GROUP 3

PROTEIN

Submitted by:

Estareja, Ralph Nikko

Eusebio, Janine Renzel Mae

Labrada, Eunice Micah

Lacar, Bathsheba

Latoja, Jan Dosedrick

BS PSYCHOLOGY 3-1

Submitted to:

Prof. Evelyn Matchete

BIOCHEMISTRY

PROTEIN
 ABSTRACT

The experiment aims to isolate the intact protein which is gluten from wheat flour by difference
in solubility and to analyze the chemical group responsible for color reaction and explain the
principle involved in each test. Gluten is a protein composite that appears in foods processed from
wheat. It gives elasticity to dough, helping it to rise and to keep its shape, and often giving the final
product a chewy texture. The intact protein was isolated by washing the dough with water which
removes starch

Casein protein is a milk protein extract recognized for its excellent amino acid profile, slow
digestion, and interesting peptides (casomorphins, casokinins, casoxins, etc.). In this experiment,
casein was isolated, hydrolyzed and neutralized from whole milk.

A xanthoproteic test is a test for the detection of proteins in which concentrated nitric acid reacts
with the proteins to form a yellow color that is intensified to orange. The objective of this
experiment is to look for certain proteins by using nitric acid.

The experiment aimed to show different ways on how to look for the presence of proteins.

Key Words: Casein, Gluten, Xanthoproteic test

INTRODUCTION

Proteins, from the Greek proteins, meaning first, are a class of organic compounds
which are present in and vital to every living cell. In the form of cell, hair, callus, cartilage,
muscles, tendons, ligaments, proteins hold together, protect, and provide structure to the body of a
multi-celled organism. In the form of enzymes, hormones, antibodies, and globulins, they catalyze,
regulate and protect the body chemistry. In the form of hemoglobin, myoglobin, and various
lipoproteins, they affect the transport of oxygen in the other substances within an organism.

Protein isolation is a process that is intended to isolate a single type of protein from a more
complex mixture this technique is critical for characterizing the function, structure and interaction
of the protein of interest. An analytical purification generally uses three distinct properties to
separate proteins, including the isoelectric technique.

The total protein component of milk is compost of numerous specific proteins. The primary
group of milk protein is the caseins. There are three or four caseins in the milk of most species, the
different caseins are distinct molecules that are similar in structures. All other proteins found in
milk are grouped together under the name of whey proteins.

Casein protein, like other protein sources, provides a rich amino acids supply to the body. Current
data suggest that exercise can increase protein needs and that increase protein intakes can improve
the response to exercise training.
Casein protein makes up approximately 80% of the protein in milk.

Gluten can be defined as rubbery mass that remains when wheat dough is washed to remove
starch granules and water soluble constituents. It is a mixture of proteins not readily soluble in
water that occurs in wheat and most other cereal grains. Its presence in flour make production of
leavened baked goods possible because the chain-like gluten molecules form elastic networks that
traps carbon dioxide gas and expand with it.

OBJECTIVE

 To learn the methods of protein precipitation and to relate the solubility of protein with its
structure.

 To learn the methods of isolation of casein from milk and to determine the percentage of
casein presented in the (powdered) milk.

MATERIALS

Quantity
Concentrated nitric acid
8 Test tubes
2 Beaker (250ml)
1 Test tube rack
2 Dropper
1 pH meter
50grams Flour
50ml Whole milk
Muriatic acid
Table 1. this shows the materials used in the experiment
 METHODOLOGY

In isolation of casein, 50ml whole milk was added to 50ml of distilled water, initial pH level of the
solution was noted. Dilute HCI or 10% HCI was added to the diluted milk until the pH level of the
milk mixture reaches 4.6. If the pH level goes lower than 4.6, it can be adjusted by adding 10%
NaOH. Stand the mixture until precipitate settle at the bottom of the beaker. Then carefully decant
the upper liquid portion of the mixture in order to collect the settled precipitate and then discard
the liquid portion after. Using cheesecloth, squeeze the precipitate to dry. The resulting solid is the
protein casein.

For isolation of gluten, a dough ball must be prepared. Approximately 50 grams of all-
purpose flour and small amount of water shall be mixed and knead. The dough must be knead
thoroughly and then allow it to rest for five minutes. After resting, gently knead the dough ball in
running water and take note of the white washing removed from it. Continue doing the process
until the white washings has been completely removed. The brown pliable mass that was left is the
protein gluten.

RESULT AND DISCUSSION

1. Isolation
1.1 Isolation of casein

pH level
pH of diluted milk 7.59
pH of diluted milk with muriatic acid 4.60

Table 2. this shows the result of adding muriatic acid in the diluted milk. The pH level of
the diluted milk decreases after adding muriatic acid.

For the isolation of the protein casein, 50 ml of whole milk and 5o ml of distilled water
was mixed in a 250 ml beaker and took its initial pH level. After that, a drop of muriatic
acid was continuously added until the pH level reached 4.60. the diluted milk with
muriatic acid was set aside to precipitate then filtered to remove the excess liquid. Cheese
cloth was used to get the solid casein protein.
.
Figure 2 this shows the initial pH level Figure 1 shows the pH level of diluted milk
of diluted milk using pH meter turned to 4.60 after the muriatic acid was
added.

Figure 4. The diluted milk with Figure 3. The filtering of the diluted milk to
muriatic acid precipitates and separate the precipitate to the remaining liquid.
settled at the bottom of the beaker
 1.2 Isolation of gluten

In the isolation of the protein gluten, a dough ball was prepared by needing 50 grams of
flour and water and set the dough ball aside for 10 minutes. After that, the dough ball was washed
in running water until the brown pliable product was remained and that is the protein gluten.

Figure 5. The dough ball

Figure 6. The protein gluten after
washing the dough ball

2.4 Xanthoproteic test

Four test tubes were prepared and labeled each with “negative control”, “positive control”,
“casein”, and “gluten”. For the negative control test tube, 0.5 mL of distilled water was added.
Then, 0.5 mL of albumin solution was added in the positive control test tube. For casein and gluten
test tubes, a pea-size amount of casein and gluten respectively.

To determine the amount of protein soluble in a solution, 5 drops of concentrated nitric

acid was added to all test tubes. Few minutes after, for the negative control it was observed that
there were no changes. Therefore, there is an absence of protein in distilled water. In casein test
tube the filtered mixture became yellow which means that there is a presence of proteins, while in
gluten test tube it turned into bright orange which means that it has a lot amount of proteins present
in the solution.
 CONCLUSION

In the experiment, casein was successfully isolated from the whole milk through isoelectric
precipitation. The isolated casein was subjected to acid and alkaline hydrolysis which yielded
visible changes comparing the appearance of the isolate before and after autoclaving it. Through
the use of xanthoproteic test, as for the positive control it was observed that there is a presence of
protein in casein, gluten and positive control solution. Moreover, as for the negative control there
is no presence of protein in distilled water.

The nitric acid reacted to all the solution that makes the casein, gluten and positive control
solution to turn in yellow color. For the xanthoproteic test, it was observed that protein
suspension gave positive results, and that its components are peptide bond nitrogen. Proteins are
made of amino acids. The amino acid reacts due to its amphoteric nature and the R-group or side
chain, it means that it is capable of reacting to both acids and bases. What determines the intensity
of the product color is the side chain or functional groups present in amino acid. The more
saturated its hue it became if it has a greater number of functional groups that reacted.

RECOMMENDATION

Make sure that all the materials are washed and cleaned before using to make sure that
there are no unnecessary chemical will be mixed on the solution because any mistakes will lead to
a wrong result. For the isolation of casein, use a cow milk or whole milk because whole milk is
high in natural protein. In the isolation of gluten, knead the dough well for when washed, more
protein gluten you’ll get.

REFERENCE

 https://www.medicalnewstoday.com/articles/318606.php

 https://www.healthline.com/nutrition/casein-protein-is-highly-underrated

 https://www.cancerquest.org/cancer-biology/biological-building-
blocks?gclid=EAIaIQobChMI4uDowqin4AIV2AcqCh3IUgfYEAAYASAAEgJe6_D_BwE

 https://idahomilkproducts.com/resource-center/what-proteins-are-present-in-idapro-milk-proteins/

 https://en.wikipedia.org/wiki/Xanthoproteic_reaction
CONTRIBUTION

Estareja, Ralph Nikko

Eusebio, Janine Renzel Mae

Labrada, Eunice Micah

Lacar, Bathsheba

Latoja, Jan Dosedrick