Methodology:

The experiment was divided into 3 parts. First are protein Isolation which is divided into 2 subparts; isolation of cassein and Isolation of Gluten. The second one is qualitative test which is
divided into 6 sub-parts; general format, ninhydrin test, lead acetate test, xanthoproteic, biuret
and sakaguchi test. The last part is Quantitative Tests (Soluble Proteins in the Liquid Endosperm
of Cocos nucifera Linn) which is divided into 4 sub-parts; preparation of protein standards, assay
proper, plotting of data, estimation of coconut water soluble protein content.
For isolation of Cassein, the first process is to add 50 mL distilled water to 50 mL whole milk
and take note of the initial pH of the solution.Then add dilute HCl or 10% HCl to to the diluted
milk until the pH of the milk mixture reaches 4.6.if the pH goes lower than 4.6 adjust it by
adding 10% NaOH. Stand the mixture until precipitates settle at the bottom of the beaker. Decant
the upper liquid portion of the mixture to collect the settled precipitate. Discard the liquid portion
and squeeze the precipitate to dry. The resulting solid is the protein casein.

For the isolation of gluten, the first process is to prepare a dough ball by kneading approximately
50 grams of all-purpose flour in small amount of water. Knead the dough thoroughly and allow
the dough ball to stand for 5 minutes. After resting, gently knead the dough ball in running water.
Take note of the white washings removed from the dough ball. Continue washing the dough ball
in running water until the white washings has been completely removed. The brown pliable mass
is the protein Gluten.
The general formula should be done first before preceding to sub-parts of qualitative test. The
first process in general format is to prepare 4 test tubes. Next, Label each test tube as follows:

"Negative Control", "Casein", "Gluten" and "Positive Control. For the "Negative Control" tube,
add 0.5 mL distilled water .For the "Positive Control" tube, add 0.5 mL albumin solution. For
"Casein" and "Gluten" test tubes, add a pea-size amount of casein and gluten respectively.
For the Ninhydrin test, the first process is to add 0.5 mL 10% NaOH to all test tubes. Next, heat
the test tubes for 2 minutes in a boiling water bath then add 0.5 mL Ninhydrin solution after
heating. The last part is to heat the test tubes for 5 minutes. Caution: Ninhydrin solution produces
an idelible stains on skin.
For lead acetate test add 0.5 mL 10% NaOH to all test tubes. Next, heat the test tubes for 2
minutes in a boiling water bath then add 0.5 mL Lead Acetate solution after heating. The last part
is to heat the test tubes for 10 minutes. Caution: Lead is a heavy-metal. DO NOT dispose in the
sink. Dispose in a designated waste bottle.
For xanthoproteic add 5 drops of concentrated HNO3 (nitric acid) to all test tubes. Caution:
Nitric acid is corrosive. Wash skin in running water in case of contact.
For the biuret test, the first process is to add 1 mL Biuret solution to all test tubes . Shake and
stand for 5 minutes.
For the sakaguchi test add 0.5 mL 10% NaOH followed by 1 mL alcoholic alpha-napthol
solution to all test tubes. Next, heat the test tubes for 2 minutes in a boiling water bath then add
Zonrox dropwise until a cherry-red color solution ofr precipitate appears in the test tube.
Caution: Zonrox is a bleaching solution. It can discolor your clothes even with your lab coats on.
The Preparation of protein standards should be done first before preceding to sub-parts of
quantitative test. The first process in preparation of protein standard is to prepare egg albumin

solutions 2.5, 5.0, 10.0 and 20.0 mg/mL. Start preparing the 20.0 mg/mL solution by dissolving
50.0 mg (0.0500 grams) of egg albumin in ~ 150 mL deionized water. Stir until all the albumin
has been dissolved. Do not apply heat. Transfer the dissolved albumin in a 250-mL volumetric
flask and add deionized water q.s. (reaching the 250-mL mark). Prepare egg albumin solutions
2.5, 5.0 and 10.0 by serial dilution. Example, the 10.0 mg/mL albumin standard solution should
be prepared accurately by diluting 50 mL of 10.0 mg/mL solution up to 100 mL with deionized
water.
For assay proper the first process is to prepare 8 test tubes. Label the tubes 1, 2, 3, 4, 5, and A, B
and C). In each tube 1, add 2.5 mL deionized water. In test tubes 2-5, add 0.1 mL of the prepared
standard solutions 2.5-20 mg/mL respectively. To test tubes A-C, add 0.1 mL of Coconut Water.
After preparing this array, 2.5 mL of the Biuret Reagent (12 mM copper sulphate pentahydrate,
32 mM potassium sodium tartrate, 36 mM potassium iodide and 200 mM sodium hydroxide) will
be added to all test tubes. Allow the solution to stand at room temperature for 5 minutes. Note:
DO NOT APPLY HEAT. Read the absorbance of the solution at 540 nm.
For plotting of data establishment of a standard curve the first process is to prepare a scatter plot
of the albumin standard concentration (x-axis, test tubes 1-5) and Absorbance (y-axis) in a
spreadsheet (MS Excel),. Next is to add a trendline and set intercept to zero. Obtain the y=mx
formula and the R-squared. This could be computed manually by linear regression.
For estimation of the coconut water soluble protein content, the first step is to do the y=mx
formula using the standard curve. Next, compute for the concentration (x) of the coconut water
by substituting the absorbance readings of test tubes A, B and C. Then report the average and
standard deviation of the three readings.