Chemical and Physical Process of Digestion 1
1
1
1
Trixie Pineda, Pierre Mikael Santiago, Jermaine Rose Serrano, Aina Elise Sutingco, and
1
Maria Felicia Tuazon
1
Department of Biological Sciences, College of Science, University of Santo Tomas
Abstract
!"#$%&'() !"#$%& ()*+,$" -%+" .,+/$ 01%2+*3"1%4"$
The Digestive system or gastrointestinal system, consists of the digestive tract or the gastrointestinal gastrointest inal 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. monosaccharai des. Pepsin is an enzyme that breaks down proteins into smaller smal ler peptides. peptid es. Triglycerides Triglycerid es are an a n ester derived from glycerol and an d three fatty acids. acids . Bile salts, which are secreted in the small intestine, help aid this difficulty by physically emulsifying the clumps of lipids.
tract, nutrients are absorbed and moves from
Introduction
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
and accessory glands that secretes enzymes
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 journeys
down
and bile, d.) absorption of end products of
the
digestion,
gastrointestinal tract, at various points of the Chemical and Physical Process of Digestion
a
liver, gallbladder and pancreas—to turn food
smaller molecules with the aid of the
food
has
assisting organs like the salivary glands,
the process of breaking down food into
as
tract
variety of functions, one is working with
ad fluids needed for digestion. Digestion is
continues
gastrointestinal
1
e.)
movement
of
APRIL 2016
undigested
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,
conformational
a
adding
water
breaking
double
sugar,
into the
disaccharide,
lipolysis
is
most
efficient
rearrangement
when
formed of two glucose units while pepsin,
contraction occurs with the lipid/water
breaks
interface.
down
proteins
into
smaller
fragments.
While
in
plants,
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 or even multiple peptide chains. During
Materials and Methods
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
2
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 37°C for 60
iodine/potassium iodide solution to half
mins.
were added for the IKI test and 2-3 drops of Benedict’s solution to the other half were Small amounts of the mixture per
added for Benedict’s test. The test tubes
test tube were transferred into small assay
were incubated at 37ºC 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 Benedict’s reagent was dispensed in each test tube with the
Tube # 1 2 3
Reagent 1 Amylase Amylase Amylase
Reagent 2 Starch Glucose Cellulose
Reagent 3 pH 7.0 pH 7.0 pH 7.0
4 5 6
Cellulose Peptidase Bacteria
Water Starch Cellulose
pH 7.0 pH 7.0 pH 7.0
remaining mixture. These test tubes were then
boiled,
and
color
changes
were
observed. The data were recorded for analysis.
Activity 2: Exploring Amylase Substrate 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
3
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.
Activity 4: Assessing Lipase Digestion of Fat
FIGURE 3- Results of the IKI test.
Starch is a storage molecule found exclusively
Six test tubes containing 7 different Chemical and Physical Process of Digestion
4
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 negative
result
since
starch
was
iodine complex.
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 Benedict’s 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
5
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 Benedict’s 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
6
APRIL 2016
down. Test tube 1 demonstrates that amylase
the positive control set up for Benedict’s
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 Benedict’s test is performed by
These results verify that amylase is an
introducing a mixture of copper sulfate
enzyme
(CuSO4),
sodium
peptidase is specific to proteins. Test tube 6
carbonate, Benedict’s 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.
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
of protozoans are: Trichomonas vaginalis,
sodium
these
are:
copper
citrate,
glucose,
ions that
and
fructose,
will oxidize
specific
Test
tube
to
carbohydrates
3
and
demonstrates
and
that
reducing sugars and this reaction yields
Trichonympha,
Parasbasalia.
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 and hydrogenases, in the gut of termites to
Three set ups tested positive for Benedict’s,
degrade cellulose (Okhkuma, 2008).
namely: 1, 2, and 6 (Figure 4). Test tube 2 is Chemical and Physical Process of Digestion
7
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
8
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 hydrolysis has occurred meaning that Tube 2
TABLE 5- Optical Density of the Test tubes.
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
9
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 hydrolases—that 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
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
aid
this
difficulty
by
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
1
Lipase
Vegetable Oil
Bile salts
pH 7.0
60
37
6.21
2
Lipase
Vegetable Oil
Water
pH 7.0
60
37
6.72
3
Lipase
Water
Bile salts
pH 9.0
60
37
9.00
4
Water
Vegetable Oil
Bile salts
pH 7.0
60
37
7.00
5
Lipase
Vegetable Oil
Bile salts
pH 2.0
60
37
2.00
6
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.
produces
yellow
For
dye
many
when
it
enzymes,
is
this
corresponds to pH values of around 7. For pepsin, which is active in the stomach, the
Conclusion
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 Benedict’s 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] Benedict’s 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
Benedict’s 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, d’Arcy 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/tm ps/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