Amylase + Starch Simple Sugarsindicator: Benedict S Reagent

Digestion Lab

Enzymes are a very important part of digestion. Digestion begins in the mouth. The first enzyme to break down food is salivary amylase found in the saliva. Salivary Amylase breaks down starches into simple sugars. Next, the food moves to the stomach where more chemical digestion takes place. The stomach has specialized cells called chief cells that release an inactive enzyme called pepsinogen. Pepsinogen is activated by hydrochloric acid produced in the stomach by parietal cells. It is important to note that enzymes in the stomach will have a very acidic optimal pH. The stomach is where protein digestion begins as a result of pepsin. Next as the food is propelled to the duodenum, the pancreas will release pancreatic juice. This juice has a more basic pH of around 8 to neutralize the contents of the stomach and contains proteases that break down protein, lipases that break down fats, and amylase that breaks down starches. Bile is also made in the liver and stored in the gall bladder. The gall bladder releases this bile into the duodenum to emulsify the fats. Emulsification allows the fats to be absorbed. In our experiment today, we will use indicators to determine if our enzyme has broken down the perspective substrate. Benedict’s reagent is used to detect reducing sugars. Amylase will break starch into simple sugars. Benedict’s contains Cu+2 that will be reduced to Cu+1 and will show a precipitate in the form of red Copper (I) oxide if simple sugars are present. Biuret will be the indicator used to test for proteins. It is purple in the presence of proteins and turns brown and disappears as they are broken down to amino acids. Phenol red is a pH indicator that will be used to determine if lipase breaks the milk fat into fatty acids and glycerol. Fat is neutral and becomes more acidic as fatty acids are hydrolyzed. The phenol red will change color from red to orange and sometimes yellow as the pH lowers. This indicator has a pH range of 6.8-8.

Amylase + starch → simple sugars Indicator: Benedict’s reagent

Pepsin + albumin → amino acids Indicator: Biuret

Lipase + milk → fatty acids Indicator: Phenol red

Materials: 6 test tubes Amylase Hot water bath of 37◦C Pepsin Boiling water Lipase Starch solution Benedict’s reagent Albumin solution Biuret Milk Phenol red Methods:

 Label the tubes, A, AC, P, PC, L, and LC. This stands for the enzymes and their controls. Amylase  Place 10 drops of amylase into tube labeled A. Do not put enzyme into the test tube labeled AC.  Place 10 drops of starch solution into both test tubes (same amount in both tubes).  Place 10 drops of Benedict’s reagent into each of the two test tubes.  Place test tubes in hot bath for about 45 min.  Amylase test tubes will need to be boiled for 2 min after hot bath. Pepsin  Place 10 drops of albumin (component of egg white) into both test tubes.  Place 10 drops of biuret into both test tubes.  Place 5 drops of pepsin into the test tube labeled P. NO enzyme goes into the test tube labeled PC.  Place test tubes in hot bath for 45 min. Lipase  Place 10 drops of milk into both tubes  Place 1 drop of phenol red into both test tubes  Place 1 drop of soap into both test tubes  Place 5 drops of Lipase into the test tube labeled L. No enzyme goes into the test tube labeled LC.  Swirl test tubes and place test tubes in hot water bath for 45 min.

Results: Test Tubes: Description of reaction A (Amylase)

AC (Amylase Control)

P (Pepsin)

PC (Pepsin Control)

L (Lipase)

LC (Lipase Control) Questions:

1. Why do we run controls in the experiment?

2. What ion in Benedict’s reagent is reduced to cause a positive test?

3. What are proteins broken down into?

4. What causes the pH to lower as lipase is added to milk?

5. Why do we add soap to the lipase to aid in the digestion of fat?

6. What is the optimal pH of pepsin?

7. What is in the contents of pancreatic juice?

8. How does the liver help the digestive process?

9. What is the optimal pH of enzymes in the pancreatic juice?

10. What is the purpose of biuret?