Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Prepare 600 mL of an acetate buffer, pH 5.
   1. Use a wax pencil or marker to label a 1-L Erlenmeyer flask “acetate buffer.”
   2. Use a graduated cylinder to measure 216 mL of 0.1 M acetic acid into the “acetate buffer” beaker.
   3. Use a graduated cylinder to measure 384 mL 0.1 M sodium acetate into the “acetate buffer” beaker and mix.
2. Prepare 100 mL of a 5% pectinase solution: Using a graduated cylinder, add 100 mL acetate buffer to the pectinase in the bottle and mix.
3. Prepare 100 mL of a 5% cellulase solution: Using a graduated cylinder, add 100 mL acetate buffer to the cellulase in the bottle and mix.

Safety Precautions

Dilute acetic acid and sodium acetate solutions are skin and eye irritants. Avoid contact of all solutions with skin and eyes. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Remind students to use cautions when cutting with knives. All food-grade items that have been brought into the lab are considered laboratory chemicals and are for lab use only. Do not taste or ingest any materials in the lab. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All of the waste solutions may be disposed of according to Flinn Suggested Disposal Method #26b, down the drain with excess water. The apple solid may be disposed of according to Flinn Suggested Disposal Method #26a, solid waste disposal in a landfill.

Lab Hints

• Enough meterials are provided in this kit for 30 students working in groups of three or for 10 groups. This laboratory activity can reasonably be completed in one 50-minute period.
• Any variety of apples should work. The procedure was tested using Red Delicious™ apples. Some sources cite problems with Golden Delicious™ apples. Apple sauce may contain starch and preservatives that will interfere with the production of juice in this experiment.
• The teacher may want to speed up the chopping of apples in order to allow the students collect juice for 20 minutes rather than 15 minutes. This can be done by chopping the apples in a food processor as the students label their glassware.
• Cake pans may be substituted for dishpans to make the water baths.
• All enzymes have optimal temperature and pH ranges—for pectinase and cellulase the optimal temperature is 40–50 °C and the optimal pH is about 5. At 60 °C, heat begins to alter the structure of the enzyme, denaturing it so that it can no longer break down pectin or cellulose molecules. If the pH of the enzyme’s environment is too low, H⁺ ions may protonate basic amino acid side chains on the enzymes and possibly alter their activity. If the pH is too high, OH^– ions may deprotonate acidic side chains on the enzyme that may be needed for enzyme activity.

Teacher Tips

• Extend the activity by having students test the enzymes on various food items. All food-grade items that have been brought into the lab are considered laboratory chemicals and are for lab use only. Do not taste or ingest any materials in the laboratory and do not remove any remaining food items after they have been used in the lab.
• Link to National Standard G: History and Nature of Science. Have students research the use of genetic engineering in the production of enzymes, which began in 1973 when the Food and Drug Administration approved the use of engineered chymosin to make cheese.
• The Enzyme Technical Association lists many commercial and industrial uses of enzymes. See their website at http://www.enzymetechnicalassoc.org/benefit_paper.pdf (accessed April 2007) for more information.

Answers to Prelab Questions

1. Define the term enzyme.

   An enzyme is a biochemical catalyst that accelerates the rate of a chemical reaction without being permanently altered in the process.

2. Based upon the information in the Background and Procedure sections, predict which sample will produce the most juice.

   Students will likely predict that the sample containing both pectinase and cellulase will produce the most juice. It is unlikely that anyone will predict the acetate buffer will produce more juice than the enzyme samples.

Sample Data

Answers to Questions

1. Graph the data for the amount of juice produced for each mixture. For this graph you will need to identify the dependent and independent variables. Note: Plot all of the data on the same graph using different colors or different data point shapes for each mixture.

   The amount of juice produced is the dependent variable. The type of enzyme used is the independent variable.

2. What other variables might have affected the amount of juice produced. How were these variables were controlled during the experiment?

   The amount of apple, the amount of solution, the type of filter paper, and the temperature were identical in each trial. The acetate buffer was used to make the enzyme solutions and served as the solution in the control trial.

3. Which enzyme treatment produced the most juice? Explain.

   The sample with the pectinase produced the most juice. More (5 mL) of the pectinase enzyme was present to digest pectin, releasing the juice from the apple’s cells.

4. Enzymes are expensive. In your opinion, does the cost of enzymes justify the amount of juice produced for the enzyme-treated mixtures?

   Students may determine that the addition of cellulase to the apples does not justify the cost. However, pectinase does significantly increase the amount of apple juice produced in this lab.

Introduction

Enzymes are complex proteins that act as biochemical catalysts. Enzymes are produced by all living cells. Human beings have used harvested enzymes produced by bacteria, plants, fungi and even other animals for thousands of years in numerous applications. Two food industry enzymes are investigated in this laboratory.

Concepts

• Catalyst
• Enzymes

Background

A catalyst is any substance that speeds up the rate of a chemical reaction without being permanently altered in the process. Thus a single molecule of catalyst can perform the same reaction thousands of times a second. Catalysts cause slow reactions that occur infrequently to occur more often by lowering the activation energy necessary for the reaction to occur.

Enzymes are specialized catalysts that perform specific functions for a living organism. Most enzymes are globular, three-dimensional proteins whose specific characteristic shape allow only a few specific chemicals to temporarily bond with the protein. This exclusive nature of enzyme/substrate binding implies that living organisms must contain thousands of different enzymes to catalyze all the different biochemical reactions that must occur. One type of enzyme may break a molecule into two or more product molecules (digestion), while a different type of enzyme may combine two or more molecules together to build a more complex product (synthesis).

One enzyme, pectinase, is excreted by fungi like Aspergillus niger to digest plant pectin. Pectin is found in plant cell walls and in between cells in the middle lamella. Pectin, along with cellulose, provide support for the plant. A. Niger secretes pectinase to break apart the plant’s cells so it can absorb nutrients from the plant. Pectinase only digests pectin, so A. Niger also excretes the enzyme cellulase. Cellulase digests the cellulose found in the plant.

In the juice industry, enzymes are used in two processes—peeling and juicing. The enzymes cellulase and pectinase are added to vats of tough-skinned fruits such as oranges and peaches. The fruits are soaked in an enzyme solution to digest the cellulose and pectin in the peel. The fruit-enzyme mixture is strained to remove the peel artifacts and the seeds. More pectinase and cellulase are added to the pulp to break down the fruits’ cell walls before the pulp is squeezed. Adding enzymes to the peeled fruit produces up to 20% more juice than would be produced by squeezing alone. The resulting juice is more appealing to consumers, as it is clearer.

For more than 3000 years, enzymes produced by bacteria and fungi have been isolated by fermentation. Fermentation involves the exponential growth of microorganisms in a fermentation vessel. After fermentation, the microorganisms are destroyed and the enzymes are isolated for commercial use. Pectinase and cellulase are easily harvested from A. Niger, a common black mold that secretes enzymes outside its cells to digest fruits and vegetables such as grapes and onions.

Experiment Overview

The purpose of this activity is to investigate the effects of adding different enzymes on the amount of juice obtained from an apple.

Materials

Prelab Questions

1. Define the term enzyme.
2. Based upon the information in the Background and Procedure sections, predict which sample will produce the most juice.

Safety Precautions

The acetate buffer solution contains acetic acid and sodium acetate and is a skin and eye irritant. Avoid contact of all solutions with skin and eyes. Wear chemical splash goggles and chemical-resistant gloves. Use caution when cutting with a knife. Always cut away from your body. All food-grade items that have been brought into the lab are considered laboratory chemicals and are for lab use only. Do not taste or ingest any materials in the lab. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

1. Use a wax pencil or marker to label each of the four 100-mL beakers and four 25-mL graduated cylinders with one of the following terms—pectinase, cellulase, both and buffer (control).
2. Place a funnel into each 25-mL graduated cylinder.
3. Fold each filter paper in half and then in half again. Tear off a small corner (see Figure 1). Place one filter paper into each funnel.
   {10825_Procedure_Figure_1}
4. Use a knife or apple slicer to cut an apple into 0.5-cm or smaller pieces.
5. Using a balance, measure 50 g of apple pieces into a weighing dish.
6. Add 50 g of apple pieces into the buffer (control) beaker.
7. Repeat steps 5 and 6 for the pectinase, cellulase and both beakers.
8. Use a clean, 10-mL graduated cylinder to measure 5 mL of acetate buffer. Pour the buffer into the buffer (control) beaker. Rinse the graduated cylinder with buffer.
9. Repeat step 8 add 5 mL of 5% pectinase into the pectinase beaker, 5 mL of 5% cellulase into the cellulase beaker and 2.5 mL of 5% pectinase and 2.5 mL of 5% cellulase into the beaker labeled “both.”
10. Cover the beakers with plastic wrap.
11. Fill a dishpan about ¼ full with 45 °C tap water bath. Add hot water to the water bath as necessary to maintain the water bath at about 45 °C during the experiment.
12. Place the four beakers into the 45 °C water bath and incubate for 20 minutes.
13. Use a wood craft stick to mash the apple mixture in each beaker.
14. Pour the buffer (control) mixture into the funnel in the buffer 25-mL graduated cylinder.
15. Repeat step 14 filtering each mixture into the appropriately labeled graduated cylinder.
16. Measure the amount of juice filtered into each graduated cylinder every 5 minutes for 15 minutes. Gently stir the apple pieces occasionally with a wood craft stick to release juice that becomes caught among the apple pieces.

Student Worksheet PDF

10825_Student1.pdf