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Project Summary

Difficulty	5  –  7
Time required	Average (about one week)
Prerequisites	None
Material Availability	Readily available
Cost	Average ($50 - $100)
Safety	No issues

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Abstract

Objective

The objective is to explore the biochemical basis for lactose intolerance. You will add the enzyme lactase to solutions containing the milk sugar lactose, and then test for one of the reaction products, glucose, using glucose strips.

Introduction

The inability to digest lactose leads to lactose intolerance, which is a very unpleasant reaction to the presence of lactose in the digestive system, characterized by cramps, bloating, gas, and diarrhea.

Lactose is a key constituent of breast milk, so it is essential that babies are able to digest it, and they do. It accounts for approximately 40 percent of the total calories provided by breast milk. Babies are able to digest lactose because they produce lactase. Lactase is an enzyme that is present in the baby's digestive tract. Enzymes are protein molecules that function as catalysts, which vastly speed up the rates of chemical reactions.

Lactase catalyzes the breakdown of lactose into glucose and galactose. See Figure 1. Unlike lactose, glucose and galactose are readily absorbed by the small intestine.

Figure 1. The enzyme lactase catalyzes the breakdown of lactose into the smaller sugars, galactose and glucose.

For reasons that are not all that clear, people stop producing lactase after about age 2. Once the production of lactase stops, drinking milk or eating milk products, such as cheese, can cause uncomfortable digestive problems. In people with low levels of the enzyme lactase, the lactose sugars in milk pass through the gastrointestinal tract undigested or are partially digested by enzymes produced by intestinal bacterial flora to yield short-chain fatty acids, hydrogen, carbon dioxide, and methane. These undigested lactose molecules and products of bacterial digestion result in the symptoms of lactose intolerance.

For those who have trouble digesting food containing dairy products, the lactase enzyme is available in tablet form. The tablets are taken with the first bite of dairy food. The lactase enzyme in the tablet breaks down lactose in the dairy product, making the food easy to digest. The lactase enzyme is also available as a liquid. Adding a few drops of the enzyme to milk reduces the level of lactose, making the milk more digestible for people with lactose intolerance. Lactose-reduced milk is available at most grocery stores. The milk contains all of the nutrients found in regular milk, but the level of lactose has been reduced.

Lactose intolerance is not a rare syndrome; it is, in fact, "normal" in the sense that the majority of people around the world are lactose intolerant. Lactose tolerance—the ability to digest dairy products—is present in a minority of the world's population, and is associated primarily with people whose ancestry is derived from western or northern Europe.

Lactose tolerance is due to a genetic change that occurred a few thousand years ago in northern Europe. This genetic change resulted in the maintenance of lactase production into adulthood. The current scientific consensus is that this mutation was advantageous and thus able to spread rapidly through the population of Europe.

Between 30 and 50 million Americans are lactose intolerant (total population: ~ 305 million) and certain ethnic and racial populations are more affected than others. Up to 80 percent of African Americans, 80-100 percent of American Indians, and 90-100 percent of Asian Americans are lactose intolerant. The condition is least common among people of northern European descent.

Lactose intolerance is a fascinating subject because it involves a number of areas of scientific inquiry, including genetics, anthropology, and enzymology. In this human biology science fair project, you will use lactase to catalyze the breakdown of lactose in milk into glucose and galactose. The level of glucose that is formed by the activity of the lactase enzyme depends on the initial level of the lactose sugar in the milk. In other words, to determine the level of lactose in the milk, you will first convert it to glucose and galactose, then measure the level of glucose. You will use glucose strips, available at most drug stores, to test the level of glucose formed by the breakdown of lactose. In the variations, you can explore the enzymology of lactase activity in more detail.

Terms, Concepts and Questions to Start Background Research

• Lactose
• Lactose intolerance
• Lactase
• Enzyme
• Catalyst
• Glucose
• Galactose
• Hydrolysis
• Lactose tolerance
• Enzymology
• Positive control
• Negative control

Questions

• What fraction of the world's population is lactose intolerant?
• What is the genetic basis for lactose tolerance?
• What are some theories as to why humans become lactose intolerant after infancy?
• The glucose strip changes color in the presence of glucose. What happens on the strip to cause this change in color?
• How is regular milk treated to make it lactose-free?

Bibliography

• Patterson, K.D. (2008). Lactose Intolerance. The Cambridge World History of Food. Retrieved July 14, 2008 from http://www.cambridge.org/us/books/kiple/lactose.htm
• Sibley, E. (2008). Lactose Intolerance. National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. Retrieved July 15, 2008 from http://www.digestive.niddk.nih.gov/ddiseases/pubs/lactoseintolerance/

Materials and Equipment

• Test tubes (4), should hold about 1 ounce (oz) of fluid; alternatively, use a measuring cup to measure the amounts and pour the liquids into small plastic or glass cups.
• Glucose (also called dextrose); 4-gram glucose tablets are available at most drug stores. Note: Table sugar is sucrose, not glucose.
• Spoon
• Permanent market
• Masking tape
• Glucose test strips for urinalysis (8); available at most drug stores and at some grocery stores (Note: these strips must be for urinalysis. These are not the strips used to test blood.)
• Lab notebook
• Teaspoon
• Regular milk (the percent fat should not matter, but you should use the same percent fat for the regular and lactose-free milk)
• Lactose-free milk (the percent fat should not matter, but you should use the same percent fat for the regular and lactose-free milk), should be 100% lactose free; available at most grocery stores
• Lactase drops; available at most grocery or drugstores
• Stopwatch
• Water

Experimental Procedure

The first step is to make positive and negative control solutions. The positive control solution is 2% glucose in water. You will use the positive control to make sure the glucose strips are able to detect glucose. The negative control is just water. You will use the negative control to make sure the glucose test strips don't react to plain water.

1. Make the positive control solution of 2% glucose in water.
   1. Using a test tube, dissolve 2 g of glucose in 100 milliliters (mL) of water, or grind one glucose tablet with a spoon in 200 mL of water.
   2. Label the container 2% with a piece of masking tape and your permanent marker.
2. Add 100 mL of water to another test tube and label it Negative Control.
3. Test the positive and negative control solutions with the glucose strips.
   1. Dip separate strips into the glucose solution (positive control), and water (negative control).
   2. Wait for the length of time specified by the strip directions.
   3. Record any color changes of the strip and compare them to the key on the bottle to determine glucose concentration of the tested fluid. Record your observations in your lab notebook.
   4. You may be able to cut the strips in half to double the number available, depending on the type of strips you have.
4. You should see a clear positive reaction for the 2% glucose control and a clear negative reaction for the water control. If not, check your solutions and the glucose strips and try steps 1-3 again.

   The next step is to determine the level of glucose in regular milk and the level of glucose in lactose-free milk. Can you predict which one will have a higher glucose level?

5. Pour about 1 teaspoon (tsp) of regular milk and 1 tsp of lactose-free milk into clean, separate test tubes (or small glass or plastic containers). Label the test tubes accordingly.
6. Determine the glucose concentration in the regular milk sample.
   1. Following the directions that came with the glucose test strips, dip a strip into the milk sample. Wait for the length of time specified by the strip directions.
   2. Compare the color of the strip after dipping it in the milk with the color-coded key on the side of the bottle to determine the concentration of glucose in the milk.
7. Determine the glucose concentration in the lactose-free milk, using a fresh test strip.
8. Record your results in your lab notebook.

   What will happen when you add the enzyme lactase to the regular milk and to the lactose-free milk?

9. Add one drop of the lactase solution to the regular milk sample.
10. Warm the milk by rolling the tube back and forth in your hands for 2 minutes. Use the stopwatch to time yourself.
11. Repeat the glucose test with a fresh test strip.
12. Record your result in your lab notebook.
13. Add one drop of the lactase solution to the lactose-free milk sample.
14. Again, warm the milk by rolling the tube back and forth in your hands for 2 minutes. Repeat the glucose test with a fresh test strip.
15. Record your results in your lab notebook.
16. Repeat steps 5-16 two more times so that you have three trials for each milk sample, both before and after adding lactase. Be sure to clean out your test tubes carefully before you begin new trials. Always use fresh test strips.
17. Explain your results.
    1. Is there a difference in glucose concentration between regular milk and lactose-free milk before addition of the lactase? Why?
    2. Do the glucose concentrations of regular milk and lactose-free milk change after the addition of the lactase? Why?

Variations

• Besides cow's milk, what other foods (goat's milk, soy milk, etc.) have lactose? Design tests to determine if the products actually contain lactose.

  The following variations depend on getting accurate, quantitative readings for the glucose level in solutions.

• You can use the calibration strip that came with your glucose strips to estimate glucose concentration.
  • Alternatively, you can make a series of glucose solutions that can function as standards. You can then measure the glucose in your test solutions by comparing the color change in the test solution to the standards (make your best guess when the test color is in between two standards). For example, you could make standard solutions with 0, 0.25%, 0.5%, 1.0%, 2.0% and 4.0% glucose. The positive controls should bracket your test solution. So if the test solution (milk treated with lactase, for example) has 0.5% glucose, make sure your standard solutions have concentrations both above and below this concentration. The standard solutions can be made in water (or in glucose-free milk).
• Take careful note of the time between dipping the glucose strip and reading the result. Control for other factors, such as pH, temperature, etc, to get accurate results. If you need to pipette small volumes accurately, ask your science teacher for help in obtaining good pipettors.
• How does the glucose concentration in regular milk change with time after you add the lactase? For example, what is the glucose concentration at 30 seconds, 1 minute, 2 minutes, and 5 minutes? Draw a graph of glucose concentration vs. time. When would you predict the rate of glucose production is fastest?
• Design an experiment where you determine how the reaction rate for glucose production depends on temperature. Vary the temperature, but keep everything else the same. What temperature would you predict is optimal for enzyme activity? At what temperature is the enzyme inactivated?
• Design an experiment where you determine how the reaction rate depends on pH.

• For more science project ideas in this area of science, see Biotechnology Project Ideas.

Credits

David Whyte, PhD, Science Buddies

This project is based on the following project: Kalumuck, K. (n.d.). Milk Makes Me Sick. Exploratorium Snacks. Retrieved July 10, 2008 from http://www.exploratorium.edu/snacks/milk_makes-me_sick/index.html

Last edit date: 2008-10-06 00:00:00

Career Focus

If you like this project, you might enjoy exploring careers in Biotechnology.

	Biochemist Growing, aging, digesting—all of these are examples of chemical processes performed by living organisms. Biochemists study how these types of chemical actions happen in cells and tissues, and monitor what effects new substances, like food additives and medicines, have on living organisms.			Agricultural Technician As the world's population grows larger, it is important to improve the quality and yield of food crops and animal food sources. Agricultural technicians work in the forefront of this very important research area by helping scientists conduct novel experiments. If you would like to combine technology with the desire to see things grow, then read further to learn more about this exciting career.
	Biological Technician What do the sequencing of the human genome, the annual production of millions of units of life-saving vaccines, and the creation of new drought-tolerant rice varieties have in common? They were all accomplished through the hard work of biological technicians. Scientists may come up with the overarching plans, but the day-to-day labor behind biotech advances is often the work of skilled biological technicians.

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