 DNSA :

This test primarily identifies C=O group hence is useful in

determining the presence of reducing sugars. 3, 5 dinitrosalicylic
acid (DNSA) is a aromatic compound that condenses with reducing
sugar like glucose in alkaline conditions. Glucose being a reducing
sugar reduces 3, 5 dinitro salicylic acid to 3 amino 5 nitro salicylic
acid and it itself gets oxidised to gluconic acid. DNSA is yellow in
oxidised state and orange- red in reduced state, this colour shift is
observed as the reaction progresses. The intensity of color is
directly proportional to the concentration of reducing sugar. The
optical density is measured at 530nm.

 Resorcinol:
The reagent Resorcinol is a phenolic compound. Generally ketoses
are more rapidly dehydrated than aldoses, this is the base of this
experiment. First ketose like fructose, is dehydrated into
hydroxymethyl furfural by a strong acid like HCl. This derivative
reacts with resorcinol in a series of condensation reaction to
produce a deep cheery red coloured complex. Thio-urea assists in
stabilizing this colour. This reaction is specific for fructose, the
intensity of colour is directly proportional to the concentration of
fructose. The optical density is measured at 530nm.

 Phenol H2SO4:
Phenol sulphuric acid can aid in detection of virually all classes of
carbohydrates, it can also help in identifying all glycopolymers and
glycol-conjugates. It is a simple and rapid colorimetric method. A
polyhydroxy aldehyde like glucose undergoes dehydration in the
presence of concentrated mineral acid like H2SO4. This
dehydration results in formation of 5 hydroxymethyl furfural,
which reacts with phenol and forms bright coloured complex. The
intensity of colour is directly proportional to the concentration of
glucose. The optical density is measured at 470 nm.

 Anthrone method:
This method can be used to identify saccharide moiety present
in any given sample. it is a simple and rapid colorimetric method
for identification of mono, di and polysaccharides. First the
polysaccharides are hydrolysed to their monomeric forms.
Dehydration of these monosaccharides forms hydroxymethyl
furfural, which reacts with the Anthrone which is a tricyclic
aromatic ketone, to give a bluish-green coloured complex. The
optical density is measured at 620nm.

 Isolation of starch from potato:

Starch is a homopolysaccharide made of repeating units of alpha-
glucose monomers liked by alpha 1 -> 4 glycosidic linkage . It
consists of amylose and amylopectin. It is main storage
polysaccharide in green plants. Isolation of starch form potato
began in the early 5os of the last century. Potato starch is a fine,
white powdery substance that contains properties like hight water
binding capacity and low gelatinization tenperatios and a tendency
to form relatively clear viscous pastes hence, it is used in textile,
petroleum mining, feed and food industries. It is insoluble in cold
water and it is slurry is a classic example of non- Newtonian fluid.
Isolation of starch, its characterization and its confirmation can be
done by performing various tests like solublity test, iodine test and
enzymatic test.
 Biuret test :
Biuret is a compound formed by heating urea at 180*C. the ma,e
of the test comes from the reagent used in it which contains 1%
solution of CuSO4. In alkaline condition the Cu2+ ions present in
the reagent forms complex with the peptide bond, and a purple
coloured cupric potassium biuret complex is formed. The reason
behind the colour is formation of chelate complex of copper co-
ordination complex. This reaction is positive for molecules that
have more than 2 peptide bonds. The intensity of colour is
directly proportional to the number of peptide bonds. The
optical density is measured at 530nm.

 Lowry’s method:
The principle behind this method lies in the reactivity of
nitrogen in peptide bonds to the copper sulphate in alkaline
condition and the subsequent reduction of Folin-Ciocalteay
phosphomolybdic or phosphotungstic acid to
heteropolymolybdenum blue by copper-catalysed oxidation of
aromatic amino acids or phenolic compound. The intensity of
colour is directly proportional to protein concentration. The
optical density is measured at 660nm. This method is sensitive
to pH changes hence the pH should be maintained between 10-
10.5.

 Bradford assay:
This assay is based on the absorbance shift between three
forms of Coomassie blue G-250 dye. Under acidic condition it is
in double protonated form showing red colour, upon binding to
protein it is in more stable unprotonated form showing blue
colour. The two bond interactions taking place in this assay are
as follows:
1. Red form of the dye donates electrons to the ionisable group
of the protein which exposes its hydrophobic pockets.
2. These hydrophobic pockets on the protiens non-covalently
bind to the non-polar region of the dye via Van der Waal’s
forces.
When the dye and the protein bind in acidic conditons its
maximum absorbance shifts from 465 to 595.

 Caesin-
Casein is a conjugate protein- phosphoprotein majorly found in
milk, the salt of casein that can be encountered in milk is
calcium caseinate. In pure form it is amorphous, white, solid and
odourless. It exists in 4 subtypes as alpha 1 and 2, beta and k
form. It is insoluble in water. The isoelectric point of this protein
is 4.5 and the pH of milk is 6.6, casein possess negative charge
at this point and hence is solubilized as salt. The protein begins
to aggregate and precipitates at its isoelectric point.

 Estimation of amino acid by Ninhydrin:

Almost all amino acids except proline and hydroxyproline
posses primary amine group. Ninhydrin which is originally
yellow reacts with the amine group and forms purple coloured
complex called Ruhenan’s purple discovered by Seigfried
Ruhenan. The intensity of colour is directly proportional to
number of amino acids. Optical density is measured at 530nm.
 Cholesterol:
Cholesterol is an animal sterol required for membrane
structure and formation of steroids, hormones, vitamin D,
bile acids etc. serum cholesterol estimation is an integral part
of lipid profiling as age progresses. It is soluble in strong
organic solvents like ethanol in presence of strong
dehydrating agents like H2SO4. When cholesterol reacts with
ferric chloride in presence of phosphoric acid it undergoes
intramolecular rearrangement and gives brick red colour. The
intensity of colour is directly proportional to the
concentration of cholesterol. The optical density is measured
at 530nm.

 Acid value of fat:

The acid value measures the extent to which the glycerides inn
the oil have been hydrolysed. These glycerides can be
hydrolysed by water in presence of oxygen in air and by certain
bacteria, this leads to peroxide formation and the fat becomes
rancid. During this, fatty acids are liberated and these fatty acids
can be estimated by simple titrating it with KOH. Hence, acid
value can be defined as miligrams of KOH required to neutralize
free fatty acids present in 1gm of oil. This helps in determining
the age and quality of the oil. Lower the acid value better the
quality of oil.

 Vit. C by DCPIP:
It is the titrometric assay of vit.C or ascorbic acid, based on
simple oxidation-reduction reaction. Vit.C is an important
dietary factor that is not synthesized by the body. In this the
ascorbic acid reduces 2,6 dicholorophenol indophenol dye to
colourless leuco base, it itself gets converted to anhydrous Vit. C
or dehydro ascorbic acid. The dye is pink in oxidised form which
indicates the end point.

SEM - II

 Alpha amylase:
Alpha amylase is a glycosidic hydrolase, majorly produced by the
salivary glands and pancreas. it catalyses the cleveag of of
a 1 -> 4 glycosidic bonds of starch which is a
homopolysaccharide and gives maltose, maltotriose , glucose
and limit dextrins. The activity of alpha amylase is measured by
colorimetric method using 3,5 dinitro salicylic acid reagent. The
amount of starch hydrolysed is directly proportional to the
enzyme activity.

 Temperature:
as with many chemical reactions, the rate of enzyme catalysed
reaction increases with increase in temperature. However, at
higher temperatures (which for most of the enzymes is 50*C)
the rate decreases due to denaturation of the enzymes as they
are proteins in nature, this is caused by breakage of hydrogen
bonds and change in arrangement of the active site and they
lose their function . The temperature at which maximum
velocity is reached is called the optimum temperature. The
velocity increases with increase in temperature and it begins to
drop drastically at temperature above its optimum value and
bell shaped curve is observed.

 pH and enzyme activity:

slight change in the pH may alter the shape of the enzyme’s
active site. Each enzyme shows maximum catalytic activity at a
specific pH value referred to as its optimum pH. Drastic change
is observed in the pH value above or below the optimum range.
Any change in The H+ ion concentration leads to change in
affinity, solublity and structural property. As the pH approaches
its optimum value there is a steady rise in catalytic activity but
once it passes the optimum pH there is a drastic drop observed,
this gives us a bell shaped graph.

 Metal ion:
Metal ions usually act as cofactors to certain enzymes, when
present in trace amounts these metal ions have shown to assist
in the enzyme activity. They usually work as the prosthetic
group. But, when present in larger amounts they may interfere
with the catalytic efficiency of the enzyme by reducing the rate
or all together halting the reaction. Hence when the
concentration of metal ions increases the catalytic activity is
decreased. These bind to the site other than the active site and
inhibit the enzyme from functioning.

 Amyloglucosidase:
Amyloglucosidase is an enzyme which acts primarily on starch
and glycogen to split them in to their monomeric forms that is
glucose. This enzyme is more efficient that the salivary amylase
as it can cleave both a 1 -> 4 and a 1 -> 6 linkages. The most
common source for amyloglucosidase is A. niger. The activity
can be detected by DNSA method. The action of enzyme is
directly proportional to the activity of enzyme and the optical
density is measured at 530 nm.

 Invertase:
Invertase also known as B- fructofuranosidase is named as such
because, it irreversibly hydrolyses sucrose into fructose and
glucose, it shows high activity over wide range of pH. It is
mostly found in yeast species. Its activity is given as ug of
reducing sugar/ ml/ min. The enzyme activity is determined by
DNSA method. The intensity of coloured is directly proportional
to the concentration of reducing sugar. The optical density is
measeured at 530 nm.

 Chromatography:
Paper chromatography is a planar partition chromatography
technique used to separate compounds based on the
differential solublity in the stationary phase and mobile phase.
Stationary phase usually is a water ( atmospheric moisture) this
is held within the fibers of the filter paper mobile phase is
usually some sort of volatile liquid. All of the proteins in nature
comprise of chain sequence of made of 20 different amino
acids, all these are similar but differ in their R group. These can
be sperated based on that factor. The mixture separated
mighrates at different points on the paper which can be
identified using the RF value.

 Estimation of inorganic phosphate:

Phosphorus isn’t found in nature in free form, it is usually in
conjugated form. In the Fiske- Subarao method the inorganic
phosphate reacts with the ammonium molybdate to form
phosphomolybdic acid. Amino napthol sulfuric acid or ANSA
reduces this phosphomolybdic acid to produce, deep blue
coloured complex. The intensity of colour is directly
proportional to the amount of phosphate in sample and the
optical density is measured at 660 nm.

 Saccharomyeces cerevisiae:
In order to study the activity of enzyme invertase produced by
yease called saccharomyces cerevisiae, it is necessary to first
immobilize the enzyme. It can be done by performing various
methods but the one used here is carried out by matrix
entrapment method using sodium alginate that reacts with
CaCl2 to form the beads which are further used for studying the
activity by using sucrose. The activity Is determined by DNSA
method.