiry
Applicabletominceorsausagemeat
Precipitation test
Mostvaluableone-based-development-certainantibodies-bloodserumfrom
anotheranimal.
Blood serum - specific immunized rabbits mixed - test tube - filtered extract -
suspectedmeat.
Turbidity - solution first occurs - specified meat - present & followed - definite
precipitation.
Precipitationtestisaproteinreactionisofvaluefordetectionofflesh,organfat
orintestines.
Greatestvaluewithrawmeat-ifmeat-wellboiledorfried-impossibletoextract
proteinfromit
Complement fixation
Utilizes - normal component - serum complement - thermolabile mixture -
substancescapable-reacting-anyantigenantibodysystemorlyses-antigen
agglutinated-antibody.
Certain antigenantibody reaction - not produce any visible result e.g.
precipitation,agglutination-detected-factthattheyuseorfixcomplementthus
making-nolongeravailableforotherreactions
Complement - derived from guinea pig serum & indicator system normally
consists of sheep red cells and rabbit serum (heated to destroy its own
complementbeforeuseinthetest).

ELISA
InELSIAtechniquerelies-abilityofantibodiestobindwithspecificantigens.
Used-bloodgroupingtissuestypingandbacteriologicalworktheELISAtest
-applied-meatidentificationbecause-differencesinproteincomposition-
foodanimalsaregenerallyslight.
ELISA-applied-detectionofboartaintandsoyainavarietyofproducts.
An extract (containing antigens) made from the meat sample under
investigation-placed-petridishortesttube&addingdifferentspecificsera
(containingantibodies).
Antibody - binds - corresponding antigen forms a complex - afterwards
recognizedandbound-secondantibodycontaininganenzymecatalyses-
reaction-produceacolour.
If-horsemeat-reactionpresence-specifichorseserum.
Positive identification - carried out - short time - 6 h & able to detect low
levelsofsuspectmeats,-aslowas3%.
Diagnostic kit - routine use - commercial practice is available - uses
polystyrene tubes and gives a marked colour reaction in positive cases &
colour-moreaccuratelymeasuredusingaspectrophotometer.
Possibilityofadaptingthetestforcookedsamples-beinginvestigated
Other techniques - species identification include electron microscopy,
electrophoresis, various chemical procedures, agar gel diffusion and
gas-liquid chromatography.

Enzyme Immunoassay
Enzyme Linked Immunosorbent Assay (ELISA)
Enzyme assay
Succinic dehydrogenase - enzyme available in specific or fixed
quantities in different species. Assessed in enzyme assay. Semi
quantitative determination of antigens by simple immunodiffusion is
only possible by subjective comparison of results between various
dilutions of sample extract and those of standards; more objective
data can be derived from more sophisticated immuno precipitation
techniques. Enzyme-linked immunosorbent assay (ELISA) is one of
several powerful quantitative immunological procedures, which are
not monitored by precipitation. Here the antibodyantigen interaction
occurs in a monomolecular layer immobilized on an inert surface and
is followed by means of an enzyme chemically bonded to one of the
immuno reagents. ELISA is a well-established technique, particularly
in clinical laboratories where it provides rapid analysis of biological
fluids and tissues for many antigens and antibodies both in qualitative
screening and quantitative diagnostic procedures. In food control
laboratories, it has been applied with some success to the
determination of soya protein in meat products, which serves as a
model for all food proteins..

ELISA has therefore been applied to species recognition by choosing


species specific proteins; in particular, the blood proteins tested by
immunodiffusion have been demonstrated to be appropriate for uncooked
samples in several laboratories using somewhat different ELISA
procedures. The initial immobilized species may be the standard itself or
the antibody. While qualitative results can be obtained in a few hours,
attempts to interpret quantitative date in terms of individual meat species
have not been successful evidently the residual blood levels are too
variable to correlate with the corresponding meat. Nevertheless, the
specificity of immunological recognition implies that more suitable
speciesspecific or organspecific antigens may be made a future basis
of both source identifications and level of determination of meats and
offals.
ELISA - employed - study biological fluids & tissues for many antigens and
antibodies both in qualitative and quantitative diagnostic procedures.
Recently - important tool in biochemical research on meat speciation
Interaction - antigen and antibody occurring in a monomolecular layer
immobilized - inert surface (polystyrenes; solidphase) made this procedure a
rapid and convenient method of determining antigens of meat foods.
Three forms of ELISA - meat speciation - recognize homologous species
proteins indirect.
Indirect ELISA, Double Antibody Sandwich (DAS) and Competitive ELISA.

Indirect ELISA
This technique involves the applications of speciesspecific antibodies on to the proteins (test antigens)
coated onto the plastic surface of micro titre plates.
The recognition of antigens results in the formation
of antigen antibody complexes, which are detected
by either enzyme linked anti-IgG or proteinA
(antibodydetector)
producing
visible
colour
reaction with added substrate. The colour intensity
is measured objectively at a specified wavelength in
a micro ELISA plate reader as absorbance values.
This is an adequate basic system for presumptive
meat speciation of about 3.0 per cent adulteration.

DAS ELISA - immunometric ELISA


This form of ELISA (immunometric ELISA) requires two
speciesspecific antibody reagents. The plastic surface of
the micro titre wells is pre-coated with mono specific
antibodies to capture antigens from sample extracts during
two or three repeat incubations. The second (detector)
speciesspecific antibodies are then applied to from the tophalf of the sandwich. The detector antibodies (enzyme
conjugated antibody, anti-IgG or proteinA) subsequently
produce colour with added substrate. The DASELISA has
improved specificity and sensitivity and is employed to
detect 0.5 percent adulteration of meat. The commercially
available Australian checkmate system, distinguished closely
related species like the cattle/ buffalo, sheep/goat or
horse/donkey.

Competitive ELISA
The third variant of ELISA is the competitive form. This form
involves the application of pre-incubated meat extracts (test
antigens incubated with known amount of specific antibodies
is PBST) on to the antigen-coated plates. Pre-incubation
sensitizes only homologous antibodies and will still be free
to interact with the plate-bound antigen. Subsequently, the
assay is completed in the usual way by adding enzymeconjugated anti IgG and substrate for the colour
development.
In this form of ELISA, the maximum colour development
indicates negative response as against the positive with little
colour. This is quick and sensitive form of ELISA provided
the anti-species antisera are made mono specific, since the
separate antigen-antibody interaction is at the monovalent
level, which obeys the laws of mass action where
affinity/avidity considerations are important for specificity.

Isoelectric Focusing
pH at which proteins will be precipitated is the isoelectric point - also species
specific.
Electrophoretictechnique-utilizes-chargesurface-protein-drive-througha
gradientgel.Polymericcompounds-ampholytessetup-pHgradient.
Proteinsapplied-gelreachesapointwheretheirsurfacecharges-neutral,the
isoelectric point. Subsequent fixing makes the protein precipitated and fixed -
samepointasbands.
Pattern of protein bands thus resulted for a particular meat extract is species
specific.
Nature - pattern specificity - protein bands - utilized - identify meat species -
determining-location,densityandarea-bands.
Widerangeofanimalspecies-identified-cookedmeatsbystainingisoelectric
focusing gels - band pattern of the enzyme adenylate kinase (AK) & creatine
kinase(CK)providedthemeat-notheatedabove120C(AK)or105C(CK).
Electrophoretogram - heated horsemeat & beef persisted - feature
differentiation
Proteinsalmostlost-configurationmakingmixedspeciesinterpretationdifficult.
IEF patterns - cooked meat mixtures - more complex & make specific
interpretationdifficult.

ELECTROPHORETIC TECHNIQUES
Apowerful technique - protein separation - characteristic band
patterns - supporting gel - differentiate meat species - basis -
electrophoreticmobility(agargelelectrophoresisAGE,starch
gel electrophoresis SGE or polyacrylamide gel
electrophoresisPAGE),isoelectricfocusingIEF(ampholine
gelsIEF PAGE or SDS-PAGE) or bothmobility and PI (two
dimensionalPAGE)ofproteins.
Sodium dodecyl sulphate PAGE (SDS PAGE)
Variant PAGE - used - separating protein sub-units &
determining-molecularweights.
Polypeptide dissociated - proteins (by heating) bind - SDS &
SDS-polypeptidecomplexeshavingsamecharge/massration
- subjected - sieving polyacrylamide gel migrate according -
molecularweights-polypeptides.
SDS PAGE permitted identification of meats in meat mixture
andincommercialsausages.

Two-dimensional electrophoresis

Utilizes two completely independent parameters


(PI & molecular weight) - separate complex
mixturesorproteins.
First dimension - electro focusing - performed -
ampholinePAGEplate
Stained strip containing proteins - interest in
incubated in buffer having SDS & laid directly -
moulded trench - SDSgradient gel for second
dimensionseparation.
In - 2-D proteins - further separated (high
resolution) - basis - sub-unit molecular weight -
permittingidentificationbetweenproteins-similar
isoelectricpoint.

Immobiline gels and Immunoblotting


Technique - electro focusing - immobiline gels -
complementary to traditional electro focussing methods
using carrier ampholytes - immobilines are acrylamide
derivatives designed to co-polymerise with gradient is
based-relativeconcentrationsoftheimmobilineateach
positions.
Covalently bound immobiline gradient (narrow pH
gradient) allows higher resolution than the conventional
carrierampholyte-basedpHgradients.
Lowconductivity-gelpermits-application-highvoltage
&pHgradientnotaffected-longfocusingtimes.
Diffusional or electrophoretic transfer of focused proteins
from-polyacrylamidegelontonitro-cellulosemembranes
- order - make them accessible - identifying reaction -
termedimmunoblotting.

IMMUNOCHEMICAL METHODS
Homologous responses of coctoprecipitins (antibodies against
cooked/heatdenaturedproteinsofmeat)-assessedbyvarious
immunochemical techniques - microhaemagglutination,
immunodiffusion
(agar
gel
diffusion-AGID),
rocket
immunoelectrophoresis&counterimmunoelectrophoresis(CIE)
& enzyme immunoassays (different formsof ELISA) recognize
thespeciesoforigin.

Antiserum
Biologicalproductraised-phylogenticallydistantanimalagainst
-specificprotein(immunogen).
Containsantibodiesderived-lymphocytes.
Large number - different & distinct lymphocytes - stimulated -
responses-antigenicsites-immunogen.
Consequently antisera containngi a heterogenous mixture -
different antibodies - varying specificity & affinity (polyclonal
antibodies)

Heterogeneous antiserum - high dilution may behave -


homogenous reagent since antibodies - highest affinities will
onlybereacting.
Homogeneity - antiserum - increased - purification (direct
absorption) - cross-reactions, affinity chromatography &
inhibitionbyblockingandconcentration.
Homogenic antisera containing monospecific antibodies -
variedapplications-immunoassays-detect,distinguishquality,
sensitizeorneutralizehomologousantigens.

Monoclonal Antibodies
Techniques - producing monoclonal antibodies involve
immunization mice - specific protein & harvesting - spleen
when-mouse-optimallysecretingantibodies.
Spleen - homogenized - produce - sterile cell suspension -
active lymphocytes (B lymphocytes). Fusion - lymphocytes &
myeloma cells - induced - polyethylene glycol & fused cells -
selected - unfused - HAT (Hypoxanthine Aminopteridin
Thyronidine)medium-culturesonlyhybridomas.

o Survivinghybridomas-platedout-agar&cultured-givesingle
cellsclones.
o Clones - cultured & cultured medium - tested - presence -
antibodies.
o Positiveclones-recloned&cultured-furtherinvestigation
o Selected hybridoma cell culture - derived form a single cell -
antibodysecreted-predefinednature&specificity.

Immunodiffusion
Agar gel immunodiffusion
Ouchterlonys(1994)methods-doublediffusioninvolving-use
- agar coated plates - wells - both antigens & antibodies -
employed-recognizehomologousantigens.
Known&unknownsamples-meat&mixture-meat-different
dilutions-testedagainst-raisedantisera.
Reactants diffuse - gel & positive reaction - indicated -
formation - immuno precipitates (opaque lines) - point -
equivalence-eachantigen-antibodypair.

Counter Immunoelectrophoresis (CIE)

Principle-technique-similartoAGID
Diffusion of antigen and antibody is facilitated
bytheapplicationoflowvoltagecurrent.
Electro-osmosis created - alkaline agar gel
changes - charge on gamma globulins
(antibodies)&movesthemtowardscathode.
Meat proteins moving towards anode produce
precipitinlines-point-equivalence.
Samples-meatdilutedupto1in300-readily
detected-CIE&utilized-detectlowlevelsof
adulteration

Laurels Rocket Immunoelectrophoresis (RIE)

Assay - migration - antigens - electrophoretic


field - gel containing antibody - basis - electro
immuno diffusion or sometimes called electro
immunoassay.
Agarose containing - antibody - poured - give -
gel-uniformthickness.
Wells - punched out - gel at one end - various
samples migrated - gel & formed precipitation
linesample-characteristicrocketshapebase
-height-antigen.
Greater concentration - antigen will migrate
further - gel before reaching a point of
equivalence.