The Isolation and Hydrolysis of Gluten from Wheat flour

Mayor, Reynelle Meg B.

Group 5, 2F-PH, Faculty of Pharmacy, UST, Manila

Abstract

The Isolation and Hydrolysis of proteins are two separate methods. The method that was first done was the
isolation of proteins. The sample used was wheat flour and the protein that is needed to be isolated is gluten.
Isolation of gluten from the wheat flour is accomplished by washing the starch out until it achieves a gummy-like
appearance. The isolate also known as the intact protein was the first medium used extracted from the sample
.This isolate will be used in the qualitative analysis of the protein in the latter part of the experiment. There are
different methods of isolation base on the kind of proteins that is to be isolated and the isolation of the
parentheses depends on the physio-chemical characteristics of proteins. A protein could be isolated by (a)
precipitation {isoelectric and induced} and by (b) difference in solubility {selective dissolution}. In this
expereiment, selective dissolution, is the method of choice for the isolation. The negative result in the iodine
solution test of the dough washing indicates that the starch is all washed out. A yellow color will indicate a
negative result. The second method was the hydrolysis of the isolated protein. Alkaline hydrolysis was used in this
experiment where 4 M NaOH was added on the intact protein and then autoclaved. With 1 M HCl, the autoclaved
hydrolysate was neutralized. The hydrolysate was also then used in the qualitative analysis of the protein extracted
from the wheat flour. There are 3 kinds of hydrolysis (a) Acid hydrolysis that uses hydrochloric acid (b) Alkaline
hydrolysis that uses Sodium hydroxide and the (c) Enzymatic hydrolysis that uses protease. The hydrolysis is
accomplished by autoclaving the intact protein soaked in the solution of either acid, base or enzyme depending on
what kind of hydrolysis should be done

Introduction

There are 20 known and common amino acids.
These amino acids have their own unique physical
and chemical characteristics, which, in turn
determine the isolation and purification method that
is to be used to extract a particular protein. To be
able to isolate a protein successfully, one must be
knowledgeable of the proteins molecular structure,
molecular weight, solubility in different solvents,
isoelectric pH and heat stability. Isolation and
Hydrolysis of protein is an intrinsic part of the
experiment because the isolate and the hydrolysate
that were the product of these methods are the
samples that are used in the qualitative and
quantitative analysis.

Methodology A. Isolation of Protein (Gluten from Wheat

flour)
Materials: 1. Add enough water in 1 cup of wheat flour.
Make a thick dough.
1 cup of wheat flour
Cheesecloth
0.01 M iodine solution
2. Wrap the dough in a cheesecloth. Remove
the starch to get the pure gluten. Place the
wrapped dough under running water until
the all starch is removed.

B. Alkaline Hydrolysis of Gluten

1.
3. To determine if the starch is all washed out,
test the dough washings in a 0.1 M iodine
solution. Negative result on iodine solution
that gives a yellow solution to the washings
indicates that the dough is already pure
gluten. It also must be a gummy-like in
appearance.
2.
Add 10 mL of 4M sodium hydroxide (NaOH)
into 0.5 g of isolated protein in a hard glass
test tube and then label the test tube as
follows:
Year/Section/Course
Group no.
OH- Hydrolysis
Protein Isolated
Cover the test tube with cotton and submit
to instructor for autoclaving for (24 hours)

4. Collect the gluten (the insoluble material)

for hydrolysis and qualitative protein
analysis.
3. Take note of the appearance of the mixture
after autoclaving. Add 10 mL of distilled
water. Transfer the mixture into a 250 mL
beaker.
 A. Isolation of Gluten:
4. Neutralize the mixture with 1M HCl. The
neutralized mixture will be used as a sample Dough Upon addition
for the characterization tests and of water in 1
chromatography cup of wheat
flour
Results and discussion Gummy-like appearance Upon washing
the dough
Gluten is a family of proteins found in grains like under running
wheat, rye, spelt and barley. water
Dirty white
Of the gluten-containing grains, wheat is by far the Insoluble
most commonly consumed. The two main proteins material
in gluten are glutenin and gliadin. Washings added with 0.1 Yellow solution
M iodine solution gives of iodine test
The glutenin often used to make seitan, a meat yellow solution indicates a
negative result
alternative used by vegetarians to make faux
which means all
chicken, faux beef and other vegetarian foods. of the starch is
Gluten is sometimes added to other foods to washed out.
increase their protein content.

While gliadin is responsible for most of the negative B. Hydrolysis of intact protein:
health effect, it can cause problems for people with
certain health conditions. This includes celiac 10 mL of 4M NaOH Clear solution
disease, gluten sensitivity, wheat allergy and some added to 0.5 grams The isolate is
isolate still visible
other diseases
After autoclaving the Yellow orange
When flour is mixed with water, the gluten proteins isolate Hydrolysate solution
The isolate
form a sticky network that has a glue-like
disappeared
consistency.

This glue-like property makes the dough elastic, and

gives bread the ability to rise when baked. It also
provides a chewy, satisfying texture.
Conclusion
I therefore conclude that isolation and hydrolysis of
intact protein is so important for us to be able to
study and further understand the structures and
composition of a protein both physically and
chemically. I have concluded that isolation of
proteins comes in different methods depending on
what kind of protein you are going to extract.
Sufficient knowledge about a particular proteins
composition is very much needed. One must be
familiar of a proteins molecular structure, molecular
weight, solubility in different solvents, isoelectric pH
and heat stability because method of isolation
depends on the proteins characteristics. A protein
could be isolated by (a) Isoelectric precipitation. The
first method makes use of the Isoelectric pH of a
certain protein. A protein renders insoluble when
their pH is at isoelectric point (IpH). When the
protein is at their IpH and is already insoluble, the
protein of interest is already isolated. The principle
behind this is that for a salt to be soluble in water , it
has to have charge on the surface (Na+ or Cl-); the
solution is formed by ionic interactions, or ion-dipole
interactions. In milk the casein is in a micelle form
which is calcium caseinate ( a salt). At the normal pH
of milk, the net negative charge of the micelle(or the
casein) will help in solubilizing. At isoelectric point
(pI), the net charge of a protein is zero, so at a pH of
4.6 (i.e lowering the pH , by adding acid) will bring it
closer to IpH, thereby precipitating the protein. This
is the method associated in the casein isolation. The
second method is (b) difference in solubility
{selective dissolution}. It is the process by which you
dissolve or corrode the unnecessary components of
a particular sample by washing it and the insoluble
material that will be left from washing the sample is
the protein of interest. This method is employed in
gluten extraction. And lastly is the salt induced
precipitation. There are two kinds of salt induced
precipitation, the salting in precipitation that makes
use 0.3-0.35 grams ammonium sulfate and the
salting out precipitation that makes use 70%
ammonium sulfate solution that is then used in
myoglobin extraction. Adjusting the salt
concentration in a solution containing a mixture of
proteins to just below the precipitation point of the
protein to be purified eliminates many unwanted
proteins from the solution. Then, after removing the
precipitated proteins by filtration or centrifugation,
the salt concentration of the remaining solution is
increased to precipitate the desired protein. The
precipitation of desired protein is then dissolved in
water to make a solution of this protein. This
procedure results in a significant purification and
concentration of large quantities of protein. These
processes are of importance because the resulting
product of these methods are the protein of
interest. Then later these proteins are to be used
and analyzed
Hydrolysis of the intact protein is also an essential
part of this experiment. There are 3 types of
hydrolysis that was introduced in this experiment.
First, acid hydrolysis that uses hydrochloric acid.
Acid hydrolysis is an important chemical
modification that can significantly change the
structural and functional properties of protein
without disrupting its granular morphology. During
acid hydrolysis, amorphous regions are hydrolyzed
preferentially, which enhances the crystallinity and
double helical content of acid hydrolyzed protein.
The second one is the alkaline hydrolysis which uses
sodium hydroxide. Alkaline hydrolysis leads to the
random breaking of nearly 40% of all peptide bonds
in proteins. The alkaline hydrolysis of gluten in 4M
NaOH and autoclaving it for 24 hours helps sterilize
the solution. And lastly is the enzymatic hydrolysis
that used protease that also breaks the proteins. The
hydrolysis of the intact protein is important for it
kills all the microbes, spores and viruses that may be
present in the sample. In this way, a more pure
protein could be extracted from the sample.
References:
https://www.ncbi.nlm.nih.gov/pubmed/171127
96
http://bitesizebio.com/853/5-laboratory-
sterilisation-methods/
http://info.gbiosciences.com/