jhfdsXavier University Ateneo de Cagayan

Salivary Digestion
(Laboratory Report No. 11)

In Partial Fulfillment
Of the Requirements for Chemistry 153L

Submitted by:
Daniel Sotto
Lew Carynn Tacandong
Lloyden Adonnis Tan

BSN – 1C

AY 2018-2019
Analysis

The digestive process contains many different steps that take place in many
different organs. The first step of digestion begins at the mouth, where the food is
subjected to the mechanical process of grinding by the teeth and the tongue. Saliva is
secreted by the three paired salivary glands namely the parotid glands, the
submandibular glands, and the sublingual glands. These glands are located in the oral
cavity and secretes serous, mucus, and enzymes like ptyalin and amylase that helps in
keeping the food soft, moisten the food, and digest starch into maltose and dextrin. The
secretion of mucus of salivary glands keeps the lining of the oral cavity moist and helps
obviate dental caries by cleansing the mouth of cariogenic carbohydrates and by
neutralizing lactic acid. In addition, the enzymes also provide lubrication to help in
chewing and swallowing of the food.

Amylase is an enzyme that help digest carbohydrates. This enzyme is secreted

by two glands: pancreas and the salivary glands. First, salivary amylase initiates protein
digestion by breaking large molecules of starch into maltose – a smaller carbohydrate.
This is evident when a sweet taste is detected after chewing starchy foods like rice,
sweet potatoes, and pastas. Second, the digestion of starch is finished when the
pancreas release amylase (called pancreatic amylase) and release it into the duodenum
papillae of the duodenum of the small intestine through the pancreatic duct. After
breaking down maltose, the smallest unit of starch is yielded: glucose. Starch needs to
be broken down into its smallest form because large molecules cannot be absorbed by
the small intestine. The mechanical digestion helps chemical digestion by breaking the
foods and thus increasing their surface area.

Therefore, the objectives of this experiment are to identify the product formed on
the salivary digestion of starch, trace the salivary digestion of starch through iodine
solution, components present in saliva, the factors that affect enzymatic activity and to
give the different functions a constituent play on both the digestion process. Qualitative
tests – test for mucin, test for protein, test for inorganic matter, test for chlorides, test for
phosphates, test for calcium, and test for thiocyanides -, the separation of the products
of salivary digestion, the digestion of starch paste, influence of free acid, and the
influence of alkali were all done to further analyze and study the properties, the products
formed, and the different composition of the saliva. Various results were obtained and
some color reaction were also noted.
Qualitative Test Observation
Microscopical Examination Sketch of the unfiltered saliva thru a
microscope

Unfiltered saliva stained with methylene

blue and viewed thru the microscope

Salivary Microscope Constituents

o Enzymes
o Mucus
o Protein
o Electrolytes
o Glycoproteins
o Lysozyme
o

pH determination Estimated pH of saliva

7
Test for mucin Identify the precipitate formed

Cloudy and white in color

What is the function of this precipitate

Lubrication of the mouth

Prevents bacteria build up

Test for Protein (biuret test) Blue to purple in color

Test for inorganic matter

5 mL of saliva + 1 drop 6N HNO3, heated

to boiling and filtered
A). test for chlorides Color: Cloudy white
Filtrate + AgNO3 Name of ppt: silver chloride
Equation: AgNO3 + Cl− → AgCl(s) + NO3

B.) test for phosphates Color: Yellow

Filtrate + (NH4)2MoO4 Name of ppt: ammonium
 phosphomolybdate
Equation: 12(NH4)2MoO4 + H3PO4 + 21HNO3
→ (NH4)3PMo13O40(s) + 21NH4NO3 + 12H2O

C.) test for sulfates Color: Achromatic

Filtrate + BaCl2 Name of ppt: Barium Sulfate
Equation: sulfate + barium chloride —–>
Barium sulfate + chloride white precipitate
D.) test for calcium Color: Achromatic
Filtrate + (NH4)2C2O4 Name of ppt: Calcium oxalate

Equation: Calcium + ammonium oxalate

—–> calcium oxalate + ammonium
Test for Thiocyanate (FeCl3 test) Color: light yellow
a.) Saliva + dil. FeCl3 + dil. HCL till
acidic
b.) + 1 drop HgCl2 Color: light yellow
c.) Saliva + distilled water Color: light yellow
Comparing results from a and c Same color was observed
Source of thiocyanate in saliva: Hydrogen cyanide, cabbage, broccoli

Microscopical Examination and pH level

Cheek cells of epithelial cells are easily shed off from the walls of the oral cavity.
These cells are eukaryotic, which insinuates that it contains nucleus and organelles
enclosed in a membrane. Cheek cells are achromatic in nature; hence, there will be a
difficulty observing these cells under the microscope. Staining these cells is necessary
for it to be visible. In the experiment, methylthioninium chloride was added to the
collected saliva. Methylene blue stained the epithelial cells present in the saliva; this
indicator has a string affinity to Deoxyribonucleic acid (DNA) and Ribonucleic acid
(RNA) – the carriers of genetic information of an individual. As observed, there are
regions of the cell that are darker in color than its surrounding – especially on the center
of the cell. It is found that the darker area is the nucleus of the cell, where genetic
information like DNA and RNA are housed.

The normal pH of a resting whole saliva ranges from 5.6 to 7.9; and it may
change according to various factors. pH depends on the concentration of salivary
proteins – bicarbonate ions and phosphate ions that act as buffers. Besides bicarbonate
ions and phosphate ions, there are also other ions that are significant to maintain the
slightly hypotonic osmolarity of saliva. Further, pH level can be affected by the food and
drink consumed by an individual. The pH level obtained of the collected saliva is 7
which is neutral.
Test for mucin and protein

Mucin is present in the collected saliva due to the precipitate. It is a

mucopolysaccharide or a large glycoprotein that is chief constituent of mucus. Its
function is to lubricate the alimentary canal from acid, microorganisms, pathogens, and
mechanical trauma and to prevent bacteria build up. By observing the alteration of
mucin expression, the detection of the development of cancer and cellular growth,
differentiation, adhesion, invasion, transformation and immune surveillance can be
possible. This is why mucins are used as markers for cancer (breast, lung, pancreas,
and other tissues). Its gels hold biologically active molecules that indicates molecular or
physical breach of the mucin layer.

Biuret test is the test for the presence of peptide bonds or a compound
containing two CONH2 joined either directly together or through a N or C atom (C – N).
From the peptide bond, the 4 nitrogen atom’s lone pair or unshared electrons coordinate
with alkaline CuSO4 ions that forms a violet – colored complex. The color indicates the
presence of proteins, specifically the peptide bonds. The intensity of the color of this test
is directly proportional to the number of peptide bonds or linkage present in the solution.
The collected saliva obtained a positive test for biuret, which means that the saliva
contains proteins. There are number proteins present in the saliva and these includes
the different enzymes like amylase, histatins, lactoperoxidase, immunoglobulins, and
statherin.

TEST FOR INORGANIC MATTER

The test for inorganic matter consists of the test for chloride, phosphate, sulfate,
and calcium. The collected saliva resulted positive for chloride due to the formation of
silver chloride from the reaction between the filtrate and AgNO 3. Chloride in the saliva
maintains osmotic pressure balance in the mouth.

AgNO3 + Cl− → AgCl(s) + NO3

 12(NH4)2MoO4 + H3PO4 + 21HNO3 → (NH4)3PMo13O40(s) + 21NH4NO3 + 12H2O

Aside from chloride, the test for phosphates also resulted positive. A yellow
precipitate (ammonium phosphomolybdate) formed from the filtrate + Ammonium
orthomolybdate reactants. Phosphate is crucial maintaining the structure of the tooth
and it also acts a buffer to prevent sudden fluctuation of pH level in the oral cavity. The
test for sulfates and calcium had a negative result indicating that these ions are not in
the composition of the collected saliva or that these may be present but in minute
amounts. These ions are found in dried fruits, bottled lemon juice, bottled lime juice,
wine, and some leafy green vegetables.

TEST FOR THIOCYANATE

SCN- + H2O2→ OSCN- + H2O

Thiocyanate, or also known as rhodanide, is a general test for smoking.

Thiocyanate ion (SCN-) is converted into hypothiocyanite (OSCN -) by peroxidase; the
concentration of hypothiocyanite is due to the diet and smoking. OSCN - can block
glucose uptake, inhibit amino acid transport, and disrupt electrochemical gradients in
the body. Large amounts of thiocyanate are generated from people with a high intake of
cyanide from smoking cigarettes, foods rich in cyanide, or even from industrial pollution
of the environment with cyanide. Humans, when exposed to high levels of thiocyanate,
are in risk of having thyroid disease and developmental and iodine deficiency disorders.
The risks can be prevented by increasing iodine uptake. By this mechanism
thiocyanate is one of the most important environmental compounds influencing the
occurrence of thyroid disease. In the experiment, the test for thiocyanate resulted
negative; hence, it is safe to say that the proprietors of the collected saliva do not
smoke nor eaten foods rich in cyanide.
DIGESTION OF STARCH PASTE
Time Color of I2 + saliva
1st time Dark Blue
Turned lighter after few minutes
2nd time Blue
Turned lighter after few minutes
3rd time Blue
Color is a bit lighter than the preceding
minute
4th time Light Blue

5th time Light Blue

6th time Achromatic

In the experiment, a blue colored solution was observed after adding a drop of I 2.
Also, as time passes, the color of the solution compared to its precedent is lighter in
color. The formation of the blue color indicates that starch’s structure (coiled) was not
yet altered. As time passes, the color of each saliva + I 2 gets lighter and lighter until the
achromatic point reached. Achromatic point is the point where the solution no longer
has color or is achromatic. Once the solution reaches achromatic point, all starch is
already digested into glucose units. So as the color gets lighter and lighter, it insinuates
that saliva is digesting or hydrolyzing the starch. The solution becomes negative to
iodine test but is now positive to benedict’s test.

DIGESTION OF STRARCH PASTE

Condition
Fehling’s test at achromatic point
What is the reduction for?
Time it took for complete transformation
of starch to sugar
Observation
Color: a color shift from blue to orange/
red at achromatic test was observed
To breakdown starch into maltose
10 – 20 minutes

The Fehling’s test resulted positive. At achromatic point, starch is completely

digested by the saliva, this means that starch is reduced into glucose units like maltose
and erythrodextrin. Maltose is a reducing sugar, it is capable of reducing copper II ions
to copper I ions. Which is the principle of Fehling’s test in the detection of reducing
sugars. From the data, the saliva is successful in digesting the starch.
SEPARATION OF THE PRODUCTS OF SALIVARY DIGESTION
Condition Observation
25mL of 1% starch paste + 1mL saliva, Color:
stirred, + iodine, + 50mL of 95% alcohol,
allowed to stand undisturbed
Residue
a.) Phenylhydrazine test
b.) Benedict’s test
c.) Dextrin ppt. subjected to iodine
test

INFLUENCE OF FREE ACID

Condition Observation
Tt # 1 4mL 0.2% CL + 1mL starch paste + 1 drop Dark Blue liquid;
saliva The formation of the
(acidity = 0.16%) mixture was the quickest
compared to the other
Tt # 2 4mL 0.5% CL + 1mL starch paste + 1 drop Dark blue liquid
saliva Faster than tt # 3 & 4
(acidity = 0.04%)
Tt # 3 4mL 0.0125% CL + 1mL starch paste + 1 drop Dark Blue liquid;
saliva Blue liquid submerged at
(acidity = 0.01%) the bottom of the test tube;
Faster than tt # 4
Tt # 4 4mL 0.006% CL + 1mL starch paste + 1 drop Dark Blue liquid
saliva Blue solution mixed the
(acidity = 0.0048%) slowest
INFLUENCE OF FREE ACID
Condition Observation
Tt # 1 4mL 2% Na2CO3 + 1mL starch paste + 1 drop Dark Blue liquid settled on
saliva top
Tt # 2 4mL 1% Na2CO3 + 1mL starch paste + 1 drop Dark Blue liquid settled on
saliva top
Tt # 3 4mL 0.5% Na2CO3 + 1mL starch paste + 1 drop A dark blue liquid settled
saliva incompletely in the whole
in the whole solution
Tt # 4 4mL 0.05% Na2CO3 + 1mL starch paste + 1 A maroon – colored liquid
drop saliva settled on top incompletely

Conclusion
Sources
https://www.sciencedirect.com/science/article/pii/0378434785800402
https://gopzonfire.wordpress.com/2015/09/06/salivary-digestion/
https://www.microscopemaster.com/cheek-cells-microscope.html
https://healthyeating.sfgate.com/functions-amylase-protease-lipase-digestive-enzymes-
3325.html
https://chemistry.stackexchange.com/questions/24195/is-saliva-an-acid-or-base
https://www.nature.com/articles/nrc1251/
https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/thiocyanate