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Posted at 10:10 PM on January 16, 2010 in USTories, USTedyante.
Qualitative Color Analysis and Isolation of Intact
Protein, Gluten, from Wheat Flour by means of Selective Dissolution
Authors: Lim,
L.B.R., *Luna, C.A.B.,
Magadia, D.P.B., Manalastas, M.G.S., Mendoza, M.K.D.
Group #5 2C-MT
ABSTRACT
Gluten
is a protein present in wheat, rye, barley and to a lesser degree in oats. It binds the dough in foods such as bread and
other baked goods and also contributes to the spongy consistency of baked
products. In this experiment, wheat flour was used as a sample to extract gluten. To be able to isolate the intact protein, the
wheat flour was made into dough and was purified by washing away the associated
starch since gluten is largely insoluble water. After several washings, gluten was obtained and it appeared to be an
elastic, rubbery, dirty white material. The principle involved in this experiment is difference in solubility
and the isolation technique used to extract the gluten from wheat flour is
selective dissolution. The reason for
the insolubility of gluten proteins in water is basically due to the hydrogen
bonds of the amino acids present in gluten. Another factor that makes the
intact protein, gluten, insoluble to water is its peculiar amino acid
composition. After the isolation of the extracted gluten, it was tested
quantitatively to detect for the presence of specific amino acids that are
present in the intact protein. The protein
give positive results to biuret, ninhydrin, xanthoproteic, millon’s, Hopkins-cole,
test for amides, and fohl’s test.
INTRODUCTION
According
to Sansted and Blish, the gluten of wheat flour has always been of outstanding
importance and interest to the wheat industry in general and to the cereal
chemist in particular. Gluten is an
elastic, rubbery protein usually found on wheat, rye, barley, and to a lesser
degree in oats. It binds the dough in
foods such as bread and other baked goods and also contributes to the spongy
consistency of baked products.
Based
on experiments and countless researches, gluten is said to be composed of two
distinct and individual storage proteins, glutenin and gliandin (http://www.jbc.org/content/85/1/195.full.pdf). Gliandin gives gluten elasticity while
glutenin gives its strength. The table below summarizes some properties of
glutenin and gliandin.
|
Table 1. Comparison of
Wheat Gluten Composition |
|
|
Gliandin |
Glutenin |
|
High extensible |
Less extensible |
|
Less elastic |
Highly elastic |
|
Soluble in alcohols |
Insoluble in alcohols |
|
Low in molecular weight |
High in molecular weight |
|
Intramolecular bonds |
Intra- and intermolecular
bonds |
Gliandin
and glutenin exist, conjoined with starch, in the endosperm of some
grass-related grains, notably wheat, rye, and barley. Being insoluble in water, they can be
purified by washing away the associated starch. Of these two proteins, glutenin is of more importance because it is
solely responsible for variations among the colloidal properties of glutens
from different flours, and that these variations may in turn be important
causes of differences among the bread-making characteristics of the flours in
question.
Gluten
proteins are very high in glutamic acid, about 35% of the total protein, and
are notably low in the basic amino acids. The gluten proteins therefore have no potential negative charges and
little potential positive charges, resulting in a low charge density.
A
number of processes have evolved over the years to produce gluten. Generally, gluten extraction from wheat flour
is based on either a dough system or a batter system. The processes have varied considerably in
terms of starting material/s and other parameters, for example, whole wheat or
flour, hard or soft wheat: consistency of wheat flour/water mixture (dough vs.
batter); dispersion method (water or other solvent): and types of equipment for
achieving starch and gluten separation (Ponte and Kulp, 2000).
The
principle under the isolation of gluten in this experiment is difference in
solubility. The solubility of proteins in aqueous buffers depends on the
distribution of hydrophilic and hydrophobic amino acid residues on the
protein’s surface. Hydrophobic residues predominantly occur in the globular
protein core, but some exist in patches on the surface. Proteins that have high
hydrophobic amino acid content on the surface have low solubility in an aqueous
solvent. Charged and polar surface residues interact with ionic groups in the
solvent and increase solubility. Knowledge of amino acid composition of a
protein will aid in determining an ideal precipitation solvent and method.
Gluten
is made from protein and proteins are composed of hundreds of amino acids
linked by peptide bonds, forming a peptide chain. Certain functional groups in
amino acids and proteins can react to produce characteristically colored
products. The color intensity of the
product formed by particular group varies among proteins in proportion to the
number of reacting functional, or free, groups present and their accessibility
to the reagent. In this experiment,
various color-producing reagents (dyes) were used to qualitatively detect the
presence of certain functional groups in amino acids and proteins.
This
experiment aims to isolate and describe the intact protein gluten from wheat
flour by means of selective dissolution through the principle of difference in
solubility and test for qualitative analysis.
EXPERIMENTAL
Isolation
of Gluten
A
cup of wheat flour was mixed with water to create dough. The mixture was thoroughly massaged to create
tough dough. Afterwards, it was wrapped on cheesecloth and placed on running
water to wash away the starch on the dough. Each and every member took turns on washing the dough because the group
was informed that it will took a long time to completely isolate the gluten
from the wheat flour. After several washings, every 10 minutes, the water from
the washings was collected and dropped with iodine solution to check for the
presence of starch. Upon the addition
of iodine solution to the washings, a color change of dark yellow to violet
indicates the presence of starch. The dough was then perpetually washed until
the next iodine test created a yellow to light yellow color change. It
approximately took an hour and a half to completely remove the starch from the
dough. Next, the wrapped dough was
opened and an elastic, rubbery, dirty white material was obtained. The extracted crude gluten from the wheat
flour was continually washed until the elastic, rubbery, dirty white substance
was completely isolated.
Qualitative
Color Analysis
Before
the extracted crude gluten was placed in test tubes for qualitative analysis,
an intact protein solution was made first. This was done by placing 0.5 g of protein in 1 ml of distilled
water. Consequently, the prepared intact
protein solution and 0.5 ml of hydrolyzed sample was placed in separate test
tubes.
Biuret Test
In
this test, sample was dropped with 20 drops of 2.5 M NaOH and was mixed
thoroughly. Next, an addition of 2-3
drops of 0.1M CuSO4 was placed in the sample and the color of the
solution was noted.
Ninhydrin Test
The
diluted sample in this test was placed with 6-10 drops of Ninhydrin
solution. Afterwards, it was heated in a
boiling water bath and the appearance of blue-violet coloration was observed.
Xanthoproteic Test
The
samples in this were slowly dropped with 12M HNO3 and the color of
the solution was noted. After slowly
mixing the mixture, 10 drops of 12M NaOH were placed in the sample and the
color of the solution was noted again.
Millon's Test
In
this test, 5 drops of Millon's reagent were place in the diluted samples and a
change in color was noticed.
Hopkins-Cole Test
The
sample in this test was slowly added with 20 drops of Hopkins-Cole reagent and
was mixed well. Next, the test tube was inclined and was added with 20 drops of
12M H2SO4 and the color at the interface was observed.
Sakaguchi Test
A
mixture of 10 drops each of NaOH and 0.2% Naphthol solution was dropped into
the sample. It was mixed and let stand for 3 minutes. Then, an addition of 3 drops of 2% NaOBr was
placed in the sample and the color produced was noted.
Nitroprusside Test
Initially,
the sample was poured with 0.5ml of 3M NaOH. Next, it was placed with 0.25ml of
2% Nitroprusside solution and the formation of red solution was observed.
Fohl's Test
In
this test, 5 drops of 30% NaOH and 2 drops of 5% Pb(OAC)2 was dropped in the
sample. Subsequently, it heated in a
water bath and a dark (black or brown) sediment appearance was noticed.
Test for Amides
In
a test tube with 10 drops of sample, an ml of 20% NaOH was added. Consequently,
the test tube was placed in a boiling water bath. Then, evolution of gas was
tested during heating by placing moistened red and blue litmus paper over the
mouth of the test tube and the results were noted.
Pauly Test
This
test requires the preparation of diazo reagent. It was done by mixing 3-5 drops of 1% sulfosalicylic acid with 3 drops
of 5 % NaNO2. Afterwards, 5
drops of the sample and 3-5 drops of 10% Na2CO3 were
added to the diazo reagent and the appearance of red coloration was observed.
RESULTS AND DISCUSSION
When
the gluten was successfully extracted from the wheat flour, it was isolated
from impurities by further washing it into running water. After each washing, the water from the
washings was again collected and was dropped with iodine solution. Such procedure was continually done until the
iodine test completely give a negative result of presence of starch. The substance that was obtained from the
washings was recorded as an elastic, rubbery, dirty white substance.
In
this experiment, the technique used to isolate the gluten was selective
dissolution which means a particular substance will only dissolve in a
particular solvent. The principle behind such isolation technique was
difference in solubility. Such method of isolation was used in this experiment
because the gluten from wheat flour is highly insoluble in water that's why
it's continually washed under running water.
The
reason for the insolubility of gluten proteins in water is basically brought
about by its peculiar amino acid composition. Gluten contains about 30% glutamine, the amide group of which can act
both as hydrogen donor and as an acceptor in hydrogen bond formation. The presence of about 10% proline with
restricted rotation hinders the formation of a helix. This and the high protein concentration in
dough favor the formation of intermolecular rather than intramolecular hydrogen
bonds. The insolubility of gluten is due
to these hydrogen bonds.
The
viscoelasticity of hydrated wheat gluten protein is attributed to several
factors, including its water compatibility and ability to swell and undergo
physicochemical interactions. As the
gluten takes up water, it goes through a glass transition where the proteins
change from a hard, glassy stage to that is rubber and elastic. An unusual property of gluten that sets it
apart from other plant proteins is the low level of polarity of its amino acid
structure (Kulp and Ponte, 2000).
Another
factor that differentiates wheat gluten from other flour proteins is its
structure. Disulphide bonds basically
play a key role in determining the structure and properties of wheat gluten
proteins. The well-known effects of oxidizing or reducing agents on the
rheological properties of dough and gluten are undoubtedly due to changes of
the thiol/disulphide structure of gluten proteins. About 95 % of total
cysteines in wheat flour are present in the disulphide (SS) form. Most α- and
γ-type gliadins have only intramolecular disulphide bonds located in the
C-terminal domains. LMW and HMW subunits of glutenin form both intra- and
intermolecular disulphide bonds and occurs in an aggregated state. About 5 % of
total cysteines in flour are present in the thiol (SH) form. Only small amounts
of free SH groups (0.5 %) are present in low-molecular-weight compounds, mainly
in glutathione and cysteine, most being present in flour proteins (4.5 %)
(Antes and Wieser, 2009).
Also,
according to previous experiments and researches conducted by different
biochemists, wheat gluten is a complex mixture of proteins (75-80%), starch
(15-20%), and fat (5-7%). The two main proteins are gliadin and glutenin.
Gliadin gives gluten elasticity while glutenin gives it strength. Together, the
proteins permit yeast-raised breads to maintain carbon dioxide gas produced
during fermentation and thereby result in breads with a desired predetermined
volume (Grace and Ye, 2000).
Below
is the tabulated result of the qualitative color reaction tests performed to
the extracted crude gluten.
|
Table 2.
Qualitative Test Results of Extracted Crude Gluten |
|
|
Qualitative
Test |
Results
(Intact Protein, Gluten) |
|
Biuret Test |
Turbid
blue-violet coloration |
|
Ninhydrin
Test |
Blue-violet
coloration |
|
Xanthoproteic
Test |
Dark-yellow
precipitate |
|
Millon's Test |
Red
precipitate |
|
Hopkins-Cole
Test |
White turbid
solution with red-violet coloration |
|
Sakaguchi
Test |
Yellow
precipitate |
|
Nitroprusside
Test |
Dark-yellow
solution |
|
Fohl's Test |
Formation of
black/brown sediment |
|
Test for
Amides |
Red litmus
paper turned to blue |
|
Pauly Test |
No reaction |
Ninhydrin
Test
The amino acids in the storage proteins of gluten contain free amino group and a free carboxylic acid group that react together with ninhydrin to produce a colored product. An amino group is attached to alpha carbon; the amino group's nitrogen atom is part of blue-purple product. Also, such proteins contain free amino groups on the alpha-carbon that reacted with ninhydrin to yield a blue purple product. Figure 1 show the reaction mechanism involved in the formation of a blue-purple product. The basic principle involved in this qualitative test is to test for ɑ-amino acids.
