Speciation of meat

INTRODUCTION
 Speciation of meat is the differentiation of meat of species.
The differtiation of meat of species wise is important to
prevent and control the adulteration of meat. Adulteration
of meat is the bad practice, which involves substitution or
mixing of flesh of cheaper variety with other meat, for over
use of bad money.
 The substitutions that may be practiced are, that of
horseflesh for beef, mutton for Chevon, mutton for venison,
beef for mutton and occasionally the flesh of the cat for that
of hare or rabbit.
 It is not difficult to differentiate the flesh and fat of these
animals in the carcass form or in joints by means of
anatomical conformation.
 But, their recognition in minced meat or their products
depends on chemical and biological tests.

METHODS OF DIFFERENTIATION

 By three methods.
o Physical

o Chemical tests and

o Biological tests.

 These methods are also classified as follows,

Physical
 Carcass shape, meat and fat colour
 Anatomical differences
  Refractive index
Chemical methods
 Glycogen content
 Iodine value
 Lenoleic acid content
 Fatty acid pattern,
 Histidine di-peptides and
 Direct probe mass spectrometry.
Biological tests
 Immunological techniques
o Agar Gel Immuno Diffusion (AGID)

o Counter Immuno Electrophoresis (CIE)

o Enzyme Linked Immuno Sorbent Assay (ELISA)

 Electrophoretic Techniques
o Polyacrylamide gel electrophoresis (PAGE)

o Polyacrylamide gel isoelectric focusing (PAGIF)

 Novel methods
 DNA analysis , PCR and gene probes.

PHYSICAL METHODS
 CHARACTERISTICS OF THE MEATS

Meat Colour Consistency Odour

Mutton Pinkish Firm Faint
Goat meat Pale red Firm Goaty
Beef Brisket red to red Fairly firm -
Buffalo meat Red to dark red Firm -
Pork Pinkish grey Soft Urine like
Veal Pale to pink Not firm -
Horse Dark red Soft Sweetish
Poultry White Firm -
CHARACTERISTICS OF BODY FAT
Meat Colour Consistency Unique features
Mutton White Fairly firm Inter muscular fat
present
Chevon White Inter muscular fat absent
Beef Yellowish Very firm Inter muscular
White fat present
Buffalo Pure white Firm -
Pork White Soft and S/C and Intramuscular
greasy fat
Veal White Firm Negligible fat content
Horse Yellow soft and No inter muscular fat
greasy
Poultry Yellow Loose
Mostly in the abdominal
cavity and in S/C area
ANATOMICAL VARIATION

Dentition
  Species Temporary Permanent
 Cattle, Sheep, Goat 2 (0 0 3 0) = 20 2 (0 0 3 3) = 32
 and Buffalo (4 0 3 0) (4 0 3 3)
 Pigs 2 (3 1 3 0) = 28 2 (3 1 4 3) = 44
 (3 1 3 2) (3 1 4 3)
 Horse 2 (3 0 3 0) = 24 2 (3 1 3 3) = 40
 (3 0 3 0) (3 1 3 3)
 Dog 2 (3 1 3 0) = 26 2 (3 1 1 2) = 32
 (3 1 2 0) (3 1 2 3)
 Cat 2 (2 1 3 0) = 24 2 (3 1 3 1) = 30
 (3 1 2 0) (3 1 2 1)
Vertebral column
Species Vertebral column
 Cattle, Buffalo C7 T13 L6 S5 Cy 18 to 20
 Sheep, Goat C7 T13 L6 S4 Cy 15 to 18
 Horse C7 T18 L6 S5 Cy 15 to 21
 Pigs C7 T14-15 L6-7 S4 Cy 20 to 23
 Rabbit C7 T12 L7-8 S3-4 Cy 14 to 20
 Chicken C15-17 T7 L+ S14 Cy 5 to 6
Paired ribs
 Species Ribs in pairs Sternal ribs
 Cattle, Buffalo 13 8
 Sheep, Goat 13 8
 Pigs 14-15 7
 Horse 18 8
 Dog 13 9
 CHARACTERISTICS OF MEAT AND FAT FROM
DIFFERENT SPECIES

Beef
 It is light red in young and dark and coarser in older
animals due to testosterone secretion.
 Intramuscular fat (marbling) is well marked. In old bulls
the dried surface of meat appears darker.
 Fat is yellowish in colour due to carotene pigment and is
firm in consistency.
 In older animas the fat tends to be more yellowish and
loose in consistency.
Veal
 In very young calves are pale in colour, watery in
consistency and fat is white in colour and jelly like.
Carabeef (buff)
 Generally buffalo meat is darker (more reddish brown) and
the fibers are coarser and looser in structure than beef.
 The odour of the buffalo meat and fat resembles that of
musk and if boiled in strong acidified (H2SO4) water, it
develops a disagreeable odour similar to that of cattle
manure.
 The fat of buffalo is strikingly white and drier and less
sticky than in cattle.
Mutton
  Mutton is light red in colour with fine firm muscle fibers
with little or no marbling.
 Fat is deposited in the fat depots and is white in colour,
odour less, firm.
 Feathering, a characteristic phenomenon of fat distribution
in intercostal muscles is a feature of mutton carcass.
Chevon
 Chevon is almost like mutton but pale in colour with no
marbling but depot fat is marked white colour and has
goaty odour.
 Kidney fat is abundant. Goat meat is more lean, dry and
firm and easy to cook.
Pork
 It is light to dark in colour, varying from whitish gray to
red, less firm in consistency and has a strong boar odour.
 Subcutaneous fat deposition is predominant which white in
colour and greasy in texture.
 Pork becomes nearly white on cooking while other meats
become darker.
Chicken
 Meat of a broiler chicken is pale brown in colour fat is
greasy and shows marbling.
 The subcutaneous fat is abundant in broiler than desi type.
Meat gives a pleasant odour.
Turkey meat
  Meat is more pinkish than broiler poultry and has a pleasant
meaty odour. Dressed turkey has more breasts and less
thigh meat than other table birds.
Rabbit meat
 Farm-raised rabbit is lean, slightly sweet meat with a
closely textured flesh that has virtually no fat and is very
high in protein Rabbit meat does not have a very strong
flavour.
 It is comparable to, but not identical with chicken.
Tenderness varies with muscle age, and depends on
changes in the proportion and type of connective tissue
supporting the muscle fibers.
 The younger rabbits are slaughtered, the tender the meat
will be. On the other hand, flavour tends to develop with
age.
Horse meat
 Horseflesh is dark red or even bluish in colour and has
more fascia (due to more exercise) with sweet or somewhat
repulsive odour, which develops rusting on keeping.
 There is no marbling and fat is yellow and greasy in
character.
Deer meat or Venison
 In deer, the subcutaneous layer of fat is not as well
developed as in sheep.
 The meat is poor in fat and possesses the odour of venison
which is easily distinguishable from the odour of sheep.
Dog meat
 The colour of dog meat is very dark than pork and is easily
made out in cooked meat.
 The muscles of the dog are smeary and the fat is more oily
than hog fat. The odour of the dog meat is repulsive.
See excel sheet
DIFFERENTIATION OF CARCASSES OF HORSE AND
OX
DIFFERENTIATION OF CARCASS OF SHEEP AND
GOAT

CHEMICAL METHODS

 Meat composition
o The intra muscular fat content in mutton is higher

(13%) and the least is buffalo (1.0%). Beef contains

2.5%, Goat 3.5% and Pork 4.5%.
o Vitamin A content is significant in beef and mutton

but highly insignificant in buffalo meat, Chevon and

pork.
o The glycogen content is high in Horse, by at least 10

times more ( compared to other food animals).

o Myoglobin content in horseflesh is maximum (0.7%)

(compared to other food animals).

 Body fat composition
 o Refractive index – The refractive index value for horse
fat is 53.5 when in pork fat it is less than 50 and beef
fat it is less than 40.
o Iodine number – Horse fat (70 to 85) is the highest
compared to lard (50 to 70), ox and sheep fat (at 35 to
46).
o Carotene content in buffalo fat is zero.
o Linoleic acid content – horse fat contains 1 to 2%
linoleic acid where as all other fat contain less than
0.1%.
o See word file (landscape table) horse vs cattle meat
and sheep vs goat meat

BIOLOGICAL METHODS - ELECTROPHORESIS

This is a powerful technique of protein separation as

characteristic band patterns in a supporting gel, is used to
quantify meat species on the basis of electro phoretic
mobility.
o Starch gel electrophoresis: Separation of proteins on
the basis of their mobility in a supporting gel of starch
at a constant pH and electrical field is known as starch
gel electro phoresis.
o Isoelectric focussing: It is an electrophoretic
technique, which utilizes the change at the surface of a
protein to drive it through a pH gradient gel.
o Polymeric compounds, the ampholyte, set up the pH
gradient.
o The proteins applied on the gel reach a point where
their surface charges become neutral, the isoelectric
point. Subsequent fixing makes the proteins to get
 precipitated and fixed at the same point as bands, this
pattern is species specific.
o By determining the location, density and the area of
the pattern specific bands, the meat species can be
quantified up to the extent of 10% pork in heated
mixture of beef and pork.

IMMUNOCHEMICAL METHODS

 The homologous response of coctoprecipitins (antibodies

against cooked/heat denatured proteins of meat) have been
assessed by various immunochemical techniques such as,
o Immunodiffuson (Agar gel Immunodiffusion-AGID

and dry disc test).

o Immunoelectrophoresis (Counter

immunoelectrophoresis-CIE and rocket

immunoelectrophoresis) and
o Enzyme immunoassays (different forms of ELISA).

BASIC PRINCIPLE

 The basic principle in these methods of meat species

identification is the reaction of the homologous meat
extract (antigen) with specific antiserum raised against the
particular species meat.
Raising of antisera
 Freeze dried water extracts of skeletal muscles are to be
reconstituted in physiological saline to get 75 mg/ml
solution.
  This has to be emulsified with an equal volume of Freunds
complete adjuvant. For immunization, healthy rabbits of 4
to 6 months of age (1.25 to 1.50 kg) are to be used.
 The antigen and the adjuvant mixture should be
administrated intramuscularly in the hind legs.
 This should be followed with booster doses of antigen but
with Freunds incomplete adjuvant at weekly intervals for
three weeks.
Assessing the potency of antisera
 Separate the serum from 3ml of blood collected from the
immunized rabbits one week after the last injection to
assess the potency of the antiserum by interfacial ring test.
 Here the known antigens are layered carefully over the
antiserum in precipitin tubes to form two distinct layers.
 When homologous response is rapid (white precipitate
formation at the junction of two layers following 30 min
incubation at room temperature) and the heterologous
response is absent (no precipitation even after 2 hr), the
antiserum can be considered to be of good titer.
Harvesting of antiserum
 The blood collected from the immunized rabbits should be
allowed to clot at room temperature and placed at 4ºC for
18hrs.
 Then the antiserum should be decanted and centrifuged at
3000 rpm for 10min to remove the remaining blood cells.
 Merthiolate should be added to this to get a concentration
of 1:10000 and the same should be stored at –20ºC.
  By tube agglutination test the antisera should be tested for
reaction with heterologous and homologous species
antigens.
 The unabsorbed antisera are to be rendered species specific
by absorption of heterologous antibodies.
 Absorbed antiserum can be prepared by adding small
amount (about 8.0 mg/ml) of freeze dried antigenic proteins
of the species with which they cross react.
 The mixture is mechanically shaken for 4hrs at room
temperature can be held overnight at 4ºC. later the same
should be centrifuged at 3000 rpm to eliminate antigen-
antibody complex formed.
 The species specific antiserum thus obtained can be used
for species identification of meats by serological
techniques.
Preparation of antigens
 Initially 100g of sample of meat should be minced
thoroughly for two minutes, then mixed in 100ml of normal
saline solution and refrigerated for 12 hrs to facilitate the
separation of maximum quantity of meat proteins.
 The decanted extract then should be filtered through
Whatman No.4 and stored under refrigeration.
 This would be useful for AGID, CIE and dry disc
immunodiffusion tests.

METHODS

Agar Gel Immunodiffusion (AGID) or Agar Gel Precipatation

Test (AGPT)
  By this method known and unknown samples of meat and
meat mixtures can be tested against the raised antisera.
 The two reactants diffuse in to the gel during 24 hr of
incubation at 37ºC and a positive reaction is indicated by
the formation of immunoprecipitates (opaque lines) at the
point of equivalence for each antigen-antibody pair.
 Research have reported detection of chicken flesh at 1, 3
and 5% levels in heated beef sausages following this
method.
 The AGID (or AGPT) is a sample test but long duration of
incubation (of 24 to 48 hrs) is a disadvantage.
Dry disc test
 It is based on the principles of AGID. Antigens and
antibodies are absorbed on to the filter paper discs and
freeze dried for stability.
 Known anti species anti body discs and reference meat
discs should be placed on the agar surface of the petri
dishes.
 Blank sample discs are to be soaked in drip/extracts of meat
samples and gently deposited on parallel test positions.
 These petridishes are to be incubated overnight at room
temperature for precipitation lines.
 The tests for fresh meat are called as Overnight Rapid
Bovine Identification Test (ORBIT), Poultry Rapid
Overnight Identification Method (PRIME).
 The detection level of adulteration is reported to be 3 to
5%.
Counter immunoelectrophoresis (CIE)
  The diffusion of antigen and antibody is facilitated by the
application of low voltage current.
 In addition, the electro osmosis created by the alkaline agar
gel moves the gamma globulins (antibodies) towards
cathode.
 The meat proteins moving towards anode produce immuno-
precipitates with homologous gamma globulins at the point
of equivalence.
 Adulteration at 1:300 dilutions can also be detected by CIE.
The main advantage of CIE is its rapidity (just 1hr) and
high sensitivity, compared to AGID.
Rocket electrophoresis
 The protein assay by the migration of antigens in an
electrophoretic field in to a gel containing antibody is the
basis of electroimmuno diffusion referred to as
electroimmunoassay.
 Agarose containing an antibody is produced to get a gel of
uniform thickness.
 Wells are punched out of the gel at one end in to which
various samples can be introduced.
 On electrophoresis the various samples migrate in to the gel
and form precipitation line and each sample will have a
characteristic rocket shape of lines and the height of rocket
would be proportional to the concentration of the antigen.
Enzyme immunoassay
 Enzyme linked immuno sorbent assay (ELISA) can be
employed in the diagnostic procedures to study both
 qualitative by as well as quantitatively the antigen and
antibodies.
 The interaction of antigen and antibody occurring as a
monomolecular layer immobilized on an inert surface
rendered this procedure a rapid and convenient method of
determining antigens of meat food origin. Three forms of
ELISA can be employed in meat speciation to recognize
homologous species proteins viz., Indirect, Double
antibody sandwich (DAS) and Competitive ELISA.
o Indirect ELISA
 It involves the application of species specific

antibodies and test proteins coated on to the

plastic surface of microtiter plates.
 The antigen-antibody complexes are detected by

either enzyme linked anti IgG or Protein-A

(antibody detector) producing visible color
reaction with added substrate.
 The color intensity when measured objectively at

a specified wavelength in a micro ELISA plate

reader as absorbance values quantifies the results
which can detect 3% adulteration.
o DAS ELISA
 This immunometric ELISA requires two species

specific antibody reagents. In this the plastic

surface of the microtiter wells is precoated with
monospecific antibodies (capturing antibodies) to
capture antigens from test samples during two or
three repeated incubations.
  The second (detecting antibody) species specific
antibodies are then applied to form the top half of
the sandwich.
 This enzyme conjugated antibodies, anti IgG or
Protein-A, subsequently produce colour with
added substrate.
 The DAS ELISA has improved specificity and
sensitivity and can conveniently detect
adulteration even at 0.5%.
 DAS ELISA is the most suitable type of ELISA
for detecting adulterated meat.
o Competitive ELISA
 This form of ELISA involves application of pre

incubated meat extracts (test antigens incubated

with known amount of specific antibodies) on to
the antigen coated plates.
 Pre incubation sensitizes only the homologous

antigens and hence the antispecies antibodies will

still be free to interact with plate bound antigens.
 Subsequently addition of conjugated anti IgG and

substrate brings about colour development.

 Electrophoretic techniques such as SGE and IEF-

PAGE and Immunochemical methods viz.,

AGID, dry disc test, CIE, Rocket IE and different
forms of ELISA can be successfully employed to
detect various levels of adulteration in meats and
meat products.
There is variation in anatomical and organoleptic (pertaining
to chemical) characteristics in meat of different species of
food of animals . Some of the salient features are:
Organ Ox Sheep/goa Pig Horse Dog
t
Tongu Black , Longitudi Tip is Spatula Media
e prominen nal grove comparativ shaped n
ce on present on ely grove
dorsum , the center rounded, no presen
filliform of the dorsum t, no
papillae tongue dorsu
present m
Liver Reddish Same as 5 lobes 3 lobes 5
brown 3 cattle but no gall lobes
lobes lobe is less bladder which
right and pointed are
left and divide
caudal. d,
Lobes gall
pointed bladde
r in
the
middl
e lobe
Splee Elongate Triangular Bean Flat or Sickle
n slightly shaped curved shape
convex sickle d
shaped
Kidne Lobulate Smooth on Bean Right Bean
y d on the the surface shaped , not triangul shape
 surface not lobulated ar and d flat
lobulated . ,flattened left and
Bean bean thick
shaped shaped
smooth
on the
surface
Organ Ox Sheep/goa Pig Horse Dog
t
Lungs Trachea As in Left has 2-3 Left 2 Left 3
has a left cattle lobes lobes lobes
lung has and and
3 lobes right 3 right
right 4-5 lobes 4-5
lobes lobes

a. Organoleptic(w.r.t.chemical) qualities of different meats (

physical and chemical properties )
b.
s. Quantiti Buffal Cattle Shee Goa Pig Horse
n es o( p t
o beef
/kara
beef)
Fat
1 Firm Firm Mode Fir Finely Soft
1 a) Co and and rately m granul greas
nsis white yello firm and ar soft y and
ten wish white whit and yello
cy e pure wish
and white
 col
or
b) Iodi 38-46 35-45 35- 50-70 71-86
ne 45
val
ue
c) Ref <40 41.5 41.5 <51.95 53.5
ract
ive
ind
ex
c. Ma Less Well Lit No
No No
rbli mark tle mar
marbli marbl
ng ed or blin
ng ing
no gsubcut
aneous
fat
deposi
tion on
feather
ing
2 Meat Dark Light Light Dar Boary Dark
color red red in to k odour red
fine youn dark pink even
textur g and pink goat bluish
ed dark fine yod in
less color musc our colou
tender in old le r
ed firm
palata musc
ble le
 and odour
less -
light less
red in
young
juicy
3. Carcass Less More 45- 45- 70- -
yield due to 45- 55% 55 80%
more 60% %
weigh
t of
hide
4. Lean Less More More
meat
a. Per
cen
tag
e
b. Dre
ssin
g
cut
Acceptib
5 Less More More Mor More Less
ility
. e
5

Composition of different meat

Table ( per cent basis)

 Content Beef Lamb Pork Broiler
Water % 60 55.8 42 71.2
Calories 273 317 457 151
Protein ( 17.5 15.7 11.9 20.2
gm)

Fat ( gm) 22.2 27.7 45 7.5

Ash ( gm) 0.9 0.8 0.6 1.1
Ca (mg.) 10.9 7.0 14 -
P ( mg) 150 157 117 200
Fe ( mg) 2.6 2.4 1.8 1.5
Thiamine 0.08 0.4 0.58 0.08
( mg)
Riboflavin 0.16 0.2 0.14 0.8
( mg)
Niacin 4.2 4.5 3.1 10.2

End of lecture dated 27-10-18 2019