ACTIVITY 7 SALIVARY DIGESTION A.) Objectives At the end of this activity, students are expected to: a.

) Explain the nature of saliva b.) Discuss the principles/rationale of each test B.) Procedure a.) Reaction of Saliva Place a few drops of resting saliva in three test tubes.

Test the reaction with Phenolphthalein, Blue Litmus and Congo red.

From the colors produced, estimate the approximate pH of your saliva. Use a pH paper.

Repeat experiment after stimulating the flow of the saliva by chewing paraffin vigorously for five minutes.

b.) Inorganic Matter Place 5 ml of saliva in a test tube

Acidify with concentrated Nitric acid and heat to boiling to remove all the proteins.

Filter, divide into four test tubes and test the filtrate for:

Chloride by adding 1 drop of AgNO3 solution to test tube 1

Phosphates by adding 1 drop of Ammonium Molybdate to test tube 2

Sulfates by adding 1 drop of BaCl3 solution to test tube 3

Calcium By adding one drop of Ammonium of Potassium oxalate

c.) Influence of Temperature on Ptyalin Activity

Place 1 ml of starch solution to each of the four test tubes

Immerse one test tube in an ice-water mixture,

Keep the second at room temperature

Place the third in a water bath at 38

Add to these test tubes 10 drops of saliva and shake well.

To the contents of the fourth tube, add 10 drops of boiled saliva.

At 15 minutes interval, remove a sample from each test tube, test with iodine solution and benedicts solution.

Allow the test tubes to stand for an hour.

d.) Influence of Dilution of Ptyalin Activity

Place 9 ml of water to each of the 6 test tubes.

To the first, add 1 ml of saliva and mix thoroughly

Transfer 1 ml of this mixture to the second tube and mix

Take 1ml of the mixture from the second tube and transfer to the third tube.

Take 1 ml of the mixture from the third tube and transfer to the fourth tube

Take 1 ml of the mixture from the fourth tube and transfer to the fifth tube

Take 1 ml of the mixture from the fifth tube and transfer to the sixth tube.

To each of the test tubes add 1 ml of 1% starch solution.

Shake well and place in water bath at room temperature of about 40 C for 20 minutes

Now and then test a portion with Iodine test and another portion with Benedicts reagents.

C.). Results and Observation a.) Influence of Temperature to Ptyalin Activity

SAMPLE Test tube 1

IODINE All the samples for the Iodine test turned black. Ideal result would be lightening of the samples. But due to procedural

BENEDICTS Sky blue; No color change

Test tube 2

All the samples for the Iodine test turned black. Ideal result would be lightening of the samples. But due to procedural

Sky blue; No color change

Test tube 3

All the samples for the Iodine test turned black. Ideal result would be lightening of the samples. But due to procedural

Sky blue; No color change

Test tube 4

All the samples for the Iodine test turned black. Ideal result would be lightening of the samples. But due to procedural

Sky blue; No color change

b.) Influence of dilution on Ptyalin activity

SAMPLE First Test Tube

IODINE TEST From brownish/yellow color, it turned light clear blue.

BENEDICTS TEST From blue to light brown turbid yellow Remained blue

Second Test Tube

From brownish yellow to very pale yellow

Third Test Tube

Brownish yellow to pale yellow

Remained blue

Fourth Test Tube

Light green

Clear

Fifth Test Tube

Light green

Light blue/remained blue

Sixth Test Tube

Light green

Remained blue

D.) Analysis and Conclusion: 1.) How did you know that digestion has taken place in the mouth? Digestion is the process by which food is broken down by enzymes through hydrolysis. That is, water is added to a large molecule, which splits into smaller molecules that can be absorbed into the bloodstream. For example, water is added until proteins are broken down into amino acids, starch is broken down into glucose, and fates broken down into glycerol and fatty acids. Enzymes are necessary for digestion, just as they are required for chemical reactions in the body. Salivary amylase hydrolyzes starch. Starch is digested by salivary amylase in the mouth, a process described by the following reaction: Starch + water (catalyzed by amylase (enzyme)) = maltose So a positive result for Benedicts test, a test for the presence of sugars e.g. maltose, indicates digestion. 2.) Explain how temperature influences the process of digestion. Like most chemical reactions, the rate of an enzyme-catalyzed reaction, in this case, digestion, increases as the temperature is raised. A ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to 100%. Variations in reaction temperature as small as 1 or 2 degrees may introduce changes of 10 to 20% in the results. In the case of

enzymatic reactions, this is complicated by the fact that many enzymes are adversely affected by high temperatures. As shown in Figure 13, the reaction rate increases with temperature to a maximum level, then abruptly declines with further increase of temperature. Because most animal enzymes rapidly become denatured at temperatures above 40C, most enzyme determinations are carried out somewhat below that temperature. Over a period of time, enzymes will be deactivated at even moderate temperatures. Storage of enzymes at 5C or below is generally the most suitable. Some enzymes lose their activity when frozen. Influence of temperature on Ptyalin Activity If digestion has not taken place, the iodine test for starch will be positive. If starch is present, a blue-black color immediately appears after a few drops of iodine are added. If digestion has taken place, a test for sugar (maltose) will be positive. A color change of blue to green to yellow to orange to red indicates the presence of maltose. Boiling the test tube is necessary for the Benedicts reagent to react. When there is an increase in temperature, there is an increase in kinetic energy which then increases effective collision. When effective collisions are increased, there is an increase in the velocity of molecules which then increase the rate of chemical reaction in Ptyalin. But, the optimum temperature for reaction is 40 degrees Celsius. Anything above or below the optimum temperature, the rate of reaction would decrease due to the inactivation of Ptyalin. In the experiment, 4 measures were made for the four test tubes; the ice water, the room temperature, the 40 degree warm bath and the boiling saliva. It is expected that in the Benedicts test, the one dipped in the ice water would become negative and in the Iodine test, it would show a positive outcome. This shows that the salivary amylase is more reactive in the Iodine test because even at low temperatures, a reaction can still occur. Possibly, a higher temperature is needed in order for a reaction to take place in the Benedicts test. In the room temperature, it is expected that both the Benedicts test and Iodine test would display positive results. As weve said before, even in low temperatures, Iodine is already reactive. This means that it is even more reactive when temperature is increased because increasing temperature also increases the rate of reactivity of a solution (as explained above in the previous sentences). Now, since temperature is now increased compared to that of the cold bath, Benedicts test is now more reactive. This is why we can expect a positive result. Benedicts test would display a positive result in the 38 degree water bath while the Iodine test will display a negative result. This is because the Benedicts test becomes more reactive when temperature increases especially when it reaches the boiling point. Having said this, we could expect that the Benedicts test would react positively to the boiling saliva.

Influence of dilution on Ptyalin activity With the same amount of substrate, ptyalin activity is directly proportional to enzyme concentration, so as the amount of enzyme concentration increases, the rate of reaction also enzyme increases. If the enzyme concentration was kept constant and the substrate concentration was varied, increasing the amount of substrate increases the velocity of the reaction until it reaches the maximum activity of the enzyme. E.) Pictures