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

Materials:

1 cup of wheat flour A. Isolation of Protein (Gluten from

Cheesecloth Wheat flour)
0.01 M iodine solution
1. Add enough water in 1 cup of is
wheat flour. Make a thick dough.

2. Wrap
the

already pure gluten. It also must be

a gummy-like in appearance.

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.

4. Collect the gluten (the insoluble

material) for hydrolysis and
qualitative protein analysis.

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
 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.

Year/Section/Course
Group no.
OH- Hydrolysis
Protein Isolated

B. Alkaline Hydrolysis of Gluten

1. Add 10 mL of 4M sodium hydroxide
(NaOH) into 0.5 g of isolated
protein in a hard glass test tube 4. Neutralize the mixture with 1M HCl.
and The neutralized mixture will be
then used as a sample for the
label characterization tests and
the chromatography
test
tube Results
as and

follows:

discussion

Gluten is a family of proteins found in

grains like wheat, rye, spelt and barley.

Of the gluten-containing grains, wheat is

2. Cover the test tube with cotton and by far the most commonly consumed. The
submit to instructor for autoclaving two main proteins in gluten are glutenin
for (24 hours) and gliadin.
The glutenin often used to make seitan, a Dirty white
meat alternative used by vegetarians to Insoluble
make faux chicken, faux beef and other material
vegetarian foods. Gluten is sometimes Washings added Yellow
added to other foods to increase their with 0.1 M iodine solution of
protein content. solution gives iodine test
yellow solution indicates a
While gliadin is responsible for most of the negative
negative health effect, it can cause result which
problems for people with certain health means all of
conditions. This includes celiac disease, the starch is
gluten sensitivity, wheat allergy and some washed out.
other diseases

When flour is mixed with water, the gluten B. Hydrolysis of intact protein:
proteins form a sticky network that has a
glue-like consistency. 10 mL of 4M NaOH Clear
added to 0.5 grams solution
This glue-like property makes the dough isolate The isolate is
elastic, and gives bread the ability to rise still visible
when baked. It also provides a chewy, After autoclaving Yellow
satisfying texture. the isolate orange
Hydrolysate solution
The isolate
disappeared

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
A. Isolation of Gluten: methods depending on what kind of
protein you are going to extract. Sufficient
Dough Upon knowledge about a particular proteins
addition of composition is very much needed. One
water in 1 must be familiar of a proteins molecular
cup of wheat structure, molecular weight, solubility in
flour different solvents, isoelectric pH and heat
Gummy-like Upon stability because method of isolation
appearance washing the depends on the proteins characteristics. A
dough under protein could be isolated by (a) Isoelectric
running precipitation. The first method makes use
water 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/
17112796
http://bitesizebio.com/853/5-
laboratory-sterilisation-methods/
http://info.gbiosciences.com/