Experiment One: Salivary Digestion

Mae Ann A. Derecho

Department of Chemistry, University of San Carlos Talamban Campus, Talamban, Cebu City,
Philippines
July 15, 2016
Saliva is viscous fluid with a pH around 5.6-7.9 is rich in protein such as mucins, amylase and
others. In this experiment, the collected saliva sample were treated in a series of test to determine the
component that were present. Mucins tested positive together with the inorganic constituents specifically
chlorides and phosphates. This is determined through precipitating out their colored product formed.
However, there are also components that tested negative despite being there and yet due to it being in
minute amounts qualititave measures failed to detect these components. Presumptive tests were also
performed and defined that ferric chloride test can be used detect the presence of thiocyanate/cyanide
obtained from food or cigarettes. Factors affecting enzymatic activity were also identified using test the
showed the effect of acid and base to salivary digestion.

INTRODUCTION
Digestion begins in the mouth where the food is
subjected to the mechanical process of grinding
to break it up into smaller particles thus enabling
the digestive juices to get at the food more
readily. This also aids in mixing the saliva of the
mouth with the food. Saliva moistens the
mucous membranes of the mouth and helps to
prevent tooth decay by cleansing the mouth of
cariogenic carbohydrates and by neutralizing
lactic acid. In addition, the salivary glands
secrete an enzyme known as salivary amylase.
This enzyme begins the breakdown of starches,
it is responsible in the digestive function of
saliva. Amylase is the responsible in breaking up
immune complexes that are known to cause
body system disruption. Saliva plays a very
important role in the digestive process. Its
effectiveness is increased with proper and
careful chewing of food. Therefore, the
objectives of this experiment is to identify the
product formed on the salivary digestion of
starch, components present in saliva, trace the
salivary digestion of starch through iodine
solution, the factors that affect enzymatic
activity and to give the different functions a
constituent play on both the digestion process
and on the human body.

METHODOLOGY
A. Test for Mucin
In a test tube, 3 mL of saliva
were added with 1-2 drops of 0.1 M
acetic acid.
B. Test for pH
Two drops of universal indicator
were added to 3 mL of saliva sample.
C. Test for Proteins
Two mL of the saliva sample
were added with Biuret reagent/
D. Inorganic Matter
Acidified 20 mL of the saliva
was added with 2 drops of acetic acid,
heated to boiling and filtered to remove
the protein. Obtained filtrate were tested
for the presence of inorganic matter
following the table below for the
reagents added:

Table 1. Reagents added for the

test of inorganic matter in saliva
samples
Ions
Chlorides
Sulfates
Phosphates
Calcium

Reagents
HNO3, AgNO3
HNO3, BaCl2
HNO3, (NH4)2MoO4
HNO3, K2C2O4

E. Test for Thiocyanates (Ferric chloride

test)
In a small porcelain dish, 1 mL
of saliva was added with a few drops of
ferric chloride before it was slightly
acidified with dilute HCl, forming a red
solution
of
ferric
thiocyanate.
Confirmatory test for the product
formed was done through adding a drop
of mercury (II) chloride that turned the
solution colorless. Also, a control set up
using distilled water instead of saliva
was prepared to compare the obtained
result.
F. Digestion of Starch
Two different set ups were prepared for
this part of the experiment. The first set
up consist of 1 mL saliva and 5 mL
distilled water mixed together in a test
tube and set aside at room temperature.
The other set up was composed of 1 mL
saliva and 25 mL of 1% starch solution,
homogenized well in a 100 mL beaker
and submerged in a water bath on a
maintained temperature of 40C. A spot
plate that contained two drops of iodine
in potassium iodide solution to each
well were then prepared. On the first
well of the spot plate, a drop of starch
solution was added and observe. A drop
of the saliva-starch mixture was added

to the second well of the spot plate and

observed in intervals between 30
seconds for every addition of the
mixture to the succeeding wells until the
yellow color of the iodine remained
persistent. Lastly, a 1 ml saliva-starch
mixture was collected and subjected for
the presence of reducing sugar using
Benedicts test.
G. Effect of Acid and Base
Table 2 showed the solutions prepared
in two separate test tubes before it was
heated at 40C for an hour and tested
for the effects of acid and base to the
digestion process.
Table 2. Solution preparation for the
effects of acid and base in saliva digest
Test tube no.
1
2

Saliva
3
3

Two drops of digested were collected

each from the set up and transferred to
clean spot plate. A drop of iodine
solution was then added to each wells
and observed for any changes that
occurred.

RESULTS AND DISCUSSION

Table 3 showed the experimental results
gathered on the first part of the experiment.
Mucin, a glycoprotein, is identified present on
the saliva sample after it is precipitated out at
around ph 4.5. Increase in mucin production can
be consider as a symptom to any abnormalities
in the body such as cancers of the pancreas,
lung, breast and other tissues.

Solutions to be
0.5% (w/w)
0
3

Table 3. Qualitative test for the components of saliva

Test
Mucin
pH
Proteins
(Biuret
Test)
Inorganic Matter
Chlorides
Phosphate
s
Sulfates
Calcium
Thiocyanates
(Ferric
Chloride
Test)

Result
cloudy soln with white ppt
yellow green soln with yellow green ppt
clear, colorless soln with indigo ppts

turbid soln with cloudy white ppt

clear, colorless soln with pale yellow ppt
slightly turbid soln
clear, colorless soln

The soln turned pale orange soln with pale orange curds/ ppt formation. The soln turned
colorless after the addition of mercury (II) chloride. In comparison to the control set up,
the soln turned milky white with white ppt formation after the confirmatory test using
the mercury (II) chloride solution.
With the saliva solution turning to yellow green
ammonium phosphomolybdate precipitate (refer
with precipitate formation after the addition of
to equation 2). The function of inorganic
the universal indicator, the normal pH range for
phosphate includes: contributes to solubility
saliva is considered to be 5.6 to 7.9. This
product of calcium phosphate, which is crucial
explains why the pH balance in the body is close
in maintaining tooth structure, important as a
to neutral. However, several factors influence
buffer and an essential nutrient for oral
the pH nature of the saliva and varies depending
microflora for metabolic pathways.
to the food and drinks intake of an individual.
12(NH4)2MoO4 + H3PO4 + 21HNO3
Qualitative determination of salivary proteins
(NH4)3PMo13O40(s) + 21NH4NO3 + 12H2O
can be performed through the use of Biuret
reagent. Biuret test is a specific to test the
(eqn. 2)
presence of peptide bones which is also present
The presence of calcium in saliva can be
in proteins. The bluish complex formation of
investigated through reacting it with potassium
copper hydroxide confirms the presence of
oxalate soln in slightly acidic medium. The
proteins in saliva. There are number of proteins
presence of the ion based from the obtained
present in saliva such as amylases, defensins,
result was not determined and may be
cystatins, histatins, immunoglobulins, statherin,
influenced by several factors. The content of
lactoperoxidase, lysozyme and lactoferin.
calcium can be related to the tooth enamel of an
The inorganic constituents of saliva includes
individual. A study conducted by Hubbell and
Na+, K+, Ca2+, Cl-, HCO3 and HPO4. Chloride
Bunting (1932) showed that as the number of
was tested to be present in the saliva through
carious teeth increases, the calcium of the saliva
precipitating out as AgCl (refer to equation 1) in
decreases; supporting the absence of calcium on
white form after silver nitrate is added. The
the experimental result. The last ion, sulfates,
chloride ions help in maintaining the osmotic
investigated showed a negative result indicating
balance in the mouth preventing any excess
that the ion is in minute amount on the saliva
inflow or outflow of water from water tissues.
sample. Sources of sulfates can be from dietary
sulfates and is essential to the humans as sulfates
AgNO3 + Cl AgCl(s) + NO3 (eqn. 1)
contribute to the protein structure of the saliva.
Inorganic phosphates were also identified to be
present in saliva through the formation of the

Thiocyante is an endproduct of
detoxification of hydrogen cyanide present in
cigarette smoke. Thiocyanate can also be present
in several foods such as cabbage and broccoli,
hence, its presence in the saliva. Test for
thiocyante are now widely used to identify
individuals who are cigarette smoker through
salivary test to which will give a white ppt in a
soln as a positive result (refer to equation 3).
3CNS- + Fe3+ Fe(CNS)3

indicating the presence of maltose (reducing

sugar) on the salivary digest.
The degree of visible result for the
Benedicts test subjected before achromatic
point can be possible however differs with the
ones obtained after. This is seen through the
color ranging from green to orange.

(eqn. 3)

Table 4. Variables tested in saliva

Test
Digestion of starch

Result

Effect of acid and base

At 40C, the salivary amylase is able to digest

the starch molecule. The formation of the deep
blue coloration after the addition of I2 (see Table
4) denotes that starch reacts the iodine molecule
an indicating that it is still a coiling
polysaccharide compound made up of several
glucose units. On the other hand, the presence of
the yellow solution after achromatic point
showed that starch is already digested and
broken down to its individual glucose.
Achromatic point is the moment wherein the
solution is without color. It is the point where
starch does not five a colored solution during I2
test. Benedicts test can be used to evaluate the
presence of reducing sugar; this is observed
through the brick red precipitate that will form

Hydrochloric acid is naturally produced

in the stomach and one of its function is to
change the pH to acidic. Salivary amylase
(enzyme) can work in optimum condition when
it is in alkaline medium, resulting for the
enzymatic activity to slow down until it comes
into halt. This explains the negative result
obtained to the digest treated with 0.01 M HCl
when I2 was added.

CONCLUSION
Saliva contains 98% water and essential
substances such as electrolytes, mucus and
various enzymes. The digestive functions of
saliva include moistening food and helping the
task of swallowing a lot easier. It contains
several components that play essential role in
humans body system, particularly the inorganic
matter inherent to man. These ions maintain the
osmotic balance in our mouth and the structure
of the tooth enamel, however, qualitative
measure for the presence of these ions varies to
each individual. As a result, some test gave
negative result. Saliva can also be used as
presumptive test to determine any symptoms of
serious illness such as cancer and the
identification of a cigarette smoker. Factors that
affect the enzymatic activity in salivary
digestion were also defined; the normal pH
range for saliva was identified to be close to
neutral, hydrochloric acid was known to be
naturally produced in our stomach that also
alters the pH to the acidic side disallowing the
enzymatic activity to run at optimum conditions.

http://healthyeating.sfgate.com/starch-changedsaliva-mouth-11883.html (Date accessed July 6,

2016)
REFERENCES
Online Websites
http://cariology.wikifoundry.com/page/Composit
ion+of+saliva (Date accessed July 6, 2016)
http://www.livestrong.com/article/488214starch-and-digestion/ (Date accessed July 6,
2016)
http://jn.nutrition.org/content/5/6/599.extract
(Date accessed July 6, 2016)

http://amrita.olabs.edu.in/?
sub=79&brch=18&sim=236&cnt=1
accessed July 6, 2016)
http://chestofbooks.com/health/naturalcure/The-Hygienic-SystemOrthotrophy/Salivary-Digestion.html
(Date accessed July 6, 2016)
http://www.wnho.net/role_of_saliva.htm
(Date accessed July 14, 2016)

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