Chemical and Physical Process of Digestion

Trixie Pineda,

Maria Felicia Tuazon

Department of Biological Sciences, College of Science, University of Santo Tomas

Keywords:
Pepsin
Amylase
Bile Salts
Triglycerides

Pierre Mikael Santiago,

Jermaine Rose Serrano,

Aina Elise Sutingco, and

Abstract
The Digestive system or gastrointestinal system, consists of the digestive tract or the
gastrointestinal tract and accessory glands that secretes enzymes ad fluids needed for
digestion. Amylase is an enzyme that breaks down carbohydrates like starch from

polysaccharide into disaccharides and/or monosaccharaides. Pepsin is an enzyme that breaks down
proteins into smaller peptides. Triglycerides are an ester derived from glycerol and three fatty acids. Bile
salts, which are secreted in the small intestine, help aid this difficulty by physically emulsifying the
clumps of lipids.

Introduction

tract, nutrients are absorbed and moves from

the GI tract into the circulatory system so

The

Digestive

system

the nutrients can be transported throughout

or

the body.

gastrointestinal (GI) system, consists of the

digestive tract or the gastrointestinal tract

The gastrointestinal tract has a

and accessory glands that secretes enzymes

variety of functions, one is working with

ad fluids needed for digestion. Digestion is

assisting organs like the salivary glands,

the process of breaking down food into

liver, gallbladder and pancreasto turn food

smaller molecules with the aid of the

into small molecules that the body can

enzymes in the digestive tract it also

absorb and use. Some of the other functions

comprises a number of interdependent rate-

of the gastrointestinal tract includes: a.)

limited processes, which culminate in the

ingestion, b.) transport of ingested food, c.)

absorption of unit (Lucas, 2004). The

secretion of digestive enzymes, acid, mucus

digestive process starts in the mouth and

and bile, d.) absorption of end products of

continues as food journeys down the

digestion, e.) movement of undigested

gastrointestinal tract, at various points of the

Chemical and Physical Process of Digestion

APRIL 2016

material and, f.) elimination of digestive

emulsions, it is the action of pepsin

waste products (Lentle et al. 2011). The

(Macierzanka et al. 2009).

digestive enzymes are called hydrolases,

Gastric

these enzymes break down organic food

immediately after eating, when the pH of the

molecules

by

the

proximal stomach lumen is high because

molecular

bonds,

bonds

gastric lipase is stable over a pH range of 3-

between the monomers. The most common

7 (Carriere et al. 1993). It may continue in

enzyme that is part of the digestive system is

the more alkaline conditions of the small

the salivary amylase, an enzyme produced

intestine. Gastric lipase, like peptidase

by the salivary glands and secreted in the

which is a pancreatic enzyme that digests

mouth. It is composed of water, mucin,

peptides, possesses an amphiphilic peptide

amylase, bicarbonate and lysozyme. The

loop covering the active site like a lid or flap

amylase breaks down starch down into

(Wrinkler et al., 1990) that undergoes

maltose, a double sugar, disaccharide,

conformational

formed of two glucose units while pepsin,

contraction occurs with the lipid/water

breaks

interface.

adding

down

water

breaking

proteins

into
the

into

smaller

fragments.

lipolysis

While

is

most

efficient

rearrangement
in

plants,

when
the

polysaccharide starch is present, where it is

used to store energy. Plants also have the

Peptides are two or more amino

cellulose, a polysaccharide that provides

acids linked together by a peptide bond.

rigidity to their cell walls.

Proteins can consist of a large peptide chain

Materials and Methods

or even multiple peptide chains. During

digestion, pepsin hydrolyzes peptide bonds,
it

is

noteworthy

that

intragastric

Activity 1: Assessing Starch Digestion by

destabilization and consequent flocculation

Salivary Amylase

of protein stabilized emulsions within the

gastric lumen may be transient, with the

8 test tubes containing different

return of the lumen pH to acidic fasting

substances namely: amylase, starch, maltose,

levels

on-going

pH 2.0, pH 7.0, pH 9.0 and deionized water

intragastric digestion, notably the action of

were prepared. The subtances in each test

lipase, and in the case of protein stabilized

tube can be seen in Table 1. Test tube 1 was

with

the

effects

of

Chemical and Physical Process of Digestion

APRIL 2016

TABLE 1 - Substances dispensed in each test tube for activity 1.

boiled while test tube 2 was frozen prior to

clean

test

tubes.

2-3

drops

of

incubating all 8 test tubes at 37C for 60

iodine/potassium iodide solution to half

mins.

were added for the IKI test and 2-3 drops of

Benedicts solution to the other half were
Small amounts of the mixture per

added for Benedicts test. The test tubes

test tube were transferred into small assay

were incubated at 37C for 60 minutes and

tubes. One drop of IKI was dispensed on

observed for any change in color.

each small assay tube and the tubes were

TABLE 2 Reagents mixed in each test
tube for activity 2

inspected to check a blue-black color

change. Five drops of the Benedicts reagent
was dispensed in each test tube with the

Tube #

remaining mixture. These test tubes were

1
2
3
4
5
6

then boiled, and color changes were

observed. The data were recorded for
analysis.
Activity 2: Exploring Amylase Substrate

Reagent
1
Amylase
Amylase
Amylase
Cellulose
Peptidase
Bacteria

Reagent Reagent
2
3
Starch
pH 7.0
Glucose
pH 7.0
Cellulose pH 7.0
Water
pH 7.0
Starch
pH 7.0
Cellulose pH 7.0

Specificity
Activity 3: Assessing Pepsin Digestion of
Protein

The following reagents were added

in each test tube (Table 2). The mixtures

Six test tubes were placed in an

were divided into half and transferred to

incubation unit. Different substances were

Chemical and Physical Process of Digestion

APRIL 2016

added per test tube Test Tube 1 and 2 with

substances namely: lipase, water, vegetable

pepsin, BAPNA, and pH 2.0 buffer, Test

oil, pH 7.0 buffer, pH 2.0 buffer, pH 9.0

Tube 3 has pepsin, deionized water, and pH

buffer, and bile salts were prepared. The

2.0 buffer, Tube 4 with deionized water,

substance in each test tube can be seen in

BAPNA, and pH 2.0 buffer, Tube 5 has

table 4. The test tubes were incubated at

pepsin, BAPNA, and pH 7.0 buffer, and

room temperature for 1 minute. Afterwards,

Tube 6 with pepsin BAPNA, and pH 9.0

the pH of each solution were measured in

buffer.

the Assay Cabinet and recorded.

FIGURE 1 Setup for the Assessment

of the Pepsin Digestion of Protein.
FIGURE 2 Setup for the assessment of
lipase digestion of fat.

Tube 1 was descended into the

incubation unit and was boiled. After boiling

Results and Discussion

tube 1, the tubes were incubated at 37oC for

60 minutes. The incubation unit gently
agitated the test tube rack so that the

Activity 1: Assessing Starch Digestion by

contents of the tubes were evenly mixed.

Salivary Amylase

The

tubes

then

were

placed

in

the

spectrophotometer to obtain the optical

density of each mixture. The data were
recorded for analysis.
FIGURE 3- Results of the IKI test.

Activity 4: Assessing Lipase Digestion of Fat

Starch is a storage molecule found

exclusively

Six test tubes containing 7 different

Chemical and Physical Process of Digestion

in

plants.

Starch

APRIL 2016

can

be

separated into amylose and amylopectin;

neither does cellulose or disaccharides such

natural starch is 10-20% amylose and 80-

as sucrose.

90% amylopectin. Amylose consists of long

polymer chains of glucose units connected
by an alpha acetal linkage.
From the results of the IKI test, we
can see that starch is detected in 4 out of 8 or

FIGURE 4- Results of the Benedict's test.

50% of the test tubes. Test tube 1 yielded a

positive result since the boiling of the

Starch and glycogen form helical

solution cause the denaturation of the

coils and the iodine atoms can fit into the

enzyme

helices to form a starch-iodine or glycogen-

amylase

which

inhibited

the

breakdown of starch. Test tube 2 yielded a

iodine complex.

negative result since starch was still

hydrolyzed by amylase since freezing did

Carbohydrates can be divided into

not affect the enzyme. Test tubes 3 yielded a

two categories based on the complexity of

negative result since starch was broken

their structure. Simple carbohydrates can

down given the optimum condition (pH 7).

form either a single ring structure or a

Test tube 4, 5, and 6 yielded a negative

double

result given the absence of starch, amylase,

carbohydrates are chains of many bonded

and starch respectively. Test tubes 7 and 8 to

simple

gave a positive result given that the pH was

expended for energy storage. These include

not ideal for the enzymatic activity of

starch, cellulose, and glycogen. A test for

amylase.

the presence of many simple carbohydrates

ring

structure.

carbohydrates,

and

Complex
are

often

is the Benedict's test. A color change from

The use of Lugol's iodine reagent

turquoise to yellow or orange is exhibited

(IKI) is useful to distinguish starch and

when the reagent reacts with reducing

glycogen

sugars.

from

other

polysaccharides.

Lugol's iodine yields a blue-black color in

For the Benedicts test, the results

the presence of starch. Starch amylopectin

can be seen in table 1. Test tubes 1,4, and 5

will not react to cause a color change;

Chemical and Physical Process of Digestion

APRIL 2016

show negative results.

The starch in test

Activity 2: Exploring Amylase Substrate

tube 1 was not hydrolyzed given that the

Specificity

enzyme was denatured through the process

of boiling. Test tube 4 did not contain starch

Amylase is an enzyme that breaks

to be broken down into maltose, and test

down

carbohydratres

tube 5 did not contain amylase to break

polysaccharide into disaccharides and/or

down starch. Meanwhile test tubes 2,3 and 6

monosaccharides.

have highly positive results. An orange color

peptidase is responsible for breaking down

shows that the sample contains more sugar

peptide bonds in proteins. In this activity,

than the green sample. This is given by the

the

optimum conditions for starch breakdown in

particularly amylase and peptidase, was

test tubes 2 and 3, while test tube 6

tested. These were verified through two

contained maltose to begin with. Test tubes

chemical tests, namely: Iodine/Potassium

7 and 8 yielded positive results although not

Iodide test (IKI), and Benedicts test.

substrate

On

like
the

specificity

starch
other

of

from
hand,

enzymes,

as strong as the aforementioned test tubes

since the conditions at these test tubes were

Iodine/Potassium Iodide test (IKI)

not the optimum conditions for starch

determines the presence of polysaccharides,

breakdown.

like starch and cellulose, in a sample. It is

performed

by

introducing

an

The Benedict's reagent starts out

iodine/potassium iodide solution and a

aqua-blue. As it is heated in the presence of

positive result will yield a blue-black color.

reducing sugars, it turns yellow to orange. In

Based from the results (Table 3), test tubes

general, blue to blue-green or yellow-green

3, 4, and 5 yielded positive results from IKI.

is negative, yellowish to bright yellow is a

These mixtures still have polysaccharides

moderate positive, and bright orange is a

present, which means that amylase, water,

very strong positive.

and peptidase are not capable or breaking it

EQUATION 1- Chemical Reaction of the Benedict's reagent with a Reducing Sugar.

Chemical and Physical Process of Digestion

APRIL 2016

down. Test tube 1 demonstrates that amylase

the positive control set up for Benedicts

is capable of breaking down starch. Test

test, since it contains glucose, which is a

tube 4 is the positive control of the IKI test

reducing sugar. Test tube 1 demonstrates

since it demonstrates what a positive result

that amylase is able to break down starch, a

for IKI should look like and it does not

polysaccharide,

contain any enzymes in the mixture. Test

monosaccharaides that gave a positive result

tube 5 affirms and verifies that peptidase

in the test. Furthermore, test tube 5 gave a

cannot break down carbohydrates.

negative result, which means that peptidase

into

disaccharides

and

is not able to break down polysaccharides.

The Benedicts test is performed by

These results verify that amylase is an

introducing a mixture of copper sulfate

enzyme specific to carbohydrates and

(CuSO4),

sodium

peptidase is specific to proteins. Test tube 6

carbonate, Benedicts solution, to the sample

demonstrates that some bacteria are capable

and heating it. This test is utilized to

of breaking down polysaccharides like

determine the presence of reducing sugars

cellulose. Plants possess cellulose, which are

and it will yield an orange color or red

compounds that humans are not able to

precipitate if positive. Reducing sugars

digest. Test tube 3 demonstrates that

possess aldehyde groups and some examples

amylase cannot break down cellulose, which

of

and

affirms that humans cannot digest it. On the

galactose. In the presence of heat and basic

other hand, some animals and insects are

solution, reducing sugars produce endiols.

able to digest cellulose due to the presence

These are reducing compounds that will

of symbiotic microbes (bacteria, archaea,

further react with the solution. CuSO4

protozoa) living in their gut. Some examples

provide copper ions that will oxidize

of protozoans are: Trichomonas vaginalis,

reducing sugars and this reaction yields

Trichonympha,

carboxylic acid and copper (I) oxide, which

Protozoans present in termite gut are closely

is the red precipitate that indicates positive

associated with bacteria and these work

(Figure 4).

hand in hand with enzymes like, cellulases

these

sodium

are:

citrate,

glucose,

and

fructose,

and

Parasbasalia.

and hydrogenases, in the gut of termites to

Three set ups tested positive for Benedicts,

degrade cellulose (Okhkuma, 2008).

namely: 1, 2, and 6 (Figure 4). Test tube 2 is

Chemical and Physical Process of Digestion

APRIL 2016

EQUATION 2- Breakdown of Poypeptide into Polypeptide fragments via Pepsin.

Based from the findings of this

Activity 3: Assessing Pepsin Digestion of

activity, it can be concluded that enzymes

Protein

are substrate specific. Its specificity is due to

the

three-dimensional

structure

of

the

Pepsin is an enzyme that breaks

enzyme-active site that corresponds to the

down proteins into smaller peptides. It is

transition state of a reaction (Hedstrom,

produced in the stomach and is one of the

2010). The most common metaphor for

main digestive enzymes in the digestive

enzymes and substrate is the lock and key

systems of humans and many other animals,

(Figure 5). A specific enzyme has its own

where it helps digest the proteins in food.

substrate that is perfectly fit for it to push

Pepsin is most active in acidic environments

through with other processes. It cannot

between 37 C and 42 C. Accordingly, its

degrade a compound when the required

primary site of synthesis and activity is in

substrate for it to bind on is not present.

the stomach (pH 1.5 to 2). Pepsin exhibits

maximal activity at pH 2.0 and is inactive at
pH 6.5 and above, however pepsin is not
fully denatured or irreversibly inactivated
until pH 8.0. Therefore, pepsin in solution of
up to pH 8.0 can be reactivated upon reacidification. The specificity of pepsin can
be

identified

as

structural

or

group

specificity. Pepsin is an endopeptidase

FIGURE 5- Enzyme specificity

mechanism.

enzyme, that hydrolyzes central peptide

bonds in which the amino group belongs to
aromatic amino acids (e.g. tyrosine and
tryptophan)

Chemical and Physical Process of Digestion

APRIL 2016

TABLE 3- Reagents in each test tube and processes they were subjected to.

BAPNA on the other hand is a synthetic

peptide that releases a yellow dye product

There were negative controls used in

when hydrolyzed, it was used as a substrate

the activity those were Tubes 3 and 4. Given

to assess pepsin activity.

these negative controls a negative result was

expected to validate the experiment because

The spectrophotometer was used to

negative controls are used to determine

measure the amount of yellow dye produced

whether

there

are

any

by each mixtures this is to quantify the

substances in the reagents.

contaminating

pepsin activity in each test solution. The

spectrophotometer exposed light through the

Test tubes 2 & 5's mixtures turned

sample and measured how much light did

yellow and the optical density recorded for

the solution absorbed. The fraction of light

these two tubes were greater than zero.

absorbed is expressed as the sample's optical

These yellow solutions showed that the

density.

BAPNA has been hydrolyzed however the

greater the optical density means the more

TABLE 5- Optical Density of the Test

tubes.

hydrolysis has occurred meaning that Tube 2

has the most activity in all of these tubes.
Colorless solutions, do not absorb light and
has an optical density of 0. In conclusion
the more the enzyme activity there is on a
mixture the optical density increases.

Chemical and Physical Process of Digestion

APRIL 2016

Activity 4: Assessing Lipase Digestion of Fat

In Table 6, tube no. 5 the pH is too

low, so a decrease in pH might be difficult

Triglycerides are an ester derived

to detect. Also, the buffer used is too acidic

from glycerol and three fatty acids. Fats and

which may cause the enzyme to be inactive

oils are poorly soluble in water. Since

or be destroyed. This is because according to

lipases are hydrolasesthat is, it break

Go et al. (1972), lipase is irreversibly

bonds using water it is hard to digest fats

inactivated below pH 3.5 (as cited in

and oils because they tend to clump

Rommel, Goebell, & Bohmer, 1975). In the

together, leaving only the molecules on the

case of tube no. 6, little reaction is present

surface exposed to these enzymes. Bile salts,

because the buffer used is too basic.

which are secreted in the small intestine,

Furthermore, tube no. 3 showed no change

help aid this difficulty by physically

in pH from the buffer used (pH 9.0) which

emulsifying the clumps of lipids. They act

means that there is no lipase activity since

like detergents separate clumps into minute

there is no substrate (vegetable oil) to digest.

triglyceride droplets thereby increasing the

Tube no. 4 also did not show a change in

surface are that is exposed to the lipases.

pH, but this time, it is because there is no

This process produces a monoglyceride and

lipase present in the solution and the role of

two fatty acids.

bile salts is solely to increase the amount of

TABLE 4- Reagents in test tubes and results of assessing lipase digestion of fat.
Tube

Reagent 1

Reagent 2

Reagent 3

Reagent 4

Time

Temp.

pH

Lipase

Vegetable Oil

Bile salts

pH 7.0

60

37

6.21

Lipase

Vegetable Oil

Water

pH 7.0

60

37

6.72

Lipase

Water

Bile salts

pH 9.0

60

37

9.00

Water

Vegetable Oil

Bile salts

pH 7.0

60

37

7.00

Lipase

Vegetable Oil

Bile salts

pH 2.0

60

37

2.00

Lipase

Vegetable Oil

Bile salts

pH 9.0

60

37

8.97

No.

Chemical and Physical Process of Digestion

10

APRIL 2016

lipids that is to be exposed to the lipases.

produce cellulase.

Lastly, in tube 1 and 2, a decrease in pH is

observed. Tube 1 (pH 6.21) showed a

Peptidase, like pepsin, hydrolyzes

greater decrease in pH than in tube 2 (pH

peptide bonds. BAPNA is used as a

6.72). The difference is due to the presence

substrate to indicate pepsin activity because

of bile salts in tube no. 1, which increases

it

the amount of lipids exposed to the lipases

hydrolyzed. Pepsin only hydrolyzes peptide

as compared to tube no. 2 wherein bile salts

bonds. The optimum pH of a particular

are absent, therefore, the lipids are still in

enzyme corresponds to the pH of its natural

clumps and the surface area is very little.

environment. For many enzymes, this

produces

yellow

dye

when

it

is

corresponds to pH values of around 7. For

Conclusion

pepsin, which is active in the stomach, the

optimum pH is 2 (the pH of the stomach).

The appropriate chemical tests were

performed to determine whether digestion

The pH decreases when lipases

occurred. With it, the group learned that

activity is present. The hydrolysis product of

salivary

to

fat digestion as monoglycerides and two

maltose. IKI detects the presence of starch

fatty acids. Bile serves to mechanically

while Benedicts indicates that the starch is

break up large globules of fat and produce

hydrolyzed by reacting to its product,

small droplets that effectively increases the

maltose or glucose.

surface area of the lipids. It is difficult to

amylase

hydrolyzes

starch

measure digestion in different pH because

the enzymes are active only on a certain

Enzymes are very specific, only one

range of pH only.

kind of substrate will fit into the active

site. Cellulose is the most common organic

References:

molecule and major structural unit of plants

and cannot be digested by humans while
starch is the storage form of carbohydrate.

[1] Benedicts test for reducing sugar.

The usual substrate for peptidase is peptides

(2015). Retrieved from

and proteins. Bacteria can aid in digestion

http://allmedicalstuff.com/benedicts-test/

by breaking down cellulose which we do not

Chemical and Physical Process of Digestion

11

APRIL 2016

Benedicts test. (n.d.). Retrieved from

gastrointestinal proteolysis of -casein and

http://www.harpercollege.edu/tm-

-lactoglubin. Soft Mattter 5:538-550

ps/chm/100/dgodambe/thedisk/carbo/bened/

[6] Ohkuma, M. (2008). Symbioses of

benedict.htm

flagellates and prokaryotes in the gut of

lower termites. Trends in Microbiology,

[2] Carriere F., Laugier R., Barrowman J.A.,

16(7), 345-352.

Douchet I., Priymenko N., Verger R. (1993)

Gastric and pancreatic lipase levels during a

[7] Winkler F.K, dArcy A., Hunziker W.

test meal in dogs. Scand J Gastroenterol

(1990) Structure of human pancreatic lipase.

28:443-454

Nature 343:771-774

[3] Hedstrom, L. (2010). Enzyme Specificity

and Selectivity. In: eLS. John Wiley & Sons
Ltd, Chichester. http://www.els.net [doi:
10.1002/9780470015902.a0000716.pub2]
Iodine/Potassium Iodide test. (n.d.).
Retrieved from
http://www.harpercollege.edu/tmps/chm/100/dgodambe/thedisk/carbo/iki/iki.
htm
[4] Lentle, R. G., & Janssen, P. W. (2011).
The Physical Processes of Digestion.
London: Springer New York.
Lucas, P. (2004), Dental functional
morphology. Cambridge University Press,
Cambridge
[5] Macierzanka A, Sancho A.I., Mills,
E.N.C, Rigby N.M., Mackie A.R. (2009)
Emulsification alters simulated
Chemical and Physical Process of Digestion

12

APRIL 2016