Polytechnic University of the Philippines

College of Science
Department of Biology

Activity 2: Digestive System

Bobos, Ailyn B.1 De Leon, Bryan D. R. Doria, Wendylyn A.2 Mangol, Maria Sheika L.3
Morales, Janelle C.4
Group 6, BS Biology 4-1, BS Biology 4-4

ABSTRACT

The digestive system is made up of the gastrointestinal tract, also called the GI tract or
digestive tract with the accessory structures which are liver, pancreas, and gallbladder. The
pancreas consists of exocrine tissue that produces pancreatic enzymes that break down
carbohydrates, fats, and proteins. In this investigation, saliva is the variable in the enzymatic
activity, saliva is a combination of serous and mucous secretions from the various salivary
glands and contains enzymes called salivary amylase. The activity of enzymes is strongly
affected by several factors, such as temperature and pH. The saliva undergone a series of test.
First, the spot test, the starch is the substrate and the salivary amylase is the enzyme and only test
tube C obtained a blue-violet color. The enzyme activity on this experiment is high because the
starch had short time to hydrolyzed. In the case of Benedict's test, all the test tube A, test tube B
and test C gave negative results, it’s supposed to give a positive result because starch is being
converted into maltose which is a reducing sugar. The Biuret test is a chemical test used for
detecting the presence of peptide bonds. Based on the results test tube A has negative result
while test tube B and C has positive result indicating a blue violet coloration. The absorbance of
each enzyme concentration was determined using photometric analysis at 540 nm. The chelate
complex absorbs light at 540 nm too appears violet. The absorbance of enzyme gives a negative
relationship which indicates that peptide bonds is low. In able to conclude the activity,
understanding the importance and properties of an enzyme is of great importance in discovering
behind all the chemical reactions happening in the digestive system.

Keywords: enzymes, salivary amylase, absorbance, concentration

Introduction

The organs of the digestive system are divided into 2 main group: The gastrointestinal tract (GI
tract) and accessory structures. GI tract is a continuous tube extending through the ventral cavity from the
mouth to the anus –it consists of the mouth, oral cavity, oropharynx, esophagus, stomach, small intestine,
large intestine, rectum, structure (Ebneshahidi, 2006). The digestive system functions in many ways, its
major functions are ingestion, mastication, propulsion, mixing, secretion, digestion, absorption, and
elimination. Three major types of glands are associated with the intestinal tract: (1) unicellular mucous
glands in the mucosa, (2) multicellular glands in the mucosa and submucosa, and (3) multicellular glands
(accessory glands) outside the digestive tract. The oral cavity or mouth is the part of the digestive tract
bounded by the lips anteriorly, the fauces (throat; opening into the pharynx) posteriorly, the cheeks
laterally, the palate superiorly, and a muscular floor inferiorly. The oral cavity is divided into two regions:
(1) the vestibule (entry), which is the space between the lips or cheeks and the alveolar processes, which
contain the teeth; and (2) the oral cavity proper, which lies medial to the alveolar processes. The oral
cavity is lined with moist stratified squamous epithelium, which provides protection against abrasion. A
considerable number of salivary glands are scattered throughout the oral cavity. Three pairs of large
multicellular glands exist: the parotid, the submandibular, and the sublingual glands. In addition to these
large consolidations of glandular tissue, numerous small, coiled tubular glands are located deep to the
epithelium of the tongue (lingual glands), palate (palatine glands), cheeks (buccal glands), and lips (labial
glands). The secretions from these glands help keep the oral cavity moist and begin the process of
digestion. All of the major large salivary glands are compound alveolar glands, which are branching
glands with clusters of alveoli resembling grapes. They produce thin serous secretions or thicker mucous
secretions. Thus, saliva is a combination of serous and mucous secretions from the various salivary glands
(McGraw-Hill, 2004). Saliva consists of 99.5% water, the remaining 0.5% is dissolved substances
including amylase enzyme (for chemically digesting carbohydrate), bicarbonate ion (HCO3-; maintains
pH of saliva at 6.5-7.5), and many electrolytes (Ebneshahidi, 2006). Thus, this activity aims to investigate
the effect of enzyme concentration in different tests to observe the chemical enzymatic activity and to
know the absorbance of saliva at 540nm in different concentrations.

Methodology

The Effect of Enzyme Concentration

 10 mL Crude Enzyme (salivary amylase)
 100 mL distilled water
 Test tubes
 8 mL 1% Starch Solution
 10 mL NaCl
 Biuret Reagent
 Iodine Solution
 Benedict’s Reagent

Procedure:

The activity was conducted by preparing a crude enzyme obtained from one of the researcher’s
mouth. The saliva donor was asked to wash his/her mouth several times prior to saliva donation. After
gathering 10 ml of salivary amylase, it is diluted on 100 ml of distilled water. Three (3) test tubes was
then prepared and labelled test tube A, B and C respectively. 8 ml 1% starch solution and 10 ml NaCl
solution was introduced to each of the test tubes. Varying volumes of distilled water and salivary amylase
was put upon the test tubes, 0.5 ml distilled water and 5 ml saliva was introduced in test tube A, 3.5 ml
distilled water and 3 ml saliva for test tube B, 6 ml distilled water and 0.5 ml saliva for test tube C. Biuret
Test was then performed on each of the test tubes. It was done by adding 1 ml of reaction mixture from
each of the test tubes to a separate test tube contained with Biuret reagent. A change of coloration to blue-
violet was the observed and noted. Spot test was also performed by taking a drop of each reaction mixture
into a drop plate after each minute the test tubes. A drop of iodine solution was added to the spot where
the reaction mixture resides and reaction which resulted to change in coloration was observed. The spot
test was repeated every minute for 15 minutes. The success of each spot test in developing a reaction was
noted. Upon failure of developing the color of the iodo-starch complex reaction, Benedict’s test was
performed; 1 ml of the reaction mixture was introduced to 1 ml Benedict’s reagent on a separate test tube
and was heated for 5-15 minutes. The data gathered upon conduction of the experiment was related to the
human digestion system.

Absorbance of Enzyme
 Test tubes
 Deionized Water
 Salivary amylase
 Biuret Reagent

Procedure:
Absorbance of enzyme was tested by first preparing five (5) test tubes and labeling them with 1,
2, 3, 4, 5 respectively. Dilution involving saliva and deionized water was performed. Each test tube was
filled with 2.4 ml deionized water. 5 ml deionized water was added to test tube 1. 0.1 ml saliva of
different concentrations was used and introduced to the test tubes 2-5. 2.5% saliva for test tube 2, 5%
saliva for test tube 3, 10% saliva for test tube 4 and 20% saliva for test tube 5. 2.5 ml Biuret Reagent was
then added to each of the test tubes. The absorbance of each enzyme concentration was determined using
photometric analysis at 540 nm. The results were noted.

Effect of Enzyme Concentration

Benedict’s Biuret
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
test test
A - - - - - - - - - - - - - - - - -
B - - - - - - - - - - - - - - - - +
C - - - - + + + + + + + + + + + - ++
Results
Table 1 Shows the effect on enzyme concentration after performing spot test, Benedict’s test, and Biuret test

Based on the table, the reaction mixture of test tubes A and B has negative results in spot test,
while test tube C, obtained positive results from the 5th to the 15th spot which means it developed a blue-
violet coloration. Since the reaction mixture of A and B, and some in C failed to develop the color of
Iodo-starch complex, Benedict’s test was performed. However, negative results were also obtained from
the Benedict’s test since it did not change color after heating. On the other hand, the Biuret test showed
positive results for B and C only which showed a color change into blue-violet, reaction mixture C
obtaining a darker color than B.

Standard Protein
Concentration Absorbance
X Y
0 0
2.5 0.067
5 0.079
 10 0.065
20 0.065
Table 2 Shows the absorbance of different enzyme concentrations at 540 nm

Enzyme concentration and absorbance is directly proportional which means as concentration

increases, so as absorbance. From a concentration of 2.5 to 5, the absorbance is increasing however it
decreases when it reached a concentration of 10 and 20, obtaining an absorbance of both 0.065 which is
the lowest absorbance.

Enzyme Absorbance
0.1
0.08
Absorbance

0.06
y = -0.0004x + 0.0729
0.04
R² = 0.2302
0.02
0
0 5 10 15 20 25
Enzyme Concentration

Figure 1 Shows the linear regression of enzyme absorbance including slope and R-squared

In the figure above, the linear regression shows a downward trend, while indicates a negative
relationship, as well as negative slope (m= -0.0004). Thus, as the concentration of enzyme increases, the
absorbance decreases.

Discussion

Chemical reactions within cells are aided by enzymes that increase the rate at which reactions
take place (Alberts et.al, 2010). Enzymes are biological catalysts that work by binding themselves to
substrate molecules and lowering the activation energy of a reaction (Alberts et.al, 2010 p.90). Enzymes
are highly specific and highly efficient which make them essential for life as it exists (Wiseman, 1971,
p.31). Saliva is rich in an enzyme called amylase. This enzyme is responsible for converting amylose and
amylopectin in starch. Amylase coats and surrounds each starch molecule in the mouth. Then the enzyme
deconstructs complex starch molecules through hydrolysis, or chemical breakdown, turning them into
smaller, more manageable particles. The end result of this conversion step is maltose, maltotriose and
dextrins, which are all considered simple types of sugar carbohydrates.

Like all catalysts, enzymes increase the rate of chemical reactions by lowering the amount of
energy needed to initiate a particular reaction. The digestive system produces numerous enzymes to
facilitate the biochemical reactions that transform ordinary food into the substances that nourish human
life. Enzymes break down the substances we eat. That means enzymes break down proteins, cellulose,
starches and other foodstuffs. This makes it possible for the intestines to absorb nutrients. Enzymes begin
the digestive process in the mouth, as they’re secreted by salivary glands. They work to break down
starch into sugars. The three or so pounds of bacteria living in the gut (mostly in the large intestine) help
us digest all manner of food. It plays a fundamental role in human digestion by breaking down sugar
polymers to simple glucose.

Based on the results obtained, no color change were observed on test tube A and B while there is
a blue-violet coloration on test tube C. Iodine is an indicator for the presence of starch. There are fewer
enzymes present as you move from test tube A-C thus the starch will not be broken down. When there is
an insufficient amount of enzyme present the reaction will not progress as quick as it would because the
active sites present are occupied. If the concentration or amount of enzymes is increased then this would
make provision for an increase in reaction rate. Reaction rate would increase due to the fact that there will
be more active sites that are unoccupied. However, if there is an excess of enzyme molecule, the rate
would not increase if more is added but it would reach at a point where it would level off.

And also after the amylase reacted with the starch there will be a discharge of maltose which is a
disaccharide. Less starch will be present as time proceeds and more maltose will be present. In addition
less starch will be available to react with iodine thus the blue/black colour will decrease. The more
enzymes available, the quicker the reaction will occur until the substrate is all used up. More substrates
will also mean quicker activity, until the enzyme is fully saturated so that it cannot continue increasing its
activity.

The Biuret test is a chemical test used for detecting the presence of peptide bonds. Based on the
results test tube A has negative result while test tube B and C has positive result indicating a blue violet
coloration. Biuret test is based on the ability of Cu (II) ions to form a violet-coloured chelate complex
with peptide bonds (-CONH- groups) in alkaline conditions. Lone electron pairs from 4 nitrogen atoms in
the peptide bond coordinate a copper (II) ion to form the chelate complex. The chelate complex absorbs
light at 540 nm so appears violet. Hence a color change from blue to violet indicates that proteins are
present. The greater the concentration of peptide bonds, the greater the color intensity. If the
concentration of peptide bonds is low such as when short-chain peptides are present the color change is
from blue to pink.

Benedict’s test is used to indicate presence of reducing sugars including all monosaccharides and
some disaccharides including lactose and maltose. In the results no color change was observed, therefore
sugar is absent. Benedict's reagent starts out aqua-blue. As it is heated in the presence of reducing sugars,
it turns yellow to orange. The "hotter" the final color of the reagent, the higher the concentration of
reducing sugar. In general, blue to blue-green or yellow-green is negative, yellowish to bright yellow is a
moderate positive, and bright orange is a very strong positive. Starch do not react or react very poorly
with Benedict's reagent, due to the relatively small number of reducing sugar moieties, which occur only
at the ends of carbohydrate chains. Starch is a polysaccharide and it does not have active carbonyl group
to react with Benedict solution.

Digestion (mechanical) begins in the mouth as the teeth tear and grind food into small bits and
pieces that can be swallowed without choking The muscular walls of the esophagus, stomach, and
intestines continue mechanical digestion, pushing the food along, churning and breaking it into smaller
particles while chemical digestion occurs at every point in the digestive system, beginning when we see
or smell food. These sensory events set off nerve impulses from the eyes and nose that trigger the release
of enzymes and other substances that will eventually break down food to release the nutrients inside. The
body then burns these nutrients for energy or uses them to build new tissues and body parts.
 There are also factors that affect digestion first which is chewing, the more thoroughly food is
chewed, the more its surface area is increased. Second are liquids, with or following meals dilute
digestive enzymes, thus increasing digestion time. Next are condiments, flavorings such as salt, vinegar,
pepper, spices, and monosodium glutamate has effects beyond causing thirst after meals. One additional
effect is that they prompt us to swallow food after insufficient chewing. Their strong flavors stimulate the
sense of taste, giving the false impression that enough of the nutrients in the food have been extracted
during the mastication process. Frequency of Meals is also one of the factors, if food is eaten before the
previous meal has been sufficiently digested, one of two undesirable events occurs: (a) the stomach
empties prematurely, releasing partly digested food into the intestines. This action results in the
absorption of partly digested proteins and burdens the immune system, which must remove them from the
blood stream. (b) the new, undigested food mixes with the partly digested food. The combined food mass
now takes longer to digest, thus allowing increased putrefaction and fermentation of it. Next is eating
within digestive limitations, overeating expands and stretches the stomach, causing irritation and
exacerbating prior harm. And the last one is food combinations, combining foods for optimal digestion is
a very important topic.

There are two types of stomach the monogastric and ruminant. A monogastric organism has a
simple single-chambered stomach while a ruminant organism has a four-chambered complex stomach.
The differences are r uminants are always herbivores while monogastrics show all types of food habits.
The digestive system of ruminants is more efficient than the monogastric system in breaking down food
and absorbing nutrients. Ruminants regurgitate the ingested food during digestion, but monogastrics do
not. Ruminants are foregut fermenters while monogastric herbivores are hindgut fermenters. The number
of monogastric species is higher than ruminant species. Monogastric does not chew their cud while
ruminant animals chew their gut. Ruminants have a more efficient digestive system as compared to the
digestive system of monogastrics. Monogastrics do not ingest food during digestion while ruminants do
and monogastric animals are much more in numbers as compared to the ruminant animals. In the lining of
the stomach, gastric juice is secreted. It has four main components, hydrochloric acid (HCL) which helps
in killing germs and harmful bacteria and also in activating pepsinogen. Pepsinogen which when activated
produces pepsin, and pepsin for the breakdown proteins into amino acids. Mucus that forms the protective
layer to prevent corrosion of stomach walls from hydrochloric acid that is concentrated in nature.

Enzyme assays are laboratory methods for measuring enzymatic activity. In spectrophotometric
assays, the course of the reaction is measured by a change in how much light the assay solution absorbs.
If this light is in the visible region there is a change in the color of the assay, and these are called
colorimetric assays. It is using light to measure the presence, amount or activity of an analyte which is a
protein with catalytic function. The results are called absorbance and have no units. Beer’s Law is used in
order to determine the concentration. Beer’s Law is that the absorbance, through a known length, is
directly proportional to the concentration of the solution. In other words, as long as we know how far the
light travelled through the sample, then we can determine the concentration of the solution based on the
absorbance. In spectrophotometric enzyme assay, a reactant becomes the product and there is an enzyme
causing this to happen and it is also the catalyst. Enzymes can be present and not active and to know if the
enzyme is there and actively doing something the reactant can absorb the light, when the enzyme is acting
the reactant can be lose, the product can also select and thus the enzyme acting, more product can be
created.

Conclusion
In this activity, understanding the importance and properties of an enzyme is of great importance
in discovering the reason behind all the chemical reactions happening in the digestive system. Enzymes
are proteins that helps catalyses or accelerates chemical processes. Enzymes are essential for healthy
digestion and a healthy body. They work with other chemicals in the body, such as stomach acid and bile,
to help break down food into molecules for a wide range of bodily functions . In the case of carbohydrates
are needed for energy, while protein is necessary to build and repair muscle, among other functions.

REFERENCES CITED

Anatomy and Physiology (Sixth ed.). (2004). The McGraw-Hill Companies.

Ebneshahidi, D. A. (2006). The Digestive System. Pearson Education, Inc.

Starch Hydrolysis by Amylase. (n.d.). Retrieved from https://eng.umd.edu/~nsw/ench485/lab5.htm

(n.d.). Retrieved from http://samson.kean.edu/~breid/enzyme/enzyme.html#BENEDICTS

Biuret Test for Protein. (n.d.). Retrieved from http://brilliantbiologystudent.weebly.com/biuret-test-for-

protein.html

Spectrophotometric Enzyme Assays. (n.d.). Retrieved from https://www.creative-

enzymes.com/resource/spectrophotometric-enzyme-assays_5.html