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
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.
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.
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
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:
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
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.
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
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..
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.
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
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)
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.
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