ABSTRACT

Carbohydrates are the most abundant class of organic compounds found in living
organisms. The objective of this experiment are to : (a) compare the products of the isolated
glycogen from chicken liver after acid and enzymatic hydrolyses, (b) illustrate the specificity
of the - amylase on the hydrolysis of the isolated polysaccharide, (c) prepare dialyzing bag
used inseparating the products of enzymatic hydrolysis and explain the principle behind it,
lastly, (d) examine microscopically the mucic acid crystals.The glycogen was first isolated
from the chicken liver. The sample produced was described as light yellow solution. The
isolate produced was then subjected to acid hydrolysis. Five mL of concentrated HCl were
added to the isolate and was placed in a boiling water bath. The acid hydrolysate was
described as clear yellow solution and is not viscous. Furthermore, the isolated glycogen
was also subjected to enzymatic hydrolysis. About 2.3 mL of saliva were added to the isolated
carbohydrate. The solution was then introduced to a dialyzing bag ang was suspended
overnight in a small flask filled with 50 mL distilled water. It was then subjected to an open
flame and was concentrated to a volume of 10 mL. The enzymatic hydrolysate was described
as whitish yellow solution and is not viscous. Both the acid and enzymatic hydrolysate were
used in qualitative tests which are done to determine the identity of the monosaccharides
present in the solution. The hydrolysates tested negative on most of the qualitative tests
except the Seliwanoffs test. The Mucic Acis test, which is a test specific for the
determination of galactose, was also performed. Three drops of galactose and lactose and 3
drops of concentrated HNO 3 were mixed in a glass slide. Then, it was passed over an alcohol
lamp in order to dry. The mixture was then cooled down at room temperature. The crystals
formed on the glass slides were examined under the microscope. The two types of sugars,
glucose and lactose exhibited positive results in this test.

linked via -1,4-glycosidic bonds in a

INTRODUCTION
[1]
Carbohydrates, or saccharides are
biological molecules composed of three
molecules namely, carbon, hydrogen and
oxygen. [3] These biological molecules, like
nucleic acids and proteins, play a variety of
roles in different processes such as enery
metablosim, molecular recognition, cellular
protection and cell signaling.
[2]
The simplest carbohyrates are known
as monosaccharides. These are small,
monomeric molecules composed of three to
nine carbon atoms. Another type of
carbohydrates is oligosaccharide.
Oligosaccharides can be formed by linking
monosaccharides together. Polysaccharides,
on the other hand, are long polymers of the
monosaccahrides.
Glycogen is a polysaccharide that is the
principal storage form of glucose (Glc) in
animal and human cells. The liver cells, also
known as hepatocytes have the highest
concentration of it . However, glycogen can
also be found in the mucscle. Furthermore,
[3]
glycogen is consist of glucose monomer
helical structure. As the principl storage of
form of glucose, glycogen play a vital role
in glucose cycle.
The objectives of this experiment are to
: (a) compare the products of the isolated
glycogen from chicken liver after acid and
enzymatic hydrolyses, (b) illustrate the
specificity of the - amylase on the
hydrolysis of the isolated polysaccharide,
(c) prepare dialyzing bag used inseparating
the products of enzymatic hydrolysis and
explain the principle behind it and (d)
examine microscopically the mucic acid
crystals formed by galactose and lactose.
METHODOLOGY
I. Acid Hydrolysis
Five drops of concentrated HCl were
added to a test tube filled with the isolated
carbohydrate from chicken liver. It was then
covered with a marble and was boiled in a
water bath for 30 minutes. The test tube was
removed and cooled down before subjecting
to the qualitative tests.
II. Enzymatic Hydrolysis
Ten mL of the isolated carbohydrate
was placed in a beaker. Then, 2.3 mL of
saliva was added. The solution was left to
stand at room temperature for about 30
minutes. The change in its viscosity was
recorded. The solution was then introduced
to a dialyzing bag. The bag was suspended
overnight in a small flask filled with 50 mL
of distilled water. It was then removed and
the dialyzing bag was discarded. Using an
open flame, the solution was concentrated to
a volume of 10 mL. The enzymatic
hydrlysate was subjected to different
qualitative tests to confirm its identity.
Figure 1. Acid Hydrolysate
III. Qualitative tests
The procedure performed in
each qualitative test were repated to
determine the identity of the hydrolysates.
IV. Mucic Acid Test
Three drops of galactose and lactose
and 3 drops of concentrated HNO 3 were
mixed in a glass slide. Using an alcohol
lamp, it was passed over a small flame until
it is almost dry. The mixture was then
cooled down at room temperature. The
crystals formed on the glass slides were
examined under the microscope. The
appearence of the mucic crystals were
noted. In case no crystals appeared, the
glass slides were let stand until the next
period.
RESULTS AND DISCUSSION
Glycogen was succesfully isolated from
the chicken liver. The isolated carbohydrate
was described as light yellow solution.
Hydrolysis of carbohydrates is a
process wherein disaccharides and
polysaccharides are broken down into their
constituent monosaccharides. The
glycosidic bond which connects each
monosaccharide unit is broken down in a
reaction with water and presence of a
catalyst. Hydrolysis can be achieved by the
reaction of the carbohydrate with acid, or by
using catalytic enzymes that are specific for
each carbohydrate.
Figure 2. Mechanism of Hydrolysis of
Carbohydrates (Maltose)
In this experiment,acid hydrolysis was
performed by adding a strong acid ,
concentrated HCl, to the isolated glycogen
inside a test tube. The solution was then
subjected to a boiling water bath for about
30 minutes. Acid hydrolysis of
polysaccharides are randomnly claved so it
will first produce smaller polysaccharides
and disaccharides, but eventually, given
sufficient time, will produce the constituent
monosaccharides.The acid hydrolysate was
decribed as a clear yellow solution and is
not viscous.
 Table 1. Hydrolysis of Polysaccharides
HYDRO- DESCRIP- BENEDICT
LYSATE TION S TEST
Acid Clear yellow; Blue green
not viscous solution
Enzymatic Whitish No change in
yellow ; not the color of
viscous the solution

Enzymatic hydrolysis, on the other

hand,is more specific with the cleavage of Figure 3. Enzymatic Hydrolysis of
the bonds. It is because of the several Glycogen
specific enzymes that can be used such as
lactase,amylase and sucrase. In this Table 2. Qualitative Tests for
experiment, alpha- amylase , which can be Carbohydrates
found in human saliva is used. The alpha- CARBO BENEDIC BARFOE SELI- BIALS
amylase catalyze the rapid,random HY- TS DS WANOF TEST
hydrolysis of internal alpha-1,4 bonds. DRATE TEST TEST FS
However, they do not hydrolyze the alpha- SOLN TEST
1,6 linkages and hydrolyze maltose. Thus, Acid Blue green No Cherry No
the glycogen was initially separated by solution change red change
alpha-amylase into dextrins which are then precipitat
further hydrolyzed into the disaccharide e
maltose and then to glucose. In our bodies Enzymat No change No Cherry No
the hydrolysis of polysaccharides (except ic change red change
cellulose) is carried out by amylase which is precipitat
found in our saliva or secreted from our e
pancreas.The enzymatic hydrolysate was
found to be a whitish yellow solution and Both the acid and enzymatic
not viscous. The figure above is a diagram hydrolysate were subjected to different
that shows what happens to polysaccharides various tests to determine its identity. The
when subjected to enzymatic hydrolysis. acid hydrolysate exhibited a change of color
when subjected to Benedicts Test.
However, this change is not the desired
result since the positive result of Benedicts
test is the appearance of a brick-red
precipitate. Moreover, the acid hydrolysate,
exhibited negative results on Barfoeds and
Bials Test. Enzymatic hydrolysate, on the
other hand, tested negative on Benedicts,
Barfoeds and Bials Test. The two
hydrolysate produced a cherry red
precipitate in Seliwanoffs test.

Figure 3. Qualitative Tests of Acid and

Enzymatic Hydrolysate
 solution then crystallizes out from water.
The resulting isomer is called mucic acid.
Mucic acid was formed from the galactose
because of the oxidation of both aldehyde
and alcohol group at C1 & C6.

Figure 4. Structure of Mucic Acid

The acid and enzymatic hydrolysate In this test, the nitric acid first
tested positive in Seliwanoffs test. The catalyzed the breakdown of lactose into
Seliwanoffs is a distinguishing test between glucose and galactose. Then, it oxidized the
aldoses and ketoses. The presence of cherry two sugars into tetrahydroxyadipic acids
red precipitate indicates the presence of a which eventually precipitates as insolluble
ketohexose. Consequently, Ketopentoses crystals after cooling down at room
yield blue-green series while aldoses and temperature.
disaccharides give no shading change. The
presence of cherry red precipitate indicates
that the hydrolysates contain a ketohexose,
specifically fructose. Meanwhile, the color
change in the acid hydrolysate suggests the
presence of trace amounts of reducing
sugars. [3] Glycogen is a polysaccharide
consist of glucose monomers linked via -
1,4-glycosidic bonds in a helical structure.
Glucose is an example of a reducing sugar.
Sugars thatcontain aldehyde groups that are
oxidised to carboxylic acids are reducing
sugars.

The Mucic Acid Test, also called

Galactaric acid test, is a distinguising test Figure 5. Results of Mucic Acid Test in
for the presence of galactose. The presence Galactose
of crystals under a microscope indicates a
positive result. As from the results above,
both galactose and lactose exhibited positive
results in this test.
The concentrated HNO3 oxidizes
galactose to an isomer of
tetrahydroxyadipic acid. This solution then
crystallizes out from water. The resulting
isomer is called mucic acid. Galactose is a
monosaccharide and a aldohexose. The
disaccharide lactose can be formed by the
combination of galactose with glucose via
dehydration reaction. The concentrated
HNO3 oxidizes galactose to an isomer of
tetrahydroxyadipic acid. The galactose is Figure 6. Results of Mucic Acid Test in
then converted to saccharic acid. This Lactose
REFERENCES

Appling, D. R., Anthony-Cahill, S. J., &

Matthews, C.K.
(2016). Biochemistry : Concepts and
Connections. England: Pearson Education
Limited .
Campbell, M. K., & Farrell, S. O.
(2015). Biochemistry. Stamford:
Cengage Learning.
Sharma, D. K. (2010). Biochemistry.
Oxford: Alpha Science International
Ltd.

N. (2013, February 21). Lab review 1.

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