Group Members: Group No.

YB2C
1. Leslie Jane B. Jala Section: YB2
2. Shimea Pagurayan Date Submitted: November
10, 2020

Expt. 8 Salivary Digestion

I. Objectives Member Member
1 2
• To prepare saliva sample Pre-lab Data
• To identify the components Data and
present in saliva Observations
• To identify the product Lab Report
formed on the salivary Sub-total
Online Act
digestion of starch.
Participation
• To detect the salivary Total Score
digestion through iodine
solution
• To identify factors that affect enzymatic activity

II. Apparatus III. Symbols of Chemicals

• Stirrer • Saliva sample
• Dropper • C16H18CIN3S
• Microscope • CH3COOH
• Microscope slides • CuSO4
• pH paper strip • NaOH
• Test tubes • HNO3
• Beaker • AgNo3
• Hot plate • (NH4)2MoO4
• Funnel • BaCl2
• Filter paper • (NH4)2C2O4
• Porcelain crucible • HCl
• Spot plate • FeCl3
• Graduated cylinder • HgCl2
• Calibrated timer • Starch Paste
• Test tube rack • I2
• thermometer • H2O
• Fehling’s reagent A
 • Fehling’s reagent B
• C2H5OH
• Benedict’s reagent
• Na2CO3
IV. Procedure:

V. Data and Observations

QUALITATIVE TEST OBSERVATION

Microscopial Examination Sketch of the unfiltered saliva viewed thru a

microscope
Unfiltered saliva stained with
methylene blue and viewed thru the
Microscope

Salivary microscope constituents

• Electrolytes
• Enzymes
• Mucus
• Proteins
• Epithelial cells
• water

pH Determination Estimated pH of saliva: 7

Test for mucin Identify the precipitate formed?

3mL saliva + 1-2 drops dil. HC2H3- • Green-blue precipitate
O2

What is the function of this precipitate?

• lubricate and hydrate the oral
structures in the mouth
• assist in swallowing
• prevent bacteria build-up
Test for protein (Biuret test) Blue-violet precipitate

Test for Inorganic Matter

5 mL of saliva + 1 drop of 6N
HNO3,
Heated to boiling and filtered Color: Cloudy white ppt.
Name of ppt: silver chloride
a) Test for chlorides Equation
Filtrate + AgNO3
Cl- (aq) + AgNO3 (aq) → AgCl (s) + NO3

Color: Yellow ppt

b) Test for phosphates Name of ppt: ammonium phosphomolybdate
Filtrate + (NH4)2MoO4 Equation:
PO43- + (NH4)2MoO4 → (NH4)3PO4(MoO3)12 +
H2O

Color: clear solution

c) Test for sulfates Name of ppt: barium sulfate
Filtrate + BaCl2 Equation:

BaCl2 (aq) + SO42- (aq) → BaSO4 (s)

Color: clear solution

d) Test for calcium Name of ppt: calcium oxalate
Filtrate + (NH4)2C2O4 Equation

Ca2+ + C2O42-+ ←→ CaC2O4

Test for thiocyanate (FeCl3 test)

a) saliva + dil. FeCl3 + dil. HCl till acidic Color: Clear Yellow Solution

b) + 1 drop HgCl2 Color: Clear Yellow Solution

c) saliva +distilled water Color: not shown in the video

Comparing results from a) and c) Same color was observed

Source of thiocyanate in saliva: Hydrogen cyanide, smoking and

cyanide-containing food like cabbage
and broccoli
DIGESTION OF STARCH PASTE

TIME COLOR of I2 + SALIVA

1st minute Blue-Black

2nd minute Brownish-Blue

3rd minute Brownish-Blue

4th minute Dark Brown

5th minute Brown

6th minute Achromatic

DIGESTION OF STARCH PASTE

CONDITION OBSERVATION

Fehling’s Test at achromatic point Color: Deep Blue solution with Red
precipitate

What is the reduction due to? Due to the reducing sugars (maltose)
present in hydrolyzed starch

Time it took for complete transformation 10 – 20 minutes

of starch to sugar

SEPARATION OF THE PRODUCTS OF SALIVARY DIGESTION

CONDITION OBSERVATION

25 mL of 1% starch paste + 1 mL saliva, Color: Colorless

stirred, + iodine, +50 mL of 95% alcohol,
allowed to stand undisturbed.
Residue White Precipitate

a) Benedict’s test blue turned into an orange solution

b) Dextrin ppt. subjected to iodine Not performed in video
test
INFLUENCE OF FREE ACID

CONDITION OBSERVATION

tt #1 4 mL 0.2% HCl + 1 mL starch Dark Blue Solution

paste + 1 drop saliva
(acidity = 0.16%)

tt #2 4 mL 0.5% HCl + 1 mL starch Dark Blue Solution

paste + 1 drop saliva
(acidity = 0.04%)

tt #3 4 mL 0.0125% HCl + 1 mL starch Dark Blue Solution

paste + 1 drop saliva
(acidity = 0.01%)

tt #4 4 mL 0.006% HCl + 1 mL starch Dark Blue Solution

paste + 1 drop saliva
(acidity = 0.0048%)

INFLUENCE OF ALKALI

CONDITION OBSERVATION

tt #1 4 mL 2% Na2CO3 + 1 mL starch Dark Blue Solution

paste + 1 drop saliva

tt #2 4 mL 1% Na2CO3 + 1 mL starch Blue Solution

paste + 1 drop saliva

tt #3 4 mL 0.5% Na2CO3 + 1 mL starch Dark Blue Solution

paste + 1 drop saliva

tt #4 4 mL 0.05% Na2CO3 + 1 mL Red Solution

starch paste + 1 drop saliva
 SCHEMATIC DIAGRAM

Saliva Preparation
Rinse mouth with water

saliva in water
Collect 50-60 ml

1 drop unfiltered
saliva saliva in water

+ 1 drop methylene blue Determine pH

View thru microscope

Filter saliva

Test for Mucin

test tube

+ 3 mL filtered saliva

+ 1-2 drops dilute CH3COOH

observe

Test for Protein

Test tube

Alkalinized saliva sample

+ 2 drops CuSO4
shake
Observe
 Test for Inorganic Matter

5 mL saliva in test
tube

+ 1 drop 6N HNO3
Heat to boiling
Filter
filtrate

Test for Test for Test for Test for

chlorides phosphates sulfates calcium

+ 5 drops saliva + 5 drops saliva + 5 drops saliva + 5 drops saliva

+ 5 drops (NH4)2MoO4
+ 5 drops AgNO3 .+ 5 drops 0.1 M + 5 drops 0.1 N
Warm for 10 min.
BaCl2 NH4C2O4
Allow to stand
Observe & record Observe & record Observe & record Observe & record

Test for Thiocyanate

Porcelain
Crucible

+ 2 drops of FeCl3

+ 2 drops of Saliva

Mixture is acidified

+ 1 drop HCl
+ a drop of HgCl2
Observe the results
Digestion of Starch Paste

Beaker

+ 25 mL starch paste

+ 5 drops filtered saliva

Stirred thoroughly

Spot
Plate

A drop of the mixture

Every one minute, a drop of I2
Repeat until 5 minutes
Observe the color

Digestion of Starch Paste – Fehling’s Reagent

Test
Tube

+ 0.5 mL Fehling’s Reagent A

+ 0.5 mL Fehling’s Reagent B

+ 1 drop starch paste

+ Few drops of saliva

Warmed in a water bath

Observe the reaction
Digestion of Starch Paste – Benedict’s Reagent

Saliva is dissolved in
50 mL CH3CH2OH.

Precipitate is filtered out

Filtrate is evaporated to dryness

in an evaporated dish

The residue is tested using

Benedict’s test

Test Test
Tube 1 Tube 2

+ 1 drop of the Benedict’s reagent

+ 1 drop starch paste

Heated to a water bath

Observe the reaction

Influence of Free Acid

Test Tube Test Tube Test Tube Test Tube

0.2% HCl 0.05% HCl 0.0125% HCl 0.2% HCl

+ 1 mL starch paste
+ drop of saliva

Water bath at 40℃ for 20 min.

+ 5 drops of I2
Observe and record.
Influence of Alkali

Test Tube Test Tube Test Tube Test Tube

2% Na2CO3 1% Na2CO3 0.5% Na2CO3 0.05% Na2CO3

+ 1 mL starch paste
+ drop of saliva

+ 5 drops of CH3COOH
Water bath at 40℃ for 20 min.
+ 5 drops of I2

Observe and record.

Laboratory Report on Experiment 8
Salivary Digestion
Introduction

One of the most recognizable digestive enzymes in human saliva is amylase

(BD Editor, 2019). This enzyme is able to break down the starch in our food to
simpler and more easily digestible sugars like glucose and maltose (BD Editor,
2019). Saliva is an aqueous fluid in the oral cavity playing a fundamental role in the
preservation and maintenance of oral health (Maddu, 2019). It acts in relation to
taste, mastication, bolus formation, enzymatic digestion, and swallowing (Maddu,
2019). Saliva has a number of functions within the digestive system beyond breaking
down starches, it keeps our mouth and digestive tract lubricated, which ensures it
functions properly (BD Editor, 2019). The protective functions of saliva including
maintenance of dental and mucosal integrity indirectly influence the digestive
process (Maddu, 2019). It is a mixture of water, mucus, antibacterial substances,
and digestive enzymes (BD Editor, 2019).

Saliva is essential in formation of the pellicle, which protects the tooth after
eruption (Maddu, 2019). Furthermore, normal salivary composition, flow, and
function are extremely important on a daily basis (Maddu, 2019). It occurs in
quantities, large or small, and recognition should be given to the many contributions
it makes to the preservation and maintenance of health (Maddu, 2019). Thus, the
purpose of this experiment is to prepare saliva sample as well as to identify and
detect the components present in saliva, the product formed on the salivary digestion
of starch, salivary digestion through iodine solution, and factors that affect enzymatic
activity. Qualitative tests, digestion of starch paste, separation of the products of
salivary digestion, and influence of free acid and alkali were all performed to further
analyze and study the properties, composition and products formed of the saliva and
amylase enzymatic activity.
Discussion

A. Saliva Preparation

The first part of the experiment is to get the saliva sample wherein the mouth
is rinsed well with water to loosen as many cells from the mouth as possible. Next is
to collect 50-60 ml saliva in a water. Chewing a small piece of pure paraffin wax can
also help stimulate secretion of saliva. Afterwards, microscopial examination is
performed by adding one drop of methylene blue to small amount of unfiltered saliva.
Then the sample is placed under a microscope. Methylene blue is a popular alkaline
stain used to view microscopic life in brilliant color and thus help identify the
sample’s components and parts using light microscopes (Microscope LCC, 2020).

Saliva is composed of a variety of electrolytes (bicarbonate, phosphate,

calcium, potassium, sodium and magnesium), immunoglobulins, enzymes (salivary,
amylase), proteins, mucins and nitrogenous products (urea and ammonia)
(Humphrey & Williamson, 2001). These components interact with one another to do
the following functions: (1) proteins and mucins work together to aggregate, cleanse
and attach oral microorganism and contribute to dental plaque metabolism; (2)
enzymes, immunoglobulins and proteins help in antibacterial activity to reduce tooth
decay by inhibiting growth of some bacteria; (3) urea, bicarbonates and phosphates
serve to modulate buffering capacity and pH of saliva; (4) proteins, calcium and
proteins function as an anti-solubility factor and modulate remineralization and
demineralization (Humphrey & Williamson, 2001). In addition, the salivary amylase in
saliva begins hydrolyzing starch, a long chain of repeating glucose molecules, into
shorter polysaccharide chains, dextrins, and the disaccharide maltose (Boundless,
2020)

The pH of the saliva was determined by a piece of pH paper which was

dipped into the saliva sample. The determined pH was 7 which is as expected as the
normal pH of saliva is 6-7 and the range of pH in salivary flow is 5.3 (low flow) to 7.8
(peak flow), thus making saliva slightly acidic (Humphrey & Williamson, 2001). This
is also the optimum pH for the enzymatic activity of salivary amylase, as this enzyme
is most active at pH 6.8 (Amrita Olabs, 2015). Below and above this pH causes the
reaction rate to decrease as the enzymes get denatured (Amrita Olabs, 2015). This
is the reason why salivary amylase is present in the mouth and its isoenzyme is
present in the intestine but not in the stomach because of the high-level acidity
present in this area that causes the enzyme to inactivate and denature (Amrita
Olabs, 2015).

B. Qualitative Test
I. Test for Mucin

Mucins are high-molecular-weight glycoproteins that coat the hard and soft
tissues serving to lubricate and hydrate the oral structures in the mouth, prevent
bacteria build-up, and assist in swallowing (Tabak, 1990). Furthermore, mucins also
function to protect the surface of oral mucous membranes from various types of
irritants and toxins in food or stimulants (Kubala et al., 2018). In this test, a clear
solution with blue-green precipitate was observed in the test. This indicates that
mucin is present in the collected saliva as precipitate formed upon addition of 2
drops of dilute acetic acid which is about pH 4.5 (Svensson, 2008). The test for
mucin is used clinically as diagnostic markers between normal and disease
conditions and can be used in many carcinomas such as cancer of the breast, colon,
stomach, lung, ovary, pancreas, prostate and other tissues (Rachagani et al., 2009).

II. Test for Protein

The biuret test is used in this part of the experiment to detect the presence of
proteins in the saliva sample. An alkalinized sample of saliva was mixed with two
drops of copper (II) sulfate forming a blue-violet precipitate in a clear solution. Thus,
a protein is present in the saliva sample which is as expected as saliva contains
proteins such as mucin, amylases, defensins, cystatins, and histatins (Loo et al.,
2010).

The principle of this test is that the peptide structure in protein containing at
least two peptide links produces a violet color when treated with alkaline copper (II)
sulfate (Aryal, 2020). The reagent’s copper ions bind itself to the nitrogen atoms
present in the protein peptides and the copper (II) undergoes reduction and is
converted to copper (I) (BYJUS, 2020). Then four nitrogen atoms donate lone pairs
to form coordinate covalent bonds with cupric ions, forming a chelate complex
(BYJUS, 2020). This complex has the ability to absorb light with a wavelength of 540
nm, which imparts a purple color to the solution (BYJUS, 2020). The intensity of the
color is directly proportional to the number of peptide bonds present in the protein
molecule and the number of protein molecules present in the reaction system (Aryal,
2020).

O HN NH
R R
2+ 2+

NH
+ Cu O Cu O

NH NH
(Blue)
R R R
O O
n

(Violet)
Figure 1. Chemical reaction in the Biuret Test

III. Test for Inorganic Matter

In this test, the first step was to prepare the reagent, wherein 5 mL of saliva is
acidified with one drop of the 6M nitric acid solution. The saliva sample is acidified to
remove residual matrix components that interfere in the detection of inorganic matter
(Matusiewiez, 2017). The mixture was then boiled and filtered to remove protein
residues. This is because most proteins are insoluble in boiling water and are
denatured by it and is irreversibly converted into an insoluble material (Koshland &
Haurowitz, 2020). The filtrate was then used for the test for chlorides, phosphates,
sulfates, and calcium.

1. Test for Chlorides

Chlorides are a type of electrolyte that help control the amount of fluids and
balance of acids and bases in the body (Medline Plus, 2020). It is often measured
along with other electrolytes to monitor conditions such as high blood pressure, heart
failure, kidney disease and liver disease (Medline Plus, 2020). In the test, five drops
of silver nitrate were added to the sample of filtered saliva. The test for chlorides is
based on the precipitation of chloride salt (Government of Canada, 2018). When a
few drops of a solution containing silver ions (Ag+), such as silver nitrate, is added to
a slightly acidic aqueous solution containing chloride ions (Cl-), a white precipitate of
silver chloride (AgCl) will form unless concentration of chloride ions is very low
(Government of Canada, 2018). The reaction is written as:

Cl- (aq.) + AgNO3 (aq.) → AgCl (s) + NO3

Furthermore, the higher the starting concentrations of chloride and silver ions
are, the more precipitate will form (Government of Canada, 2018). In the test, a milky
white precipitate is formed indicating the presence of chloride. This is as expected as
saliva contains chlorine which maintains osmotic pressure balance in the mouth
(Humphrey & Williamson, 2001).

2. Test for Phosphates

Phosphate tests are utilized in measuring phosphate levels in malnourished

people, and diagnosing for digestive system conditions, ketoacidosis and kidney
failure (NHS, 2018). In this test, 5 drops of ammonium molybdate were mixed with 5
drops of filtered saliva. The mixture is then warmed in a water bath for 10 minutes
and is allowed to stand. The test for phosphates is based on the precipitation of
phosphate salt (Nipuna, 2019). Phosphate residues in phospholipids will react with
ammonium molybdate to form ammonium molybdophosphate to produce a yellow
precipitate (Nipuna, 2019). Gentle heating can also facilitate in the appearance of the
precipitate (Nipuna, 2019). The reaction is written as:

PO43- + (NH4)2MoO4 → (NH4)3PO4(MoO3)12 + H2O

In the test, a yellow precipitate was formed in the yellow solution indicating the
presence of phosphates. This is as expected as phosphate is a component of saliva
that function as a buffer that prevent sudden fluctuation of pH level in the oral cavity
and are needed for remineralization of tooth surfaces (GC Asia Dental Pte Ltd,
2008).
 3. Test for Sulfates

In this test, 5 drops of barium chloride were added to 5 drops of filtered saliva.
The test for sulfates is based on the precipitation of sulfate salt. When barium
chloride is added to an acidified solution that contains sulfate ions, a white
precipitate of barium sulfate forms (Goalby, 2016). The reaction is written as:

BaCl2 (aq) + SO42- (aq) → BaSO4 (s)

An acidified sample or acidified barium chloride is needed to remove

carbonate impurities that are often found in salts that would form a white barium
carbonate and give a false result (Goalby, 2016). In the test, there was no formation
of white precipitate as the solution remained clear upon addition of the reagent.
Although saliva does contain sulfates, the negative result could be due to low
concentrations of inorganic sulfate (SO4) in the saliva sample (Cole & Landry, 2002)

4. Test for Calcium

Measurement of salivary calcium concentration can be used as a screening

tool for osteoporosis (Rabiei et al., 2012). In this test, 5 drops of ammonium oxalate
is added to five drops of filtered saliva. The test for calcium is based on the
precipitation the carbonates of calcium (Robolab Technologies, 2020). In this test,
calcium ion reacts with ammonium oxalate to form a white precipitate of calcium
oxalate and it imparts a brick red color to the flame (Robolab Technologies, 2020).
The reaction is written as:

Ca2+ + C2O42-+ ←→ CaC2O4

In the test, the white precipitate did not form which is unexpected as calcium
is a component of saliva that help to modulate remineralization and demineralization
of tooth surfaces (Humphrey & Williamson, 2001). The negative result could be due
to not enough concentration of calcium in saliva for the reaction to occur.
IV. Test for Thiocyanate

This test is used for identifying individuals who are cigarette smokers as
thiocyanate is an end product of detoxification of hydrogen cyanide in cigarette
smoke (Luepker et al., 1981). A positive test is indicated by formation white
precipitate in a solution (Oxford Reference). Thiocyanate ion (SCN-) is converted into
hypothiocyanite (OSCN-) by peroxidase; the concentration of hypothiocyanite is due
to food consumption and smoking (Sotto, 2019). Furthermore, OSCN- can block
glucose uptake, inhibit amino acid transport, and disrupt electrochemical gradients in
the body (Sotto, 2019). Large amounts of thiocyanate are generated from people
with high intake of cyanide from smoking cigarettes, foods rich in cyanide, or even
from industrial pollution of the environment with cyanide (Sotto, 2019). Humans,
when exposed to high levels of thiocyanate, are in risk of having thyroid disease as
well as developmental and iodine deficiency disorders (Sotto, 2019). The risks can
be prevented by increasing iodine uptake (Sotto, 2019). The reaction is written as:

SCN- + H2O2 → OSCN- + H2O

In this experiment, the reagents used were hydrochloric acid (3 M HCl), Iron
(III) chloride (0.1 M FeCl3), and Mercury (II) chloride (0.1 M HgCl2). In a porcelain
crucible 2 drops of FeCl3 were added to 2 drops of saliva. The mixture is acidified
with 1 drop of HCl and then a drop of HgCl2 was added. As for the results, it gave a
negative yellow solution which means thiocyanate is absent in the saliva and the
person being tested does not smoke.

C. Digestion of Starch Paste

In this test, 25 mL of starch paste is prepared in a beaker and 5 drops of

filtered saliva was added, which was then stirred thoroughly. At one-minute intervals,
a drop of the mixture is placed on a spot plate and is tested with Iodine solution. The
principle of the test relied on the concept that the amylase present in saliva
hydrolyses the α (1→4) linked D-glucose units in starch which can form the
disaccharide maltose with the intermediate formation of various dextrins (Cheng,
1992). The reaction of starch and dextrin with iodine solution will yield a blue color,
intermediate dextrin will give a reddish-brown color, while maltose and lower dextrins
will have no reaction with iodine (Cheng, 1992). Thus, the action of amylase can be
observed by following the time taken when the color reaction of starch with iodine will
no longer occur, this is called the achromic point (Cheng, 1992). Once the solution
reaches achromatic point, this means all the starch is already digested into
disaccharide or monosaccharide units (Sotto, 2019). Therefore, as the color gets
lighter and lighter, it insinuates that saliva is digesting or hydrolyzing the starch
sample (Sotto, 2019).

salivary amylase
Starch Paste Maltose/Trose/Maltotriose

The result of the test shows the following: (1) at the 1st minute, after a drop of
the Iodine solution, a blue-black colored solution was formed; (2) at 2nd minute and 3
minute the solution turned to brownish-blue color; (3) in the last 4th and 5th minute, it
formed a dark brown and brown solutions respectively. This is as expected as the
longer the reaction between starch and amylase in saliva, the more the starch
undergoes hydrolysis and forms lighter color with the iodine solution (Cheng, 1992).
In this test, the 5th minute can be considered as the achromic point as the blue-black
color no longer formed. Although, the 6th minute that was not performed in the
experiment could also be the achromatic point wherein no color reaction is produced
after adding the reagent. The monosaccharide or disaccharide produced in this
experiment was detected by the Benedict’s test and Fehling’s test (Cheng, 1992).

I. Digestion of Starch Paste – Fehling’s Test

The Fehling’s test comprises of two reagents; the Fehling’s solution A and
Fehling’s solution B (Karki, 2018). Fehling’s A is a blue aqueous solution of copper
sulphate and Fehling’s B is a clear and colorless alkaline solution of sodium
potassium tartrate (Rochelle salt), (Karki, 2018). Rochelle salts (sodium potassium
tartrate) present in the reagent acts as the chelating agent in this reaction (Karki,
2018). In this test, 0.5 mL of Fehling’s Reagent A and B together with 1 drop of
hydrolyzed starch paste were mixed in a test tube with a few drops of saliva added.
The mixture is then warmed in a water bath. On heating an aldehyde or reducing
sugar with Fehling’s solution, some sugars can be oxidized and losses their electron
while the Fehling’s mixture can obtain the electrons (reduced) (Karki, 2018). This
reaction will give a reddish-brown precipitate (Karki, 2018). Formation of red
precipitate of cuprous oxide denotes the presence of reducing sugars (Karki, 2018).

Figure 1. Reaction of Fehling’s Test

In this test, the results showed the formation of a deep blue solution with a red
precipitate indicating a positive result for reducing sugars. This is expected as at the
achromatic point, starch is completely digested by the saliva which means that
starch is hydrolyzed into glucose units such as maltose and erythrodextrin, wherein,
maltose is the reducing sugar that reduced copper II ions to copper I ions forming the
red precipitate (Sotto, 2019).

II. Digestion of Starch Paste – Benedict’s Test

Benedict’s reagent contains potassium thiocyanate and is used to determine

how much reducing sugar is present (Aryal, 2019). This solution forms a copper
thiocyanate precipitate which is white and can be used in a titration (Aryal, 2019).
When Benedict’s solution and simple carbohydrates are heated, the color of the
solution changes from green to dark red or rusty brown, depending on the amount
and type of sugar present (Aryal, 2019). This reaction is caused by the reducing
property of simple carbohydrates (Aryal, 2019). The copper (II) ions in the Benedict’s
solution are reduced to Copper (I) ions, which causes the color change (Aryal, 2019).
Complex carbohydrates such as starches do not react positively with the Benedict’s
test unless they are broken down through heating or digestion (Aryal, 2019).

H O
HO O
+ 2 CU+2 + 5 OH- + 2 CU2O + 3 H2O
R
R

Aldehyde Cupric ions Carboxylic acid Cuprous oxide

(brick-red precipitate)

Figure 2. Benedict’s Test Reaction

In this test ethanol (CH3CH2OH) was used as a solvent and Benedict’s

solution as the reagent. First, the hydrolyzed saliva is dissolved in 50 mL of ethanol
to precipitate the sugars present. After standing, the precipitate is filtered out. Then
the filtrate is collected in an evaporating dish and evaporated to dryness. The
residue is tested using the Benedict’s test wherein, 1 drop of the Benedict’s reagent
was added to 1 drop of hydrolyzed starch paste and heated to a water bath. The
Benedict’s test turned the sample into an orange-brown colored solution. Thus, this
color change implies that 1 to 1.5 percent of sugar is present in the sample (Aryal,
2019).

D. Influence of Free Acid

Salivary amylase hydrolyzes bonds of starch to the disaccharide maltose and

moderate length oligosaccharides called limit dextrins (Amrita Olabs, 2015).
Although most enzymes are only active at a narrow pH range and in this case,
salivary amylase is active at pH 6-7 (Amrita Olabs, 2015). Above and below this
range causes the reaction rate to decrease as the enzymes get denatured (Amrita
Olabs, 2015).

In this experiment, the reagents used were Hydrochloric acid (HCl) and Iodine
solution (I2). A series of different hydrochloric acid concentrations are prepared first;
0.2% HCl, 0.05% HCl, 0.0125% HCl, and 0.006% HCl. Then, 1 mL of starch paste is
added to each test tube together with a drop of saliva, after which the test tubes are
agitated. These are then placed in a water bath at 40℃ for 20 minutes. Afterwards, 5
drops of Iodine solution was added to each test tube. Based from results of the
experiment, the more acidic the solution, the faster the coloration. Test tube #1 with
acidity of 0.16% showed a very high acidity as it spread quickly in the test tube.
Followed by the test tube #2 with the acidity of 0.04%, #3 with the acidity of 0.01%
and lastly is #4 with the acidity of 0.0048% which has the most diluted acidity among
the four test tubes. As all the test tubes formed a blue solution, this indicates the
presence of starch, which means that there was no occurrence of digestion or
hydrolysis. This is due to the acidity brought by the hydrochloric acid as in acidic
solutions, the rate of reaction decreases or would lead to inactivation of the enzyme
(Amrita Olabs, 2015). This is what happens in the stomach wherein, the hydrochloric
acid that is naturally produced in the stomach causes the salivary amylase to
denature by breaking down its structure which result to inactivating the enzymatic
activity of the enzyme to run at optimal conditions (Amrita Olabs, 2015).

E. Influence of Alkali

In this experiment, the reagents used were Hydrochloric acid (HCl), Sodium
carbonate (Na2CO3), and Iodine solution (I2). A series of different Sodium carbonate
concentrations are also prepared; 2% Na2CO3, 1% Na2CO3, 0.5% Na2CO3, and
0.05% Na2CO3. Then 1 mL of starch paste is added to each test tube together with a
drop of saliva, after which the test tubes are agitated. Then, 5 drops of the acetic
acid was added to acidify the solution for the Iodine test. The acetic acid has the
greatest inhibiting power as this solution disrupts the alkalinity which is necessary for
buffering capability. The test tubes are then placed in a water bath at 40℃ for 20
minutes. Then 5 drops of Iodine solution was added to each test tube. Based from
the results, the only variables are the percentage of acidity. Test tube #1 which has
the sodium carbonate concentration of 2% and test tube #2 which has the sodium
carbonate concentration of 1% showed the same movement of the mixture which
spreads slowly forming a dark blue solution. Test tube #3 also showed a dark blue
solution, which has the sodium carbonate concentration of 0.5%. But in test tube #4
which has the sodium carbonate concentration of 0.05%, instead of a blue solution a
red colored solution formed meaning that starch is no longer present in this solution
as it was already hydrolyzed by the saliva into the disaccharide maltose (Amrita
Olabs, 2015). Most enzymes work only within a narrow pH level and the pH at which
enzymes function optimally is called the pH optimum (Amrita Olabs, 2015). Thus, in
this test, the sodium carbonate solution of 0.05 % has the pH wherein the enzymatic
activity of salivary amylase works, digesting the starch paste (Amrita Olabs, 2015).

Conclusion

Saliva is becoming more popular as an easily available noninvasive

diagnostic tool to internists, pediatricians, pharmacologists, pathologist,
psychologists, immunologist, endocrinologists, and dentists (Humphrey &
Williamson, 2001). As such new and fast-growing technology have broadly widened
possibilities of saliva testing which can be used in determining symptoms of serious
conditions in the body (Humphrey & Williamson, 2001).

Thus, the goal of this experiment which was preparing saliva sample and
identifying components present in saliva, the product formed on the salivary
digestion of starch, salivary digestion through iodine solution, and factors that affect
enzymatic activity was accomplished successfully. Saliva sample was prepared and
subjected to the following qualitative tests: (1) mucin test which yielded a positive
result forming a green-blue precipitate; (2) Biuret test which gave a positive result
with the formation of a blue-violet precipitate (3) Test for Inorganic matter which
yielded a positive result for chlorides and phosphates forming a white and yellow
precipitate respectively while sulfates and calcium tested negative with no formation
of precipitate which may be due to in their low amount or concentration in the saliva
sample; (4) and lastly, test for thiocyanate gave a negative result forming a yellow
solution indicating that the person tested does not smoke. Then the digestion of
starch paste was monitored with the iodine test to determine its achromatic point.
Afterwards, the hydrolysis product was tested using the Fehling’s and Benedict’s test
giving out a positive result for both tests forming a red precipitate and orange
solution respectively. Then lastly, factors that affect enzymatic activity (free acid and
alkali) were tested with the iodine test. It revealed that the enzymatic activity of
salivary amylase in saliva works at a narrow pH range of pH 6 to 7. Below or above
this pH would cause the enzyme to denature and inactivate.
References

Amrita Olabs (2015). Action of salivary amylase on starch. Retrieved November 6,

2020 from https://amrita.olabs.edu.in/?brch=18&cnt=1&sim=236&sub=79

Aryal, S. (2020). Biuret Test for Protein. Microbenotes. Retrieved November 7, 2020
from https://microbenotes.com/biuret-test-for-protein/

Aryal, S. (2019). Benedict’s Test- Principle, Composition, Preparation, Procedure

and Result Interpretation. Microbiology Info.Com. Retrieved November 7,
2020 from https://microbiologyinfo.com/benedicts-test-principle-composition-
preparation-procedure-and-result-interpretation/

BD Editors. (2019). Salivary Glands. Biology Dictionary. Retrieved November 7,

2020 from https://biologydictionary.net/salivary-glands/

Boundless (2020). Digestive System; Mouth and Stomach. Retrieved November 6,

2020 from https://bio.libretexts.org

BYJUS (2020). Biuret test. Retrieved November 7, 2020 from

https://byjus/chemistry/biuret-test/
Cheng, C. (1992). Laboratory experiments on the actions of digestive enzymes.
Biochemical Education, 20(1), pp. 52-55. Retrieved from
https://onlinelibrary.wiley.com/doi/pdf/10.1016/0307-4412(92)90025
Cole, D. & Landry D. (2002). Determination of inorganic sulfate in human saliva and
sweat by controlled anion chromatography: Normal values in adult humans.
Journal of Chromatography B: Biomedical Science and Applications, 337, pp
267-278. Retrieved from https://doi.org/10.1016/0378-4347(85)80040-2

Encyclopedia Britannica (2020). human digestive system | Description, Parts, &

Functions. Retrieved November 7, 2020 from
https://www.britannica.com/science/human-digestive-system

GC Asia Dental Pte Ltd (2008). Saliva testing. Retrieved November 7, 2020 from
http://www.gcaustralasia.com/upload/product/pdf/97/brochure-Saliva-Check-
BUFFER.pdf&ved
Goalby, N. (2016). Group 2. Retrieved November 6, 2020 from
https://chemrevise.files.wordpress.com/2016/08/2-2-revision-guide-group-
2.pdf
Humphrey, S. & Williamson, R. (2001). A review of saliva: Normal composition, flow
and function. The Journal of Prosthetic Dentistry, 85(2), pp. 162-169.
Retrieved from https://doi.org/10.1067/mpr.2001.113778
Government of Canada (2018). How to test for chloride ions in iron treatment
solutions using silver nitrate. Retrieved November 6, 2020 from
https://www.canada.ca/en/conservation-institute/services/conservation-
preservation-publications/canadian-conservation-institute-notes-test-chloride-
ions-iron-treatment-silver-nitrate.html#a3

Karki, G. (2018). Fehling’s Test: Objective, Principle, Reagents, Procedure and

Result. Online Biology Notes. Retrieved November 7, 2020 from
https://www.onlinebiologynotes.com/fehlings-test-objective-principle-reagents-
procedure-and-result/

Koshland, D. & Haurowitz, F. (2020). Inhibition of enzymes. Britannica. Retrieved

November 7, 2020 from www.britannica.com/science/protein/Inhibition-of-
enzymes

Kubala, E. et al. (2018). A review of selected studies that determine the physical and
chemical properties of saliva in the field of dental treatment. Biomed Research
International. Retrieved from https://doi.org/10.1155/2018/6572381

Leupker, R. et al. (1981). Saliva thiocyanate: a chemical indicator of cigarette

smoking in adolescents. Am J Public Health, 71(12), pp. 1320-1324.
Retrieved from https://doi.org/10/2105/ajph.71.12.1320

Loo, J. et al (2010). Comparative human salivary and plasma proteomes. Journal of

Dental Research, 89(10), pp. 1016-1023. Retrieved from
https://doi.org/10.1177/0022034510380414

Maddu, N. (2019). Functions of Saliva | IntechOpen. IntechOpen. Retrieved

November 7, 2020 from https://www.intechopen.com/books/saliva-and-
salivary-diagnostics/functions-of-saliva

Matusiewiez, H. (2017). Sample preparation for inorganic trace element analysis.

Physical Science Reviews, 2(5). Retrieved from https://doi.org/10.1515/psr-
2017-8001

Medline Plus (2020). Chloride blood test. Retrieved October 6, 2020 from
https://medlineplus.gov/lab-tests/chloride-blood-test/

Microscope LCC (2020). How to use basic stains. Retrieved November 6, 2020 from
https://www.microscope.com/education-center/how-to-guides/basic-stains/

NHS (2018). Phosphate Test. Retrieved November 7, 2020 from

https://www.nhs.uk/comditions/phophate-test/
Nipuna, H. (2019). Phosphate ion testing, phosphoric acids, compounds, reactions.
Chemistry School.
https://www.chemistryscl.com/advancedlevel/inorganic/qualitative_analysis/te
sting-for-phosphate-
ion/main.html#:%7E:text=ammonium%20molybdate%20test%20for%20identif
ying%20phosphate%20ion%20Add,That%20yellow%20precipitate%20will%2
0dissolve%20in%20ammonia%20solution.

PhD Essay. (2020). Project on chemistry: study of digestion of starch by salivary

amylase - PHDessay.com. Free Essays - PhDessay.Com.
https://phdessay.com/project-on-chemistry-study-of-digestion-of-starch-by-
salivary-amylase/

Rabiei, M. et al. (2012). Salivary calcium concentration as a screening tool for

postmenopausal osteoporosis. International Journal of Rheumatic Diseases,
16(2). Retrieved from https://doi,org/10.1111/1756-185X.12003

Rachagani, S. et al. (2009). Current Status of mucins in the diagnosis and therapy of
cancer. Biofactors, 35(6), pp. 509-527. Retrieved from
https://doi.org/10.1002/biof.64

Robolab Technologies (2020). Test for the Calcium Ion. Retrieved October 7, 2020
from https://www.robolab.in/tests-for-the-calcium-ion/

Oxford Reference (n.d.). Scott's test. Retrieved 5 Nov. 2020 from

https://www.oxfordreference.com/view/10.1093/oi/authority.201108031004493
15.

Sotto, D., Tacandong, L. C., & Tan, L. A. (2019). Salivary Digestion. CourseHero.
Retrieved November 7, 2020 from
https://www.coursehero.com/file/p4lmb6t/Salivary-Microscope-Constituents-o-
Enzymes-o-Mucus-o-Protein-o-Electrolytes-o/

Svensson, O. (2008). Interactions of mucin with biopolomers and drug delivery

particles. Health and Society Doctoral Dissertations, 8, pp. 1-81. Retrieved
from www.diva-portal.org

Tabak, L. (1990). Structure and function of human salivary mucins. Oral Biology and
Medicine, 1(8), pp. 229-234. Retrieved from http://cro.sagepub.com