physioex digestion (fin)

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Chemical and Physical Process of Digestion APRIL 20161

Chemical and Physical Process of Digestion1 Trixie Pineda,

1 Pierre Mikael Santiago,

1 Jermaine Rose Serrano,

1 Aina Elise Sutingco, and

1 Maria Felicia Tuazon

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

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

The Digestive system or

gastrointestinal (GI) system, consists of the

digestive tract or the gastrointestinal tract

and accessory glands that secretes enzymes

ad fluids needed for digestion. Digestion is

the process of breaking down food into

smaller molecules with the aid of the

enzymes in the digestive tract it also

comprises a number of interdependent rate-limited processes, which culminate in the

absorption of unit (Lucas, 2004). The

digestive process starts in the mouth and

continues as food journeys down the

gastrointestinal tract, at various points of the

tract, nutrients are absorbed and moves from

the GI tract into the circulatory system so

the nutrients can be transported throughout

the body.

The gastrointestinal tract has a

variety of functions, one is working with

assisting organs like the salivary glands,

liver, gallbladder and pancreas—to turn food

into small molecules that the body can

absorb and use. Some of the other functions

of the gastrointestinal tract includes: a.)

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

secretion of digestive enzymes, acid, mucus

and bile, d.) absorption of end products of

digestion, e.) movement of undigested

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material and, f.) elimination of digestive

waste products (Lentle et al. 2011). The

digestive enzymes are called hydrolases,

these enzymes break down organic food

molecules by adding water into the

molecular bonds, breaking the bonds

between the monomers. The most common

enzyme that is part of the digestive system is

the salivary amylase, an enzyme produced

by the salivary glands and secreted in the

mouth. It is composed of water, mucin,

amylase, bicarbonate and lysozyme. The

amylase breaks down starch down into

maltose, a double sugar, disaccharide,

formed of two glucose units while pepsin,

breaks down proteins into smaller

fragments.

Peptides are two or more amino

acids linked together by a peptide bond.

Proteins can consist of a large peptide chain

or even multiple peptide chains. During

digestion, pepsin hydrolyzes peptide bonds,

it is noteworthy that intragastric

destabilization and consequent flocculation

of protein stabilized emulsions within the

gastric lumen may be transient, with the

return of the lumen pH to acidic fasting

levels with the effects of on-going

intragastric digestion, notably the action of

lipase, and in the case of protein stabilized

emulsions, it is the action of pepsin

(Macierzanka et al. 2009).

Gastric lipolysis is most efficient

immediately after eating, when the pH of the

proximal stomach lumen is high because

gastric lipase is stable over a pH range of 3-

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

the more alkaline conditions of the small

intestine. Gastric lipase, like peptidase

which is a pancreatic enzyme that digests

peptides, possesses an amphiphilic peptide

loop covering the active site like a lid or flap

(Wrinkler et al., 1990) that undergoes

conformational rearrangement when

contraction occurs with the lipid/water

interface. While in plants, the

polysaccharide starch is present, where it is

used to store energy. Plants also have the

cellulose, a polysaccharide that provides

rigidity to their cell walls.

Materials and Methods

Activity 1: Assessing Starch Digestion by

Salivary Amylase

8 test tubes containing different

substances namely: amylase, starch, maltose,

pH 2.0, pH 7.0, pH 9.0 and deionized water

were prepared. The subtances in each test

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


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boiled while test tube 2 was frozen prior to

incubating all 8 test tubes at 37°C for 60

mins.

Small amounts of the mixture per

test tube were transferred into small assay

tubes. One drop of IKI was dispensed on

each small assay tube and the tubes were

inspected to check a blue-black color

change. Five drops of the Benedict’s reagent

was dispensed in each test tube with the

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

The following reagents were added

in each test tube (Table 2). The mixtures

were divided into half and transferred to

clean test tubes. 2-3 drops of

iodine/potassium iodide solution to half

were added for the IKI test and 2-3 drops ofBenedict’s solution to the other half were

added for Benedict’s test. The test tubes

were incubated at 37ºC for 60 minutes and

observed for any change in color.

Activity 3: Assessing Pepsin Digestion of

Protein

Six test tubes were placed in an

incubation unit. Different substances were

Tube # Reagent

1

Reagent

2

Reagent

3

1 Amylase Starch pH 7.0

2 Amylase Glucose pH 7.0

3 Amylase Cellulose pH 7.0

4 Cellulose Water pH 7.0

5 Peptidase Starch pH 7.0

6 Bacteria Cellulose pH 7.0

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

TABLE 2 – Reagents mixed in each test

tube for activity 2


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added per test tube Test Tube 1 and 2 with

pepsin, BAPNA, and pH 2.0 buffer, Test

Tube 3 has pepsin, deionized water, and pH

2.0 buffer, Tube 4 with deionized water,

BAPNA, and pH 2.0 buffer, Tube 5 has

pepsin, BAPNA, and pH 7.0 buffer, and

Tube 6 with pepsin BAPNA, and pH 9.0

buffer.

Tube 1 was descended into the

incubation unit and was boiled. After boiling

tube 1, the tubes were incubated at 37oC for

60 minutes. The incubation unit gently

agitated the test tube rack so that the

contents of the tubes were evenly mixed.

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

Six test tubes containing 7 different

substances namely: lipase, water, vegetable

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

buffer, and bile salts were prepared. The

substance in each test tube can be seen in

table 4. The test tubes were incubated at

room temperature for 1 minute. Afterwards,

the pH of each solution were measured in

the Assay Cabinet and recorded.

Results and Discussion

Activity 1: Assessing Starch Digestion by

Salivary Amylase

Starch is a storage molecule found

exclusively in plants. Starch can be

FIGURE 2 – Setup for the assessment of

lipase digestion of fat.

FIGURE 3- Results of the IKI test.

FIGURE 1 – Setup for the Assessment

of the Pepsin Digestion of Protein.


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separated into amylose and amylopectin;

natural starch is 10-20% amylose and 80-

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

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

positive result since the boiling of the

solution cause the denaturation of the

enzyme amylase which inhibited the

breakdown of starch. Test tube 2 yielded a

negative result since starch was still

hydrolyzed by amylase since freezing did

not affect the enzyme. Test tubes 3 yielded a

negative result since starch was broken

down given the optimum condition (pH 7).

Test tube 4, 5, and 6 yielded a negative

result given the absence of starch, amylase,

and starch respectively. Test tubes 7 and 8 to

gave a positive result given that the pH was

not ideal for the enzymatic activity of

amylase.

The use of Lugol's iodine reagent

(IKI) is useful to distinguish starch and

glycogen from other polysaccharides.

Lugol's iodine yields a blue-black color in

the presence of starch. Starch amylopectin

will not react to cause a color change;

neither does cellulose or disaccharides such

as sucrose.

FIGURE 4- Results of the Benedict's test.

Starch and glycogen form helical

coils and the iodine atoms can fit into the

helices to form a starch-iodine or glycogen-

iodine complex.

Carbohydrates can be divided into

two categories based on the complexity of

their structure. Simple carbohydrates can

form either a single ring structure or a

double ring structure. Complex

carbohydrates are chains of many bonded

simple carbohydrates, and are often

expended for energy storage. These include

starch, cellulose, and glycogen. A test for

the presence of many simple carbohydrates

is the Benedict's test. A color change from

turquoise to yellow or orange is exhibited

when the reagent reacts with reducing

sugars.

For the Benedict’s test, the results

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


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show negative results. The starch in test

tube 1 was not hydrolyzed given that the

enzyme was denatured through the process

of boiling. Test tube 4 did not contain starch

to be broken down into maltose, and test

tube 5 did not contain amylase to break

down starch. Meanwhile test tubes 2,3 and 6

have highly positive results. An orange color

shows that the sample contains more sugar

than the green sample. This is given by the

optimum conditions for starch breakdown in

test tubes 2 and 3, while test tube 6

contained maltose to begin with. Test tubes

7 and 8 yielded positive results although not

as strong as the aforementioned test tubes

since the conditions at these test tubes were

not the optimum conditions for starch

breakdown.

The Benedict's reagent starts out

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

reducing sugars, it turns yellow to orange. 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.

Activity 2: Exploring Amylase Substrate

Specificity

Amylase is an enzyme that breaks

down carbohydratres like starch from

polysaccharide into disaccharides and/or

monosaccharides. On the other hand,

peptidase is responsible for breaking down

peptide bonds in proteins. In this activity,

the substrate specificity of enzymes,

particularly amylase and peptidase, was

tested. These were verified through two

chemical tests, namely: Iodine/Potassium

Iodide test (IKI), and Benedict’s test.

Iodine/Potassium Iodide test (IKI)

determines the presence of polysaccharides,

like starch and cellulose, in a sample. It is

performed by introducing an

iodine/potassium iodide solution and a

positive result will yield a blue-black color.

Based from the results (Table 3), test tubes

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

These mixtures still have polysaccharides

present, which means that amylase, water,

and peptidase are not capable or breaking it

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


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down. Test tube 1 demonstrates that amylase

is capable of breaking down starch. Test

tube 4 is the positive control of the IKI test

since it demonstrates what a positive result

for IKI should look like and it does not

contain any enzymes in the mixture. Test

tube 5 affirms and verifies that peptidase

cannot break down carbohydrates.

The Benedict’s test is performed by

introducing a mixture of copper sulfate

(CuSO4), sodium citrate, and sodium

carbonate, Benedict’s solution, to the sample

and heating it. This test is utilized to

determine the presence of reducing sugars

and it will yield an orange color or red

precipitate if positive. Reducing sugars

possess aldehyde groups and some examples

of these are: glucose, fructose, and

galactose. In the presence of heat and basic

solution, reducing sugars produce endiols.

These are reducing compounds that will

further react with the solution. CuSO4

provide copper ions that will oxidize

reducing sugars and this reaction yields

carboxylic acid and copper (I) oxide, which

is the red precipitate that indicates positive

(Figure 4).

Three set ups tested positive for Benedict’s,

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

the positive control set up for Benedict’s

test, since it contains glucose, which is a

reducing sugar. Test tube 1 demonstrates

that amylase is able to break down starch, a

polysaccharide, into disaccharides and

monosaccharaides that gave a positive result

in the test. Furthermore, test tube 5 gave a

negative result, which means that peptidase

is not able to break down polysaccharides.

These results verify that amylase is an

enzyme specific to carbohydrates and

peptidase is specific to proteins. Test tube 6

demonstrates that some bacteria are capable

of breaking down polysaccharides like

cellulose. Plants possess cellulose, which are

compounds that humans are not able to

digest. Test tube 3 demonstrates that

amylase cannot break down cellulose, which

affirms that humans cannot digest it. On the

other hand, some animals and insects are

able to digest cellulose due to the presence

of symbiotic microbes (bacteria, archaea,

protozoa) living in their gut. Some examples

of protozoans are: Trichomonas vaginalis,

Trichonympha, and Parasbasalia.

Protozoans present in termite gut are closely

associated with bacteria and these work

hand in hand with enzymes like, cellulases

and hydrogenases, in the gut of termites to

degrade cellulose (Okhkuma, 2008).


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Based from the findings of this

activity, it can be concluded that enzymes

are substrate specific. Its specificity is due to

the three-dimensional structure of the

enzyme-active site that corresponds to the

transition state of a reaction (Hedstrom,

2010). The most common metaphor for

enzymes and substrate is the lock and key

(Figure 5). A specific enzyme has its own

substrate that is perfectly fit for it to push

through with other processes. It cannot

degrade a compound when the required

substrate for it to bind on is not present.

FIGURE 5- Enzyme specificitymechanism.

Activity 3: Assessing Pepsin Digestion of

Protein

Pepsin is an enzyme that breaks

down proteins into smaller peptides. It is

produced in the stomach and is one of the

main digestive enzymes in the digestive

systems of humans and many other animals,

where it helps digest the proteins in food.

Pepsin is most active in acidic environments

between 37 °C and 42 °C. Accordingly, its

primary site of synthesis and activity is in

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 re-

acidification. The specificity of pepsin can

be identified as structural or group

specificity. Pepsin is an endopeptidase

enzyme, that hydrolyzes central peptide

bonds in which the amino group belongs to

aromatic amino acids (e.g. tyrosine and

tryptophan)

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


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BAPNA on the other hand is a synthetic

peptide that releases a yellow dye product

when hydrolyzed, it was used as a substrate

to assess pepsin activity.

The spectrophotometer was used to

measure the amount of yellow dye produced

by each mixtures this is to quantify the

pepsin activity in each test solution. The

spectrophotometer exposed light through the

sample and measured how much light did

the solution absorbed. The fraction of light

absorbed is expressed as the sample's optical

density.

TABLE 5- Optical Density of the Test

tubes.

There were negative controls used in

the activity those were Tubes 3 and 4. Given

these negative controls a negative result was

expected to validate the experiment because

negative controls are used to determine

whether there are any contaminating

substances in the reagents.

Test tubes 2 & 5's mixtures turned

yellow and the optical density recorded for

these two tubes were greater than zero.

These yellow solutions showed that the

BAPNA has been hydrolyzed however the

greater the optical density means the more

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.

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


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Activity 4: Assessing Lipase Digestion of Fat

Triglycerides are an ester derived

from glycerol and three fatty acids. Fats and

oils are poorly soluble in water. Since

lipases are hydrolases—that is, it break

bonds using water— it is hard to digest fats

and oils because they tend to clump

together, leaving only the molecules on the

surface exposed to these enzymes. Bile salts,

which are secreted in the small intestine,

help aid this difficulty by physically

emulsifying the clumps of lipids. They act

like detergents separate clumps into minute

triglyceride droplets thereby increasing the

surface are that is exposed to the lipases.

This process produces a monoglyceride and

two fatty acids.

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

low, so a decrease in pH might be difficult

to detect. Also, the buffer used is too acidic

which may cause the enzyme to be inactive

or be destroyed. This is because according to

Go et al. (1972), lipase is irreversibly

inactivated below pH 3.5 (as cited in

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

case of tube no. 6, little reaction is present

because the buffer used is too basic.

Furthermore, tube no. 3 showed no change

in pH from the buffer used (pH 9.0) which

means that there is no lipase activity since

there is no substrate (vegetable oil) to digest.

Tube no. 4 also did not show a change in

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

lipase present in the solution and the role of

bile salts is solely to increase the amount of

Tube

No.

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



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


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lipids that is to be exposed to the lipases.

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

observed. Tube 1 (pH 6.21) showed a

greater decrease in pH than in tube 2 (pH

6.72). The difference is due to the presence

of bile salts in tube no. 1, which increases

the amount of lipids exposed to the lipases

as compared to tube no. 2 wherein bile salts

are absent, therefore, the lipids are still in

clumps and the surface area is very little.

Conclusion

The appropriate chemical tests were

performed to determine whether digestion

occurred. With it, the group learned that

salivary amylase hydrolyzes starch to

maltose. IKI detects the presence of starch

while Benedict’s indicates that the starch ishydrolyzed by reacting to its product,

maltose or glucose.

Enzymes are very specific, only one

kind of substrate will “fit” into the active

site. Cellulose is the most common organic

molecule and major structural unit of plants

and cannot be digested by humans while

starch is the storage form of carbohydrate.

The usual substrate for peptidase is peptides

and proteins. Bacteria can aid in digestion

by breaking down cellulose which we do not

produce cellulase.

Peptidase, like pepsin, hydrolyzes

peptide bonds. BAPNA is used as a

substrate to indicate pepsin activity because

it produces yellow dye when it is

hydrolyzed. Pepsin only hydrolyzes peptide

bonds. The optimum pH of a particular

enzyme corresponds to the pH of its natural

environment. For many enzymes, this

corresponds to pH values of around 7. For

pepsin, which is active in the stomach, the

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

The pH decreases when lipases

activity is present. The hydrolysis product of

fat digestion as monoglycerides and two

fatty acids. Bile serves to mechanically

break up large globules of fat and produce

small droplets that effectively increases the

surface area of the lipids. It is difficult to

measure digestion in different pH because

the enzymes are active only on a certain

range of pH only.

References:

[1] Benedict’s test for reducing sugar.

(2015). Retrieved from

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


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Benedict’s test. (n.d.). Retrieved from

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

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

benedict.htm

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

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

Gastric and pancreatic lipase levels during a

test meal in dogs. Scand J Gastroenterol

28:443-454

[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

gastrointestinal proteolysis of "-casein and

"-lactoglubin. Soft Mattter 5:538-550

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

flagellates and prokaryotes in the gut of

lower termites. Trends in Microbiology,

16 (7), 345-352.

[7] Winkler F.K, d’Arcy A., Hunziker W.

(1990) Structure of human pancreatic lipase.

Nature 343:771-774

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