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ANALYSIS OF HUMAN SALIVA

Zanthope Czarina B. Perez

Mrs. Emilie Franje

CHEM 23A
MF 7:00-10:00 AM

Analysis of Human Saliva

I.

Introduction
Saliva in humans is a mouth fluid possessing several functions
involved in oral health and homeostasis, with an active protective role
in maintaining oral healthiness. Saliva helps bolus formation by
moistening food, protects the oral mucosa against mechanical damage
and plays a role in the preliminary digestion of food through the
presence of - amylase and other enzymes. [1]
In humans, oral fluid originates mainly from three pairs of major
salivary glands (parotid, sublingual and submandibular) and from a
large number of minor salivary glands. Parotid glands are entirely
serous glands since their secretion lacks mucin, whereas
submandibular and sublingual glands are mixed sero-mucous. Minor
salivary glands are mainly Von Ebner glands (entirely serous organs
situated in the connective tissue below the circumvallatae papillae)
and Blandin-Nhm mucous glands [2].
Hence, this experiment aims to test if there is a presence of
amylase, mucins, calcium and inorganic phosphate in the saliva which
can be seen as colored precipitates.

II.

Methodology

A. Materials and Apparatus


The chemicals used in this experiment are as follows; 1%
acetic acid, 10% NaOH solution, 0.5% NaCl solution, 2% Potassium
oxalate solution, starch solution, KI solution, human saliva and 0.5%
CuSO4 solution. The apparatus used in this experiment are as
follows; beaker, test tubes, test tube rack, spot plate, alcohol lamp,
droppers and 10-mL graduated cylinder.
B. Procedure
1. Collection of saliva
About 3mL of saliva was collected and placed in a clean
test tube. Time was taken after the required amount was
collected. The saliva flow rate was then determined by its unit
mL/min. The 3mL saliva was then diluted with 15mL of 0.5%
NaCl solution.

2. Test for Amylase


On each spot of the spot plate, 1 drop of KI solution was
added. On the first spot, 1 drop of starch solution was added.
The color was noted. This first spot served as blank. Time was
then noted. Meanwhile, 5mL of the 1% starch solution was
mixed in a test tube with 1 mL of the diluted saliva solution.
The mixture was then shaken. 2 drops of the mixture was then
added to another spot every minute. The color change was
noted. Adding of the mixture on every spot was continued
until the color no longer changed. Time was then noted after
the color stopped changing. It was noted that if the color has
not changed from blue-black to brown to yellow in 4 minutes,
the amylase activity is slow, in this case more saliva is added.
But if the change is done in 2 minutes, the rate is too fast,
thus, more NaCl solution or the diluted saliva mixture is
added. The procedure was repeated but this time after
chewing a biscuit.
3. Test for Mucins
2 mL of saliva fluid was added with 1% acetic acid drop by
drop until something changes.
4. Test for Calcium
2 mL of salivary fluid was added with 5 drops of 1% acetic
acid and 5 mL of 2% potassium oxalate solution.
5. Test for Proteins
2 mL of salivary fluids was added with 1 mL of 10% NaOH
solution to a separate test tube. 1-2 drops of 0.5% CuSO 4
solution was then added. The mixture was shaken and results
were noted.

III.

Results
A. Collection of Saliva
Amount of saliva collected: 3 mL
Time after required amount was collected: 11 mins.
Salivary flow rate: 3mL / 11 mins. = 0.273 mL/min

B. Test for Amylase


Time (mins.)
1
2
3
4
5
6
7
8
9
10
11

Before chewing a After chewing a


biscuit
biscuit
Blue-black
Blue-black
Brick-red
Blue-black
Orange
Blue-black
Yellow
Blue-black
Dark violet
Brick red
Brick red
Brick red
Faint brick red
Orange
orange
Yellow

C. Test for Mucin


Separation/ thread-like precipitate within 18 drops
D. Test for Calcium
Cloudy white precipitate
E. Test for Protein
Dark violet solution

IV.

Discussion
The result for the salivary flow rate was 0.273 mL/min. In this
case, the salivary flow rate is low. This means that the saliva is not
functioning to its full potential and the oral health is at risk. The
saliva may not flow and protect surfaces effectively and the general
acidity in the mouth will be favoring demineralization and/or
increasing colonization of aciduric bacteria. (Dawes, 1996)
Furthermore, individuals with low salivary flow rate may experience
dry lip. (Hansel, et. al, 2000). The extended use of saliva flow rate
test would probably lead to the earlier detection of hypo salivation
and thereby in many cases the early detection of underlying
diseases.

Saliva was rich in the enzyme amylase which is the main


enzyme to break starch. Amylase hydrolases -1,4 glycosidic bonds
in starch at random. According to the results, the rate of time that
the starch was broken down was fast but after chewing a biscuit,
the rate went slow. Amylase is an enzyme that carries out specific
reaction of breaking down starch into simple sugar. Starch is made
up of many molecules of glucose joined in a chain. When we say
that amylase can catalyze the same reaction many times over, we
mean that it can break the bond between two glucose molecules in
starch over and over without losing its activity. (Fulop, B., et al.,
2014) In this case, the rate of the amylase to break down starch
was fast. But when we say that an enzyme catalyzes a specific
reaction we mean that it is designed to do one thing very well, to
the point where it cant do other things. Example would be that
Amylase cannot break down proteins, which are chains of amino
acids. (Noriega, T., 2014) Hence, causing the rate of break down
after chewing the biscuit to be slow.
A long thread-like precipitate was formed which indicates the
presence of mucins. Mucin is a glycoprotein constituent of mucus.
The precipitate was formed because of the acetic acid which is
acidic. Test of mucins was performed to know if there are any
symptoms of abnormalities that is happening to the body.
Increased mucin production occurs in many adenocarcinomas
including cancer the pancreas, lung, breast, ovary, colon and other
tissues. (McPherson, RA., et al., 2011)
On the test for calcium, it showed a cloudy white ppt. in this
case, the saliva has a high content of calcium. The precipitate was
formed due to the calcium ions precipitated as potassium oxalate
under neutral or slightly acidic conditions. One probable reason for
having such high content of calcium is eating or drinking too much
food rich in calcium. Eating too much calcium inhibits the absorption
of magnesium, and eating too much magnesium inhibits the
absorption of calcium (Hoffer & Saul, 2000).
Meanwhile, A dark violet colored solution occurred in the test
for proteins. This result indicates a high content of protein. The
copper atoms of Biuret solution (CuSO 4 and NaOH) will react with
several peptide bonds on polypeptides, producing a color change
from blue to a deep violet or blue color. Furthermore, the major

factors affecting the protein concentration and composition of whole


saliva are the salivary flow rate, protein contributions of the
glandular saliva and crevicular fluid proteins. Thus, the elevated
protein levels are most likely due to enhanced synthesis and
secretion by the individual glandular saliva. ( Pai, M., et al., 2013).

V.

Summary and Conclusion


Therefore, these particular test for the different
components of the saliva which gave a positive result on all test,
this generally indicate that these components are always present
in the saliva like the amylase. Without the presence of it, starch
in food we eat will never be break down because starch is
necessary to break down into glucose monomer by amylase.
Positive results are in the form of dark violet which indicates the
presence of protein, while a thread-like structure/precipitate was
formed in the test for mucin which indicates the presence of
mucins and a cloudy white precipitate for the presence of
calcium.

VI.

References

[1] McPherson RA, Ben-Ezra J. Basic examinationof saliva. In: McPherson


RA, Pincus MR,eds.
Henry's Clinical Diagnosis and Management by Laboratory Meth
ods
22nd ed.Philadelphia, PA: Elsevier Saunders; 2011:chap28.

[2] Dawes, C., Ong, BY. (1973). Circadian rhythms in the flow rate
and proportional
contribution of parotid to whole saliva volume in man. Arch
Biol 18:1145-53.
[3] Dawes, C. (1996). Factors influencing salivary flow rate and
composition. In: Saliva and oral health. WM Edgar and DM
OMullane editors. London: British Dental Association, pp.
27-41.

[4] Fox, PC. (1996). Differentiation of dry mouth etiology. Adv Dent
Res 10:13-6
[5] Hoffer, R., Saul, E. (2000). Oral status of 81 subjects with eating
disorders. Eur J Oral Sci 07:157-63
[6] Pai, M., et al. (2013). Viscosity of whole saliva. Acta Odontol
Scand 56:210-4
[7] Fulop, B., et al. (2014). Amylase-exploring digestion. Retrieved
at www.seplessons.org/mode
[8] Noriega, T. (2014). Amylase in saliva. Retrieved at
www.seplessons.org/mode/2443