If the amylase enzyme is effective in breaking down the cracker, would you expect to see a positive or negative result for the presence of starch?

Preparation for Lab

Read the lab procedure and answer the embedded questions.  You may need to consult your textbook as well as the supplementary materials posted on Canvas.  Note that you will not be turning in the embedded questions as part of your lab write-up.  Instead, use these embedded questions to guide your preparation for the experiments.

1. Covalent bonds occur when atoms share…
2. Building a molecule from simpler components is called anabolism. Breaking a substance down is called…
3. The protein in gelatin that allows it to form a gel is called…
4. An enzyme in pineapple capable of digesting the protein in gelatin is called…
5. For each of the following substances, identify whether it is a carbohydrate, lipid, protein, or “other”.
   1. vegetable oil
   2. lactase

1. starch
2. lactose
3. distilled water

 

1. Each of the following statements applies to one of the tests we will perform in Part III of this lab. Give the name of the appropriate test.
2. Uses strips that change color in the presence of the test substance
3. Requires that the test solution be heated

• A very dark colored solution indicates that a lot of starch is present

 

1. Developing predictions: If the lactase enzyme is effective in breaking down lactose, would you expect to see a positive or negative result for the glucose test (“positive” = glucose is present)?

 

1. Developing predictions: If the amylase enzyme is effective in breaking down the cracker, would you expect to see a positive or negative result for the presence of starch?

 

 

 

Courtesy of Grace Sparks, Seattle Central Community College, and J. Walny, A. Rubenstein, and E. Matthews, Michigan State University.

 

Carbohydrates, lipids, and proteins are relatively large molecules that play many important roles in living organisms.  The basic framework or “backbone” of all these macromolecules consists of carbon atoms.

• Why is carbon a particularly useful element to form large molecules?

 

In lab, we will use a suite of chemical tests to 1) detect the types of molecules present in various food items, and 2) study how enzymes affect the molecules present in food items.

 

 

Learning Objectives

• Make accurate conclusions on the macromolecule content of food based off of your experimental results.
• Learn and apply the proper terminology to describe the molecular structure of the 4 major categories of biological macromolecule.
• Learn how to utilize positive and negative controls in the interpretation of experimental results.

 

Background:  Biological Macromolecules

Carbohydrates come in several basic forms.  The simplest sugars are called monosaccharides, and they contain 3-7 carbon atoms, often bonded together in a ring structure.  Glucose is a very common 6-carbon sugar.  When two monosaccharides bond together, they form a disaccharide, such as sucrose.  Many monosaccharide subunits bonded together forms a polysaccharide, a much more complex carbohydrate.  Starch, cellulose, and glycogen are common examples of polysaccharides.  Our test methods will allow us to easily detect starch and also reducing sugars (detected when copper is reduced to give a red color).  Glucose and fructose should test positive, but sucrose (a disaccharide) may not.

• If the disaccharide sucrose is made of a glucose bonded to a fructose, do you think copper will give a red color after sucrose is broken down by heat or acid?

 

There are many different types of lipids.  They are all grouped together as lipids because at least some part of the molecule does not dissolve in water.  Oil and vinegar salad dressing requires vigorous shaking because the oil and vinegar (which is a water-based solution) do not mix well.  We will use a simple test to help determine whether or not lipids are present in our samples.

 

Most proteins are very large molecules with complex shapes.  These shapes are formed by the interactions among many amino acids subunits bonded in a long chain.  Amino acids join to one another by a specific type of covalent bond called a peptide bond.

Proteins serve many functions, including structures, signaling, and facilitating chemical reactions.  Each function is dependent upon the protein’s folded shape.  Proteins that are chemical catalysts are called enzymes.  Please note that not all enzymes are proteins, but most are!

 

Read the following protocols to understand how we will test for various biological macromolecules and observe how enzymes can break them down.

 

 

Lab Protocol

1. Enzymatic activity of saliva.

Background:  As food is broken down by the teeth by way of chewing (the process of mastication), saliva is provided by the salivary glands.  This is where chemical digestion begins.  Saliva, among other important substances, contains an enzyme called amylase that aids in the breakdown of the polysaccharide starch.  Enzymes are strings of amino acids, joined by peptide bonds, that are responsible for catalyzing both synthesis (anabolic) and decomposition (catabolic) reactions.  Amylase breaks up long starch molecules into smaller sugar molecules.

 

Objective:  Investigate enzyme activity for an enzyme that breaks down polysaccharides.

 

Materials:

 

• Fresh Saltine Crackers
• Iodine Test Reagent
• Benedicts Test Reagent
• Hot Water Bath (90°)
• Test Tube Holder (1 per group)
• Test Tubes (4 per group)
• Sample Cups (2 per group)
• Spatula
• Razor Blade
• positive and negative controls for both tests (at instructor’s station)

 

 

Procedure:

Now you will test both an unchewed cracker and a chewed cracker for the presence of sugar and the presence of starch.  Here’s how:

 

1. First, chew a cracker for one full minute, then spit it into one of the sample cups. Add enough water to bring it to at least 1 mL.

 

1. In a new sample cup, add an unchewed cracker (the same size as the chewed one), and break it up using a spatula. Add enough water to bring it to at least 2 mLs.

 

1. Using a 1 mL pipette with the end cut off 0.5 cm from the tip (your instructor can show you how), transfer 1 mL of each sample to two labeled test tubes, one for the Benedict’s test and one for the starch test. You will need to do this for each of the two samples.  When you are finished, you should have a total of 4 tubes to which you will now be ready to add the test reagents.

 

1. Testing for Sugar (Benedict’s Test)
   1. Using a 1 mL pipette, add 1 mL of Benedict’s solution to each of the cracker sample tubes.

 

1. Heat the test tubes (use test tube holder) for 3 minutes in a hot water bath.

 

1. Compare with the class positive and negative controls to determine whether the polysaccharide has been broken down into monosaccharides. A cloudy precipitate will form that varies from green to yellow to orange to red to brown in color depending on the concentration of monosaccharides.

 

1. Testing for Starch (Iodine Test)
   1. Add 2 drops of iodine to a tube with 1 mL of chewed or unchewed cracker (the ones you set up in step 3).

 

1. Compare with the class positive and negative controls to determine whether any starch is still remaining (i.e. not yet broken down to sugar). If it changes color to dark blue or black, starch is present.  If the color is brown, no starch is present.

 

1. Answer the questions for this week’s lab write-up.

 

2.Pineapple flavored Jello?

Background:  Gelatin is obtained from selected pieces of calf and cattle skins, demineralized cattle bones (ossein), and pork skin.  Contrary to popular belief, hoofs, horns, hair, feathers, or any keratin material is not a source of gelatin.  There are two forms of gelatin: Type A, derived from acid processed materials—primarily pork skin; and Type B, derived from alkaline or lime processed materials—primarily cattle or calf hides and ossein.

 

Gelatin is made from a protein called collagen, a long, fibrous protein which comes from the joints of animals.  Gelatin dissolves in hot water and congeals (jells) at cooler temperatures.  As the dissolved gelatin mixture cools, the collagen forms into a matrix that traps the water.  As a result, the mixture turns into the jiggling, wriggling pseudo-solid that we all know and love as Jell-O™.

 

The pineapple belongs to a group of plants called Bromeliads.  The enzyme in fresh pineapple that is responsible for the breakdown of collagen is bromelain.  The process of canning pineapple denatures (unfolds) the bromelain in such a way that it can no longer catalyze the breakdown of gelatin.

 

Objective:  Investigate enzyme activity for an enzyme that breaks down proteins.

 

Materials:

• Sample Cups (2 per group)
• Jell-O™ Liquid (sugar free)
• Fresh Pineapple (1 piece per group)
• Canned Pineapple (1 piece per group)

 

Procedure:

1. Observe the sample cups containing the canned pineapple and fresh pineapple. These were prepared 24 hrs ago by adding a piece of either fresh or canned pineapple to a cup of Jell-O.

 

1. Answer the questions for this week’s lab write-up.

3.Lipids, water, and soap!

Background:  One of the most important characteristics of fats and lipids, in general, is their insolubility in water due to their non-polar  nature.  Lipids are made of long chains of hydrocarbons with relatively little oxygen atoms.  As a result, they tend to be non-polar and therefore do not dissolve in polar substances such as water.  (“Like dissolves like.”)  Polar or charged substances can be dissolved in polar substances and non-polar substances can be dissolved in non-polar substances.

 

In our digestive systems, lipids are, in part, broken down by bile, which is produced by the liver and aids in the digestion of fats in the small intestine.  Bile is not an enzyme, but it does help the enzymes do their job.  Bile helps create microscopic fat globules (a process called emulsification).

 

 

Emulsification is important because it allows lipases (important digestive enzymes that break down fats) to attack and break down the smaller fat globules.  Larger fat globs would mean that the lipases could not access the fats (lipids) on the interior of the lipid globs.

 

 

In this lab, you will use soap to mimic the action of bile.  Soap is unique in that a soap molecule has a polar (charged) end and a non-polar (non-charged) end.  The non-polar end interacts with and dissolves grease, oil, or fat, while the polar end interacts with a polar substance such as water molecules.  In this way, it can separate lipid molecules.

 

Objective:  Investigate the behavior of lipids and use soap to mimic the action of bile.

 

Materials

• Clean Test Tubes (2 per group)
• Water
• Vegetable Oil
• Dish Soap

 

Procedure:

1. Obtain two clean test tubes (no soap or oil).

 

1. Fill each 1/3 full with water.

 

1. Fill each 1/3 full with oil.

 

1. Add about 1 drop of soap to one of the test tubes.

 

1. In your lab notebook, draw a “before” picture of the two test tubes.

 

1. Cover the openings of the test tubes with hands/fingers and shake them vigorously.

 

1. In your lab notebook, draw an “after” picture of the two test tubes.

 

1. Answer the questions for this week’s lab write-up.

 

 

1. Lactose intolerance and glucose.

Background:  Lactose is the sugar found in milk and therefore has the common name “milk sugar.”  Lactose is a disaccharide composed of glucose and galactose sugar subunits.  When humans ingest milk, lactose must be broken down into glucose before it can be used as an energy source.  The enzyme responsible for breaking down or “digesting” lactose is called lactase.

 

 

Normally, all people are born with the ability to make lactase and can easily digest the lactose in mother’s milk and later in dairy products.  However, for some people, increasing age means loss of the production of lactase.  Loss of lactase production can begin as early as two years of age in some individuals and appears to occur more frequently and earlier in individuals of African or Asian heritage.  Individuals who do not produce lactase cannot break down the sugar lactose into its component parts.

 

Since only glucose passes from the intestines into the blood, lactose sugar remains in the intestinal tract until it leaves the body in the feces.  The lactose, however, is used as an energy source by the fermentative bacteria present in the intestines.  As a result of the bacteria’s fermentation, gas is released.  This can cause bloating, cramps, and diarrhea.  Lactaid and similar over-the-counter medications contain the enzyme lactase.  When these pills are taken in sufficient amounts with dairy products, people who are normally unable to enjoy dairy products can digest lactose and avoid the uncomfortable side effects they normally experience.

 

Diabetics have a problem where excess glucose appears in the blood and urine, causing damage to organs like the eyes and kidneys.  To monitor this glucose, test strips are sometimes used to test the urine for excess glucose.  A chemical indicator on the end of the dipstick changes color in the presence of glucose.  The glucose test strips that you will be using turns from pink (no sugar) to dark purple (presence of sugar).  Note: An indicator is a substance that changes color in the presence of a particular chemical.  There is an additional indicator at the end of each strip that tests for the presence of albumin, a protein, but you can disregard it for this test.

 

Objective:  Investigate the enzyme activity of an enzyme that breaks down a disaccharide into two monosaccharides.

 

Materials

• Glucose Test Strips (2 per group)
• Sample Cups (1 per group)
• Lactaid Pills (1 per group)
• Milk

 

Procedure:

1. Answer the first question for this exercise in your lab write-up before proceeding with the lab activity.

 

1. Fill a sample cup 1/3 full of plain milk.

 

1. Test the plain milk with a glucose test strip and record the results in your lab notebook.

 

1. Crush up ½ of a Lactaid pill with a mortar and a pestle.

 

1. Add the pill powder to the milk.

 

1. Stir with a fresh glucose test strip (10 seconds minimum) and record the results in your lab notebook.

 

1. Answer the remaining questions for this week’s lab write-up.

 

 

Lab Write-Up:

You may work together to answer these questions in lab.  For your lab write-up, type your final answers (using complete sentences) into a separate document.

 

 

(Part 1) Enzymatic activity of saliva.

1. For the chewed cracker…
   1. What color was produced by the Benedict’s test?
   2. What color was produced by the Iodine test?

 

1. For the unchewed cracker…
   1. What color was produced by the Benedict’s test?
   2. What color was produced by the Iodine test?

 

1. According to the above results…
   1. What type of carbohydrate(s) (monosaccharides and/or polysaccharides) was (were) present in the chewed sample?
   2. What about the unchewed cracker?

 

1. Does the amylase enzyme catalyze a catabolic or anabolic reaction? Briefly explain your choice in one complete sentence.

 

 

(Part 2) Pineapple-flavored Jello-O?

1. What is the consistency (i.e. texture) of the Jell-O made with the canned pineapple compared to the Jell-O made with fresh pineapple? What does this tell you about the reaction that has occurred?

 

1. Which of the 4 types of biological macromolecule allows Jell-O™ to form a gel?

 

1. In terms of an enzymatic reaction, is collagen a substrate, an enzyme, or a product? (see figure 8.13 in your book for help)

 

1. In terms of an enzymatic reaction, is bromelain a substrate, an enzyme, or a product? (see figure 8.13 in your book for help)

 

1. Briefly describe what happens during the cooking of pineapple that affects its interaction with Jell-O™.

 

1. What class of monomers (subunits) will the collagen break down into?

 

1. Fresh pineapple is used as a meat tenderizer. Based on the results of our study, explain why.

 

 

(Part 3) Lipids, water, and soap!

1. Compare your drawings of the oil/water tube and the soap/oil/water tube before and after shaking. How are the contents of the test tubes different after shaking?

 

1. Soaps contain amphipathic molecules, molecules that have distinct polar and non-polar regions. How does this property explain your observations in the “Lipids, water, and soap” experiment?

 

1. Look up “bile” in your book. It is a substance manufactured by your gall bladder to help digest food. What category of biological macromolecule would bile help digest?

 

1. If bile is not an enzyme, then briefly describe how it helps you digest food?

 

 

(Part 4) Lactose intolerance and glucose.

1. If lactase (Lactaid) is added to milk, what should happen?

 

1. According to your tests, which had a higher concentration of glucose: milk or milk + Lactaid?

 

1. The “-ose” ending suggests that lactose is a(n) _

 

1. The “-ase” ending suggests that lactase is a(n) __

 

1. Is the reaction catalyzed by lactase catabolic or anabolic?