Lab 3. Chemistry of Food

OBJECTIVES

• Test for various biological macromolecules in food and observe how enzymes can break them down.
• Investigate the behavior of lipids, and use soap to mimic the action of bile.

PRELAB

Read the lab AND consult the textbook to answer the questions (include in lab notebook):

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”.
   • vegetable oil
   • lactase
   • amylase
   • starch
   • lactose
   • distilled water

6. Each of the following statements applies to one of the tests we will perform in this lab. Give the name of the appropriate test.

• • Uses strips that change color in the presence of the test substance
  • Requires that the test solution be heated
  • A very dark colored solution indicates that a lot of starch is present

7. 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)? Explain.
8. 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? Explain.

INTRODUCTION: Testing foods for carbohydrates, lipids, and proteins

Carbohydrates, lipids, and proteins are relatively large molecules that play many important roles in living organisms.  Carbon atoms and the many covalent bonds they can form provide the basic framework for all of these macromolecules.  In lab today, we will use a suite of chemical tests to detect the types of molecules present in various food items and how enzymes affect them.

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 as copper is reduced to give a red color). Glucose and fructose should test positive, but sucrose (a disaccharide) may not (unless it 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.  While monosaccharides are the subunits that make up polysaccharides, amino acids are the subunits of proteins.  Amino acids join to one another by a specific type of covalent bond called a peptide bond.  .

In this exercise, you will use the methods described below to test for various biological macromolecules and observe how enzymes can break them down.

Exercise A – Enzymatic Activity of Saliva

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.

Materials:

Fresh Saltine Crackers

Iodine Test Reagent

Benedicts Test Reagent

Hot water bath (90°)

Test tubes (4 per group)

Test tube holders (1 per group)

Sample cups (2 per group)

Spatula

Positive (starch and sugar solutions) and negative (water) controls for both tests

PROCEDURE

• 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 3 mL.
• 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 3 mL.
• 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, for each of the two samples (see diagram below).
  • To transfer 1 mL of your samples into the test tubes, you may need to pipet twice in 0.5 mL increments with your cut 1 mL transfer pipettes.
  • When you are finished, you should have a total of 4 tubes to which you will now be ready to add the test reagents.

4. 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.
   2. Heat the test tubes (use test tube holder) for 3 minutes in a 90 ^oC hot water bath.

• 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. Compare with the class positive and negative control to determine whether the polysaccharide has been broken down into monosaccharides.

5. Testing for Starch (Iodine Test)

• Add 2 drops of iodine to a tube with 1 ml of chewed or unchewed cracker (the ones you set up in step 3).
• If it changes color to dark blue or black, starch is present. If the color is brown or reddish (and more transparent), no starch is present. Compare with the class positive and negative control to determine whether any starch is still remaining (i.e., not yet broken down to sugar).
• Answer post-lab questions for Exercise A.

Exercise B – Pineapple-flavored Jello

Gelatin is obtained from selected pieces of calf and cattle skins, demineralized cattle bones (ossein), and porkskin. 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 porkskin; 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, 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 Bromeliaceae. Kiwi, papaya, and figs are other types of Bromeliaceaes. The enzyme in fresh pineapple that is responsible for the breakdown of collagen is bromelin. The process of canning pineapple denatures the bromelin in such a way that it can no longer catalyze the breakdown of gelatin.

Materials:

Sample cups (2 per group)

Jell-O liquid (sugar free jello)

Fresh pineapple (1 piece per group)

Canned pineapple (1 piece per group)

PROCEDURE

1. Determine which of the sample cups contains the canned pineapple and the fresh pineapple. These were prepared 24 hrs ago by adding a piece of either fresh or canned pineapple to a cup of Jell-O.
2. Answer the post-lab questions for Exercise B.

Exercise C -Lipids, water and soap!

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 minute, 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 is amphipathic – it 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 is able to separate lipid molecules.

Materials:

Clean test tubes (2 per group)

Water

Vegetable Oil

Dish Soap

PROCEDURE

1. Obtain two clean test tubes (no soap or oil).
2. Add 1/3 water and 1/3 oil to one of the test tubes, and to the other add 1/3 water, 1/3 oil, and one drop of soap.
3. In your lab notebook, draw a before picture of the two test tubes below in the group lab report.
4. Cover the openings of the test tubes with hands/fingers and shake them vigorously.
5. In your lab notebook, draw an after picture of the two test tubes on in the group lab report.
6. Answer the post-lab questions for Exercise C.

Exercise D – Lactose Intolerance & glucose

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 and as a result, gas is released. This can cause bloating, cramps, and diarrhea. Lactaid and similar over-the-counter medications contain the enzyme lactase. When 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. In order 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.

Materials:

Glucose test strips (2 per group)

Milk

Sample cups (1 per group)

Lactaid pills (1 per group)

PROCEDURE

1. Answer the first three post-lab questions for this exercise before proceeding with the lab activity.
2. Fill a sample cup 1/3 full of plain milk.
3. Test the plain milk with a glucose test strip and record the results in the table below.
4. Crush up ½ of a Lactaid pill with a mortar and a pestle.
5. Add the pill powder to the milk.
6. Stir with a fresh glucose test strip (10 seconds minimum) and record the results in the group lab report.
7. Answer the post-lab questions for Exercise D.

 POST-LAB QUESTIONS

(Exercise A) Enzymatic activity of saliva.

1. For the chewed cracker… What color was produced by the Benedict’s test? What color was produced by the Iodine test?
2. For the unchewed cracker… What color was produced by the Benedict’s test? What color was produced by the Iodine test?
3. According to the above results, what type of carbohydrate(s) (monosaccharides and/or polysaccharides) was (were) present in the chewed sample? What about the unchewed cracker? (answer in 1-2 complete sentences)
4. Does the amylase enzyme catalyze a catabolic or anabolic reaction? Explain your choice in 1 complete sentence.

(Exercise B) 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? (answer in 2-3 complete sentences)
2. Which of the 4 types of biological macromolecule allows Jell-O to form a gel?
3. In terms of an enzymatic reaction, is collagen a substrate, an enzyme, or a product?
4. In terms of an enzymatic reaction, is bromelin a substrate, an enzyme, or a product?
5. What happens during the cooking of pineapple that affects its interaction with Jell-O? (answer in 1 complete sentence)
6. What class of monomers (subunits) will the collagen break down into?
7. Fresh pineapple is used as a meat tenderizer. Based on the results of our study, explain why. (answer in 1-2 complete sentences)

(Exercise C) 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 test tubes different after shaking? (answer in 1 sentence)
2. 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? (answer in 2-3 complete sentences).
3. 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?
4. If bile is not an enzyme, then how does it help digest food? (answer in 1-2 complete sentences).

(Exercise D) Lactose Intolerance and Glucose

1. If lactase (Lactaid) is added to milk, what should happen? (answer in 1 sentence)
2. The “ose” ending suggests that lactose is a(n) ________________________
3. The “ase” ending suggests that lactase is a(n) ________________________
4. According to your tests, which of the following substances had a higher concentration of glucose? Milk, or the Milk with Lactaid added?
5. Is the reaction catalyzed by lactase catabolic or anabolic?