CHEMICAL AND PHYSICAL PROCESSES OF DIGESTION

LABORATORY - CHEMICAL AND PHYSICAL PROCESSES OF DIGESTION:
WET LAB
(Based on Marieb, 2005)
Chemical Digestion of Foodstuffs: Enzymatic Action
Because nutrients can be absorbed only when broken down to their monomers, food digestion is a prerequisite
to absorption.
Enzymes are large protein molecules produced by body cells. They are biological catalysts, meaning they
increase the rate of a chemical reaction without themselves becoming part of the product. The digestive
enzymes are hydrolytic enzymes, or hydrolases. Their substrates, or the molecules on which they act, are
organic food molecules which they break down by adding water to the molecular bonds, thus cleaving the
bonds between the subunits or monomers.
The various hydrolytic enzymes are highly specific in their action. Each enzyme hydrolyzes only one or a
small group of substrate molecules, and specific environmental conditions are necessary for it to function
optimally. Since digestive enzymes actually function outside the body cells in the digestive tract, their
hydrolytic activity can also be studied in a test tube. Such an in vitro study provides a convenient
laboratory environment for investigating the effect of such variations on enzymatic activity.
Table 1 shows the progressive digestion of proteins, fats, and carbohydrates. It indicates specific enzymes
involved, their site of formation, and their site of action. Acquaint yourself with the table before
beginning this experiment, and refer to it as necessary during the laboratory session.
Objectives
• To list the digestive system enzymes involved in the digestion of proteins, fats, and carbohydrates; to
state their site of origin; and to summarize the environmental conditions promoting their optimal
functioning.
• To recognize the variation between different types of enzyme assays.
• To name the end products of protein, fat, and carbohydrate digestion.
• To perform the appropriate chemical tests to determine if digestion of a particular foodstuff has
occurred.
• To cite the function(s) of bile in the digestive process.
• To discuss the possible role of temperature and pH in the regulation of enzyme activity,
• To define enzyme, catalyst, control, substrate, and hydrolase.
• To explain why swallowing is both a voluntary and a reflex activity.
• To discuss the role of the tongue, larynx, and gastroesophageal sphincter in swallowing.
• To compare and contrast segmentation and peristalsis as mechanisms of propulsion.
1

Table 1: Digestion and absorption of foodstuffs (from Maried, Fig. 39A.1)
Path of absorption Foodstuff Enzyme(s) and source Site of action
Carbohydrate
absorption
Product consumed Starch and disaccharides
Lactose, Maltose,
Sucrose
End-product absorbed: Galactose, Glucose and
Fructose
Protein digestion
Product consumed Proteins
Salivary amylase Mouth
Pancreatic amylase Small intestine
Brush border enzymes Small intestine
(lactase, maltase,
sucrase, and dextrinase)
Pepsin Stomach
Large Polypeptides Pancreatic enzymes
(trypsin, chymotrypsin
and carboxypeptidase)
Small polypeptides,
small peptides
End-product absorbed: Amino acids (some
dipeptides and
tripeptides)
Fat digestion
Product consumed Unemulsified fats
End-product absorbed: Monoglycerides,
glycerol, and fatty acids.
Brush border enzymes
(aminopeptidases,
carboxypeptidase, and
dipeptidases)
Emulsification by
detergent action of bile
salts ducted in from
liver
Lumen of small
intestine
Brush border of small
intestine.
Small intestine
Pancreatic lipase Small intestine
2

Materials
Part 1: Enzyme Action
• Hot plates
• IncubatorTest
• test tube clamps
Activity 2:
• Test tubes + rack (7 tubes per team)
• Spot plates
• non-sterile 5 cc. pipettes (labelled)
• droppers with bulbs (6)
Activity 3:
• Test tubes + rack (6 tubes per team)
• pipettes (labelled)
Activity 4:
• 11 Disposable test tubes and rack
• Bile salts (sodium taurocholate)
(scoops)
• pipettes (labelled)
• Litmus cream (fresh cream to which
powdered litmus is added to achieve a
blue colour) (not a dropper bottle, use
20 ml/team)
Part 2: P hys ical Pr ocess es
Activity 6:
• Spring water (water pitcher)
• Stethoscope
• wax markers
• 250-ml beaker
• Chalkboard for recording class results
• Lugol’s IKI (Lugol’s iodine) (dropper
bottle)
• Benedict’s solution (dropper bottle)
• distilled water
• alpha-amylase solution (20 cc / team)
• 1% boiled starch solution, freshly prepared
(20 cc / team)
• 1% maltose solution (5cc X #team)
• 1% Trypsin (20 cc X # teams)
• 0.1% BAPNA solution (20 cc / team)
• distilled water
• 1% Pancreatin solution
• Vegetable oil (5 cc/team)
• dropper bottle of 0.1 M HCL
• distilled water
• paper cups
• alcohol + gauze
• stop watch
3

Activities
Activit y 1: Develop ing Hypoth eses for Experimen ts on Enzymatic D igest ion
Work in groups according to instructors’ instructions, with each group taking responsibility for
setting up and conducting one of the following experiments. In each of the digestive procedures being
studied (starch, protein. and fat digestion) and in the amylase assay, you are directed to boil the contents
of one or more test tubes. To do this, obtain a 250-ml beaker and a hot plate. Add about 125 ml of water
to the beaker and bring to a boil. Place the specimen in the boiling water for the time (min.) specified in
the directions.
Upon completion of the experiments, each group should communicate its results to the rest of the class by
recording them on the chalkboard. All members of the class should observe the controls (the specimens
or standards against which experimental samples are compared) as well as the positive and negative
examples of all experimental results. Additionally, all members of the class should be able to explain the
tests used and the results observed and anticipated for each experiment.
• Develop a hypothesis for each of the enzymatic digestions studied in Activities 2-4: starch, protein,
and fat.
• Write down your hypotheses, including your reasoning behind each hypothesis.
• Carry out the following activities.
4

Activity 2: Assessing Starch Digestion by Salivary Amylase
In this experiment you will investigate the hydrolysis of starch to maltose by salivary amylase (the enzyme
produced by the salivary glands and secreted into the mouth), so you’ll need to be able to identify the presence of
starch and maltose to determine to what extent the enzymatic activity has occurred. Thus controls must be prepared
to provide a known standard against which comparisons can be made. Starch decreases and sugar increases as
digestion occurs, according to the following formula:
Amylase
Starch + Water � Maltose
Table 2: Salivary Amylase Digestion of Starch
1. Obtain a test tube rack, 7 test tubes, and a wax marking pencil. From supply area 1, obtain distilled water,
maltose, amylase, and starch solutions.
2. Prepare the controls (tubes 1A to 3A) and the experimental samples (tubes 4A to 6A).
3. Mark each tube with a wax pencil and, load the tubes as indicated in Table 2, using 3 ml of each indicated
substance.
4. Place all tubes in a rack in the incubator or cold water bath for approximately 1 hour. Shake the rack gently from
time to time to keep the contents evenly mixed.
5. (While these tubes are incubating, proceed to Activity 6: Physical Processes: Mechanisms of Food Propulsion
and Mixing.)
6. Once the incubation period is over, you are ready to carry out the amylase assay.
Amylase assay
1. From supply area 1 obtain a spot plate and dropper bottles of Lugol’s IKI and Benedict’s solution. Set up your
boiling water bath using a hot plate, and a 250-ml beaker.
2. While the water is heating, mark the spot plate A (for amylase) and number six of its depressions 1 to 6 for
sample identification.
3. Pour about a drop of the sample from each of the tubes 1A-6A into the appropriately numbered spot. Into each
sample droplet, place a drop of I Lugol’s IKI solution. A blue-black colour indicates the presence of starch and
is referred to as a positive starch test. If starch is not present, the mixture will not turn blue, which is referred
to as negative starch test. Record your results (+ for positive, - for negative) in the table and on the chalkboard.
4. Into the remaining mixture in each tube, place 5 drops of Benedict's solution. Put each tube into the beaker of
boiling water for about 5 minutes. If a green-to-orange precipitate forms, maltose is present, this is a positive
sugar test. A negative sugar test is indicated by no colour change. Record your results in the table and on the
chalkboard.
5

Activit y 3: Protein digestion by tryp sin
Trypsin, an enzyme produced by the pancreas, hydrolyzes proteins to small fragments (proteoses, peptones,
and peptides). BAPNA (N-alpha-benzoyl-L-arginine-p-nitroanilide) is a synthetic trypsin substrate consisting of a
dye covalently bound to an amino acid. Trypsin hydrolysis of BAPNA cleaves the dye molecule from the amino
acid, causing the solution to change from colourless to bright yellow. Since the covalent bond between the dye
molecule and the amino acid is the same as the peptide bonds that link amino acids together, the appearance of a
yellow colour indicates the presence and activity of an enzyme that is capable of peptide bond hydrolysis. Because
the colour change from clear to yellow is direct evidence of hydrolysis, additional tests are not required when
determining trypsin activity using BAPNA.
Table 3: Trypsin Digestion of Protein
Assessing Protein Digestion by Trypsin
1. Obtain 6 test tubes and a test tube rack, trypsin, BAPNA, distilled water and the appropriate labelled pipettes
and bring them to your bench.
2. Prepare the controls (tubes 1T and 2T) and the experimental samples (tubes 3T- to 6T), as shown in Table 3.
3. Mark each tube with a wax pencil and load the tubes as indicated in the Table 3, using 3 ml of each indicated
substance.
4. Place all tubes in a rack in the cold water bath or incubator for approximately 1 hour. Shake the rack
occasionally to keep the contents well mixed.
5. While these tubes are incubating, proceed to Activity 6: Physical Processes: Mechanisms of Food Propulsion
and Mixing.
• Once the incubation period is over, you are ready to carry out the trypsin assay.
Trypsin Assay
Since BAPNA is a synthetic colourigenic (colour-producing substrate), the presence of yellow colour indicates
a positive hydrolysis test; the dye molecule has been cleaved from the amino acid. If the sample mixture remains
clear, no detectable hydrolysis has occurred. Record the negative hydrolysis test results in Table 3 and on the
chalkboard.
6

Activit y 4: Pancreatic Lipase Digestion of Fats and th e A ct ion of Bile
The treatment that fats and oils go through during digestion in the small intestine is a bit more complicated than
that of carbohydrates or proteins – pre-treatment with bile to physically emulsify the fats is required. Hence, two sets
of reactions occur.
First: Bile
Fats/Oils �
(Emulsification)
Then: Lipase
Fat/oil droplets �
(Digestion)
Minute fat/oil droplets
Monoglycerides and fatty
acids
The term pancreatin describes the enzymatic product of the pancreas, which includes enzymes that digest proteins,
carbohydrates, nucleic acids, and fats. It is used here to investigate the properties of pancreatic lipase, which
hydrolyzes fats and oils to their component monoglycerides and two fatty acids (and occasionally to glycerol and
three fatty acids).
The fact that some of the end products of fat digestion (fatty acids) are organic acids that decrease the pH provides
an easy way to recognize that digestion is ongoing or completed. You will be using a pH indicator called litmus blue
to follow these changes; it changes from blue to pink as the test tube contents become acid.
Fig 1: Emulsification action of bile
Demonstrating the Emulsification Action of Bile
Although bile, a secretory product of the liver is not an enzyme, it is important to fat digestion because of its
emulsifying action (the physical breakdown of larger particles into smaller ones) on fats. Emulsified fats provide a
larger surface area for enzymatic activity.
NOTE: use disposable tubes with caps for this experiment.
1. To demonstrate the action of bile on fats, prepare two test tubes and mark them 1E and 2E (fig.1).
2. To tube 1E add 5 ml of water and 1 ml of vegetable oil.
3. To tube 2E add 5 ml of water, 1 ml of vegetable, and a pinch of bile salts.
4. Cap each tube, shake vigorously, and allow the tubes to stand at room temperature.
5. After 10-15 minutes, observe both tubes.
If emulsification has not occurred, the oil will be floating on the surface of the water. If emulsification has occurred,
the fat droplets will be suspended throughout the water, forming an emulsion.
In which tube has emulsification occurred?______________
7

Assessing Fat Digestion by Lipase
Table 4: Pancreatic Lipase Digestion of Fats.
Prepare the controls (1L and 2L), and set up the experimental samples (3L to 6L and 4B to 6B) as illustrated in
Table 4.
1. Obtain 9 disposable test tubes with caps, a test tube rack, lipase, litmus cream, and water from supply area 3.
2. Mark each tube with a wax pencil and load the tubes using 3 ml of each indicated solution.
3. Place a scoop of bile salts in tubes 4B and 5B.
4. Cover each tube with a cap and shake to mix the contents.
5. Remove the caps and place all tubes in a rack in water bath or incubator for approximately 1 hour. Shake the
test tube rack from time to time to keep the contents well mixed.
6. (While these tubes are incubating, proceed to Activity 6: Physical Processes: Mechanisms of Food Propulsion
and Mixing.)
7. Once the incubation period is over, you are ready to carry out the trypsin assay.
Lipase Assay
The basis of this assay is a pH change that is detected by a litmus powder indicator. Alkaline or neutral
solutions containing litmus are blue but will turn reddish in the presence of acid. Since fats are digested to fatty acids
(organic acids) during hydrolysis, they lower the pH of the sample they are in. Litmus cream (fresh cream providing
the fat substrate to which litmus powder was added) will turn from blue to pink if the solution is acid. Because the
effect of hydrolysis is directly seen, additional assay reagents are not necessary.
1. To prepare a colour control, add 0.1 N HCI drop by drop to tubes 1L and 2L (capping the tubes after each
addition and shaking to mix) until the cream turns pink.
2. Record the colour of the tubes in Table 4 and on the chalkboard.
8

Activit y 5: Reporting Results an d Con clusion s
1. Share your results with the class as directed in Activity 1
2. Suggest additional experiments, and carry out experiments if time permits.
3. Prepare a lab report for the experiments on digestion.
Activit y 6: Ph ysical p roces ses: Mechanisms of Food Propulsion an d Mixing
Although enzyme activity is a very important part of the overall digestion process, foods
must also be processed physically (churning and chewing), and moved by mechanical means
along the tract if digestion and absorption are to be completed. Just about any time organs exhibit
mobility, muscles are involved, and movements of and in the gastrointestinal tract are no
exception. Although we tend to think only of smooth muscles when visceral activities are
involved, both skeletal and smooth muscles are involved in digestion. This fact is amply
demonstrated by the simple activities that follow.
Deglutition (Swallowing)
Swallowing, or deglutition, which is largely the result of skeletal muscle activity, occurs in
two phases: bucal (mouth) and pharyngeal-esophageal. The initial phase – the bucal – is
voluntarily controlled and initiated by the tongue. Once begun, the process continues
involuntarily in the pharynx and esophagus, through peristalsis, resulting in the delivery of the
swallowed contents to the stomach.
Observing Movements and Sounds of Digestion
1. Obtain a pitcher of water, a stethoscope, a cup, an alcohol swab in preparation for making
the following observations.
2. While swallowing a mouthful of water, consciously note the movement of your tongue
during the process. Record your observations.
3. Repeat the swallowing process while your laboratory partner watches the externally visible
movements of your larynx. (This movement is more obvious in a male, who has a larger
Adam's apple.) Record your observations.
What do these movements accomplish?
Before donning the stethoscope your lab partner should clean the earpieces with an alcohol swab.
Then, he or she should place the diaphragm of the stethoscope over your abdominal wall,
approximately 2.5 cm below the xyphoid process and slightly to the left, to listen for sounds as
you again take two or three swallows of water. There should be two audible sounds – one when
the water splashes against the gastroesopheal sphincter and the second when the peristaltic wave
of the esophagus arrives at the sphincter and the sphincter opens, allowing water to gurgle into
9

the stomach. Determine, as accurately as possible, the time interval between these two sounds
and record it below.
Interval between arrival of water at the sphincter and the opening, of the sphincter: ______sec
This interval gives a fair indication of the time it takes for the peristaltic wave to travel down the 25 cm-long
esophagus. (Actually the time interval is slightly less than it seems, because pressure causes the sphincter to relax
before the peristaltic wave reaches it.)
Segmentation and Peristalsis
Although several types of movements occur in the digestive tract organs, peristalsis and
segmentation are most important as mixing and propulsive mechanisms. Peristaltic movements
are the major means of propelling food through most of the digestive viscera. Essentially they are
waves of contraction followed by waves of relaxation that squeeze foodstuffs through the
alimentary canal, and they are superimposed on segmental movements.
Segmental movements are local constrictions of the organ wall that occur rhythmically. They
serve mainly to mix the foodstuffs with digestive juices and to increase the rate of absorption by
continually moving different portions of the chyme over adjacent regions of the intestinal wall.
However, segmentation is also an important means of food propulsion in the small intestine, and
slow segmenting movements called haustral contractions are frequently seen in the large
intestine.
Sources
Marieb, Elaine. 2005. Human anatomy and physiology laboratory manual, 8 th ed. Pearson.
Exercise 39A, pp. 435-442
Hickman, Cleveland P.Jr., Larry S. Roberts, Alland, Larson, & Helen I’Anson. 2004. Integrated
principles of zoology. pp. 682-688.
10