Methodology

Isolation of Protein
-Powdered non-fat milk was dissolved with
distilled water then was heated at 40oC.
Acetic acid was added to the solution until pH
4.6 was obtained. Filtration was done to
obtain dry casein residue by air-drying.
Alkaline Hydrolysis of Intact Protein
-Extracted casein from skimmed milk was
hydrolyzed with Sodium hydroxide and was
autoclaved for 5 hours at 15 psi. After
autoclaving, addition of distilled water was
added then was neutralize by adding 6 M
Hydrochloric acid then was tested with blue
and red litmus paper.
Qualitative Color Reactions
-Alkaline Casein hydrolysate was tested with
different reagents namely (Biuret Test,
Ninhydrin Test, Xanthoproteic Test, Millons
Test, Hopkins-Cole Test, Sakaguchi Test,
Nitroprusside Test, Fohls Test, Test for
Amides, and Pauly Test) and was observed
with different color changes.

In Biuret Test, Sodium hydroxide and Copper

sulfate solution were added to the basic
casein hydrolysate.
In Ninhydrin Test, 0.1% ninhydrin solution was
added to the diluted basic hydrolysate sample
then was heated in a boiling water bath.
In Xanthoproteic Test, concentrated Nitric acid
and concentrated Sodium hydroxide were
slowly added to the diluted basic hydrolysate
sample.
In Millons Test, Millons reagent was added to
the diluted basic hydrolysate sample.
In Hopkins-Cole Test, Hopkins-Cole reagent
was added to the basic hydrolysate sample.
Inclination and slowly addition of
concentrated Sulfuric acid were done.
In Sakaguchi Test, 10% of Sodium hydroxide
and 0.02% of naphthol solution were added to
the basic hydrolysate sample and were left for
3 minutes. 2% of Sodium hypobromite was
then added to the sample.
In Nitroprusside Test, 3 M Sodium hydroxide
and 2% nitroprusside solution were added to
the basic hydrolysate sample.

In Fohls Test, 30% Sodium hydroxide and 5%

Lead acetate were added to the basic
hydrolysate sample. The sample was placed
in a boiling water bath.
In the Test for Amide, 20% Sodium hydroxide
was added to the basic hydrolysate sample.
The sample was placed in a boiling water
bath. A moistened red litmus paper was
placed over the mouth of the test tube.
In Pauly Test, diazo reagent was prepared by
mixing 1% sulfanilic acid and 5% Sodium
nitrite. 10% Sodium carbonate and diazo
reagent was added to the basic hydrolysate
sample.
Separation and Identification of Amino Acid by
Paper Chromatography
-Thin Layer Chromatography plate (12x15cm)
was prepared by marking the bottom with 1.5
cm margin. Thirteen points were marked
equidistantly placing the standards namely
(Tryptophan standard, Arginine standard,
Proline standard, Cysteine standard, Serine
standard, Aspartic acid standard, Tyrosine
standard, Histidine standard, Glycine

standard, Alanine standard) and samples

namely (Acid hydrolysate, Base hydrolysate
and Enzyme hydrolysate) with capillary tubes.
The plate was placed inside the pre-calibrated
chamber and was left undisturbed. After the
solvents ascended, the plate was removed
and was air-dried. One percent of ninhydrin
reagent was sprayed over the dried plate and
was oven for 3 minutes.