Project Chemistry

Mucosa (cui)7,#;ifrl1\\--Sublingual duels

~~,~:~~~~~~~SUbmandjbUlar ductSublingual salivary gland

Mylohyoid muscle (cui)Submandibular salivary gland

Masseter muscle

Copyrlghl 0 The McGraw-HIli Companies, rnc. Permission required lor reproduction or display.Parotid salivary gland

Parotid duct

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“Effect of salivary amylase on starch” ____________________________________________________

Salivary amylase Starch dextrin + maltose + glucose

••• Parotid glands (located in the upper part of each cheek).

•••• Submandibular glands (located in the floor of the mouth).

•••• Sublingual glands (located beneath the tongue).

“Salivary amylase” is a digestive enzyme secreted by the salivary glands. It is responsible for starting the breakdown of the insoluble polysaccharide “starch” into soluble dextrins (oligosaccharides), maltose (disaccharide) and glucose (monosaccharide) in the mouth so that starch can be absorbed (amylon is a Greek word that means “starch”). Complete digestion of starch occurs in the small intestine by the action of pancreatic amylase.

There are three pairs of relatively large and major salivary glands:

amyl opectin

~

CHtJH0 H ~CHtJH 0 H

~H H ~H H IOH 0

H OH H OH 0

CHzDH CHzOH 6!Hz CHzOH CHzDH

~

OH~H OH~H OH~H OH~H OHOH H OH H OH H OH H OH H

OH 0 0 0 0 OH2H OH H OH H OH H OH H OH

CHzOH ){f6CHzOH, CHzOH CHzOH CHzOH

~

HH5 OH~OH~OH~OHH H H H HOH H 4 OH H 1 0H H OH H OH H

OH 0 "0 0 0 OH

H OH ~3 J~ H OH H OH H OHamylose

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Starch makes up a large proportion of our food. Chemically, starch consists of two types of molecules, “amylose” (20-30%) and “amylopectin” (70-80%) which are both polymers of glucose.

Amylose is a linear chain of glucose units that are linked by α-1,4 bonds, whereas amylopectin is a branched molecule in which glucose units are

linked by α-1,4 bonds in the straight chain region in addition to α-1,6 bonds at the branching points. The relative proportions of amylose to amylopectin depend on the source of the starch.

Salivary amylase hydrolyses only the α-1,4 bonds that link glucose units.

Therefore, starch and glycogen, which consist of α-1,4 bonds, are

hydrolyzed by amylase while cellulose, which consists of β-1,4 bonds, is not hydrolyzed by salivary amylase.

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In a beaker, collect about 1 ml of your own “saliva” then add 9 ml of distilled water and 60 ml of 0.5% sodium chloride solution and gently mix. Use this “enzyme solution” to carry out the following four experiments:

I- Effect of salivary amylase on starch:

Principle:

If amylase is added to a solution of starch, starch will be digested. As the reaction proceeds, the progress of the reaction can be visualized by testing:

1. the disappearance of the substrate (starch). 2. the appearance of one of the products (the reducing sugar, maltose).

Two simple tests, “iodine test” for starch detection and “Benedict's test” for maltose detection are used for this purpose. Procedure:

(to the left: starch; to the right: starch + iodine)

2. In a test tube, put 2 ml of 2% starch solution and carry out “Benedict’s test” as follows: add 2 ml of Benedict's reagent to the content of the test tube and put in a boiling water bath for 5 minutes. The test gives negative result where Benedict's reagent retains its clear blue color and no precipitate is formed indicating the absence of maltose (starch did not undergo any hydrolysis yet).

1. In a test tube, put 2 ml of 2% starch solution and carry out “iodine test” as follows: add 3 drops of iodine solution to the content of the test tube; a blue-black color is formed (N.B. shake starch solution well before withdrawing any volume).

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3. In a test tube, put 2 ml of the previously prepared enzyme solution and add 2 ml of 2% starch solution; mix well and leave for 10 minutes.

b. To the second test tube, add 2 ml of Benedict’s reagent and put in a boiling water bath for 5 minutes. The test gives positive result (development of a precipitate with a color ranging from green, through yellow and orange to red) indicating the formation of maltose; the test depends upon the ability of the reducing sugar maltose to reduce cupric ions (blue) in Benedict's reagent to cuprous ions (green to red).

Color Amount of maltose Blue None

Green Some

Yellow More

Orange Much

Red Most

Benedict’s test

4. Divide the enzyme-starch mixture in 2 clean test tubes and carry out “Iodine test” to the first and “Benedict's test” to the second as follows:

a. To the first test tube, add 3 drops of iodine solution; the test still gives positive result (blue-black color) indicating the presence of a remaining part of starch that did not undergo hydrolysis by salivary amylase.

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II- Effect of temperature on the activity of salivary amylase:

Principle:

Salivary amylase is protein in nature; the optimum temperature for its

activity is 37°C. In extreme temperatures (cold and heat), the activity of the enzyme is inhibited (high temperature causes denaturation of the protein structure of the enzyme). Procedure:

1. Get 3 test tubes and number them “1”, “2” & “3”.

2. Put 2 ml of the previously prepared enzyme solution in each of the three test tubes.

3. To each test tube, add 2 ml of 2% starch solution and mix well.

4. Immediately put test tube “1” in an ice bath, test tube “2” in a 37°C water bath and test tube “3” in a 70°C water bath, each for 20 minutes.

5. Remove each tube to room temperature and carry out Benedict’s test to check for the presence of the starch hydrolysis product “maltose” as follows: add 2 ml of Benedict’s reagent to the enzyme-starch mixture in each tube and put in a boiling water bath for 5 minutes; Benedict’s reagent will retain its clear blue color in test tubes “1” & “3” indicating the absence of maltose as a result of inhibited enzyme activity at cold and hot temperatures, while a green to red precipitate is formed in test tube “2” indicating the formation of maltose as a result of a retained activity of salivary amylase at 37°C.

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III- Effect of pH on the activity of salivary amylase:

Principle:

The optimum pH for the activity of salivary amylase is the neutral pH (about

7). In the acidic pH of the stomach, the activity of salivary amylase stops. Pancreatic amylase, on the other hand, has an optimum activity in alkaline medium. Procedure:

2. Put 2 ml of the previously prepared enzyme solution in each of the three test tubes.

3. To each test tube, add 2 ml of 2% starch solution and mix well.

1. Get 3 test tubes and number them “1”, “2” & “3”.

4. Add 2 ml of acidic solution to test tube “1”, 2 ml neutral solution to test tube “2” and 2 ml alkaline solution to test tube “3”. Put the 3 test tubes in a 37°C water bath for 10 minutes.

5. Remove each tube to room temperature and carry out Benedict’s test to check for the presence of the starch hydrolysis product “maltose” as follows: add 2 ml of Benedict’s reagent to the content of each tube and put in a boiling water bath for 5 minutes; Benedict’s reagent will retain its clear blue color in test tubes “1” & “3” indicating the absence of maltose as a result of inhibited enzyme activity in acidic and alkaline pH, while a green to red precipitate is formed in test tube “2” indicating the formation of maltose as a result of a retained activity of salivary amylase in neutral pH.

Student Name: ………………………………………. Student number: ……………

I. Effect of salivary amylase on starch:

Test Starch solution Enzyme-starch mixture

Iodine test

II. Effect of temperature on the activity of salivary amylase:

Temperature Salivary amylase activity

0°C

37°C

70°C

III. Effect of pH on the activity of salivary amylase:

Neutral

Alkaline

“Effect of salivary amylase on starch”

Benedict’s test

pH Salivary amylase activity

Acidic

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